protease-deficient filamentous fungal cells and methods of using them

专利摘要:
protease-deficient filamentous fungal cells and methods of using them. the present invention relates to a filamentous fungal cell comprising at least three endogenous proteases having reduced activity and a recombinant polynucleotide encoding a heterologous polypeptide, as well as methods for improving the stability of a heterologous polypeptide-producing polypeptide.
公开号:BR112014016587A2
申请号:R112014016587-4
申请日:2013-01-04
公开日:2020-10-27
发明作者:Christopher Landowski；Markku Saloheimo；Anne Huuskonen；Juhani Saarinen；Ann Westerholm-Parvinen；Anne Kanerva；Jari Natunen；Anna-Liisa Hänninen；Noora Salovuori；Merja Penttilä
申请人:Novartis Ag；Glykos Finland Oy；
IPC主号:

专利说明:

[001] [001] The present invention relates to compositions and methods useful for the production of heterologous proteins in filamentous fungal cells. BACKGROUND
[002] [002] Post-translational modification of eukaryotic proteins, particularly therapeutic proteins, such as immunoglobulins, is often necessary for protein folding and proper function. Since conventional prokaryotic expression systems lack the proper machinery necessary for such modifications, alternative expression systems have to be used in the production of these therapeutic proteins. Even where eukaryotic proteins do not have post-translational modifications, prokaryotic expression systems often lack the chaperone proteins necessary for proper folding. Yeast and fungi are attractive options for protein expression, since they can be easily grown on a large scale in simple media, which allows low production costs and yeasts and fungi have post-translational machinery and chaperones that perform functions similar to those found in mammalian cells. In addition, tools are available to manipulate the relatively simple genetic makeup of yeast and fungal cells, as well as more complex eukaryotic cells, such as mammalian or insect cells (De Pourca et al., Appl Microbiol Biotechnol, 87 ( 5): 1617-31). Despite these advantages, many therapeutic proteins are still being produced in mammalian cells, which produce therapeutic proteins with post-translational modifications more similar to proteins.
[003] [003] To solve this deficiency, new strains of yeasts and fungi are being developed that produce post-translational modifications that are more similar to those found in native human proteins. Thus, there has been renewed interest in using yeast and fungal cells to express more complex proteins. However, due to the industry's focus on mammalian cell culture technology for such a long period of time, fungal cell expression systems, such as Trichoderma, are not as well established as culture of mammalian cells and therefore suffer from deficiencies when expressing mammalian proteins.
[004] [004] Thus, there remains a need in the art for improved filamentous fungal cells, such as Trichoderma fungus cells, which can steadily produce heterologous proteins, such as immunoglobulins, preferably at high levels of expression. SUMMARY
[005] [005] Compositions described here, including filamentous fungal cells, such as Trichoderma fungal cells, having little or no detectable activity of at least three proteases and which have a recombinant polynucleotide encoding a heterologous polypeptide that is produced in increased levels. Also described here are methods for improving the stability of the heterologous polypeptide and methods of preparing heterologous polypeptides in which the proteases do not have reduced activity.
[006] [006] Thus, one aspect includes filamentous fungal cells having reduced or no detectable activity of at least three products.
[007] [007] In certain embodiments, the level of expression of at least three proteases is reduced or eliminated. In certain embodiments, genes encoding the three proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain modalities that can be combined with the preceding modalities, the three genes that encode protease are pep1, tsp1 and sip1. In other embodiments, the three genes that encode protease are gap1, sip1 and pep1.
[008] [008] In certain modalities, fungal cells have little or no detectable activity of four endogenous proteases; genes encoding the four proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain modalities that can be combined with the preceding modalities, the four genes that encode proteases are pep1, tsp1, sIp1 and gap1.
[009] [009] In certain modalities that can be combined with the preceding modalities, the three or four genes that encode
[0010] [0010] In other embodiments, fungal cells have reduced or no detectable activity from five endogenous proteases; genes encoding the five proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain modalities that can be combined with the preceding modalities, the five genes that encode protease are pep1, tsp1, sip1, gap1 and pep4. In other modalities, the five genes that encode protease are pep1, tsp1, sIp1, gap1 and gap2.
[0011] [0011] In certain modalities, fungal cells have little or no detectable activity of six endogenous proteases; genes encoding the six proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain embodiments, the cell has six protease-encoding genes, each of which comprises a mutation that reduces or eliminates the corresponding protease activity and the six protease-encoding genes are pep1, tsp1, sip1, gap1, gap2 and pep4.
[0012] [0012] In certain modalities that can be combined with the preceding modalities, fungal cells have three to six proteases having little or no detectable activity, each of the three to six proteases selected from pep1, pep2, pep3, pep4, pepº5, tsp1 , sip1, sip2, sIp3, gap1 and gap2.
[0013] [0013] In certain modalities that can be combined with the preceding modalities, the cell has seven genes that encode proteases, each of which comprises a mutation that reduces or eliminates the corresponding protease activity and the seven genes that encode proteases are pep1, tsp1, sip1, gap1, gap2, pep4 and pep3.
[0014] [0014] In certain modalities that can be combined with the preceding modalities, the cell has eight genes that encode proteases, each of which comprises a mutation that reduces or eliminates the corresponding protease activity and the genes that encode eight proteases are pep1, tsp1, sip1, gap1, gap2, pep4, pep3 and pepo5.
[0015] [0015] In certain modalities that can be combined with the preceding modalities, the fungal cell has an additional protease having reduced activity, the gene encoding the additional protease comprises a mutation that reduces or eliminates the corresponding protease activity and the protease additional is selected from pep7, pep8, pep11, pep12, top1, gap2, sIp3, sIp5, sIp6, sip7 and sip8.
[0016] [0016] In certain modalities that can be combined with the preceding modalities, the heterologous polypeptide is a mammalian polypeptide. In certain embodiments, the mammalian polypeptide is glycosylated.
[0017] [0017] In certain embodiments, the mammalian polypeptide is selected from an immunoglobulin, an antibody and its antigen-binding fragments, a growth factor, an interferon, a cytokine and an interleukin. In certain embodiments, the mammalian polypeptide is an immunoglobulin or an antibody. In certain modalities, the mammalian polypeptide is selected from insulin-like growth factor 1 (Insulin-like Growth Factor 1- IGF1), human growth hormone (human Growth Hormone - hGH) and interferon alfa 2b (IFNa2b).
[0018] [0018] In certain modalities that can be combined with the preceding modalities, the heterologous polypeptide is a non-mammalian polypeptide. In certain embodiments, the non-mammalian polypeptide is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase , invertase, laccase, lipase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phosphatase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylanase.
[0019] [0019] In certain modalities that can be combined with the preceding modalities, the fungal cell additionally contains reduced or no detectable activity of ALG3, a mannosyl transferase enzyme. In certain embodiments, the gene encoding ALG3 contains a mutation that reduces or eliminates the corresponding activity. In certain modalities that can be combined with the preceding modalities, the fungal cell also contains a polynucleotide that encodes an α-1,2-mannosidase.
[0020] [0020] In certain modalities that can be combined with the preceding modalities, the fungal cell has a mutation that reduces the expression of a desired protease to have reduced activity. In certain modalities that can be combined with the preceding modalities, the mutation is a deletion in the gene that encodes the protease. In certain modalities that can be combined with the preceding modalities, the mutation is a deletion of the portion of the gene that encodes the proteinase catalytic domain. In certain modalities that can be combined with the preceding modalities, the fungal cell has a point mutation in the portion of the gene that encodes the catalytic protease domain.
[0021] [0021] In other modalities, the reduction or elimination of
[0022] [0022] In certain modalities that can be combined with the preceding modalities, the fungal cell still contains a catalytic domain of N-acetylglucosaminyl transferase | and a catalytic domain of N-acetylglucosaminyl transferase Il. In certain modalities, the catalytic domain of N-acetylglucosaminyl transferase 1 and the catalytic domain of N-acetylglucosaminyl transferase 1 are encoded by a polynucleotide. In certain embodiments, the catalytic domain of N-acetylglucosaminyl transferase | it is encoded by a first polynucleotide and the catalytic domain of N-acetylglucosaminyl transferase | 1 is encoded by a second polynucleotide. In certain modalities that can be combined with the preceding modalities, the fungal cell also contains a polynucleotide that encodes an Il mannosidase and / or a galactosyl transferase. In certain embodiments, the fungal cell contains enzymes selected from the group consisting of a-1,2 mannosidase, N-acetylglucosaminyl transferase |, N-acetylglucosaminyl transferase |, manosidase | and / or galactosyl transferase, said enzymes further comprising an objectification peptide, for example, a heterologous objectification peptide for appropriate localization of the corresponding enzyme. In certain embodiments, the targeting peptide is selected from SEQ ID NOs: 589-594.
[0023] [0023] In certain modalities that can be combined with the preceding modalities, the fungal cell is a fungal cell
[0024] [0024] In certain modalities that can be combined with the preceding modalities, the fungal cell is of the wild type for pep4 protease.
[0025] [0025] Another aspect includes methods to improve the stability of the heterologous polypeptide by: a) supplying the filamentous fungal cell of any of the preceding modalities; and b) cell culture so that the heterologous polypeptide is expressed, where the heterologous polypeptide has increased stability compared to the heterologous polypeptide produced in a corresponding parental filamentous fungal cell in which the proteases have no reduced activity, for example, do not contain mutations in the genes encoding proteases. Another aspect includes methods of preparing a heterologous polypeptide by: a) supplying the filamentous fungal cell of any of the preceding modalities; b) culturing the host cell so that the heterologous polypeptide is expressed; and c) purification of the heterologous polypeptide. In certain embodiments that can be combined with the preceding embodiments, the additional filamentous fungal cell contains a carrier protein. In certain modalities, the carrier protein is CBH1. In certain modalities that can be combined with the preceding modalities, the culture is in a medium that comprises a protease inhibitor. In certain embodiments, the culture is in a medium that has one or two protease inhibitors selected from SBT1 and chemostatin. In certain embodiments, the heterologous polypeptide
[0026] [0026] Another aspect includes Trichoderma fungal cells having reduced or no detectable activity of at least three proteins selected from pep1, pep2, pep3, pep4, pep5, tsp1, sip1, sIp2, gap1 and gap2, where the cell additionally contains a recombinant polynucleotide that encodes a mammalian polypeptide produced at a level at least twice as high as the level of production of the polypeptide in a corresponding parental Trichoderma fungal cell.
[0027] [0027] In certain embodiments, the level of expression of at least three proteases is reduced or eliminated in the Trichoderma fungal cell. In certain embodiments, the genes encoding at least three proteases each comprise a mutation that reduces or eliminates the corresponding protease activity in the Trichoderma fungal cell. In certain modalities, the Trichoderma fungal cell includes three genes that encode protease with a mutation that reduces or eliminates protease activity, which are selected from gap1, sip1 and pep1. In certain modalities that can be combined with the preceding modalities, the mammalian polypeptide in the fungal cell of Trichoderma is an antibody or its antigen-binding fragments or an immunoglobulin and the at least three proteases are selected from pep7, pep3 , pep4, tsp1, sip1, sip2, gap1 and gap2. In certain modalities, the Trichoderma fungal cell contains four genes that encode proteases, each of which comprises a mutation that reduces or eliminates the corresponding protease activity and the four genes that encode protease with such mutations are pep1, tsp1 , sip1 and gap1. In certain embodiments, the Trichoderma fungal cell has five genes that encode proteases, each of which comprises a mutation that reduces or eliminates the corresponding protein activity and the five genes that encode protease with such mutations are pep1, tsp1 , sip1, gap1 and pep4. In certain modalities that can be combined with the preceding modalities, the mammalian polypeptide in the Trichoderma fungal cell is a growth factor, interferon, a cytokine or interleukin and the three proteases with reduced activity are selected from pep1, pep2, pep3 , pep4, pep5, pep8, pep11, pep12, gap1, gap2, sIip1, sip2, slpT and tsp1. In certain embodiments, the Trichoderma fungal cell has five genes that encode proteases, each of which comprises a mutation that reduces or eliminates the corresponding protease activity and the five genes that encode protease with such mutations are pep1 , tsp1, sip1, gap1 and gap2. In certain modalities, the Trichoderma fungal cell has six genes that encode proteases, each of which comprises a mutation that reduces or eliminates the corresponding protease activity and the six genes that encode protease with such mutations are pep1, tsp1, sip1 , gap1, gap2 and pep4. In certain modalities that can be combined with the preceding modalities, the Trichoderma fungal cell has seven genes that encode proteases, each of which comprises a mutation that reduces or eliminates the corresponding protease activity and the seven genes that encode proteases are pep1, tsp1, sip1, gap1, gap2, pep4 and pep3. In certain modalities that can be combined with the preceding modalities, the Trichoderma fungal cell has eight genes that encode proteases, each of which comprises a mutation that reduces the corresponding protease activity and the eight genes that encode protease with such mutations are pep1, tsp1, sip1, gap1, gap2, pep4, pep3 and pep5.
[0028] [0028] In certain modalities that can be combined with the preceding modalities, the Trichoderma fungal cell still contains reduced or undetectable activity of one or more additional proteases. In certain embodiments, the level of expression of one or more additional proteases in the Trichoderma fungal cell is reduced or eliminated. In certain embodiments, genes encoding one or more additional proteases in the Trichoderma fungal cell each have a mutation that reduces or eliminates the corresponding protease activity. In certain modalities that can be combined with the preceding modalities,
[0029] [0029] In certain modalities that can be combined with the preceding modalities, the Trichoderma fungal cell still contains reduced or no detectable activity of ALG3. In certain embodiments, the gene encoding ALG3 in the Trichoderma fungal cell contains a mutation that reduces or eliminates the corresponding activity. In certain modalities that can be combined with the preceding modalities, the Trichoderma fungal cell also contains a polynucleotide that encodes an al, 2-mannosidase. In certain modalities that can be combined with the preceding modalities, the mutation reduces or eliminates the expression of the gene in the Trichoderma fungal cell. In certain modalities that can be combined with the preceding modalities, the mutation is a deletion of the gene in the Trichoderma fungal cell. In certain modalities that can be combined with the preceding modalities, the mutation is a deletion of the portion of the gene that encodes the protease catalytic domain of the Trichoderma fungal cell. In certain modalities that can be combined with the preceding modalities, the mutation is a point mutation in the portion of the gene that encodes the catalytic protease domain of the Trichoderma fungal cell. In certain modalities that can be combined with the preceding modalities, the Trichoderma fungal cell also contains a catalytic domain of N-acetylglucosaminyl transferase | and a catalytic domain of N-acetylglucosaminyl transferase | 1. In certain embodiments, the catalytic domain of N-acetylglucosamini! transferase | and the catalytic domain of N-acetylglucosaminyl transferase Il are encoded by a polynucleotide from the Trichoderma fungal cell. In certain embodiments, the catalytic domain of N-acetylglucosaminyl transferase | it is encoded by a first polynucleotide and the catalytic domain of N-acetylglucosaminyl transferase II is encoded by a second polynucleotide from the Trichoderma fungal cell. In certain modalities that can be combined with the preceding modalities, the Trichoderma fungal cell also contains a polynucleotide that encodes an Il mannosidase. In certain modalities, the protections each have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1, 17, 37, 58, 66, 82, 98, 118, 129, 166 and
[0030] [0030] Another aspect includes methods to improve the stability of the heterologous polypeptide by: a) supplying the Trichoderma fungal cell of any of the preceding modalities; and b) cell culture so that the heterologous polypeptide is expressed
[0031] [0031] Figure 1 represents a PAGE gel that shows fractions eluted from purification in an aspartic protein affinity column.
[0032] [0032] Figure 2 represents a PAGE gel that shows the results of incubation of IgG with aspartic proteases.
[0033] [0033] Figure 3 represents Southern blot analysis showing the generation of single strains with protease deletion M181 and M195. Figure 3A represents the expected ORF signal from pep1:> 8 kb of parental M127, no signal from transformants. Figure 3B represents the expected signal of 5'flanking pep1:> 8 kb of parental M127, 4 kb of transformants. Figure 3C represents the expected 3 'flanking signal from pep1:> 8 kb of parental M127, 4.2 kb of transformants.
[0034] [0034] Figure 4 represents Southern blot analysis showing the generation of rituximab antibody in the M182 strain with pep1 deletion. Figure 4A represents the expected ORF signal from pep1:> 8 kb of parental M169, no signal from transformants. Figure 4B represents the expected bar signal. 1.0 + 1.7 kb of transformants, 3.1 kb of pTTv41, no M169. Figure 4C represents the expected bar signal. 1.8 + 2.8 kb of transformants, 3.1 kb of pTTv41, no M169.
[0035] [0035] Figure 5 represents a protein gel showing peak fractions of aspartic protease purifications from a strain containing pep1 and a strain containing Apep1.
[0036] [0036] Figures 6A-B represent an immunoblot illustrating that the exclusion of pep2 protease from the rituximab M169 production strain enhanced the production of light (A) and (B) heavy chains in the 206A transformer (M455 strain). The bands representing the 18 kD light chain fragment and the 38 kD heavy chain fragment were more intense in the M455 strain compared to the parental M169 strain.
[0037] [0037] Figure 7 graphically represents the protease activity of the supernatant of the rituximab production strain M169 and of the transformants 98A, 116A, 198A, 201A, 206A and (M455) with pep2 protease deletion. Transformants 116A, 198A and 206A show reduced protease activity against casein in relation to their parental M169 strain.
[0038] [0038] Figure 8 represents an immunoblot showing the effects of the protease activity of PEP3 and PEP7 on the heavy chain of MABO1 and native IGF-1. Figure 8A represents the effects of protease activity on MABO1 at a pH of 5.5. Figure 8B represents the effects of protease activity on MABO1 at a pH of 4.5. Figure 8C represents the effects of protease activity on native IGF-1 at a pH of 4.5.
[0039] [0039] Figure 9 depicts a PAGE gel showing protein containing fractions purified from a SIP peptide affinity column.
[0040] [0040] Figure 10 represents an immunoblot showing SIP protease activity on the MABO1 heavy chain.
[0041] [0041] Figure 11 graphically represents the protein activity against casein with and without inhibitors.
[0042] [0042] Figure 12 represents an immunoblot showing the production levels of MABO1 heavy and light chains after deletion of each of the s / p1, sip2, slp3 and gap1 proteases. Figure 12A shows MABO1 heavy chain production. Figure 12B shows MABO1 light chain production.
[0043] [0043] Figure 13 graphically represents the times of improvement in the production of MABO1 heavy and light chains after deletion of each of the proteases sip1, sip2, sIp3 and gap1. Each bar represents the average from several of the clones shown in Figure
[0044] [0044] Figure 14 represents an immunoblot that shows the levels of MABO1 production from the M244 strain with gap2 deletion. Figure 14A shows the MABO1 Heavy Chain (HC) production. Figure 14B shows the MABO1 Light Chain (LC) production.
[0045] [0045] Figure 15 represents an immunoblot showing the MABO1 antibody levels after incubation with the Pichia supernatant containing the GAP2 protease.
[0046] [0046] Figure 16 represents an immunoblot that shows the level of protease degradation in human IgG1.
[0047] [0047] Figure 17 represents the results of a zymogram of the MABO antibody from affinity purification with an aminobenzamidine column (purified fractions) and supernatant samples (supernatant).
[0048] [0048] Figure 18 represents the generation of the M219 strain with deletion of Apep1Atsp1 double protease. Figure 18A represents the expected ORF signal of tsp1: 6.4 kb of parental M196. Figure 18B represents the expected signal of 5'flanking of tsp71: 3.9 kb of trans-
[0049] [0049] Figure 19 represents a Southern blot analysis showing the generation of the M194 strain with Apep1Atsp2 double deletion. Figure 19A represents the expected tsp1: kb ORF signal from parent M181. Figure 19B represents the expected bar signal. 1.4 + 2.5 kb of transformants, 2.9 kb of pTTv42, no M181. Figure 19C represents the expected bar signal. 1.9 + 3.2 kb of transformants, 2.9 kb of pTTv42, no M181.
[0050] [0050] Figure 20 graphically represents the normalized protease activity data from culture supernatants from each of the supernatants with protease deletion and the parental strain M124. Protease activity was measured at a pH of 5.5 in the first five strains and at a pH of 4.5 in the last three strains with deletion. Protease activity is against green fluorescent casein. The six protease deletion strain has only 6% activity of the parental wild-type strain and the 7 protease deletion strain had about 40% less activity than the 6 protease deletion strain.
[0051] [0051] Figure 21A represents the results of a MABO2 zymogram with fractions purified on aminobenzamidine from the fermentation supernatants. Figure 21B represents an SDS PAGE gel (7%) of aminobenzamidine purified fractions from fermentation supernatants.
[0052] [0052] Figure 22 represents the results of a MABO2 zymogram assay with SBTI affinity purified fractions containing proteases. The main proteolytic activities appear in white, where the protease degraded the MABO 2 antibody. Concentrated fraction 3 (cf3) and non-concentrated fractions 1-4 (fl-f4) were passed on the zymogram gel.
[0053] [0053] Figure 23 represents an SDS PAGE gel showing affinity purified fractions of SBTI containing proteases. The concentrated cf3 and cf4 fractions are shown on the gel.
[0054] [0054] Figure 24 illustrates an immunoblot showing the level of degradation of rituximab heavy chain by proteases purified in SBTI.
[0055] [0055] Figure 25 represents an immunoblot that shows the level of antibody degradation when incubated overnight with Pichia supernatants containing subtilisin. Figure 25A shows the degradation of rituximab heavy chain by protease. Figure 25B shows the MABO1 heavy chain degradation by protease.
[0056] [0056] Figure 26 represents Southern blot analysis showing the generation of the M277 strain with triple protease deletion. Figure 26A represents the expected s / p1 ORF signal: 6.5 kb of parental (M219, M228) only. Figure 26B represents the expected 5'-s / p1 flanking signal: 6.5 kb of parents, 3.3 kb of transformants, 4.4 kb of the control plasmid pTTv126. Figure 26C represents the expected 3'-s / p1 flanking signal: 6.5 kb of parents, 2.3 kb of transformants, 4.4 kb of the control plasmid pTTv126.
[0057] [0057] Figure 27 represents a MABO2 zymogram assay that shows the activity of the supernatants of the strain with a protease deletion. White regions on the colored gel indicate an area of protease activity.
[0058] [0058] Figure 28 graphically represents the total protease activity of culture supernatants with protease deletion compared to the wild type M124 activity.
[0059] [0059] Figure 29 shows Southern blot analysis showing the generation of the M307 strain with quadruple protease deletion. Figure 29A represents the expected ORF signal from parental gap1: 4 kb
[0060] [0060] Figure 30 graphically represents the total protease activity in strains with triple and quadruple deletion compared to the wild type M124 strain.
[0061] [0061] Figure 31 graphically represents the protein activity over time between the M304 strain with triple deletion and the M371 strain with quadruple deletion.
[0062] [0062] Figure 32 represents a Southern blot analysis showing the generation of the M369 strain with a five-fold protease deletion. Figure 32A represents the expected gap2 ORF signal: 4.9 kb of parental (M307). Figure 32B represents the expected 5'-gap2 flanking signal: 4.9 kb of parental, 2.3 kb of transformants, 2.3 kb of the control plasmid pTTv145. Figure 32C represents the expected 3 'gap2 flanking signal: 4.9 kb of parental, 3.8 kb of transformers, 3.8 kb of the control plasmid pTTv145. Figure 32D represents Southern blot analysis showing the generation of pyr4- from the M369 strain with a five-fold protease deletion, resulting in the M381 strain (clone 14). The expected signal is 5 'gap2 flanking: 1.5 kb from all strains, 4.1 kb from the control plasmid pTTv145. Figure 32E represents Southern blot analysis showing the generation of pyr4- from the M369 strain with five-fold protease deletion, resulting in the M381 strain (clone 14). The expected signal is 3 'gap2 flanking: 3.6 kb of M307, 2.7 kb of M369 + l / oopout clones, 3.8 kb of the control plasmid pTTv145.
[0063] [0063] Figure 33 graphically represents the protein activity
[0064] [0064] Figure 34 represents Southern blot analysis showing the generation of strains M396 and M400 with deletion of 6 proteases. Figure 34A represents the expected pep4 ORF signal: 6.3 kb from M307 and M369. Figure 34B represents the expected pep4 ORF signal: 6.3 kb from M307 and M369, no transformants signal. Figure 34C represents the expected signal of 5'flanking of pep4: 6.3 kb of M307 and M369, 4.8 kb of transformants, 4.0 kb of pTTv181. Figure 34D represents the expected 3 'pep4 flanking signal: 6.3 kb of M307 and M369, 2.1 kb of transformants, 4.0 kb of pTTv181. Figure 34E represents Southern blot analysis showing the generation of pyr4- from M396 strains with 6 protease deletion. The expected signal is 3 'pep4 flanking: 6.3 kb of M307 and M369, 2.1 kb of repurified transformers, 4.9 kb of clones / loopout.
[0065] [0065] Figure 35 represents an immunoblot showing the amount of rituximab heavy chain fragments created in vitro by supernatant proteases.
[0066] [0066] Figure 36 represents an immunoblot showing the degradation of heavy and light chain samples of supernatant from cultures treated with SBTI and untreated controls. Figure 36A shows heavy chain degradation. Figure 36B shows the light chain degradation.
[0067] [0067] Figure 37 represents an immunoblot showing the level of heavy and light chain degradation of supernatant samples from cultures treated with chemostatin and pepstatin A or from cultures
[0068] [0068] Figure 38 represents the process of purifying antibodies from T. reesei culture supernatants.
[0069] [0069] Figure 39 represents an immunoblot showing the improved stability of the heavy (Heavy Chain - HC) and light (Light Chain - LC) chains of antibody from T. reesei cells containing a pep1 protease deletion. Three model antibodies were tested on the supernatant in a large shaking flask (Apep1 and M124) and fermentation supernatant (pH 5.5; 28 O; 20 g / L of grain extract consumed, 60 g / L of lactose ).
[0070] [0070] Figure 40 shows an immunoblot showing enhanced production of the heavy chain of rituximab (Rx) from T. reesei cells containing a deletion of the tsp1 protease. Transformants 12-2A and 12-16A clearly show more heavy chain compared to the parental strain.
[0071] [0071] Figure 41 represents an immunoblot showing the reduced degradation of MABO1 heavy chain after incubation overnight with supernatant strain M277 with triple protein deletion. After overnight incubation in the culture supernatant of day 5, there was 2.5 times more heavy chain found in the supernatant with triple protease deletion compared to the supernatant of the control M124 strain, which has no protease deletions. When incubated in the 7-day culture supernatant, there was 4 times more heavy chain found in the supernatant with triple protease deletion compared to the supernatant of the control M124 strain.
[0072] [0072] Figure 42 represents a study of degradation of model proteins. Undiluted supernatant of the strain with the deletion of 6 proteases was used at a pH of 4.2 for enrichment in pure model proteins (0.05 ug / ul). 50 mM sodium citrate, pH 4.0, enriched
[0073] [0073] Figure 43 represents the stability testing of the MABO1 antibody heavy chain in supernatants of the strain with 6 protease deletion. MABO1 antibody was present in the undiluted supernatant at 0.05 µg / ul. 10 µl of each sample was loaded onto a 4-20% SDS PAGE gel. The heavy chain was stable after a 20 hour incubation at 37 ° C (C in the strain supernatant with a deletion of 6 proteases at a pH of 4.2. The heavy chain was detected with AP-conjugated IgG heavy chain antibody (Sigma tHA3188) diluted 1: 30,000 in TBST The full length heavy chain migrated to 50 kD on the gel.
[0074] [0074] Figure 44 represents samples of 4 days of human growth hormone cultures in 24 wells with and without inhibitors and supplements. 12 ul of each supernatant was loaded. Primary mouse anti-hGH antibody from Acris, catalog XAMOO0401PU-N (diluted to 2 µg / ml in TBST) and secondary goat anti-IgG mouse antibody from BioRad AP-conjugated (fH170-6520) diluted 1: 10000. HGH standard (200 ng), Abeam catalog ttab51232. The full-length hGH protein migrates to 22 kD.
[0075] [0075] Figure 45 represents a phylogeny of aspartic proteases from T. reesei, Myceliophthora thermophila, Neurospora crassa, Pekinillium chrysogenum, Aspergillus oryzae, A. nidulans and A. niger. The alignment was created with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and the tree was calculated using the average distance with BLOSUM6 2.
[0076] [0076] Figure 46 represents a phylogeny of subtilisable proteases
[0077] [0077] Figure 47 represents a phylogeny of glutamic proteases from T. reesei, Myceliophthora thermophila, Neurospora crassa, Pekinillium chrysogenum, Aspergillus oryzae, A. nidulans and A. niger. The alignment was created with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and the tree was calculated using the average distance with BLOSUM6 2.
[0078] [0078] Figure 48 represents a phylogeny of sedolinsin proteases from T. reesei, Myceliophthora thermophila, Neurospora crassa, Penicillium chrysogenum, Aspergillus oryzae, A. nidulans and A. niger. The alignment was created with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and the tree was calculated using the average distance with BLOSUM62. Since slp7 resembles sedolysin proteases, it is included in the tree. Sequences of Aspergillus fumigatus are included to assist in determining the relationships between sedolysins. The abbreviations sedA / B / C / D / E in front of each protease are based on Reichard et al. (2006) AP- PLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 72, pages 1739-1748, Figure 4, from which BLAST research with A. fumigatus sylysis for the corresponding protease was recovered.
[0079] [0079] Figure 49: A: Schematic drawings for the expression plasmids pTTv67 and pTTv99. The MABO1 heavy chain is con-
[0080] [0080] Figure 50: A: Western blot analysis of MABO1 heavy and light chain production at a pH of 5.2 in a continuous fermentation of the M507 strain of MABO1 production. The antibodies used were Sigma A3188 against the heavy chain (blot on the left) and Sigma A3813 against the light chain (blot on the right), both in dilutions of 1: 10,000. Sample codes denote the fermentation time in days. 0.1 ul of supernatant was placed in each lane in both blots. B: Western blot analysis of MABO1 heavy and light chain production in the batch fermentation of the M507 strain of MABO1 production at a pH of 5.5. The antibodies used were Sigma A3188 against the heavy chain (left blot) and Sigma A3813 against the light chain (right blot), both in dilutions of 1: 10,000. Sample codes denote the fermentation time in days. 0.1 ul of supernatant was placed in each lane in both blots.
[0081] [0081] Figure 51. Western blot analysis of MABO1 heavy and light chain production in M304 strains in the discontinuous fermentation of bio00503b at a pH of 5.5. The antibodies used were Sigma A3188 against the heavy chain and Sigma A3813 against the light chain. On day 8 from the M304 fermentation, bio00477b was included as a control. Sample codes denote the fermentation time in days. 0.1 ul of supernatant was applied to both blots. The uppermost immunoblot is the heavy chain and the lower immunoblot is the light chain.
[0082] [0082] Figure 52. The pTTv204 RNAi expression vector.
[0083] [0083] Figure 53: Immunoblot that detects MABO1 heavy chain production in strains expressing RNAi that express RNAi that causes slp2 knockdown.
[0084] [0084] Figure 54A represents the quantification of the level of expression of IFN-a 2b from the sample of 3 days of fermentation of MA401. 1 ul / 2 ul / 4 ul of the supernatant was loaded onto a 4-20% SDS PAGE gel. Immunoblot was performed with Abeam anti-| FN-a 2b antibody (ftab9386) diluted to 1 µg / ml in TBST. The goat anti-IgG mouse secondary antibody from the AP-conjugated BioRad (8170-6520) was diluted 1: 5000 in TBST. The protein patterns were loaded onto the gel corresponding to 50 ng, 100 ng and 200 ng of full-length IFN-a 2b. Densitometric quantification was performed using the Totallab Quant TL100 software. For quantification, the 2 ul sample was more representative. Control of full length IFN-a 2b (100 ng) migrates to 19.3 kD and IFN-a 2b attached to vehicle at 70 kDa.
[0085] [0085] Figure 54B describes immunoblot analysis for samples from days 3-6 of M577 and M652 fermentation cultures. 0.2 µl of growth supernatant was loaded onto a 4-20% SDS PA-GE gel. Immunoblot was performed with Abeam anti-| IFN-a 2b antibody (ttab9386) diluted to 1 µg / ml in TBST. The secondary anti-IgG mouse antibody from AP-conjugated mouse from BioRad (% 170-6520) was diluted 1: 5000 in TBST. Control of full length IFN-a 2b (100 ng) migrates to 19.3 kD and IFN-a 2b attached to vehicle at 70 kDa.
[0086] [0086] Figure 55 represents the quantification of the level of expression of IFN-a 2b from the samples of day 4 (fermentation M577) and day 3 (fermentation M652). 0.05 µl and 0.1 µl of supernatant were loaded from each sample onto a 4-20% SDS PAGE gel. Immunoblot was made with anti-IFN-a 2b antibody from Abeam (ttab9386) diluted to 1 µg / ml in TBST. BioRad AP-conjugated goat anti-IgG goat secondary antibody (f% 170-6520) was diluted 1: 5000 in TBST. The protein patterns were loaded onto the correct gel
[0087] [0087] The present invention relates to improved methods of producing recombinant heterologous polypeptides in filamentous fungal cells that have reduced or none of at least three proteases. The present invention is based, in part, on the surprising discovery that reducing the activity of a specific combination of endogenous proteases in filamentous fungal cells increases the expression and stability of a variety of recombinantly expressed heterologous proteins, such as like immunoglobulins and growth factors. Although others have created Trichoderma fungal cells with one or more inactivated proteases, they have not provided guidance on which proteases are most relevant for increasing the expression and stability of specific types of proteins, such as mammalian proteins. For example, document WO2011 / 075677 describes certain proteins that can over knock out in Trichoderma and also describes Trichoderma fungal cells that are deficient in several proteins. However, WO2011 / 075677 does not provide any guidance on which of the proteases have a negative impact on the expression and stability of mammalian proteins, such as immunoglobulins or growth factors, since no example of expression of any mammalian proteins is described therein. In addition, WO2011 / 075677 describes only heterologous expression of a single fungal protein in each of three different fungal strains deficient in a single protease. At-
[0088] [0088] The Applicants surprisingly show that several proteases are relevant for reducing total protease activity, increasing production of heterologous proteins and stabilizing heterologous proteins after expression in filamentous fungal cells, such as Trichoderma fungal cells. In particular, inventors have identified proteases that are actually expressed in Trichoderma fungal cells (as opposed to merely being encoded in the genome) by purifying these proteases and determining which ones have activities that are most relevant in de- gradation of heterologous proteins, such as mammalian proteins. Additionally, the inventors confirmed that deletion of the genes responsible for the protease activities in particular achieved a substantial reduction in total protease activity, which correlates with an increase in protein stabilization in terms of both quantity and quality of proteins produced in filamentous fungal cells containing such deletions and resulted in an
[0089] Consequently, certain aspects of the present description provide filamentous fungal cells that produce increased levels of a heterologous protein by having reduced activity or none of at least three proteases, where the cell additionally contains a recombinant polynucleotide that encodes a polypeptide heterologous produced at a level at least twice as high as the level of production of the polypeptide in a corresponding parental fungal cell in which the proteases have no reduced activity. In other words, the desired increase in the level of heterologous protein production is determinable by comparing the level of heterologous protein production in a filamentous fungal cell having reduced activity of at least three proteases with that of a cell filamentous fungus which does not have such reduced activity, but is otherwise identical to the cell that exhibits the increased level.
[0090] [0090] Other aspects of the present description provide methods to improve the stability of the heterologous polypeptide, by: a) providing a filamentous fungal cell of the present description having reduced or none activity of at least three proteases, where the cell additionally contains a recombinant polynucleotide that encodes a heterologous polypeptide; and b) cell culture so that the heterologous polypeptide is expressed, where the heterologous polypeptide has increased stability compared to a host cell that does not contain the mutations of the genes that encode the proteases.
[0091] [0091] Still other aspects of the present description provide methods of preparing a heterologous polypeptide by: a) supplying a filamentous fungal cell of the present description having reduced activity or none of at least three proteases, where the cell additionally contains a recombinant polynucleotide that encodes a heterologous polypeptide; b) culturing the host cell so that the heterologous polypeptide is expressed; and c) purification of the heterologous polypeptide.
[0092] [0092] Certain aspects of the present description also provide Trichoderma fungal cells that produce increased levels of a mammalian polypeptide because it has reduced activity or none of at least three proteases selected from pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, sIp1, sIp2, gap1 and gap2 ,, where the cell additionally contains a recombinant polynucleotide that encodes a mammalian polypeptide produced at a level at least twice the level of production of the polypeptide in one corresponding parental Trichoderma fungal cell in which proteases do not have reduced activity. In other words, the desired increase in the level of heterologous protein production is determinable by comparing the level of heterologous protein production in a Trichoderma fungal cell having the reduced activity of at least three proteases with that of a fungal cell of Trichoderma that has no such reduced activity but is otherwise identical to the cell that exhibits the increased level.
[0093] [0093] Other aspects of the present description provide methods to improve the stability of the mammalian polypeptide by: a) providing a Trichoderma fungal cell of the present description that has reduced activity of at least three proteases, where the cell additionally contains a recombinant polynucleotide encoding a mammalian polypeptide; and b) culturing the cell so that the mammalian polypeptide is expressed, where the mammalian polypeptide has increased stability compared to a host cell that does not contain the mutations of the genes encoding the proteases.
[0094] [0094] Other aspects of the present description provide methods of preparing a mammalian polypeptide by: a) providing a Trichoderma fungal cell of the present description that has reduced activity of at least three proteases, where the cell additionally contains a recombinant polynucleotide that encodes a mammalian polypeptide; b) culturing the host cell so that the mammalian polypeptide is expressed; and c) purification of the mammalian polypeptide. Definitions
[0095] [0095] As used here, an "immunoglobulin" refers to a multimeric protein containing a heavy chain and a light chain covalently coupled together and capable of specifically combining with an antigen. Immunoglobulin molecules are a large family of molecules that include several types of molecules, such as IgM, 1gD, IgG, IgA and IgE.
[0096] [0096] As used here, an "antibody" refers to intact immunoglobulin molecules, as well as fragments thereof, which are capable of binding to an antigen. These include hybrid (chimeric) antibody molecules (see, for example, Winter et al., Nature 349: 293-99225, 1991; and United States Patent No.
[0097] [0097] As used herein, a "peptide" and "polypeptide" are sequences of amino acids including a plurality of consecutive polymerized amino acid residues. For the purposes of the present invention, peptides are typically those molecules that include up to 50 amino acid residues and polypeptides include more than 50 amino acid residues. The peptide or polypeptide can include modified amino acid residues, naturally occurring amino acid residues not coded for by a codon and non-naturally occurring amino acid residues. As used herein, "protein" can refer to a peptide or polypeptide of any size. Proteases of the Invention
[0098] [0098] The invention described here relates to filamentous fungal cells
[0099] [0099] Proteases include, without limitation, aspartic proteases, trypsin-like serine proteases, subtilisin proteases, glutamic proteases and sedolysin proteases. Such proteases can be identified and isolated from filamentous fungal cells and tested to determine whether reduced activity affects the production of a recombinant polypeptide from the filamentous fungal cell. Methods for identifying and isolating proteases are well known in the art and include, without limitation, affinity chromatography, zymogram assays and gel electrophoresis. An identified protease can then be tested by deleting the gene encoding the identified protease from a filamentous fungal cell that expresses a recombinant polypeptide, such as a heterologous or mammalian polypeptide, and determining whether the deletion results in a decrease in the total protease activity of the cell, for example, to a level of 49% or less or 31% or less, of the total protease activity of the corresponding parental filamentous fungal cell; and an increase in the level of production of the expressed recombinant polypeptide,
[00100] [00100] Aspartic proteases are enzymes that use an aspartate residue to hydrolyze peptide bonds in proteins and polypeptides. Typically, aspartic proteases contain two highly conserved aspartate residues in their active site which are optimally active at acidic pH. Aspartic proteases from eukaryotic organisms, such as Trichoderma fungi, include pepsins, cathepsins and renins. Such aspartic proteases have a structure with two domains, which are believed to arise from a duplication of the ancestral gene. Consistent with such a duplication event, the global duplication of each domain is similar, although the sequences of the two domains have started to diverge. Each domain contributes one of the catalytic aspartate residues. The active site is in a gap formed by the two domains of the aspartic proteases. Eukaryotic aspartic proteases also include conserved disulfide bridges, which can assist in the identification of polypeptides as aspartic acid proteases.
[00101] [00101] Nine aspartic proteases have been identified in fungal cells of Trichoderma: pep1 (tre74156); pep2 (tre53961); pep3 (tre121133); pep4 (tre77579), pep5 (tre8 1004), pep7 (tre58669), pep8 (tre122076), pep11 (tre121306) and pep12 (tre119876)). Pep1
[00102] [00102] Examples of suitable pep1 proteases include, without limitation
[00103] Consequently, in certain embodiments, a protease of the present description, typically a pep71 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence SEQ ID NOs: 1-16, SEQ ID NOS: 491-494. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 1-16, SEQ ID NOs: 491-494.
[00104] [00104] In some embodiments, pep1 is pep1 from T. reesei. The amino acid sequence encoded by T. Reesei's pep1 is shown in SEQ ID NO: 1. In other embodiments, a protease from the pre-
[00105] [00105] Examples of suitable pep2 proteases include, without limitation, Trichoderma reesei pep2 (SEQ I | D NO: 182), T. Atroviide jgilTriat2 142040 (SEQ ID NO 183), T. virens jgilTrivicgv29 8 2153481 ( SEQ ID NO: 184), gi | l346326575 of Cordyceps militaris CMO1 (SEQ ID NO: 185), gil85111370 of Neurospora crassa (SEQ ID NO: 495) and their counterparts.
[00106] Consequently, in certain embodiments, a protease of the present description, typically a pep2 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence of SEQ ID NOs: 182-185, SEQ ID NO: 495. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 182-185, SEQ ID NO: 495.
[00107] [00107] In some embodiments, pep2 is pep2 of T. reesei. The peptide-encoded amino acid sequence of T. reesei is shown in SEQ ID NO: 182. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5% or more) with SEQ ID NO: 182. In other embodiments, the protease has 100% identity with SEQ ID NO: 182. Pep3
[00108] [00108] Examples of suitable pep3 proteases include, without limitation, Trichoderma reesei pep3 (SEQ ID NO: 17), 7. atroviride jagi | lTriat2 (SEQ ID NO: 18), T. virens jgilTriviGv29 8 2 ( SEQ ID NO: 19), gi [145583125 from Hypocrea lixii (SEQ ID NO: 20), gi / 51860175 from Trichoderma asperellum (SEQ ID NO: 21), gi | l317025164 from Aspergillus niger (SEQ ID NO: 22) , gi | 159122534 from Aspergillus fumigatus (SEQ ID NO: 23), gil 134054572 from Aspergillus niger (SEQ ID NO: 24), gi / 346318620 from Cordyceps militaris (SEQ ID NO: 25), gil810800156 from Glomerella graminicola (SEQ ID NO: NO : 26), gi | l3842871221 from Fusari- an oxysporum (SEQ ID NO: 27), gil320591121 from Grosmannia clavi- gera (SEQ ID NO: 28), gi [12002205 from Botryotinia fuckeliana (SEQ ID NO: 29), gil3846997107 from Thielavia terrestris (SEQ ID NO: 30), gi [156055954 from Sclerotinia sclerotiorum (SEQ ID NO: 31), gi [116197829 from Chaetomium globosum (SEQ ID NO: 32), gil336472132 from Neurospora tetrasperma (SEQ ID NO: 33), gil85102020 from Neurospora c rassa (SEQ ID NO: 34), Neosartorya fischeri gi 19467426 (SEQ ID NO: 35), Penicillium marneffei gil212534792 (SEQ ID NO: 36), M. thermophila gi367025909 (SEQ ID NO: 496), gi255947264 from P chrysogenum (SEQ ID NO: 497), A. oryzae gi391870123 (SEQ ID NO: 498) and homologues of the same.
[00109] Consequently, in certain embodiments, a protease of the present description, typically a protease pep3, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence of SEQ ID NOs: 17-36, SEQ ID NOs: 496-498. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 17-36, SEQ ID NOs: 496-498.
[00110] [00110] In some modalities, pep3 is pep3 of T. reesei. The amino acid sequence encoded by T. reesei pep3 is shown in SEQ ID NO: 17. In other embodiments, a protease of the present description has an amino acid sequence having 50% or more identity (for example , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 , 5% or more) with SEQ ID NO: 17. In other embodiments, the protease has 100% identity with SEQ ID NO: 17.
[00111] [00111] Examples of suitable pep4 proteases include, without limitation, Trichoderma reesei pep4 (SEQ ID NO: 37), T. virens jgilTriviGv29 8 2 (SEQ ID NO: 38), T. atroviride jgilTriat2 (SEQ ID NO: 39), Trichoderma aureoviride gil 193735605 (SEQ ID NO: 40), Aspergillus niger gil 145232965 (SEQ ID NO: 41), gi |) 70999520 of As- pergillus fumigatus (SEQ ID NO: 42), gil121705756 of Aspergillus clavatus (SEQ ID NO: 43), Nectria haematococca gi | l302899226 (SEQ ID NO: 44), Glomerella graminicola gil310796316 (SEQ ID NO: 45), Cordyceps militaris gil346322842 (SEQ ID NO: 46), gil46138535 from Gibberella zeae (SEQ ID NO: 47), gil322708430 from Metarhizium anisopliae (SEQ ID NO: 48), gi / 342882947 from Fusarium oxysporum (SEQ ID NO: 49), gil322700747 from Metarhizium acridum (SEQ ID NO: 50 ), gil346973691 from Verticilium dahliae (SEQ ID NO: 51), gil154309857 from Botryotinia fuckeliana (SEQ ID NO: 52), gil116203505 from Chaetomium globosum (SEQ ID NO: 53), gil347001590 from Thielavia terr strips (SEQ ID NO: 54), M. oryzae gil39973863 (SEQ ID NO: 55), gi / 296417651 of Tuber melanosporum (SEQ ID NO: 56), gi / l85094599 of Neurospora crassa (SEQ ID NO: 57), gi367031892 and gi255947264 by M. thermophila (SEQ ID NO: 499), gi255936729 and gi255947264 by P. chrysogenum (SEQ ID NO: 500), gil69770745 and gi255947264 by A. oryzae (SEQ ID NO: 501), gi624 nidulans
[00112] Consequently, in certain embodiments, a protease of the present description, typically a pep4 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence of SEQ ID NOs: 37-57, SEQ ID NOs: 499-502. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 37-57, SEQ ID NOs: 499-502.
[00113] [00113] In some embodiments, pep4 is pep4 from T. reesei. The pep4 amino acid sequence encoded by T. reesei is shown in SEQ ID NO: 37. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 37. In other embodiments, the protease has 100% identity with SEQ ID NO: 37. Pep5
[00114] Examples of suitable pep5 genes include, without limitation, Trichoderma reesei pep5 (SEQ ID NO: 58), T. virens TriviGv29 8 2 (SEQ ID NO: 59), T. atroviride jgilTriat21277859 ( SEQ ID NO: 60), gil322695806 from Metarhizium acridum (SEQ ID NO: 61), gi [156071418 from Fusarium oxysporum (SEQ ID NO: 62), gi | l346324830 from Cordyceps militaris (SEQ ID NO: 63), gil46124247 from Gibberella zeae (SEQ ID NO: 64), Verticilium dahliae gil346978752 (SEQ ID NO: 65), M. thermophila gi367019798 (SEQ ID NO: 503) and homologues thereof.
[00115] Consequently, in certain embodiments, a protease of the present description, typically a pep5 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence of SEQ ID NOs: 58-65, SEQ ID NO: 503. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 58-65, SEQ ID NO: 503.
[00116] [00116] In some embodiments, pep5 is pep5 from T. reesei. The amino acid sequence encoded by T. reesei pep5 is shown in SEQ ID NO: 58. In other embodiments, a protease of this description has an amino acid sequence that has 50% or more identity (for example, example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 58. In other embodiments, the protease has 100% identity with SEQ ID NO: 58. Pep7
[00117] [00117] Examples of suitable pep7 genes include, without limitation, Trichoderma reesei pep7 (SEQ ID NO: 186), Trichoderma atroviride jgi | Triat2 (SEQ ID NO: 187), jailTriviGv29 8 2 of Tricho- derma virens (SEQ ID NO: 188), Glomerella gram-cola gil310800487 (SEQ ID NO: 189), Metarhizium acridum gi | l322700577 (SEQ ID NO: 190), Thielavia terrestris gi / l347003264 (SEQ ID NO: 191 ), gil [171680938 from Podospora anserina (SEQ ID NO: 192), gil840905460 from Chaetomium thermophilum (SEQ ID NO: 193), gil346975960 from Verticillium dahliae (SEQ ID NO: 194), gil347009870 and gi367026morahthio Mycel ID5 My name : 195), gil85090078 from Neurospora crassa (SEQ ID NO: 196), gil39948622 from M. oryzae (SEQ ID NO: 197), gil16191517 from Chaetomium globosum (SEQ ID NO: 198), gil39970765 from M. oryzae (SEQ ID NO: 199), A. nidulans gi67522232 (SEQ ID NO: 504), A. niger gi350630464 (SEQ ID NO: 505),
[00118] Consequently, in certain embodiments, a protease of the present description, typically a pep7 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence of SEQ ID NOs: 186-199, SEQ ID NOs: 504-506. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 186-199, SEQ ID NOS: 504-506.
[00119] [00119] In some embodiments, pep7 is pep7 from T. reesei. The pep7 amino acid sequence encoded by T. reesei is shown in SEQ ID NO: 186. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5% or more) with SEQ ID NO: 186. In other embodiments, the protease has 100% identity with SEQ ID NO: 186. Pep8
[00120] Examples of suitable pep8 genes include, without limitation, Trichoderma reesei pep8 EGR48424 (SEQ ID NO: 507), Trichoderma virens EHK19238 (SEQ ID NO: 508), Trichoderma atroviride EHK40047 (SEQ ID NO: 509), EGO053367 by Neurospora tetrasperma (SEQ ID NO: 510), XP 003658897 by Myceliophthora thermophila (SEQ ID NO: 511), XP 965343 by Neurospora crassa (SEQ ID NO: 512), EFZ03501 by Metarhizium anisopliae (SEQ ID NO: SEQ ID NO: 512) 513), XP 003656869 from Thielavia terrestris (SEQ ID NO: 514), E-GU79769 from Fusarium oxysporum (SEQ ID NO: 515), XP 381566 from Gibberella zeae (SEQ ID NO: 516), XP 0037145401 from M. oryzae
[00121] Consequently, in certain embodiments, a protease of the present description, typically a pep8 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence of SEQ ID NOs: 507-521. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 507-521.
[00122] [00122] In some embodiments, pep8 is pep8 from T. reesei. The pep8-encoded amino acid sequence of T. reesei is shown in SEQ ID NO: 507. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 507. In other embodiments, the protease has 100% identity with SEQ ID NO: 507. Pep11
[00123] Examples of suitable pep11 genes include, without imitation, pep11 EGR49498 from Trichoderma reesei (SEQ ID NO: 522), EHK26120 from Trichoderma virens (SEQ ID NO: 523), EHK41756 from Trichoderma atroviride (SEQ ID NO: NO : 524), EKJ74550 from Fusarium pseudograminearum (SEQ ID NO: 525), EFY91821 from Metarhizium acridum (SEQ ID NO: 526), XP 384151 from Gibberella zeae (SEQ ID NO: 527), XP 003667387.1 from M. thermophila (SEQ ID NO: 528), XP 960328.1 by N. crassa (SEQ ID NO: 529) and their counterparts.
[00124] Consequently, in certain embodiments, a protease of the present description, typically a pep11 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence of SEQ ID NOs: 522-529. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 522-529.
[00125] [00125] In some embodiments, pep11 is pep11 from T. reesei. The pep11-encoded amino acid sequence of T. reesei is shown in SEQ ID NO: 522. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 522. In other embodiments, the protease has 100% identity with SEQ ID NO: 522. Pep12
[00126] [00126] Examples of suitable pep12 genes include, without limitation, pep12 EGR52517 from Trichoderma reesei (SEQ ID NO: 530), pep12 EHK188591 from Trichoderma virens (SEQ ID NO: 531), pep12 EHK45753 from Trichoderma atroviride (SEQ ID NO: 532), pep12 EKJ733921 from Fusarium pseudograminearum (SEQ ID NO: 533), pep12 XP 388759 from Gibberella zeae (SEQ ID NO: 534), pep12 EFY954891 from Metarhizium anisopliae (SEQ ID NO: 535), XP 964574.1 from N crassa (SEQ ID NO: 536), XP 003659978.1 from M. thermophila (SEQ ID NO: 537) and their counterparts.
[00127] [00127] — Consequently, in certain embodiments, a protease of the present description, typically a pep12 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
[00128] [00128] In some embodiments, pep12 is pep12 from T. reesei. The peptide-encoded amino acid sequence of T. reesei is shown in SEQ ID NO: 530. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 530. In other embodiments, the protease has 100% identity with SEQ ID NO: 530. Trypsin-type Serine proteases
[00129] [00129] —Serine trypsin-like proteases are enzymes with substrate specificity similar to that of trypsin. Trypsin-like serine proteases use a serine residue for hydrolysis of peptide bonds in proteins and polypeptides. Typically, trypsin-like serine proteases cleave peptide bonds after a positively charged amino acid residue. Trypsin-like serine proteases from eukaryotic organisms, such as Trichoderma fungi, include trypsin 1, trypsin 2 and mesotripsin. Such trypsin-like serine proteases generally contain a catalytic triad of three amino acid residues (such as histidine, aspartate and serine) that form a charge relay that serves to make the nucleophilic serine active site. Eukaryotic trypsin-like serine proteases also include an "oxyanion hole" formed by the amide hydrogen atoms of the glycine and serine skeleton, which can assist in the identification of polypeptides as being serine-type proteases trypsin.
[00130] A trypsin-like serine protease has been identified in fungal cells of Trichoderma: tsp1 (tre73897). As discussed below, tsp1 has been shown to have a significant impact on the expression of recombinant polypeptides, such as immunoglobulins.
[00131] [00131] As discussed below in Example 3, serine proteases have been purified from Trichoderma and have been shown to have multiple protease activities that degrade mammalian proteins. Of these activities, tsp1 was identified as a trypsin-like serine protease. The tsp1 protease gene was then deleted from Trichoderma fungal cells and it was demonstrated that the tsp1 deletion obtained a significant reduction in total protease activity, resulting in increased stabilization of the mammalian proteins produced by the cells.
[00132] [00132] Examples of suitable tsp1 proteases include, without limitation, Trichoderma reesei tsp1 (SEQ ID NO: 66), jgi | lTriat21298187 of Trichoderma atroviride (SEQ ID NO: 67), jagi | lTriviGgv29 8 2 (SEQID NO: 68), gil 145583579 of Hypocrea lixii (SEQ ID NO: 69), gi / 63025000 of Hypocrea lixii (SEQ ID NO: 70), gil 156052735 of Sclerotinia sclero-tiorum (SEQ ID NO: 71), gil154314937 of Botryotinia fuckeliana ( SEQ ID NO: 72), gil169605891 from Phaeosphaeria nodorum (SEQ ID NO: 73), gil3812219044 from Leptosphaeria maculans (SEQ ID NO: 74), gi / 37992773 from Verticilium dahliae (SEQ ID NO: 75), gi | 1072114 from Cochio carbonum (SEQ ID NO: 76), gi | 322695345 from Metarhizi- an acridum (SEQ ID NO: 77), gil4768909 from Metarhizium anisopliae (SEQ ID NO: 78), gij464963 (SEQ ID NO: 79), gi | 46139299 de Gibberella zeae (SEQ ID NO: 80), Metarhizium anisopliae (SEQ ID NO: 81), A. nidulans gi67523821 (SEQ ID NO: 538) and homologues thereof.
[00133] Consequently, in certain embodiments, a protease of the present description, typically a tsp1 protease, has an amino acid sequence that has 50% or more identity
[00134] [00134] In some embodiments, tsp1 is tsp1 of T. reesei. The amino acid sequence encoded by T. reesei tsp1 is shown in SEQ ID NO: 66. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 66. In other embodiments, the protease has 100% identity with SEQ ID NO: 66. Proteases Subtilisin
[00135] [00135] Subtilisin proteases are enzymes with substrate specificity similar to that of subtilisin. Subtilisin proteases use a serine residue to hydrolyze peptide bonds in proteins and polypeptides. Subtilisin proteases are generally serine proteases that contain a catalytic triad of three amino acids, aspartate, histidine and serine. The disposition of these catalytic residues is shared with the prototype subtilisin of Bacillus licheniformis. Subtilisin proteases from eukaryotic organisms, such as Trichoderma fungi, include furin, MBTPS1 and TPP2. Subtilisin proteases from eukaryotes also include including an aspartic acid residue in the oxy-ion orifice. Subtilisin s / p7 protease also resembles the top1 sedo-lysine protease.
[00136] [00136] Seven subtilisin proteases have been identified in fungal cells of Trichoderma: silp1 (tre51365); sip1 (tre123244); sip3 (tre123234); sip5 (tre64719), sIp6 (tre121495), sip7 (tre123865) and sip8
[00137] [00137] Examples of suitable s / p1 proteases include, without limitation, Trichoderma reesei sip1 (SEQ ID NO: 82), Trichoderma atroviride jgilTriat2 (SEQ ID NO: 83), Trichoderma atrovi- ride jgilTriat2 ( SEQ ID NO: 84), jagi | lTriviGv29 8 2 from Trichoderma virens (SEQ ID NO: 85), gil145583581 from Hypocrea lixii (SEQ ID NO: 86), gi / 322694632 from Metarhizium acridum (SEQ ID NO: 87), gil342877080 Fusarium oxysporum (SEQ ID NO: 88), Gibberella zeae gil46139915 (SEQ ID NO: 89), gi [170674476 from Epichloe festucae (SEQ ID NO: 90), gi / l802893164 from Nectria haematococca (SEQ ID NO: 91) , gil836266150 from Sordaria macrosporos (SEQ | D NO: 92), gil310797947 from Glomerella graminicola (SEQ ID NO: 93), gil336469805 from Neurospora tetrasperma (SEQ ID NO: 94), gil85086707 from Neurospora crassa (SEQ ID NO: 95), gil 145608997 from M. oryzae (SEQ ID NO: 96), gi | 16208730 from Chaetomium globosum (SEQ ID NO: 97), gi367029081 from M. thermophila (SEQ ID NO: 539) and homologues thereof.
[00138] Consequently, in certain embodiments, a protease of the present description, typically a s / p1 protease, has an amino acid sequence that has 50% or more identity (e.g., 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a sequence of amino acids selected from SEQ ID NOs: 82-97, SEQ ID NO: 539. In some embodiments, the protease has 100% identity with a selected amino acid sequence from SEQ ID NOs: 82-97, SEQ ID NO: 539 .
[00139] [00139] In some modalities, s / p1 is sip1 of T. reesei. The amino acid sequence encoded by T. reesei sIp1 is shown in SEQ ID NO: 82. In other embodiments, a protease from the pre-
[00140] [00140] Examples of suitable s / p2 proteases include, without limitation, Trichoderma reesei slp2 (SEQ ID NO: 98), T. atroviride jgilTriat2 (SEQ ID NO: 99), T. virens jgilTriviGv29 8 2 ( SEQ ID NO: 100), gil115111226 from Hypocrea lixir (SEQ ID NO: 101), gi / 70997972 from Aspergillus fumigatus (SEQ ID NO: 102), gi | 302915240 from Nectria haematococca (SEQ ID NO: 103), gij46105128 from Gibberel - la zeae (SEQ ID NO: 104), gi | 68165000 de / saria farinose (SEQ ID NO: 105), Glomerella graminicola gil310797854 (SEQ ID NO: 106), Epichloe festucae gil170674491 (SEQ ID NO: 107), gil322697754 from Metarhizium acridum (SEQ ID NO: 108), gi [147225254 from Acremo- nium sp. F11177 (SEQ ID NO: 109), gil1 5808807 from Ophiostoma piliforum (SEQ ID NO: 110), gil3836463649 from Neurospora tetrasperma (SEQ ID NO: 111), gil3840992600 from Chaetomium thermophilum (SEQ ID NO: 112), gil254351265 Metarhizium flavoviride (SEQ ID NO: 113), Podospora anserina gil171680111 (SEQ ID NO: 114), M. oryzae gi / l39943180 (SEQ ID NO: 115), Sclerointinia sclerotiorum gil 156058540 (SEQ ID NO: 116 ), Talaromyces stipitatus gil242790441 (SEQ ID NO: 117), M. thermophila gi367021472 (SEQ ID NO: 540), A. niger gi45237646 (SEQ ID NO: 541), gi | l689780712 by A. oryzae (SEQ ID NO: 542), P. chrysogenum gi255955889 (SEQ ID NO: 543), A. nidulans gi259489544 (SEQ ID NO: 544), N. crassa gi85084841 (SEQ ID NO: 545) and homologues of the same.
[00141] [00141] — Consequently, in certain embodiments, a protease of the present description, typically a s / p2 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a sequence selected amino acid from SEQ ID NOs: 98-117, SEQ ID NOs: 540-545. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 98-117, SEQ ID NOS: 540-545.
[00142] [00142] In some modalities, s / p2 is sip2 of T. reesei. The amino acid sequence encoded by T. reesei sip2 is shown in SEQ ID NO: 98. In other embodiments, a protease of this description has an amino acid sequence that has 50% or more identity (for example, example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 98. In other embodiments, the protease has 100% identity with SEQ ID NO: 98. Sip3
[00143] [00143] Examples of suitable s / p3 proteases include, without limitation, Trichoderma reesei slip3 (SEQ ID NO: 166), T. atroviride jgilTriat2 (SEQ ID NO: 167), T. virens (2) jgilTriviGg29 SEQ ID NO: 168), gil124295071 from Hypocrea koningii (SEQ ID NO: 169), gi [130750164 from Purpureocillium lilacinum (SEQ ID NO: 170), gil16215677 from Metarhizium anisopliae (SEQ ID NO: 171), gi / 90655148 from Hirs rhossiliensis (SEQ ID NO: 172), gil 18542429 of Tolypocladium inflatum (SEQ ID NO: 173), gil19171215 of Meta-cordyceps chlamydosporia (SEQ ID NO: 174), gi | 346321368 of Cordy-ceps militaris (SEQ ID NO: 175) ), Fusarium sp. (SEQ ID NO: 176), Neurospora tetrasperma gil336471881 (SEQ ID NO: 177), Chaetomium globosum gil16197403 (SEQ ID NO: 178), Neurospora crassa gil85084841 (SEQ ID NO: 179), gi / 56201265 de
[00144] Consequently, in certain embodiments, a protease of the present description, typically a s / p3 protease, has an amino acid sequence that has 50% or more identity (e.g., 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a sequence of amino acids selected from SEQ ID NOs: 166-181, SEQ ID NOs: 546-547, SEQ ID NOs: 222-223. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 166-181, SEQ ID NOs: 546-547, SEQ ID NOs: 222-223.
[00145] [00145] In some modalities, s / p3 is sip3 of T. reesei. The amino acid sequence encoded by s / p3 of T. reesei is shown in SEQ ID NO: 166. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 166. In other embodiments, the protease has 100% identity with SEQ ID NO: 166. SIp5
[00146] [00146] Examples of suitable s / p5 proteases include, without limitation, Trichoderma reesei slp5 (SEQ ID NO: 200), 7. atroviride jgilTriat2 (SEQ ID NO: 201), T. virens jgilTriviagv29 8 2 SEQ ID NO: 202), Hypocrea lixii gil18161442 (SEQ ID NO: 203), Fusarium oxysporum gi / 342883549 (SEQ ID NO: 204), Gibberella zeae gil46135733 (SEQ ID NO: 205), Glomerella graminicola gil310796396 ( SEQ ID NO: 206), gi / 302927954 by Nectria haematococca (SEQ ID NO: 207), gi / l346319783 by Cordyceps militaris (SEQ ID NO: 208), gi | l85094084 by Neurospora crassa (SEQ ID NO: 209) , gil836467281 from Neurospora tetrasperma (SEQ ID NO: 210), gil346971706 from Verticilium dahliae (SEQ ID NO: 211), gil3847001418 from Thielavia terrestris (SEQ ID NO: 212), gil 145605493 from M. oryzae (SEQ ID NO: 213) , M. thermophila gi367032200 (SEQ ID NO: 548), P. chrysogenum gi62816282 (SEQ ID NO: 549) and homologues thereof.
[00147] Consequently, in certain embodiments, a protease of the present description, typically a s / p5 protease, has an amino acid sequence that has 50% or more identity (e.g., 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a sequence of amino acids selected from SEQ ID NOs: 200-213, SEQ ID NOs: 548-549. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 200-213, SEQ ID NOS: 548-549.
[00148] [00148] In some modalities, s / p5 is sIp5 of T. reesei. The amino acid sequence encoded by s / p5 of T. reesei is shown in SEQ ID NO :. 200 In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO:. 200 In other embodiments, the protease has 100% identity with SEQ ID NO: 200. SIip6
[00149] [00149] Examples of suitable s / p6 proteases include, without limitation, Trichoderma reesei sip6 (SEQ ID NO: 214), 7. atroviride jgilTriat2 (SEQ ID NO: 215), T. virens (2) jagilTriviGv29 8 2 SEQ ID
[00150] Consequently, in certain embodiments, a protease of the present description, typically a s / p6 protease, has an amino acid sequence that has 50% or more identity (e.g., 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a sequence of selected amino acids from SEQ ID NOs: 214-230, SEQ ID NO: 550. In some embodiments, the protease has 100% identity with a selected amino acid sequence from SEQ ID NOs: 214-230, SEQ ID NO: 550 .
[00151] [00151] In some modalities, the s / p6 is sI / p6 of T. reesei. The amino acid sequence encoded by s / p6 of T. reesei is shown in SEQ ID NO :. 214 In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 214. In other embodiments, the protease has 100% identity with SEQ ID NO: 214. SIp7
[00152] [00152] Examples of suitable s / p7 proteases include, without limitation, Trichoderma reesei slp7 (SEQ ID NO: 231), 7. atroviride jgilTriat2 (SEQ ID NO: 232), T. virens (jgilTriviGv29 8 2) SEQ ID NO: 233), gil322710320 from Metarhizium anisopliae (SEQ ID NO: 234), gil802915000 from Nectria haematococca (SEQ ID NO: 235), gi / l347009020 and gi367024935 from Myceliophthora thermophila 236 (SEQ ID NO: 23) zeae (SEQ ID NO: 237), Thielavia terrestris gil346996549 (SEQ ID NO: 238), M. oryzae gil145610733 (SEQ ID NO: 239), A. nidulans gi67541991 (SEQ ID NO: 551), gi255933786 from P chrysogenum (SEQ ID NO: 552), A. niger gi317036543 (SEQ ID NO: 553), A. oryzae gi | 69782882 (SEQ ID NO: 554), N. crassa gi85109979 (SEQ ID NO: 555) and homologists.
[00153] Consequently, in certain embodiments, a protease of the present description, typically a slp7 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence of SEQ ID NOs: 231-239, SEQ ID NOs: 551-555. In some embodiments, the protease has 100% identity with an amino acid sequence selected from SEQ ID NOs: 231-239, SEQ ID NOs: 551-555.
[00154] [00154] In some modalities, s / p7 is sIp7 of T. reesei. The amino acid sequence encoded by T. reesei's slp7 is shown in SEQ ID NO: 231. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5% or more) with SEQ ID NO: 231. In other embodiments, the protease has 100% identity with SEQ ID NO: 231. SIp8
[00155] [00155] Examples of suitable s / p8 proteases include, without limitation, Trichoderma reesei s / p8 (SEQ ID NO: 240), T. atroviride's jgilTriat2H98568 (SEQ ID NO: 241), T. gg29 29 2133902. viruses (SEQ ID NO: 242) and their counterparts.
[00156] Consequently, in certain embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with an amino acid sequence selected from SEQ ID NOs: 240 -242. In some embodiments, the protease has 100% identity with a selected amino acid sequence of SEQ ID NOs: 240-242.
[00157] [00157] In some modalities, s / p8 is sIp8 of T. reesei. The amino acid sequence encoded by s / p8 of T. reesei is shown in SEQ ID NO :. 240 In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO :. 240 In other embodiments, the protease has 100% identity with SEQ ID NO: 240. Glutamic Proteases
[00158] [00158] Glutamic proteases are enzymes that hydrolyze peptide bonds in proteins and polypeptides. Glutamic proteases are insensitive to pepstatin A and thus are often referred to as acid proteases insensitive to pepstatin. Although glutamic proteases were previously grouped with aspartic proteases and often referred to together as acid proteases
[00159] [00159] Two glutamic proteases have been identified in Trichoderma fungal cells: gap1 (tre69555) and gap2 (trel06661). Gap1
[00160] [00160] Examples of suitable gap1 proteases include, without limitation, Trichoderma reesei gapl (SEQ ID NO: 118), T. atroviide's jgilTriat21408638 (SEQ | D NO 119), TT jgilTrivicgv29 8 21192684. virens (SEQ | D NO: 120), gi / 238499183 of Aspergillus flavus (SEQ ID NO: 121), gi | 145251555 of Aspergillus niger (SEQ ID NO: 122), gil115491521 of Aspergillus terreus (SEQ ID NO: 123), gil37154543 (SEQ ID NO: 124), gil48425531 (SEQ ID NO: 125), gil3851873 (SEQ ID NO: 126), gi / 346997245 of Thielavia terrestris (SEQ ID NO: 127), gil255940586 of Penicillium chrysogenum (SEQ ID NO: 128), M. thermophila gi367026504 (SEQ ID NO: 574), A. oryzae gi317150886 (SEQ ID NO: 575), N. crassa gi85097968 (SEQ ID NO: 576), A. niger gil31056 (SEQ ID NO: 576) NO: 577), P. chrysogenum gi255930123 (SEQ ID NO: 578), A. niger gil45236956 (SEQ ID NO: 579), A. oryzae gi | l689772955 (SEQ ID NO: 580), gij45249222 from A. niger (SEQ ID NO: 581), A. nidulans gi67525839 (SEQ ID NO: 582), A. oryzae gi / 69785367 (SEQ ID NO: 583), P. chrysogenum gi255955319 (SEQ ID NO: 584), gi36 / 7019352 by M. thermophila (SEQ ID NO: 585), gi391863974 by A. oryzae (SEQ ID NO: 586), gi367024513 by M. thermophila (SEQ ID NO: 587) and their counterparts.
[00161] Consequently, in certain embodiments, a protease of the present description, typically a gap71 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with one
[00162] [00162] In some modalities, gap1 is gap1 of T. reesei. The amino acid sequence encoded by the T. reesei gap1 is shown in SEQ ID NO: 118. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5% or more) with SEQ ID NO: 118. In other embodiments, the protease has 100% identity with SEQ ID NO: 118. Gap2
[00163] [00163] Examples of suitable gap2 proteases include, without limitation, gap by Trichoderma reesei (SEQ ID NO: 129), jgilTriat21298116 by T. atroviide (SEQ ID NO: 130), jgilTriviagv29 8 2130331 by T. virens (SEQ I | D NO: 131), jgilTriviagv29 8 21225131 (SEQ ID NO: 132 ), gi | 238499183 from Aspergillus flavus (SEQ ID NO: 133), gi | 145251555 from Aspergillus niger (SEQ ID NO: 134), gil67901056 from Aspergillus nidulans (SEQ ID NO: 135), gil121711990 from Aspergillus clavatus (SEQ ID NO: 135) ID NO: 136), gi | 70986250 from Aspergillus fumigatus (SEQ ID NO: 137), gil212534108 from Penicillium marneffei (SEQ ID NO: 138), gil242789335 from Tala-romyces stipitatus (SEQ ID NO: 139), gi | l320591529 from Grosmannia clavigera (SEQ ID NO: 140), gil19474281 from Neosartorya fischeri (SEQ ID NO: 141), gil212527274 from Penicillium marneffei (SEQ ID NO: 142), gil255940586 from Penicilium chrysogenum (SEQ ID NO: 14356), gi [13 SEQ ID NO: 144), M. thermophila gi367030275 (SEQ ID NO: 588) and homologues thereof.
[00164] Consequently, in certain embodiments, a protease of the present description, typically a gap2 protease, has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a selected amino acid sequence from SEQ ID NOs: 129-144, SEQ ID NO: 588. In some embodiments, the protease has 100% identity to an amino acid sequence selected from SEQ ID NOs: 129-144, SEQ ID NO: 588.
[00165] [00165] In some modalities, gap2 is gap2 of T. reesei. The amino acid sequence encoded by T. reesei gap2 is shown in SEQ ID NO :. 129 In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO :. 129 In other embodiments, the protease has 100% identity with SEQ ID NO: 129. Proteases Sedolysin
[00166] [00166] Sedolysin proteases are enzymes that use a serine residue to hydrolyze peptide bonds in proteins and polypeptides. Sedolysin proteases generally contain a unique catalytic triad of serine, glutamate and aspartate. Sedolysin proteases also contain an aspartate residue in the oxyanion orifice. Sedolysin proteins from eukaryotic organisms, such as Trichoderma fungi, include tripeptidyl peptidase.
[00167] [00167] Examples of suitable tpp1 proteases include, without limitation, Trichoderma reesei top1 (SEQ ID NO: 145), T. atroviride jgi | lTriat2H88756 (SEQ ID NO: 146), T. virens jgilTriviGv29 8 21217176 (T. SEQ ID NO: 147), gi) 70993168 from Aspergillus fumigatus (SEQ ID NO: 148), gi [169776800 from Aspergillus oryzae (SEQ ID NO: 149), gi [145236399 from Aspergillus niger (SEQ ID NO: 150), gi [121708799 of
[00168] Consequently, in certain embodiments, a protease of the present description, typically a tpp1 protease, has an amino acid sequence that has 50% or more identity
[00169] [00169] In some modalities, tpp1 is tpp1 of T. reesei. The amino acid sequence encoded by T. reesei tpop1 is shown in SEQ ID NO: 145. In other embodiments, a protease of the present description has an amino acid sequence that has 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5% or more) with SEQ ID NO: 145. In other embodiments, the protease has 100% identity with SEQ ID NO: 145. Homologous proteases
[00170] [00170] Other modalities of the present description refer to the reduction of the activity of proteases that are homologous to the proteases of the present description. "Homology", as used herein, refers to the sequence similarity between a reference sequence and at least a fragment of a second sequence. Homologues can be identified by any method known in the art, preferably using the BLAST tool to compare a reference sequence with a single second sequence or fragment of a sequence or with a sequence database. As described below, BLAST will compare strings based on percent identity and similarity.
[00171] [00171] The terms "identical" or "percentage" identity, in the context of two or more nucleic acid or amino acid sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid or nucleotide residues that are the same (ie, 29% identity, optionally, 30%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region or, when not specified, over all sequence), when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably, over a region that is 100 to 500 or 1000 or more nucleotides (or 20 , 50, 200 or more amino acids) in length).
[00172] [00172] For sequence comparison, typically, a sequence acts as a reference sequence with which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary and the parameters of the sequence algorithm program are designated. Default program parameters can be used or alternative parameters can be assigned. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences in relation to the reference sequence based on the program parameters. When comparing two sequences for identity, it is not necessary for the sequences to be contiguous, but any gap would entail a penalty that would reduce the overall percentage identity. For blastn, the default parameters are gap opening penalty = 5 and gap extension penalty = 2. For blastp, parameters
[00173] [00173] A "comparison window", as used here, includes reference to a segment of any of the series of contiguous positions including, but not limited to, 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, in which a sequence can be compared with a reference sequence of the same number of contiguous positions after the two sequences are perfectly aligned. Sequence alignment methods for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol 48 (3): 443 -453, by the Pearson and Lipman similarity search method (1988) Proc Natl Acad Sci USA 85 (8): 2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wis software package - consin Genetics, Genetics Computer Group, 575 Science Dr., Madison, WI) or by manual alignment and visual inspection [see, for example, Brent et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou Ed)].
[00174] [00174] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402 and Altschul et al. (1990) J. Mol Biol 215 (3): 403-410, respectively. Software for performing BLAST analysis is available to the public through the National Center for Biotechnology Information. This algorithm first involves identifying High Scoring sequence Pairs (HSPs) by identifying short words of length W in the query sequence which match or satisfy any threshold score of T of positive value when aligned with a word of same length in a database string.
[00175] [00175] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc Natl Acad Sci USA 90 (12): 5873-5877). A measure of similarity provided by the BLAST algorithm is the least probability of sum (P (N)), which provides an indication of the probability by which a correspondence between two sequences of nucleotides or amino acids would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the probability of the smallest sum in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than than about 0.01 and, even more preferably, less than about 0.001.
[00176] [00176] In addition to the percent identity of the sequence mentioned above, another indication that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide codified by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
[00177] [00177] As described herein, the proteases of the present description can also include proteases which are conservatively modified variants of proteases encoded by the protease genes described above. "Conservatively modified variants", as used herein, include substitutions, deletions or individual additions of a coded amino acid sequence that result in the substitution of one amino acid for another chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, homologous between species and alleles from the description. The following eight groups contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); 6) Phenylaniline (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, Proteins (1984)).
[00178] [00178] Figures 45-48 represent phylogenetic trees of aspartic, subtilisin, glutamic and sedolysin proteases from selected filamentous fungi. Invention Protease Activity Reduction Methods
[00179] [00179] Other aspects of the present description refer to the reduction of activity of proteases present in filamentous fungal cells that express a heterologous polypeptide, such as a mammalian polypeptide.
[00180] [00180] The activity of proteases present in filamentous fungal cells can be reduced by any method known to those skilled in the art.
[00181] [00181] In some embodiments, reduced protease activity is obtained through reduction of protease expression, for example, by modification of promoter or RNAi.
[00182] [00182] In other embodiments, reduced protease activity is obtained by modifying the gene that encodes the protease. Examples of such modifications include, without limitation, a knockout mutation, a truncation mutation, a point mutation, an antisense mutation, a substitution mutation, a frame shift mutation, an insertion mutation, a duplication mutation - tion, an amplification mutation, a translocation mutation or an inversion mutation and which results in a reduction in the corresponding protease activity. Methods of generating at least one mutation in a gene encoding the protease of interest are well known in the art and include, without limitation, mutagenesis and random selection, site-directed mutagenesis, PCR mutagenesis, insertion mutagenesis, mutagenesis chemistry and irradiation.
[00183] [00183] In certain embodiments, a portion of the gene encoding the protease is modified, such as the region encoding the catalytic domain, the coding region or a control sequence necessary for the expression of the coding region. Such a gene control sequence can be a promoter sequence or a functional part of it, that is, a part that is sufficient to affect the expression of the gene. For example, a promoter sequence can be inactivated, resulting in no expression, or a weaker promoter can be replaced with the native promoter sequence to reduce the expression of the coding sequence. Other control sequences for possible modification include, without limitation, a leader sequence, a pro-peptide sequence, a signal sequence, a transcription terminator and a transcription activator.
[00184] The protease genes encoding the present description that are present in filamentous fungal cells that express a recombinant polypeptide can also be modified using gene deletion techniques to eliminate or reduce expression of the gene. Gene deletion techniques allow partial or complete removal of the gene, thus eliminating its expression. In such methods, deletion of the gene can be carried out through homologous recombination using a plasmid that has been constructed so as to continuously contain the 5 'and 3' regions that flank the gene.
[00185] The protease genes encoding the present description that are present in filamentous fungal cells that express a recombinant polypeptide can also be modified by introducing, replacing and / or removing one or more nucleotides in the gene or a control sequence thereof necessary for transcription or translation of the gene. For example, nucleotides can be inserted or removed for the introduction of a terminal codon, the removal of the initial codon or a frame shift from the open reading frame. Such modification can be carried out by methods known in the art including, without limitation, site-directed mutagenesis and peR generated mutagenesis (see, for example, Botstein and Shortie, 1985, Science 229: 4719; Lo et al., 1985, Proceedings of the National Academy of Sciences USA 81: 2285; Higuchi et al., 1988, Nucleic Acids Research 16: 7351; Shimada, 1996, Meth. Mol. Biol. 57: 157; Ho et al., 1989, Gene 77: 61; Horton et al., 1989, Gene 77: 61; and Sarkare Sommer, 1990, BioTechniques 8: 404).
[00186] [00186] Furthermore, the protease-encoding genes of the present description that are present in filamentous fungal cells that express a recombinant polypeptide can be modified by gene disruption techniques by inserting a nucleic acid construct into the gene a fragment of nucleic acid homologous to the gene, which will create a duplication of the homology region and incorporate the DNA construct between the duplicated regions. Such a genetic disruption can eliminate gene expression if the inserted construct separates the gene promoter from the coding region or interrupts the coding sequence so that a non-functional gene product results. A disruption construct can simply be a selectable marker gene accompanied by regions 5 'and 3' homologous to the gene. The selectable marker allows identification of transformants containing the disrupted gene.
[00187] [00187] Protease-encoding genes of the present description that are present in filamentous fungal cells that express a recombinant polypeptide can also be modified by the process of gene conversion (see, for example, Iglesias and Trautner, 1983, Molecular General Genetics 189: 573-76). For example, in gene conversion, a nucleotide sequence that corresponds to the gene is mutated in vitro to produce a defective nucleotide sequence which is then transformed into a Trichoderma strain to produce a defective gene. Through homologous recombination, the defective nucleotide sequence replaces the endogenous gene. It may be desirable that the defective nucleotide sequence also contains a marker for selecting transformants containing the defective gene.
[00188] The protease genes encoding the present description that are present in filamentous fungal cells expressing a recombinant polypeptide can also be modified using antisense techniques established using a nucleotide sequence complementary to the nucleotide sequence of the gene (vi, for example, Parish and Stoker, 1997, FEMS Microbiology Letters 154: 151-157). In particular, gene expression by filamentous fungal cells can be reduced or inactivated by introducing a nucleotide sequence complementary to the nucleotide sequence of the gene, which can be transcribed in the strain and is able to hybridize with the produced mMRNA in the cells. Under conditions that allow the hybridizing antisense nucleotide sequence to complement
[00189] [00189] Furthermore, genes encoding proteases of the present description that are present in filamentous fungal cells that express a recombinant polypeptide can also be modified using established RNA interference (RNAi) techniques (see, for example) , WO 2005/056772 and WO 2008/080017).
[00190] The protease genes encoding the present description that are present in filamentous fungal cells that express a recombinant polypeptide can also be modified through random or specific mutagenesis using methods well known in the art including, without limitation, chemical mutagenesis (see , for example, Hopwood, The Isolation of Mutants in Methods in Microbiology (JR Norris and DW Ribbons, eds.) pages 363-433, Academic Press, New York, 1970). Modification of the gene can be performed by subjecting the filamentous fungal cells to mutagenesis and screening of mutant cells in which the expression of the gene has been reduced or inactivated. Mutagenesis, which can be specific or random, can be performed, for example, using a suitable physical or chemical mutagenic agent, using an appropriate oligonucleotide, subjecting the DNA sequence to the generated mutagenesis or whatever - or a combination of them. Examples of physical and chemical agents for mutagenesis include, without limitation, ultraviolet (UV), hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (N-Methyl-N'-Nitro-N- NitrosoGuanidine - MNNG), N-methyl-N'-nitrosoguanidine (N-methyl-N'- NiTrosoGuanidine - NTG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulfonate (Ethyl Methane Sulphonate - EMS), sodium bisulfite, formic acid and nucleotide analogs. When these agents are used, mutagenesis is typically performed by incubation
[00191] [00191] In certain embodiments, at least one mutation or alteration in a gene encoding protease of the present description results in a modified protease that has no detectable protease activity. In other embodiments, at least one change in a gene encoding the protease of the present description results in a modified protease that is at least 25%, at least 50% less, at least 75%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000% or a higher percentage of less protease activity compared to a corresponding unmodified protease.
[00192] [00192] In certain modalities, for example, in a Trichoderma cell, at least one mutation or alteration in a gene encoding protease of the present description results in a reduction of total protease activity to 49% or less , typically with a mutation in at least 2 distinct protease genes or 31% or less, typically with a mutation in at least 3 distinct protease genes or 13% or less, typically with a mutation in at least 4 genes different protease genes or 10% or less, typically with a mutation in at least 5 different protease genes or 6.3% or less, typically with a mutation in at least 6 different protease genes or 5.5 % or less, typically with a mutation in at least 7 distinct protease genes, of the total protease activity of the corresponding Trichoderma cell rental.
[00193] [00193] The invention also relates here to the increase in the production of heterologous polypeptides in filamentous fungal cells that express these heterologous polypeptides by reducing the activity of proteases present in the cells.
[00194] [00194] As used herein, a "heterologous polypeptide" refers to a polypeptide that is not found naturally (i.e., enogenous) in a filamentous fungal cell of the present description or that is expressed at a high level in fungal cells filamentous when compared to the endogenous version of the polypeptide. In certain embodiments, the heterologous polypeptide is a mammalian polypeptide. In other embodiments, the heterologous polypeptide is a non-mammalian polypeptide. Mammal Polypeptides
[00195] [00195] Mammalian polypeptides of the present description can be any mammalian polypeptide that has a biological activity of interest. As used herein, a "mammalian polypeptide" is a polypeptide that is expressed natively in a mammal, a polypeptide that is derived from a polypeptide that is expressed natively in a mammal or a fragment thereof. A mammalian polypeptide also includes peptides and oligopeptides that retain biological activity. Mammalian polypeptides of the present description can also include two or more polypeptides that are combined to form the encoded product. Mammalian polypeptides of the present description can further include fusion polypeptides which contain a combination of partial or complete amino acid sequences obtained from at least two different polypeptides. Mammalian polypeptides may also include naturally occurring and modified allelic variations of any of the described mammalian polypeptides and hybrid mammalian polypeptides.
[00196] [00196] The mammalian polypeptide can be a naturally glycosylated polypeptide or a naturally non-glycosylated polypeptide.
[00197] [00197] Examples of suitable mammalian polypeptides include, without limitation, immunoglobulins, antibodies, antigens, enzymes, antimicrobial peptides, growth factors, hormones, interferons, interleukins, cytokines, immunodilators, neurotransmitters, receptors, reporter proteins, structural proteins and transcription factors.
[00198] [00198] Specific examples of suitable mammalian polypeptides include, without limitation, immunoglobulins, immunoglobulin heavy chains, immunoglobulin light chains, monoclonal antibodies, hybrid antibodies, F (ab ') antibody fragments, fragments of F (ab) antibodies, Fv molecules, single chain Fy antibodies, antibody fragments, dimeric and trimeric antibody fragments, functional antibody fragments, immunoadhesins, insulin-like growth factor-1, growth hormone - ment, insulin, interferon alpha 2b, fibroblast growth factor 1, human serum albumin, antibodies and / or fragments of camelid antibodies, antibodies with a single domain, antibodies with a single multimeric domain and erythropoietin.
[00199] [00199] Other examples of suitable mammalian proteins include, without limitation, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, an catalase, glycosyl transferase, deoxyribonuclease, esterase, galactosidase, betagalactosidase, glucosidase, glucuronidase, glucuronyl esterase, haloperoxidase, invertase, lipase, oxidase, phospholipase, proteolytic enzyme ribonuclease, urokinase, albumin, collagen, tropoelastin and elastin. Non-Mammalian Polypeptides
[00200] [00200] Non-mammalian polypeptides of the present description can be any non-mammalian polypeptide that has a biological activity of interest. As used here, a "non-mammalian polypeptide" is a polypeptide that is expressed natively in a non-mammalian organism, such as fungal cells, a polypeptide that is derived from a polypeptide that is expressed natively in a non-mammalian organism or a fragment thereof. A non-mammalian polypeptide also includes peptides and oligopeptides that retain biological activity. Non-mammalian polypeptides of the present description can also include two or more polypeptides that are combined to form the encoded product. Non-mammalian polypeptides of the present description can further include fusion polypeptides which contain a combination of partial or total amino acid sequences obtained from at least two different polypeptides. Non-mammalian polypeptides may also include naturally occurring and modified allelic variations of any of the described non-mammalian polypeptides and non-mammalian polypeptides.
[00201] [00201] Examples of suitable non-mammalian polypeptides include, without limitation, aminopeptidases, amylases, carbohydrates, carboxypeptidases, catalases, cellulases, chitinases, cutinases, deoxyribonucleases, esterases, alpha-galactosidase, beta-galactosidase, gluco-galactosidase, , alpha-glucosidases, beta-glucosidases, invertases, laccases, lipases, oxidases, mutanases, pectinolytic enzyme, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases and xylanases. Heterologous Polypeptide Production
[00202] [00202] A heterologous polypeptide of interest is produced by filamentous fungal cells of the present description containing at least three proteases having reduced activity by culturing the cells in a nutrient medium for producing the heterologous polypeptide using methods known in the art. For example, cells can be cultured by shaking flask, small-scale fermentation or large-scale fermentation (including continuous, batch, batch or solid fermentation) in laboratory or industrial fermenters performed in a suitable medium and under conditions that allow the polypeptide to be expressed and / or isolated. Culture takes place in a suitable nutrient medium comprising sources of carbon and nitrogen and inorganic salts using procedures known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (for example, in catalogs of the American Type Culture Collection). The secreted polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be obtained from cell lysates.
[00203] [00203] A heterologous polypeptide of interest produced by a filamentous fungal cell of the present description containing at least three proteases with reduced activity can be detected using methods known in the art that are specific to the heterologous polypeptide. These detection methods may include, without limitation, the use of specific antibodies, high-performance liquid chromatography, capillary chromatography, formation of an enzyme product, disappearance of an enzyme substrate and SDS PAGE. For example, an enzyme assay can be used to determine the activity of an enzyme. Procedures for determining enzymatic activity are known in the art for many enzymes (see, for example, O. Schomburg and M. Salzmann (eds.), Enzyme Handbook,
[00204] The resulting heterologous polypeptide can be isolated by methods known in the art. For example, a heterologous polypeptide of interest can be isolated from the culture medium by conventional procedures including, without limitation, centrifugation, filtration, extraction, spray drying, evaporation and precipitation. The isolated heterologous polypeptide can then be further purified by means of a variety of procedures known in the art including, without limitation, chromatography (for example, ion exchange, affinity, hydrophobic, chromatofocusing and exclusion by size), electrophoretic procedures (for example, preparative isoelectric focusing (IsoElectric Focusing - IEF), differential solubility (for example, precipitation with ammonium sulfate) or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) Preparation of Polynucleotides Encoding Heterologous Polypeptides
[00205] [00205] Sequences of the heterologous polynucleotides of the present description are prepared by any suitable method known in the art including, without limitation, direct chemical synthesis or cloning. For direct chemical synthesis, formation of a nucleic acid polymer generally involves the sequential addition of 3'-blocked and 5'-blocked nucleotide molecules to the terminal 5'-hydroxyl group of a growing nucleotide chain, where each - determination is carried out by means of nucleophilic attack of the terminal 5'-hydroxyl group of the growing chain at the 3 'position of the added monomer which is typically a phosphorus derivative, such as a phosphor-triester, phosphoramidite or similar . Such a methodology is known to those skilled in the art and is described in relevant texts and literature [for example, in Matteucci et al., (1980) Tetrahedron Lett 21:
[00206] [00206] Each heterologous polynucleotide of the present description can be incorporated into an expression vector. "Expression vector" or "vector" refers to a compound and / or composition that translates, transforms or infects a host cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to cell or in a way not native to the cell. An "expression vector" contains a sequence of nucleic acids (commonly DNA or RNA) to be expressed by the host cell. Optionally, the expression vector also includes materials to assist in obtaining nucleic acid entry into the host cell, such as a virus, liposome, protein coating or the like. The expression vectors considered for use in the present description include those in which a nucleic acid sequence can be inserted, along with any preferred or necessary operational elements. In addition, the expression vector must be one that can be transferred to a host cell and reproduced in it. Preferred expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the preferred or necessary operational elements for transcribing the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known
[00207] [00207] The incorporation of individual polynucleotides can be performed using known methods that include, for example, use of restriction enzymes (such as BamHlI, EcoRI, Hhal, Xhol, Xmal and so on) to cleave specific sites in the vector of expression, for example, plasmid. The restriction enzyme produces single-stranded ends that can be heat treated with a polynucleotide having, or synthesized to have, a termination with a sequence complementary to the ends of the cleaved expression vector. Annealing is carried out using a suitable enzyme, for example, DNA ligase. As will be appreciated by those skilled in the art, both the expression vector and the desired polynucleotide are often cleaved with the same restriction enzyme and thus ensure that the ends of the expression vector and the ends of the polynucleotide complementary to each other. In addition, DNA ligands can be used to facilitate ligation of nucleic acid sequences into an expression vector.
[00208] [00208] A series of individual polynucleotides can also be combined using methods that are known in the art (for example, United States Patent No. 4,683. 195).
[00209] [00209] For example, each of the desired polynucleotides can be generated initially in a separate PCR. After that, specific primers are designed so that the ends of the PCR products contain complementary sequences. When PCR products are mixed, denatured and annealed, tapes with the corresponding sequences at their 3 'ends overlap and can act as initiators for each other. Extending this overlap by DNA polymerase produces a molecule in which the original sequences are "joined" together. In this way, a series of individual polynucleotides can be "joined" and subsequently transduced in a host cell simultaneously. Thus, expression of each of the plurality of polynucleotides is obtained.
[00210] [00210] Individual polynucleotides or "joined" polynucleotides are then incorporated into an expression vector. The present descriptive report is not limited in relation to the process by which the polynucleotide is incorporated into the expression vector. Those skilled in the art are familiar with the steps required to incorporate a polynucleotide into an expression vector. A typical expression vector contains the desired polynucleotide preceded by one or more regulatory regions, along with a ribosomal binding site, for example, a nucleotide sequence that is 3-9 nucleotides in length and located 3-11 nucleotides upstream of the initial codon in E. coli. See Shine and Dalgarno (1975) Nature 254 (5495): 34-38 and Steitz (1979) Biological Regulation and Development (ed. Goldberger, R. R), 1: 349-399 (Plenum, New York).
[00211] [00211] The term "operably linked", as used here, refers to a configuration in which a control sequence is placed in an appropriate position in relation to the coding sequence of the DNA or polynucleotide sequence, so that the control sequence directs the expression of a polypeptide.
[00212] [00212] Regulatory regions include, for example, regions that contain a promoter and an operator. A promoter is operably linked to the desired polynucleotide, thereby initiating transcription of the polynucleotide by means of an RNA polymerase enzyme. An operator is a sequence of nucleic acids adjacent to the promoter which contains a protein-binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription is initiated via the promoter. When present, protection
[00213] [00213] While any suitable expression vector can be used to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pSC101, pBR322, pBBRMCS-3, PUR, pEX, pMR100, pCRA4, PBAD24, pUC19, pRS426; and bacteriophages, such as phage M13 and phage X. Of course, such expression vectors may be suitable only for particular host cells. Those skilled in the art, however, can easily determine, through routine experimentation, whether any particular expression vector is suitable for any given host cell. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference can be made to the relevant texts and literature, which describe vectors of expression and their suitability.
[00214] [00214] Expression vectors suitable for purposes of the present invention, including expression of the heterologous polypeptide, enzyme and one or more desired catalytic domains described herein, include expression vectors containing the polynucleotide encoding the heterologous polypeptide, enzyme or domain desired catalytic (s) operatively linked to a constitutive promoter or an inducible promoter. Examples of promoters particularly suitable for the operative binding of such polynucleotides include the promoters of the following genes: goda, cbhl, TAKA amylase from Aspergillus oryzae, aspartic proteinase from Rhizomucor miehei, neutral alpha-amylase from Aspergillus niger , acid-stable Aspergillus niger alpha-amylase, Aspergillus niger glucoamylase (glaA), Aspergillus awamori glaA, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus ory phosphate triose isomerase lans, Aspergillus oryzae acetamidase, trypsin protease from Fusarium rium oxysporum, fungal endo al-arabinase (abnA), fungal aol-arabinofuranosidase A (abfA), fungal aL-arabinofuranosidase B (abfB) fungal, xylane, xylanase fungal, fungal ATP-synthase, fungal subunit 9 (oliC), fungal phosphate triosis (tpi) isomerase, fungal alcohol (adhA) dehydrogenase, fungal a-amylase (amy) fungal amyloglucosidase (glaA), fungal acetamidase (amdS), fungal glyceraldehyde-3-phosphate (gpd) dehydrogenase, yeast alcohol dehydrogenase, yeast lactase, yeast 3-phosphoglycerate kinase, yeast phosphate triose isomerase, bacteryl2 amylase and bacterial SSO. Examples of such expression vectors and suitable promoters are also described in the document POCT / EP2011 / 070956, the entire content of which is incorporated herein by reference.
[00215] [00215] In another aspect, the present invention provides a composition, for example, a pharmaceutical composition, containing one or more heterologous polypeptides of interest, such as mammalian polypeptides, produced by the filamentous fungal cells of the present description that has reduced activity of at least three proteins and also containing a recombinant polynucleotide that encodes the heterologous polypeptide, formulated in conjunction with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention can also be administered in combination therapy, that is, in combination with other agents. For example, the combined therapy may include a mammalian polypeptide of interest combined with at least one other therapeutic agent.
[00216] [00216] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and retardant absorption agents and the like that are physiologically compatible. Preferably, the vehicle is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (for example, by injection or infusion). Depending on the route of administration, the active compound, that is, the mammalian polypeptide of interest, can be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
[00217] [00217] The pharmaceutical compositions of the invention can include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not confer any undesirable toxicological effects (see, for example, Berge, SM,., Et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids , substituted phenyl alkanoic acids, alkanoic hydroxy acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as non-toxic organic amines, such as N N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline , diethanolamine, ethylenediamine, procaine and the like.
[00218] [00218] A pharmaceutical composition of the invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (Butylated HydroxyAnisole - BHA), butylated hydroxytoluene (Butylated HydroxyToluene - BHT), lecithin, propyl gallon, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylene diaminetetraacetic acid (Ethyl yleneDiamine Tetraacetic Acid - EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
[00219] [00219] Examples of suitable aqueous and non-aqueous vehicles that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, green oils minerals, such as olive oil, and injectable organic esters, such as ethyl oleate. Adequate fluidity can be maintained, for example, by using coating materials, such as lecithin, maintaining the
[00220] [00220] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.
[00221] [00221] Pharmaceutically acceptable vehicles include sterile and post-sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersions. The use of such means and agents for pharmaceutically active substances is known in the art. Except to the extent that any conventional medium or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the invention is considered. Supplementary active compounds can also be incorporated into the compositions.
[00222] [00222] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by using a coating, such as lecithin, maintaining the required particle size in the case of dispersion and using surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be obtained by including, in the composition, an agent that delays absorption, for example, monostearate salts and gelatin.
[00223] [00223] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of the ingredients listed above, as required, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional desired ingredient from a solution previously sterilized by filtration.
[00224] [00224] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the individual being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will, in general, be the amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent active ingredient, preferably from about 0.1 percent to about 70 percent percent, more preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
[00225] [00225] Dosage regimens are adjusted to give the optimal response desired (for example, a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used here, refers to physically distinct units suitable as unit dosages for the individuals to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical excipient. The specification for the unit dosage forms of the invention are guided by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved and (b) the limitations inherent in the technique of composing such active compound for the treatment of sensitivity in individuals.
[00226] [00226] For the administration of a mammalian polypeptide of interest, in particular where the mammalian polypeptide is an antibody, the dosage ranges from about 0.0001 to 100 mg / kg and, more commonly, 0, 01 to 5 mg / kg of host body weight. For example, dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or mg / kg body weight or 1-10 mg / kg range. An exemplary treatment regimen involves administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to six months. Some dosing schedules for an antibody may include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration, with the antibody being delivered using one of the following dosing schedules: (1 ) every four weeks for six doses, then every three months; (ii) every three weeks; (iii) 3 mg / kg of body weight once, followed by 1 mg / kg of body weight every three weeks.
[00227] [00227] Alternatively, a mammalian polypeptide of interest can be administered as a sustained release formulation and, in this case, less frequent administration is required. Dosage and frequency vary depending on the half-life of the substance administered to the patient. In general, human antibodies require a longer half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes necessary until the progression of the disease is reduced or stopped and, preferably, until the patient shows partial or complete improvement in the symptoms of the disease. ence. After that, a prophylactic regimen can be administered to the patient.
[00228] [00228] The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present description can be varied, in order to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a patient, composition and mode of administration in particular, without being toxic to the patient. The dosage level selected will depend on a variety of factors, including the pharmacokinetic activity of the compositions of the present invention employed in particular or the ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the compound to be be used in particular, the duration of treatment, other drugs, compounds and / or materials used in combination with the compositions employed in particular, the age, sex, weight, condition, general health and previous medical history of the patient to be treated and similar factors well known in the medical art.
[00229] [00229] An "therapeutically effective dose" of an immunoglobulin of the present description preferably results in a decrease in the severity of the symptoms of the disease, an increase in the frequency and duration of periods without symptoms of the disease or a prevention of insufficiency. - insufficiency or incapacity due to distress due to the disease. For example, for the treatment of tumors, a "therapeutically effective dose" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably at least about 60% and, more preferably, at least about 80% in relation to untreated individuals. The ability of a compound to inhibit tumor growth can be assessed in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be assessed by examining the compound's inhibition capacity by means of in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can decrease the size of the tumor or otherwise improve symptoms in an individual. Those skilled in the art will be able to determine such amounts based on factors such as the size of the individual, the severity of the individual's symptoms and the composition or route of administration.
[00230] [00230] The composition of the present description can be administered through one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Certain routes of administration of binding portions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration, for example, by injection or infusion. The phrase "parenteral administration", as used here, means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal and intraborbital.
[00231] Alternatively, a mammalian polypeptide according to the present description can be administered via a non-parenteral route, such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually. or topical.
[00232] [00232] The active compounds can be prepared with vehicles that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Biocompatible, biodegradable polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polytoesters and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art (see, for example, Sustained and Controlled Re-lease Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978).
[00233] [00233] Therapeutic compositions can be administered with medical devices known in the art. For example, in a given embodiment, a therapeutic composition of the invention can be administered with a hypodermic needle-free injection device, such as the devices described in United States Patent No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;
[00234] [00234] In certain embodiments, use of mammalian polypeptides in accordance with the present description is for the treatment of any disease that can be treated with therapeutic antibodies. Filamentary Fungal Cells of the Invention
[00235] [00235] The present invention also relates to increasing levels of production of heterologous polypeptides, such as polypeptides,
[00236] [00236] "Filamentous fungal cells" includes cells of all filamentous forms in the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). Filamentous fungal cells are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is due to hyphal elongation and carbon catalysis is mandatory aerobic. In contrast, vegetative growth by yeast, such as Saccharomyces cerevisiae, is by budding from a single-celled stem and carbon catabolism can be fermentative.
[00237] [00237] Any filamentous fungal cell can be used in the present description, as long as it remains viable after it has been transformed with a nucleic acid sequence and / or has been modified or mutated to decrease protease activity. Preferably, the filamentous fungal cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of proteins (for example, mammalian proteins) or the resulting intermediates.
[00238] [00238] Examples of suitable filamentous fungal cells include, without limitation, cells from a strain of Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium or Trichoderma. In certain embodiments, the filamentous fungal cell is from a strain of Trichoderma sp., Acremonium, Aspergillus, Aureobasidium, Crypto-coccus, Chrysosporium, Chrysosporium lucknowense, Filibasidium, Fusarium, Gibberella, Magnaporthe, Mucor, Myceliophth, Myceliophth Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia or Tolypo-cladium.
[00239] [00239] Aspergillus fungal cells of this description may include, without limitation, Aspergillus aculeatus, Aspergillus awa- mori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus japonicus, Aspergillus japonicus, Aspergillus japonicus, Aspergillus japonicus Aspergillus oryzae or Aspergillus terreus.
[00240] [00240] Neurospora fungal cells of the present description may include, without limitation, Neurospora crassa.
[00241] [00241] In certain embodiments, the filamentous fungal cell is not an Aspergillus cell.
[00242] [00242] In certain embodiments, the filamentous fungal cell is selected from the group consisting of Trichoderma (T. reeseil), Neurospora (N. crassa), Penicillium (P. chrysogenum), Aspergillus (A. nidulans, A. niger and A. oryzae), Myceliophthora (M. thermophila) and C-hrysosporium (C. lucknowense).
[00243] [00243] In certain embodiments, the filamentous fungal cell is a Trichoderma fungal cell. Trichoderma fungal cells of the present description can be derived from a wild type Trichoderma strain or a mutant thereof. Examples of suitable Trichoderma fungal cells include, without limitation, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma atroviride, Trichoderma virens, Trichoderma viride; and their alternative sexual form (ie, Hypocrea).
[00244] [00244] General methods for gene disruption and filamentous fungal cell culture are described, for example, for Penicillium in Kopke et al. (2010) Application of the Saccharomyces cerevisiae FLP / FRT Recombination System in Filamentous Fungi For Marker Re-cycling and Construction of Knockout Strains Devoid of Heterologous Genes. Appl Environ Microbiol 76 (14): 4664-74. It hurts
[00245] [00245] Certain aspects of the present description refer to filamentous fungal cells having reduced or no detectable activity of at least three proteases and having a recombinant polynucleotide that encodes a heterologous polypeptide that is produced at increased levels, for example, levels of at least two times increased. Other aspects of the present description refer to Trichoderma fungal cells that do not have detectable protease activity from at least three proteases selected from pep1, pep 2,
[00246] [00246] The reduced activity of at least three proteases in filamentous fungal cells or Trichoderma fungal cells of the present description may be the result of reduced or nonexistent protease expression. In some embodiments, reduced or nonexistent expression of at least three proteases is the result of a modification of the catalytic domain, the coding region or a control sequence necessary for expression of the coding region of the genes encoding each of the proteases . In other modalities, the reduced or nonexistent expression of the proteases is the result of the introduction, replacement and / or removal of one or more nucleotides in the genes or a control sequence of the same ones necessary for transcription or translation of the genes that encode each one of the proteases.
[00247] [00247] In other modalities, the reduced or nonexistent expression of the proteases is the result of insertion, in the genes encoding each of the proteases, of rupture nucleic acid constructs, each containing a fragment of nucleic acid homologous to each of the genes, which will create a duplication of the homology region and incorporate the DNA construct between the duplicated regions.
[00248] [00248] In some embodiments, the genes encoding proteases each contain a mutation that reduces or eliminates the corresponding protease activity. In other embodiments, the mutation reduces or eliminates the expression of each of the proteases. In other modalities, the mutation is a knockout mutation, a truncation mutation, a point mutation, an antisense mutation, a substitution mutation, a frame shift mutation,
[00249] [00249] In some embodiments, the mutation is a deletion of the gene that encodes the protease. In other embodiments, the mutation is a deletion of the portion of the gene that encodes the protease that encodes the catalytic protease domain. In still other embodiments, the mutation is a point mutation in the portion of the gene that encodes the protease that encodes the catalytic protease domain. Protease Gene Combinations
[00250] [00250] The filamentous fungal cells or Trichoderma fungal cells of the present description can contain at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more aspartic proteases, trypsin-like serine proteases, sub-lysine proteases and / or glutamic proteases. In certain embodiments, proteases are encoded by pep-type protease genes, gap-type protease genes or sip-type protease genes. In some embodiments, the pep-type protease genes are selected from pep1, pep2, pep3, pep4, pep5, pep8, pep11 and pep12. In other modalities, the gap-like protease genes are selected from gap1 and gap2. In other embodiments, the sip-type protease genes are selected from s / p1, sIlp2, sip3 and sip7; or they are selected from sip1, sip2, sIip3, sIp5, sIp6, slip7 and sip8. In certain preferred embodiments, the sip-like protease gene is sip1.
[00251] [00251] In other embodiments, proteases are encoded by genes selected from pep1, pep2, pep3, pep4, pep5, pep7, pep8, pep11, pep12, tsp1, sIip1, sip2, sIp3, sIp5, sIp6, sIp7, sip8, gap1 , gap2 and top1. In some embodiments, the filamentous fungal cell, for example, a Trichoderma cell, has reduced or nonexistent expression levels of at least three or at least four protease-coding genes selected from pep1, pep2, pep3, pep4, pepº5, pep8, pep11, pep12, tsp1, sip1, sip2, sip3, slp7, gap1 and gap2. In certain modalities, the filamentous fungal cell, for example, a Trichoderma cell, has reduced or nonexistent levels of expression of at least three protease genes selected from pep1, tsp1 and sIp1. In other modalities, the filamentous fungal cell, such as a Trichoderma cell, has reduced or nonexistent levels of expression of at least three genes that code for proteases selected from gap1, sIp1 and pep 1. In some lities, the filamentous fungal cell, for example, a Trichoderma cell, has reduced or nonexistent levels of expression of protease genes selected from sip2, pep1 and gap1. In some embodiments, the filamentous fungal cell, for example, a Trichoderma cell, has reduced or nonexistent levels of expression of genes encoding proteases selected from slp2, pep1, gap1 and pep4. In some embodiments, the filamentous fungal cell, for example, a Trichoderma cell, has reduced or nonexistent levels of expression of genes that code for proteases selected from sip2, pep1, gap1, pep4 and sip1. In some embodiments, the filamentous fungal cell, for example, a Trichoderma cell, has reduced or nonexistent levels of expression of genes encoding proteases selected from s / p2, pep1, gap1, pep4, sip1 and sip3. In some modalities, the filamentous fungal cell, for example, a Trichoderma cell, has reduced or nonexistent expression levels of genes encoding proteases selected from slp2, pep1, gap1, pep4, sIp1, sIp3 and pep3. In some embodiments, the filamentous fungal cell, for example, a Trichoderma cell, has reduced or nonexistent levels of expression of genes that encode selected protease.
[00252] [00252] In certain embodiments, the filamentous fungal cell has at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more proteases with activity reduced protease, in which the corresponding proteases with wild-type activity each have an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequences of SEQ ID NOs: 1-16; 17-36; 37-57; 58-65; 66-81; 82-97; 98-117; 118-128; 129-144; 166-181; 182-185; or SEQ ID NOs: 491-
[00253] [00253] The filamentous fungal cells or Trichoderma fungal cells of the present description contain a recombinant polynucleotide that encodes a heterologous polypeptide. In certain embodiments, the heterologous polypeptide is a mammalian polypeptide. In other embodiments, the heterologous polypeptide is a non-mammalian polypeptide.
[00254] [00254] In embodiments where the filamentous fungal cell contains a recombinant polynucleotide encoding a mammalian polypeptide, the mammalian polypeptide may be a non-glycosylated mammalian polypeptide, a glycosylated mammalian polypeptide or combinations thereof, including, without limitation , an immunoglobulin, an antibody, a growth factor and an interferon. In some embodiments, the mammalian polypeptide is an immunoglobulin or an antibody. In modalities where the filamentous fungal cell contains a recombinant polynucleotide that encodes an immunoglobulin or antibody, the filamentous fungal cell, for example, a Trichoderma fungal cell, may have reduced or nonexistent expression of at least three or at least four protease coding genes selected from pep1, pep3, pep4, pep8, pep11, pep12, tsp1, sIp1, sIp2, slp7, gap1 and gap2. In certain preferred embodiments, the cell, for example, a Trichoderma fungal cell, contains a recombinant polynucleotide that encodes an immunoglobulin or antibody and has reduced or nonexistent expression of genes encoding protease selected from s / p1, sip2, sip3, tsp1, pep1, gap1, pep4, pep3, pep2, pep5 and gap2. In certain preferred embodiments, the cell, for example, a Trichoderma fungal cell, contains a recombinant polynucleotide that encodes an immunoglobulin or antibody and has reduced or nonexistent expression of genes that encode proteases selected from pep1, tsp1, sip1 and gap1T.
[00255] [00255] In other embodiments, the filamentous fungal cell contains a recombinant polynucleotide that encodes a growth factor, interferon, cytokine or interleukin. In modalities in which the filamentous fungal cell, for example, a Trichoderma fungal cell, contains a recombinant polynucleotide that encodes a growth factor, interferon, cytokine, human serum albumin or interleukin, the filamentous fungal cell may have expression reduced or nonexistent of at least three or at least four genes that code for at least one protease selected from pep1, pep2, pep3, pep4, pep5, pep8, gap1, gap2, sip1, sIlp2, sip7 and tsp1. In certain modalities, the cell contains a recombinant polynucleotide that encodes a growth factor, interferon, cytokine, human serum albumin or interleukin and reduced expression of protease-selected genes selected from pep1, tsp1, sip1, gap1 and gap2. In certain modalities, the cell contains a recombinant polynucleotide that encodes a growth factor, interferon, cytokine, human serum albumin
[00256] [00256] In certain embodiments, the mammalian polypeptide is produced at a level that is at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 75 times, at least 80 times, at least 90 times, at least 100 times or greater than the level of polypeptide production in a corresponding parental filamentous fungal cell with no reduced protease activity. In other embodiments, the mammalian polypeptide is produced in a full-length version at a level higher than the production level of the full-length version of the poly-
[00257] [00257] In embodiments in which the filamentous fungal cell contains a recombinant polynucleotide that encodes a non-mammalian polypeptide, the non-mammalian polypeptide can be an aminopeptidase, amylase, carbohydrase, carboxypeptidase, cellulose, cellulase , chitinase, cutinase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccases, lipase, mutanase, oxidase, a pectinolytic enzyme, peroxidase, polyphenol, phospholipase, phospholipase - nol, a proteolytic enzyme, ribonuclease, transglutaminase or xylaninase. In embodiments in which the filamentous fungal cell contains a recombinant polynucleotide that encodes a non-mammalian polypeptide, the filamentous fungal cell may have reduced or nonexistent detectable expression of at least three, at least four, at least five or at least six genes that encode proteases selected from pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, sip1, sip2, sip3, gap1 and gap2. In certain modalities, the non-mammalian polypeptide is produced at a level that is at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least less than 70 times, at least 75 times, at least 80 times, at least 90 times, at least 100 times or greater than the level of polypeptide production in a corresponding parental filamentous fungal cell. In other embodiments, the non-mammalian polypeptide is produced in a full-length version at a level higher than the production level of the full-length version of the polypeptide in a corresponding parental filamentous fungal cell.
[00258] [00258] In some embodiments, the filamentous fungal cells or Trichoderma fungal cells of the present description also have reduced activity of one or more additional proteases. In certain modalities, the level of expression of one or more additional protections is reduced. In certain preferred embodiments, the genes encoding one or more additional proteases each comprise a mutation that reduces the corresponding protease activity. The one or more genes that encode protease can be pep7, top1, gap2, sip3, sIp5, sIp6, sip7 or sip8.
[00259] [00259] In certain embodiments, when the filamentous fungal cell is an Aspergillus cell, the total protease activity is reduced to 50% or less of the total protease activity in the corresponding parental Aspergillus cell in which the proteases have no reduced activity.
[00260] [00260] In certain modalities, the total protein activity is reduced in the cell of the present description, for example, a Trichoderma cell, to 49% or less, 31% or less, 13% or less, 10% or less or 6.3% less or 5.5% or less of the total protease activity in the corresponding parental fungal filamentous cell in which the proteases have no reduced activity. Additional Recombinant Modifications
[00261] [00261] In certain embodiments, the filamentous fungal cells or Trichoderma fungal cells of the present description also have reduced activity - from a doliquil-P-Man: Man (5) GICNAc (2) -PP-doliquyl mannosyl transferase. doliquil-P- Man: Man (5) GICNAc (2) -PP-doliquyl mannosyl transferase (EC 2.4.1.130) transfers an alpha-D-mannosyl residue from dolichyl phosphate D-mannose into a lipid-bound oligosaccharide of the membrane . Typically, the enzyme doliquyl-P-Man: Man (5) GIcNAc (2) -PP-doliquyl manosyl transferred is encoded by an alg3 gene. Thus, in certain modalities, the filamentous fungal cell has reduced activity of ALG3, which is the activity encoded by the a / g3 gene. In some embodiments, the a / lg3 gene contains a mutation that reduces the corresponding ALG3 activity. In certain embodiments, the a / g3 gene is deleted from the filamentous fungal cell.
[00262] [00262] In other embodiments, the filamentous fungal cells or Trichoderma fungal cells of the present description also contain a polynucleotide that encodes an a1,2-mannosidase. The polylucleotide encoding a1,2-mannosidase can be endogenous in the host cell or it can be heterologous to the host cell. These polynucleotides are especially useful for a filamentous fungal cell that expresses high mannose glycans transferred from the Golgi to the ER without effective exo-a-2-mannosidase cleavage. A1,2-mannosidase may be a mannosidase type enzyme | belonging to the 47 family of glycoside hydrolases (cazy.org/GH47 all.html). In certain embodiments, a1,2-mannosidase is an enzyme listed at cazy.org/GH47 characterized.html. In particular, a1,2-mannosidase can be an ER-type enzyme that cleaves glycoproteins, such as enzymes in the a-mannosidase enzyme subfamily | of ER (EC
[00263] [00263] In other embodiments, the filamentous fungal cells or Trichoderma fungal cells of the present description also contain a catalytic domain of N-acetylglucosaminyl transferase | and a catalytic domain of N-acetylglucosaminyl transferase Il. Such catalytic domains are useful for the expression of complex N-glycans in non-mammalian cells. N-acetylglucosaminyl transferase | (GICNAc-TI; GnTI; EC 2.4.1.101) catalyzes the reaction of UDP-N-acetyl-D-glucosamine + 3- (alpha-D-mannosyl) -beta-D-mannosyl-R <=> UDP + 3- (2- (N-acetyl-beta-D-glucosaminyl) -alpha-D-mannosyl) -beta-D-mannosyl-R, where R represents the remainder of the N-linked oligosaccharide at the glycan receptor. A catalytic domain of N-acetylglucosaminyl transferase | is any portion of an N-acetylglucosaminyl transferase enzyme | that is able to catalyze this reaction. N-acetylglucosaminyl transferase 11 (GICNAcC-TII; GnTII; EC 2.4.1.143) catalyzes the reaction of UDP-N-acetyl-D-glucosamine + 6- (alpha-D-mannosyl) -beta-D-mannosyl-R <= > UDP + 6- (2- (N-acetyl-beta-D-glucosaminyl) -alpha-D-mannosyl) -beta-D-mannosyl-R, where R represents the remainder of the N-linked oligosaccharide at the glycan receptor. A catalytic domain of N-acetylglucosaminyl transferase | is any portion of an N-acetylglucosaminyl transferase enzyme | that is able to catalyze this reaction. Examples of catalytic domains of N-acetylglucosaminyl transferase | and suitable N-acetylglucosaminyl transferase | l catalytic domains can be found in International Patent Application No. POCT / EP2011 / 070956. The catalytic domain of N-acetylglucosaminyl transferase | and the catalytic domain of N-acetylglucosaminyl transferase II can be encoded by a polynucleotide. In certain embodiments, the polynucleotide encodes a fusion protein that contains the catalytic domain of N-acetylglucosaminyl transferase | and the catalytic domain of N-acetylglucosaminyl transferase Il. Alternatively, the catalytic domain of N-acetylglucosaminyl transferase | it can be encoded by a first polynucleotide and the catalytic domain of N-acetylglucosaminyl transferase | 1 can be encoded by a second polynucleotide.
[00264] [00264] In modalities in which the filamentous fungal cell or Trichoderma cell contains a catalytic domain of N-acetylglucosaminyl transferase | and a catalytic domain of N-acetylglucosaminyl transferase II, the cell can also contain a polynucleotide encoding an Il mannosidase. Mannosidase enzymes | are capable of cleaving man5 structures from GIcNAcMan5 to generate GIcNAcMan3 and, if combined with the action of a catalytic GnTII domain, generate GO; and, also, with the action of a catalytic domain of a galactosyl transferase, they generate G1 and G2. In certain embodiments, mannosidase enzymes of type | belong to the 38 family of glycoside hydrolases (cazy.org/GH38 all.html). Examples of such enzymes include human AAC50302 enzyme, D. melanogaster enzyme (Van den Elsen JM et al (2001) EMBO J. 20: 3008-3017), those with 3D structure according to the reference 1HTY in the PDB and others cited with the catalytic domain in the PDB. For expression in ER / Golgi, the catalytic domain of mannosidase is typically fused with an N-terminal objectification peptide, for example, using the objectification peptides listed in International Patent Application No. PCT / EP2011 / 070956 or SEQ ID NOs: 589-594. Post-transformation with the catalytic domain of a type 1l mannosidase, a strain that effectively produces GIcCNAc2Man3, GIcNAcl-Man3 or GO is selected.
[00265] [00265] In certain modalities that can be combined with the preceding modalities, the filamentous fungal cell still contains a polynucleotide that encodes a UDP-GIcNAc transporter.
[00266] [00266] In certain modalities that can be combined with the preceding modalities, the filamentous fungal cell also contains a polynucleotide that encodes a B-1,4-galactosyl transferase. Generally, B-1,4-galactosyl transferases belong to the CAZy glycosyl transferase family 7 (cazy.org/GT7 all.html). Examples of useful B4GalT enzymes include B4GalT1, for example, enzyme AAA30534.1 Bos taurus beef (Shaper NL et al., Proc. Natl. Acad. Sci. U.-SA 83 (6), 1573-1577 (1986 )), human enzyme (Guo S. et al., Glycobiology 2001, 11: 813-20) and AAA37297 enzyme from Mus musculus (Shaper, NL et al., 1998 J. Biol. Chem. 263 (21), 10420 - 10428). In certain embodiments of the invention in which the filamentous fungal cell contains a polynucleotide encoding a galactosyl transferase, the filamentous fungal cell also contains a polynucleotide encoding a UDP-Gal 4-epimerase and / or UDP-Gal transporter. In certain embodiments of the invention in which the filamentous fungal cell contains a polynucleotide that encodes a transfer galactosyl, lactose can be used as a carbon source, rather than glucose, when culturing the host cell. The culture medium can be between a pH of 4.5 and 7.0 or between 5.0 and 6.5. In certain embodiments of the invention in which the filamentous fungal cell contains a polynucleotide encoding a galactosyl transferase and, optionally, a polynucleotide encoding a UDP-Gal 4-epimerase and a UDP-Gal transporter, a divalent cation, such as Mn ”, Ca ** or Mg *, can be added to the cell culture medium.
[00267] [00267] In certain modalities that can be combined with the preceding modalities, the level of alpha-1,6-mannosyl transferase activity in the host cell is reduced compared to the level of activity in a wild type host cell. In certain embodiments, the filamentous fungal cell has a reduced level of expression of an och1 gene compared to the level of expression of a wild-type filamentous fungal cell.
[00268] [00268] Another aspect includes methods of producing an N-glycan Man3GIcNAc [ie Mana3 (Mana6) ManB4GIcNAcB4GIcNAc] in a filamentous fungal cell, including the steps of supplying a filamentous fungal cell with a recombinant polynucleotide that encodes a heterologous polypeptide and a reduced level of activity in a mansoil transferase alg3 compared to the level of activity in a filamentous fungal cell of wild type and culture of the filamentous fungal cell to produce a Man3GIcNAc2 glycan, where Man3GIcNAc2 glycans constitute at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% (mol%) of neutral N-glycans secreted by the filamentous fungal cells. In certain embodiments, N-glycan Man3GIcNAc2 represents at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% less 90% or 100% (mol%) of the total N-glycans of the heterologous polypeptide.
[00269] [00269] Another aspect includes methods of producing a complex N-glycan (for example, an N-glycan comprising a terminal GIcNAc2Man3 structure), for example, Glc-NAc2Man3GIcNAc2 glycan (ie, GO, or sejay Glc-NAcB2Mana3 ( GlIcNAcB2Mana6) ManB4GIcNACB4GICNAc) in a filamentous fungal cell, including the steps of providing a filamentous fungal cell with a recombinant polynucleotide that encodes a heterologous polypeptide, a reduced level of activity of a mannosyl transferase alg3 activity compared to the level of alg3 activity compared to the level of alg3 activity filamentous, wild-type fungal fungus and further comprising a polynucleotide encoding a catalytic domain of N-acetylglucosaminyl transferase | and a polynucleotide that encodes a catalytic domain of N-acetylglucosaminyl transferase | and filamentous fungal cell culture to produce complex N-glycans, for example - GIcNAc2Man3GIcNAc2 glycan where Glc NAc2Man3GIcNAc2 glycan constitutes at least 5%, at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% (mol%) of neutral N-glycans secreted by the filamentous fungal cells. In certain embodiments, the complex N-glycan, for example, GICNAc2Man3GIcNAc glycan, represents at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 %, at least 90% or 100% (mol%) of the total N-glycans of the polypeptide. In certain embodiments, said complex N-glycans are GICNAcMan3 and / or GIcNAc2Man3.
[00270] [00270] Another aspect includes methods of producing an N- glycan G1 or G2 or a mixture thereof, for example, GalGlc- NAc2Man3GIcNAc2 (ie, G1, ie GalB4GIcNAcB2Mana3 (GIcNAcB2Mana6) ManB4GIcNAcCB4GICNACCB4GICNAC) and / or Gal2GIcNAc2Man3GIcNAc2 (ie, G2, ie GalB4GIcNAcB2Mana3 (GalB4GICNAcB2Mana6) ManB4GIcNACB4GIcN Ac) glycine in a filamentous fungal cell, including the steps of supplying a fungal polypeptide with a fungal cell reduced level of activity of a mannosyl transferase alg3ã compared to the level of activity in a filamentous fungal cell of wild type and still comprising a polynucleotide that encodes a catalytic domain of N-acetylglucosaminyl transferase |, a polynucleo-
[00271] [00271] In certain embodiments, the method of producing a complex N-glycan will generate a mixture of different glycans. Complex N-glycans or Man3GIcNAc2 can make up at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , by less than 80%), or at least 90% or more of such a mixture of glycines. In certain modalities, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% ), or
[00272] [00272] In certain embodiments, methods of producing a hybrid N-glycan are desirable. As used here, the term "hybrid" means a glycan containing both unsubstituted terminal mannose residues (as present in high mannan glycans) and mannose residues substituted with an N-acetylglucosamine bond, for example, Glc-NAcB2Mana3 [Mana3 (Mana6) Mana6] ManB4GIcNACB4GICNAc. In such modalities, a filamentous fungal cell that expresses Man5 (ie, Mana3 [Mana (Mana6) Mana6] ManB4GIcNACB4GIcCNAc), like a T. reesei strain, is transformed with a recombinant polynucleotide that encodes a heterologous polypeptide and a polynucleotide that encodes a catalytic domain of N-acetylglucosaminyl | transferase | and the filamentous fungal cell is grown to produce the hybrid N-glycan, where the hybrid N-glycan constitutes at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% (mol%) of the neutral N-glycans secreted by the filamentous fungal cell. In certain modalities, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% (mol%) of the polypeptide N-glycans consist of hybrid N-glycans.
[00273] [00273] The N-glycans Man3GIcNAc2, complex, hybrid, G1 and G2 can be linked to a molecule selected from an amino acid, a peptide and a polypeptide. In certain embodiments, the complex, hybrid, Man3GIcNAc2 N-glycans, G1 and G2 are linked to a heterologous polypeptide. In certain embodiments, the heterologous polypeptide is a glycosylated protein. In certain embodiments, the glycosylated polypeptide is a mammalian polypeptide. In certain embodiments, the mammalian polypeptide is an antibody or antigen-binding fragment thereof.
[00274] [00274] In certain embodiments, glycosyl transferase or, for example, glycosyl hydrolases GnTI, GnTII or Gal or, for example, a1,2-mannosidase or mannosidase | l, include a targeting peptide linked to the catalytic domains. The term "linked", as used here, means that two polymers of amino acid residues, in the case of a polypeptide, or two nucleotide polymers, in the case of a polynucleotide, are coupled directly next to each other or are within the same polypeptide or polynucleotide, but are separated by means of amino acid or nucleotide residues. An "objectification peptide", as used here, refers to any number of consecutive amino acid residues of the recombinant protein that are able to locate the recombinant protein in the endoplasmic reticulum (Endoplasmic Reticulum - ER) or Golgi apparatus (Golgi ) within the filamentous fungal cell. The targeting peptide can be either N-terminal or C-terminal to the catalytic domains. In certain embodiments, the objectifying peptide is N-terminal to the catalytic domains. In certain embodiments, the objectification peptide allows direct binding to the ER or Golgi membrane. Components of the objectification peptide can originate from any enzyme that normally resides in the ER or Golgi apparatus. Such enzymes include mannosidases, mannosyl transferases, glycosyl transferases, Golgi type 2 proteins and MNN2, MNNA4, MNN6, MNN9, MNN10, MNS1, KRE2, VAN1 and OCH1 enzymes. Suitable objectification peptides are described in International Patent Application No. POCT / EP2011 / 070956. In one embodiment, the GnTI or GnTIII targeting peptide is a human GnTIl enzyme. In other embodiments, the objectification peptide is derived from Kre2, of the Kre2, Och1, Anp1 and Van1 type from Trichoderma. In one embodiment, the objectifying peptide is selected from the group of SEQ ID NOs: 589-
[00275] [00275] The invention further relates to methods of using any of the filamentous fungal cells of the present description, such as Trichoderma fungal cells, which have little or no protease activity from at least three proteases and which contain a recombinant polynucleotide that encodes a heterologous polypeptide, such as a mammalian polypeptide, which is produced at increased levels to improve the stability of the heterologous polypeptide and to produce a heterologous polypeptide. Methods for measuring protein stability and for producing a heterologous polypeptide are well known and include, without limitation, all of the techniques and methods described in the present description.
[00276] [00276] Thus, certain modalities of the present description refer to methods to improve the stability of the heterologous polypeptide by: a) supply of a filamentous fungal cell of the present description having reduced or nonexistent activity of at least three proteases, where the cell additionally contains a recombinant polynucleotide that encodes a heterologous polypeptide; and b) culturing the cell so that the heterologous polypeptide is expressed, where the heterologous polypeptide has increased stability compared to a host cell that does not contain the mutations of the genes encoding the proteases. Other modalities of the present description refer to methods to improve the stability of the mammalian polypeptide by: a) providing a Trichoderma fungal cell of the present description having reduced or nonexistent activity of at least three proteases, where the cell it additionally contains a recombinant polynucleotide that encodes a mammalian polypeptide; and b) culturing the cell so that the mammalian polypeptide is expressed, wherein the mammalian polypeptide has increased stability compared to a host cell that does not contain the mutations of the genes encoding the proteases. The filamentous fungal cell or Trichoderma fungal cell can be any cell described in the section entitled "Filamentous Fungal Cells of the Invention". Methods for measuring polypeptide stability and for culturing filamentous fungal cells and Trichoderma fungal cells are well known in the art and include, without limitation, all of the techniques and methods described in the present description.
[00277] [00277] In certain embodiments, the stability of the heterologous polypeptide or mammal polypeptide is increased by at least 2 times, at least 3 times, at least 4 times, at least 6 times, at least 7 times at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 75 times, at least 80 times, at least 90 times, at least 100 times or greater compared to a heterologous polypeptide or mammal polypeptide expressed in a cell filamentous fungal or corresponding parental Trichoderma fungal cell.
[00278] [00278] Other modalities of the present description refer to methods of preparing a heterologous polypeptide by: a) supply of a filamentous fungal cell of the present description having reduced or nonexistent activity of at least three proteases, where the cell it additionally contains a recombinant polynucleotide that encodes a heterologous polypeptide; b) culturing the host cell so that the heterologous polypeptide is expressed; and c) purification of the heterologous polypeptide. Other embodiments of the present description refer to methods of preparing a mammalian polypeptide by: a) providing a Trichoderma fungal cell of the present description having reduced or nonexistent activity of at least three proteases, where the cell additionally contains a recombinant polynucleotide that encodes a mammal polypeptide; b) culture of the host cell so that the mammalian polypeptide is expressed; and c) purification of the mammalian polypeptide. The filamentous fungal cell or Trichoderma fungal cell can be any cell described in the section entitled "Invention Filament Fungal Cells". Methods of culturing filamentous fungal cells and Trichoderma fungal cells and purifying polypeptides are well known in the art and include, without limitation, all the techniques and methods described in the present description.
[00279] [00279] In certain modalities, the filamentous fungal cell or Trichoderma fungal cell is cultivated in a pH range selected from a pH of 3.5 to 7; a pH of 3.5 to 6.5; a pH of 4 to 6; a pH of 4.3 to 5.7; a pH of 4.4 to 5.6; and a pH of 4.5 to 5.5. In certain modalities, for the production of an antibody, the filamentous fungal cell or Trichoderma fungal cell is cultivated in a pH range selected from a pH of 4.7 to 6.5; a pH of 4.8 to 6.0; a pH of 4.9 to 5.9; and a pH of 5.0 to 5.8.
[00280] [00280] In some embodiments, the heterologous polypeptide is a mammalian polypeptide. In other embodiments, the heterologous polypeptide is a non-mammalian polypeptide.
[00281] [00281] In certain embodiments, the mammalian polypeptide is selected from an immunoglobulin, an immunoglobulin heavy chain, an immunoglobulin light chain, a monoclonal antibody, a hybrid antibody, an F antibody fragment (ab '), an F (ab) antibody fragment, an Fv molecule, a single chain Fv antibody, a dimeric antibody fragment, a trimeric antibody fragment, a functional antibody fragment, an antibody with a single domain, antibodies with a single multimeric domain, an immunoadhesin, insulin-like growth factor 1, a growth hormone, insulin and erythropoietin. In other embodiments, the mammalian protein is an immunoglobulin or growth factor 1 similar to insulin. In still other embodiments, the mammalian protein is an antibody. In other embodiments, the yield of the mammalian polypeptide is at least 0.5, at least 1, at least 2, at least 3, at least 4 or at least 5 grams per liter. In certain embodiments, the polypeptide is a mammalian antibody, optionally I9G1, IgG2, IgG3 or IgG4. In other embodiments, the yield of the antibody is at least 0.5, at least 1, at least 2, at least 3, at least 4, or at least 5 grams per liter. In still other embodiments, the mammalian polypeptide is either a growth factor or a cytokine. In other embodiments, the yield of the growth factor or cytokine is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 1, at least 1.5, at least 2, at least 3, at least 4 or at least 5 grams per liter. In other
[00282] [00282] In certain embodiments where the mammalian polypeptide is purified from cell culture, the culture containing the mammalian polypeptide contains fragments of polypeptides that make up a mass percentage that is less than 50%, less than 40%, less 30%, less than 20% or less than 10% of the mass of the polypeptides produced. In certain preferred embodiments, the mammalian polypeptide is an antibody and the polypeptide fragments are heavy chain fragments and / or light chain fragments. In other embodiments where the mammalian polypeptide is an antibody and the antibody is purified from cell culture, the culture containing the antibody contains free heavy chains and / or free light chains that constitute a mass percentage that it is less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the mass of the antibody produced. Methods for determining the mass percentage of polypeptide fragments are well known in the art and include measurement of signal strength from an SDS gel.
[00283] [00283] According to other modalities, the non-mammalian polypeptide is selected from an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase. chitinase, cutinase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,
[00284] [00284] In certain modalities of any of the described methods, the method also includes the stage of supplying one or more, two or more, three or more, four or more or five or more protease inhibitors. In certain embodiments, protease inhibitors are peptides that are coexpressed with the mammalian polypeptide. In other embodiments, the inhibitors inhibit at least two, at least three or at least four proteases from a protease family selected from aspartic proteases, trypsin-like serine proteases, subtilisin proteases and glutamic proteases.
[00285] [00285] In certain modalities of any of the described methods, the filamentous fungal cell or Trichoderma fungal cell also contains a carrier protein. As used here, a "carrier protein" is part of a protein that is endogenous to and highly secreted by a filamentous fungal cell or Trichoderma fungal cell. Suitable carrier proteins include, without limitation, those from mannanase | T. reesei (Man5A or MANI), T. reesei cellobiohydrolase (Cel6A or CBHII) (see, for example, Palo-heimo et al., Appl Environ Microbiol, December 2003; 69 (12): 7073-7082) or cellobiohydrolase | T. reesei (CBHI). In some modalities, the carrier protein is CBH1. In other embodiments, the carrier protein is a truncated CBH1 protein from T. reesei that includes the central region of CBH1 and part of the CBH1 binding region. In some embodiments, a vehicle, such as a cellobiohydrolase or fragment thereof, is fused to an antibody light chain and / or an antibody heavy chain. In some embodiments, a vehicle, such as a cellobiohydrolase or fragment thereof, is fused to the
[00286] [00286] It should be understood that, although the invention has been described in conjunction with certain specific modalities thereof, the preceding description is intended to illustrate and not to limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be evident those versed in the technique to which the invention belongs.
[00287] [00287] The invention having been described, the following examples are offered to illustrate the invention by way of illustration, not by way of limitation.
[00288] [00288] This example demonstrates the ability of aspartic proteases from culture supernatants of Trichoderma reesei (T. reesei) to degrade heavy chains and antibody light chains. Purification of Aspartic Protease
[00289] [00289] It was discovered that protease activity in T. reesei supernatants could be inhibited with the aspartic protease inhibitor pepstatin A. Therefore, pepstatin A (Sigma ttP2032) was linked to the agarose beads via diaminodipropylamine binder and was used as an affinity resin for purification. The T. reesei batch fermentation supernatant (15 ml) was used to bind the proteases to the resin in 35 ml of buffer containing 50 mM sodium acetate, 0.2 M NaCl, pH 3.0. The column was washed with the same binding buffer and the bound protein was removed with elution buffer (50 MM Tris-HCl, 1 M NaCl, pH 8.5). Fractions of 0.5 ml were collected. In total, 42 µg of protease was purified. The peak fraction contained 0.04 µg of protein / µl. 30 ul of each fraction were mixed with 6 ul of Laemmli sample buffer containing B-mercaptoethanol. The samples were heated to 95 ° C for 5 minutes before being loaded onto a 4-15% PAGE gel (BioRad Mini-Protean TGX pre-fused gel) along with a wide variety of pre-molecular weight markers stained (Bio-Rad). The gel was passed in continuous buffer to SDS PAGE for 100 V minutes and then stained with blue GelCode dye (Thermo Scientific).
[00290] [00290] A 42 kD double band was purified on the pepstatin A affinity column (Figure 1) and excised from the SDS PAGE gel and subjected to trypsin digestion with trypsin modified for degree of sequencing (Promega tV5111). The resulting peptides were then extracted from the gel and purified by C18 ZipTip (Milpore tZTC18M096). The purified peptides were analyzed by LC-MS / MS in an ESI-hybrid QSTAR Pulsar quadrupole TOF (AB Sciex).
[00291] [00291] This analysis resulted in the identification of 4 aspartic proteases that have very similar molecular weights. Identified proteases included: pep1 (Tre74156; 42.7 kD, 42% sequence coverage), pep2 (Tre53961; 42.4 kD, 15% sequence coverage), pep3 (Tre121133; 49 kD, 6% sequence coverage) and pep5 (Tre81004; 45 kD, 9% sequence coverage). These proteases
[00292] [00292] Protein (0.8 µg) of the peak fraction (F3) was then incubated with IgG (50 pg / ml) in sodium citrate buffer (50 mM, pH 5.5) at 37 C for 20 hours (Figure 2). The protein was incubated in the presence or absence of 10 µM pepstatin A. The antibody mixture was combined with Laemmli sample buffer and heated to 95 ° C for 5 minutes. These samples were then loaded onto a 4-15% PAGE gel (BioRad Mini-Protean TGX pre-fused gel), along with a wide variety of pre-stained molecular weight markers (BioRad). The gel was passed in continuous buffer to SDS PAGE for 30 minutes at 100 V. The IgG was not reduced before being passed on the gel. Full-size IgG migrates just above the 200 kDa marker. As can be seen in the unreduced gel in Figure 2, aspartic proteases were able to produce mild IgG degradation. In addition, IgG degradation was inhibited by pepstatin A. Aspartic protease activity was more limited at a pH of 5.5 than at an acidic pH, where it had maximum activity. Pep1 Deletion Analysis
[00293] [00293] Aspartic protease pep1 was then tested to determine its abundance in 7. reesei. This was done by purifying aspartic proteases from supernatant samples derived from the M182 strain with pep71 deletion. The M182 strain with pep1 deletion also produces the rituximab antibody.
[00294] [00294] The M181 strain with pep1 deletion made on the M124 base strain was grown in large shake flask cultures together with M124 control flasks. Cultures were grown in 300 ml of TTMM with 4 g / L of lactose, 2 g / L of grain extract consumed and PIPPS at 100 mM, pH 5.5. Three different model antibodies were incubated (final concentration 0.05 µg / µl) in the culture supernatants in a shaking flask (diluted to 2 mg / ml in sodium citrate buffer, pH 5.5) of the strain with pep1 deletion and its parental strain M124 and, as a comparison, in a fermentation culture supernatant of the parental strain. Samples of supernatant (30 µl) from cultures of day 5 containing antibody were loaded onto a 4-15% SDS PAGE gel and transferred to nitrocellulose for immunoblotting with an AP-conjugated heavy chain antibody (Sigma f4A3188 ) or AP-conjugated light anti-chain antibody (Sigma HA3813) diluted 1: 30,000 in TBST. When incubated with antibody overnight for 18 hours, the Apep1 supernatant degraded less of the heavy chain protein compared to the control strain M124 or fermentation supernatant (pH 5.5; 28 (C, 20 g / L of grain extract consumed, 60 g / L lactose) (Figure 39). The heavy chain was more susceptible to degradation compared to the light chain. The greater stabilizing effect was evident for the heavy chains of rituximab and MABO1. heavy chain, two distinct degradation products can be seen at -48 kD and -38 kD (Figure 39), there was only a slight improvement in the stability of the light chain protein in the Apep1 supernatant compared to the controls (Figure 39).
[00295] [00295] The first construct with pep1 deletion (TrelD74156) was designed to allow removal of the selection marker from the Trichoderma reesei genome after successful integration and, thus, recycling of the selection marker for subsequent inactivation of genes from protease. In this approach, the recycling of the marker, that is, removal of the pyr4 gene from the deletion construct, is similar to the so-called disintegrating cassettes developed for yeasts (Hartl, L. and Seiboth, B., 2005, Curr Genet
[00296] [00296] The TrelD number refers to the identification number of a particular protease gene from the Trichoderma reesei v2.0 genome database of the Joint Genome Institute. Primers for constructing deleted plasmids were designed "with the naked eye" or using Primer3 software (website Primer3, Rozen and Skaletsky (2000) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pages 365-386 ).
[00297] [00297] The principle of the disintegrating cassette using pyr4 as the marker gene is as follows: pyr4, which encodes T. reesei mono-phosphate (OMP) decarboxylase (OMP) (Smith, JL et al., 1991, Curriculum Genetics 19: 27-33) is necessary for uridine synthesis, strains deficient in OMP decarboxylase activity are unable to grow in minimal medium without uridine supplementation (ie, they are auxotrophic for uridine). 5-fluoro-orotic (5-FOA) in the generation of mutants lacking OMP decarboxylase activity (pyr4- strains) is based on the conversion of 5-FOA into a toxic intermediate 5-fluoro-UMP by OMP decarboxylase Therefore, cells that have a mutated pyr4 gene are resistant to 5-FOA but, in addition, they are also auxotrophic for uridine. Resistance to 5-FOA can, in principle, also result from a mutation in another gene (pyr2, orotate phosphoribosyl transferase) and, therefore, the spontaneous mutants obtained with this selection they must be checked for the pyr4- genotype by complementing the mutant with the pyr4 gene. Once it has mutated, the pyr4 gene can be used as a marker of auxotrophic selection in T. reei. In our disintegrating cassette, pyr4 is followed by a repeat
[00298] [00298] Thus, the selection marker pyr4 and the 5 'direct repeat fragment (808 bp S'UTR from pyr4) were produced by PCR using plasmid pARO502 (containing a genomic copy of pyr4 from T. reesei) as a model. PCR amplification was performed with Phusion polymerase and HF buffer or GC buffer or with Dynazyme EXT polymerase. The reaction conditions varied based on the fragment to be amplified. Both fragments contained 40 bp overlapping sequences necessary to clone the plasmid with the / oopout cassette using homologous yeast recombination (see below). To allow for possible additional cloning steps, an Ascl digest site was placed between the pyr4 marker and the 5 'and Notl direct repeat to enclose the complete disintegrating cassette.
[00299] [00299] 1066 bp from the 5 'flanking region and 1037 bp from the 3' flanking region were selected as the base of the plasmid with pep71 deletion. The fragments were produced by PCR. The products were separated by agarose gel electrophoresis and the correct fragments were isolated from the gel with a gel extraction kit (Qiagen) using conventional laboratory methods. Model DNA used to amplify the flanking regions was from the wild type T. reesei strain QM6a (ATCC13631).
[00300] [00300] For the yeast homologous recombination system used in cloning, sequences that overlap the vector and the selection marker were placed in appropriate PCR primers. To allow interruption of the marker in the construct, Notl restriction sites were introduced between the flanking regions and the selection marker. Pmel restriction sites were placed between the vector and the flanking regions to remove the vector sequence before transformation into T. reesei. The pRS426 vector backbone was digested with restriction enzymes (EcoRI and Xhol). The restriction fragments were then separated by agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods.
[00301] [00301] To construct the plasmid with deletion, the vector skeleton and the appropriate marker and fragments of the flanking region were transformed into Saccharomyces cerevisiae (strain H3488 / FY834). The yeast transformation protocol was based on the yeast homologous recombination method described in the workshop material for Colot and Collopy Neurospora knockouts (Dartmouth Neurospora Genome Protocols website) and the Gietz laboratory protocol (University of Manitoba, website from the Gietz laboratory). Plasmid DNA from yeast transformants was recovered by transformation into Escherichia coli. Some clones were cultured, the plasmid DNA was isolated and digested for screening for correct recombination using conventional laboratory methods. Some clones with correct insert sizes have been sequenced and stored.
[00302] [00302] The first plasmid with pep1 deletion (plasmid pTTv41, Table 1.1) used another selection marker, bar, a synthetic construct that brings a N-acetyl transferase from Streptomyces ssp. (GenBank ID: AFO013602.1, Sweigard et al., 1997, Fungal
[00303] [00303] To allow recycling of the selection marker and to allow rapid elimination of subsequent protease genes, pep1 was deleted from M127 (pyr4- mutant of the basic M124 strain) using the pyr4 disintegrating cassette described above. To remove the vector sequence, plasmid pTTv71 (Apep1-pyr4) was digested with Pmel and the correct fragment was purified from an agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the pep1 deletion shell was used to transform the M127 strain. Preparation of protoplasts and transformation for selection of pyr4 were performed essentially according to methods in Penttila et al. (1987, Gene 61: 155-164) and Gruber et al. (1990, Curr. Genet. 18: 71-76).
[00304] [00304] 200 clones were chosen as selective stripes. 24 fast-growing transformants such as selective streaks were screened by PCR using the primers listed in Table 1.2 for correct integration using conventional laboratory methods. Seven putative disrupted entities were purified in single cell clones. Pep1 deletion was verified by Southern analysis of these clones (Figure 3A) using conventional laboratory methods. DNA for Southern analysis was extracted with the Easy-DNA kit for isolation of genomic DNA (Invitrogen). Southern analyzes were performed essentially according to the protocol for homologous hybridizations in Sambrook et al. (1989, Molecular Cloning: A laboratory manual. 2nd Ed., Cold Spring Harbor Laboratory Press) using radioactive marking (* P), the HexaLabel Plus or DecaLabel Plus (Fermentas) kits. Digestion schemes for Southern were designed using the Sci Ed Central for Windows 95 software (Clone Manager 5 for Windows 95) or Geneious Pro 5.3.6 (Geneious website). Southern analyzes also found that four of the clones were individual members (Figures 3B and 3C). Three clones indicated multiple or inaccurate integration of the deletion cassette and were discarded. Two pure clones were designated with strain numbers M181 (9-20A-1) and M195 (9-35A-1).
[00305] [00305] To remove the vector sequence, plasmid pTTv41 (Apep1-bar) was digested with Pmel and the correct fragment was purified from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen). About 5 µg of the pep1 deletion cassette were used to transform the M169 strain (expressing humanized rituximab antibody). Protoplast preparation and transformation were carried out according to the methods described in Penttila et al. (1987) and Avalos et al. (1989).
[00306] [00306] About 100 clones were chosen as selective stripes. 24 fast-growing transformants as selective stripes were screened by PCR (using the primers listed in Table
[00307] [00307] The M182 strain was grown in minimal medium for Trichoderma (TrMM) supplemented with 20 g / l of consumed grain extract, 60 g / l of lactose and 8.1 g / l of casamino acids at pH 5.5 and 28 “C. Seven micrograms of aspartic protease were recovered from 15 ml of supernatant. When the purified fractions were passed on a 4-15% SDS PAGE gel (BioRad Mini- Protean TGX pre-fused gel), the 42 kD molecular weight band previously observed in the parental strain had disappeared (Figure 5 ). Only a weak band around 40 kDa can be seen. The 40 kD band can correspond to smaller aspartic proteases. A second purification was done from a culture supernatant where pep1 was present. The M169 strain produced rituximab and did not contain a pep1 protease deletion. The strain was grown in minimal medium for Trichoderma supplemented with 20 g / l of grain extract consumed, 60 g / l of lactose and 8.1 g / l of casamino acids at a pH of 5.5 and 28 C. 17 ug of aspartic protease were purified from 15 ml of supernatant and showed a 42 KD band on the SDS PAGE gel (Figure 5). According to this analysis, about 10 µg of pep1 protease are produced per 15 ml of culture supernatant. That is, about 60% of the total aspartic protease and only about 0.04% of the total protein content in the supernatant. These data demonstrate that pep1 is the most abundant aspartic protease in 7. review.
[00308] [00308] The deletion of pep2 showed only a slight improvement in the production of heavy antibody chain and reduction in total protease activity (Figures 6 and 7).
[00309] [00309] Therefore, pep3 and pep5 were the next important proteases to be deleted, especially in a pep1 / tsp1 / tsp1 triple-deletion strain, since they additionally contribute up to half the remaining protease activity in a supernatant. of the strain with triple deletion. Plasmid Generation with pep2 Deletion
[00310] [00310] Plasmid pTTv96 with aspartic protease deletion pep2 (TrelDO053961) was constructed essentially as described for plasmid pTTv41 with pep1 deletion above. 920 bp of the 5 'flanking region and 1081 bp of the 3' flanking region were selected based on the plasmid with pep 2 deletion. Fragments of the flanking region were produced by PCR using the primers listed in Table 1.3. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. Model DNA used in the flanking region PCR was from the wild type T. reesei strain QM6a. The bar cassette was obtained from pTTv41 through digestion with Notl. The vector skeleton was pRS426 digested with E-coRI / Xhol, as per pTTv41 above. The plasmid was constructed using the yeast homologous recombination method described for pTITv41 above. This pep2 deletion plasmid (pTTv96) results in a 1437 bp deletion at the pep2 locus and encompasses the complete coding sequence for PEP 2.
[00311] [00311] To remove the vector sequence, plasmid pTTv96 (Apep2-bar) was digested with Pmel and the correct fragment was purified from an agarose gel using the QIAquick Gel Extraction Kit (Qiagen). Approximately 6 µg of the pep2 deletion cassette was used to transform the M169 strain (which expresses humanized rituxrimab antibody). Preparation of protoplasts and transformation were carried out as described for M182 above using selection with bar.
[00312] [00312] More than 200 clones were chosen as selective stripes. 29 transformants grew as well as second stripes. The top 10 transformants that grow rapidly as selective stripes were screened for correct integration by PCR using the primers listed in Table 1.4 using conventional laboratory methods. The deletion cassette was correctly integrated in 9 of the 10 analyzed clones. The open reading frame was excluded in 9 of the 10 transformants analyzed by PCR. Five disrupted entities were purified in single cell clones. A pure transformer (206A) was designated with strain number M455. Table 1.4: Initiators for screening integration of constructs with pep2 deletion. For pTTv96 integration tracking Initiator —— Sequence Bflang fw. Vector GTARCGCCAGGGTTTTCCCAGTCACGAC - "úÚúÚÀ GTTTAAACTTCAGTTGTGGCA TCTCAGC (SEQ ID NO: 265) To32 bar Lopp CATTGTTGACCTCCACTAGC (SEQ ID NO: 266) u is ALKU CGTCACCGAGATCTGATCC to30 bar (SEQ ID NO: 267) rev 3fanaq - GCGGATAACAATTTCACACAGGAAACAG- = | GTTTAAACTCCTCACCGAAG AGCAAGTC vector rev (SEQ | D NO: 268) For ORFdepep2 deletion screening | Initiator ——— Sequence T601 pep2 fwd GACGTGGTACGACAACATCG (SEQ ID NO: 269) | T623 pep2 rev TATCAAGGTACCGGGGACAG (SEQ ID NO: 270) | Analysis of the production by ritu pep2 deletion
[00313] [00313] The M455 strain, 4 other transformers with pep2 deletion and the parental M169 strain that produces rituximab were grown in flask cultures in minimal medium for Trichoderma
[00314] [00314] The aspartic proteases pep3 (tre121133) and pep7 (tre58669) from T. reesei expressed from Pichia were also tested in vitro, measuring the degradation of MABO1 and IGF-1 antibodies. Degradation of MABO1 and IGF-1 by pep3 and pep7 was analyzed by immunoblot. Aspartic proteases were produced in Pichia supernatants. Pichia supernatants were diluted to a 1x concentration and then mixed with 50 mM sodium citrate buffer, pH 5.5. MABO1 was added to each reaction, so that the final concentration was 0.05 ua / ul. IGF-1 was added to each reaction so that the final concentration was 0.30 µg / µl. Ten microliters of each of the reaction mixtures were then collected and added to 3 μl of Laemmli sample buffer with B-mercaptoethanol. The samples were heated to 95 (C for 5 minutes before being loaded onto a 4-15% PAGE A gel (BioRad Mini-Protean pre-fused gel), along with a pre-stained All molecular weight marker Blue Precision Plus (BioRad) The PAGE gel was passed for 30 minutes at 200 V. The proteins in the gel were then electrotransferred to a 100 V nitrocellulose filter for 1 hour. The protein containing nitrocellulose filter was then, blocked with 5% powdered milk in saline solution buffered with Tris with 0.1% Tween (TBST), for 1 hour under stirring at room temperature. The blocked membranes were then hybridized with the MABO1-containing membranes were hybridized with a heavy IgG AP-conjugated anti-chain antibody (Sigma t% A3188) diluted 1: 30,000 in TBST. IGF-1 samples were analyzed using a primary anti-IGF- 1 (1: 2000 in TBST) and secondary anti-IgG AP-conjugated antibody (1: 5000 in TBST). antibody actions were performed for 1 hour at room temperature on a shaker. The membranes were then washed with 3 exchanges of TBST for 20 minutes each on the shaker. The membranes were developed with BCIP / NBT alkaline phosphatase substrate (Promega & S3771) for up to 5 minutes. As shown in Figure 8, the protease pep3 had low MABO1 degradation activity at a pH of 5.5 after overnight incubation at 37 CT, but the activity was higher at a pH of 4.5. The pep7 protease had only minimal antibody degradation activity at a pH of 4.5.
[00315] [00315] Several additional aspartic proteases were isolated from the M277 strain with triple deletion of T. reesei protease (pep1, tsp1,
[00316] [00316] 30 ul of each purified fraction was then passed on a 4-15% SDS PAGE gel (BioRad Mini-Protean TGX pre-melted gel) and stained overnight with GelCode blue (Thermo Scientific) ). The SDS PAGE gel showed predominant bands around 42 kDa and a weak band around 25 kDa (Figure 9). The bands of the gel were then cut and submitted to trypsin digestion in gel with trypsin modified for degree of sequencing (Promise tV5111). The resulting peptides were extracted from the gel and purified by C18 ZipTip (Millipore tZTC18M096). The purified peptides were analyzed by LC-MS / MS in a TOF with quadrupole E-SI-hybrid QSTAR Pulsar (AB Sciex). This analysis revealed that PEP 2,
[00317] [00317] The SIP-purified proteases were then tested for their ability to degrade the heavy chain of the MABO1 antibody. The SIP-purified proteases were incubated overnight with MABO1 at a final concentration of 0.05 µg / µl in 37% sodium citrate buffer (CC. The samples were incubated at a pH of 4.0 and a pH of 5 , 5 and in the presence and absence of a SIP inhibitor peptide Reactions were collected later.The collected samples were analyzed by immunoblot with a heavy IgG AP-conjugated anti-chain antibody (Sigma A3188) diluted 1: 30,000 in TBST. The immunoblot results showed that proteases had high protease activity against the MABO1 heavy chain when incubated at a pH of 4.0 and reduced activity at a pH of 5.5 (Figure 10). both aspartic and glutamic protease activity were inhibited by incubation with the SIP peptide (Figure 10). Analysis of SIP-purified Aspartic Proteases
[00318] [00318] Protease activity was then tested against casein in the presence and absence of protease inhibitors. Protease activity against casein was tested using the EnzChek protease assay kit (Molecular Probes% E6638, green fluorescent casein substrate). The working stock solution was prepared by diluting the stock to 10 µg / ml in 50 MM sodium citrate, pH 5.5. The purified protease fractions (10 µl) were diluted with 40 µl sodium citrate, pH 5.5. 100 μl of the diluted substrate was combined with the protease fractions diluted in a 96 well specimen plate. The plate was then covered and kept at 37 ºC for one to three hours. Fluorescence readings were taken in one, two and three hours with a Varioskan fluorescent plate reader (Thermo Scienti-
[00319] [00319] The SIP inhibitor peptide, pepstatin A, LIP peptide, SBTI and chemostatin were used as inhibitors. The SIP inhibitor peptide inhibited both aspartic and glutamic proteases; pepstatin A inhibited only aspartic proteases; LIP peptide inhibited only glutamic protease; SBT! was able to inhibit SLP2 and PEP4 and chemostatin inhibited SLP2. SIP, LIP and pepstatin A were used at a concentration of 60 UM and SBTI was used at a concentration of 200 pg / ml. To differentiate between aspartic and glutamic proteases, pepstatin A was used as an inhibitor, since it does not inhibit glutamic proteases.
[00320] [00320] When casein digestion was studied, a large part of the SIP protease activity was inhibited by pepstatin A (Figure 11). The results of casein degradation studies suggested that a large part of the activity at a pH of 5.5 in the purified fractions originates from aspartic proteases. The LIP peptide, which is the GAP1 pro-peptide, inhibited the protease activity slightly less compared to the SIP inhibitor. SBTI and chemostatin were able to inhibit the SLP2 protease in the purified sample.
[00321] [00321] These results support the conclusion that there are 4 aspartic proteases present in the SIP fraction (PEP2, PEP3, PEP4 AND PEPS5). EXAMPLE 2 - IDENTIFICATION OF GLUTAMIC PROTEASES
[00322] [00322] This example demonstrates the ability of glutamic proteases from culture supernatants of Trichoderma reesei (T. reesei) to degrade heavy chains and antibody light chains. Gap Deletion Analysis1
[00323] [00323] It was previously determined that there are four sequences of glutamic protease in the T. reesei genome. The most abundant glutamic protease is gap71 (tre69555), as determined by transcription profile characterization. Thus, the protease gap1 was purified from the T. reesei supernatant by means of SIP peptide affinity chromatography, as described in Example
[00324] [00324] A gap1 deletion was then generated using the T. reesei M244 strain that produces MABO1 antibody (Apep1). Generation of plasmid with gap1 deletion
[00325] [00325] Plasmid pTTv117 with glutamic protease deletion gap1 (TrelD69555) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1. 1000 bp 5 'flanking region and 1100 bp flanking region 3 'were selected as the base of the plasmid with gap1 deletion. Fragments of the flanking region were produced by PCR using the primers listed in Table 2.1. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. Model DNA used in the PCR of the flanking regions was from the wild type T. reesei strain QM6a. The pyr4 disintegrating cassette was obtained from pTTv71 through digestion with Notl. The vector skeleton was pRS426 digested with EcoRI / Xhol, as in Example 1. The plasmid was constructed using the homologous yeast recombination method described in Example 1. This gap1 deletion plasmid (pTTv1I7) resulted in a deletion of 1037 bp at the gap1 locus and covers the complete GAP1 coding sequence.
[00326] [00326] To generate the strain that produces MABO1 antibody for the double protease deletions, the M181 strain with pep1 deletion (Example 1) was transformed with MABO1 heavy and light chain constructs (pTTv98 + pTTv67) using hygromycin and acetamide in the selection. This strain that produces MABO1 with pep1 deletion was named M244. Removal of the p-yr4 disintegrating cassette from the pep1 locus was performed essentially as described in Example 3 below for M195 (in the generation of the M219 strain with double protease deletion). This pyr4- strain was designated with the number M285 and used as the parental strain for the subsequent deletion of
[00327] [00327] To remove the vector sequence, plasmid pTTv1 17 (Agap1-pyr4) was digested with Pmel and the correct fragment purified from an agarose gel using the QIAquick Gel Extraction Kit (Qigen). Approximately 5 µg of the gap1 deletion cassette was used to transform the M285 strain (pyr4- of the M244 strain that produces MABO1 antibody, based on the M181 Apep1 strain). Preparation of protoplasts and transformation were performed using selection with bpyr4 essentially as described for strains M181 and M195 with pep1 deletion in Example 1.
[00328] [00328] Colonies of the transformation plates were chosen as selective stripes. Rapidly growing clones as selective stripes were screened by PCR using the primers listed in Table 2.2 for correct integration using conventional laboratory methods. Putative disrupted entities were purified in single cell clones. Table 2.2: Initiators for screening gap1 integration and strain purity For screening for pTTv117 integration Primer Sequence TO52 gap1 5tracing CTCAGAAAGGTTGTAGTTGTGA (SEQIDNO: 275) =
[00330] [00330] The double deletion strain (Apep1Agap1) was grown in a 2-liter shake flask culture containing 300 ml of minimal medium for Trichoderma supplemented with 40 g / I of lactose, 20 g / I | of grain extract consumed and 9 g / l of casamino acids and buffered at a pH of 5.5 with 100 mM PIPPS. The Agap1 strain was then tested for the production of MABO1 heavy and light chain (Figure 12). Was the Agap1 strain compared to strains having deletions in each of s / p1, sp and sip3. The M244 Apep1 strain was used as a control. The samples were from cultures in a large 7-day shaking flask. The samples were analyzed by immunoblot with heavy anti-chain antibody (Sigma tA3188) or light anti-chain (Sigma 4XA3812) of IgG AP-conjugate (Figure 12). The gap1 deletion resulted in a 2-fold improvement in heavy chain production and a 1.6-fold improvement in light chain production compared to the control M244 strain (Figure 13). Gap2 Deletion Analysis
[00331] [00331] Based on the transcription profile data generated from the Trichoderma reesei strain M194, the second most abundant glutamic protease was identified as gap2 (tre106661). Thus, the protease gap2 was also deleted from the M244 strain (Apep1) using the pTTV145 deletion construct. Generation of plasmid with gap2 deletion
[00332] [00332] Plasmid pTTv145 with deletion of glutamic protease gap2 (TrelD106661) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1. 1021 bp of 5 'flanking region and 1010 bp of flanking region 3 'were selected based on the gap2 deletion plasmid. In this plasmid, the direct repeat fragment from the pyr4 disintegrator cassette was exchanged from SUTR from pyr4 to 320 bp of direct repeat from the end of the 5 'flanking region of gap2 and no Ascl sites were added between pyr4 and a direct repetition
[00333] [00333] To generate the strain that produces MABO1 antibody for double protease deletions, the strain M181 with pep1 deletion (Example 1) was transformed with MABO1 heavy and light chain constructs (pTTv98 + pTTv67) using hygromycin and acetamide in selection. The removal of the pyr4 disintegrating cassette from the pep1 locus was carried out essentially as described in Example 3 below for M195 (in the generation of the M219 strain with double protease deletion). This pyr4- strain was designated with the number M285 and used as a parent for subsequent protease deletion. Generation of M360 strains with Apep1Agap2 double deletion that produce MABO1
[00334] [00334] To remove the vector sequence, plasmid pTTv145 (Agap2-pyr4) was digested with Pmel and the correct fragment purified from an agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the gap2 deletion cassette was used to transform the M285 strain (pyr4- from the M244 strain that produces MABO1 antibody, based on the M181 Apep1 strain). Protoplast preparation and transformation were performed using selection with bpyr4 essentially as described for strains M181 and M195 in Example 1.
[00335] [00335] The colonies of the transformation plates were collected as selective stripes. Rapidly growing clones as selective streaks were screened by PCR using the primers listed in Table 2.4 for correct integration using conventional laboratory methods. Putative disrupted entities were purified in single cell clones. Table 2.4: Initiators for tracking gap2 integration and strain purity. For pTTv145 integration screening Initiator —— TO48 gap2 sequence 5 tracking F = —— GCTTGGCATCACGGAAGCT (SEQ | D NO: 287) TO26 Pyr4 orf 55rev2 - - CCATGAGCTTGAACAGGTAA (SEQ ID NO: 288) TOCC gapGAGA AGTGT : 289) TO28 Pyr4 flang rev ——— CATCCTCAAGGCCTCAGAC (SEQ ID NO: 290) For ORFdegap2 deletion screening T107 gap2 ORF FATGGATGCTATCCGAGCCAG (SEQ ID NO: 291) T108 gap2 ORF RCTATTCATACTCAACAGTCACA NO. with double deletion Apep1Agap2 that produces MABO1
[00336] [00336] Several deletion transformants have been produced. The culture supernatants from these transformants were passed on a 4-15% SDS PAGE A gel, and then the MABO1 antibody heavy chain was analyzed by immunoblot with an anti-AP heavy-chain antibody (Sigma tHA3188 ) and the light chain was detected with an AP-conjugated light anti-chain antibody (Sigma HA3812). The results of the immunoblot demonstrate that the gap2 deletion resulted in a multiple-fold increase in the production of heavy and light MAbO1 chains (Figure 14). Gap2 analysis expressed in Pichia
[00337] [00337] Pichia supernatants containing Trichoderma reesei gap2 were also studied in vitro. The supernatant containing gap2 and MABO1 antibody were diluted in sodium citrate buffers adjusted to a pH of 4.0, 4.5, 5.0 and 5.5 and incubated for 20 hours at 37 ° C (C. Samples were collected a 0 minutes and after 20 hours MABO1 heavy chain production was analyzed by immunoblot using an AP-conjugated IgG heavy chain antibody (Sigma HA3188) .The results of the immunoblot demonstrate that gap2 had maximum proteolytic activity against the chain heavy MABO1 at a pH of 4.0 (Figure 15). Although the protease gap2 activity has been reduced at a pH of 5.5 (Figure 15), over 4 days it was able to demonstrate an activity protease gap2 produced degradation products around 25 kDa, indicating that it has proteolytic activity in the hinge region of the heavy chain Example 3 - IDENTIFICATION OF SERINE PROTEASES
[00338] [00338] This example demonstrates the ability of the serine proteases of Trichoderma reesei (T. reesei) to degrade heavy chains and antibody light chains. Purification of Serine Protease
[00339] [00339] Serine proteases include a large family of proteases that have been identified as antibody-degrading enzymes. In this way, serine proteases were purified from the Trichoderma supernatant. Serine proteases were first affinity purified from fermentation culture supernatants with a fast-flowing p-aminobenzamidine Sepharose 4 resin (GE Healthcare ft117-5123-10). 15 ml of the batch fermentation culture supernatant was bound to the resin in 35 ml of binding buffer (0.05 M Tris-HCI, 0.5 M NaCl, pH 7.4). After packaging and washing the column with the same binding buffer, the column was eluted with 0.05 M glycine, pH 3.0. The fractions were then neutralized with 1 M Tris-HCl, pH 8.8.
[00340] [00340] A total of 1.7 mg of protein has been purified from the affinity column. When the peak fractions were passed on a 4-15% SDS PAGE gel, several major bands (- 110 kD, 53 kD, 39 kD, 29 kD) and many other smaller bands were observed. The peak fraction protein mixture (F4) was then tested for protease activity by incubating a sample of F4 with human IgG1 in sodium citrate buffer (50 mM, pH 5.5) at 37 (C for 20 hours. Samples were incubated in the presence and absence of the serine protease inhibitor PMSF (5 mM). The incubated samples were then analyzed by immunoblot with an anti-heavy chain antibody of IgG AP-conjugate (Sigma% A3188) and an anti-IgG AP-conjugated light chain antibody (Sigma t% A3812) diluted 1: 30000 in TBST. The results of the immunoblot showed that the purified protein F4 fraction it completely degraded IgG (Figure 16), in addition, treatment with PMSF was able to inhibit most of the degradation, which indicates that the protease activity in the F4 fraction responsible for IgG degradation was predominantly the activity of serine protease.
[00341] [00341] In order to identify the proteins in which the purified fractions exhibited protease activity, the peak fractions were passed on a SDS PAGE for IgG zymogram gel (0.5 mg / ml MABO2) ( 12%). The purified fractions and samples of unpurified supernatants were passed on the zymogram gel under denaturing conditions. After passing the gel, the proteins in the gel were renatured by incubating the gel in 1% Triton X-100
[00342] [00342] There were two clear bands visible on the IgG zimogram gel of about 29 kD and 65 kD. However, the 29 kD band was much more prevalent, suggesting that it may be responsible for most of the serine protease activity in the sample. These bands were the only two visible in the sample of unpurified supernatant and were more pronounced in the purified fractions (Figure 17). When the protease sample was pretreated with PMSF, a known serine protease inhibitor, the light white bands appeared gray or were not visible, which indicates that the bands correspond to the serine protease enzymes (Figure 17) .
[00343] [00343] Identification of 29 kD sIp1 serine protease
[00344] [00344] From a corresponding SDS PAGE gel without MABO 2, the 29 kDa band was cut from the gel and subjected to trypsin-in-gel digestion with trypsin modified for degree of sequencing (Promega t & V5111). In the purified fractions, the 29 kD band was observed as a distinct protein band. This distinctive band was then isolated. The resulting peptides were extracted from the gel and purified by C18 ZipTip (Millipore & XZTC18M096). The purified peptides were analyzed by LC-MS / MS in a TOF with ESI-hybrid QSTAR Pulsar (AB Sciex). The resulting mass analysis clearly identified the 29 kDa band as the sIp1 trypsin-like serine protease (tre73897, 35% sequence coverage). SIp1 deletion analysis
[00345] [00345] The slp1 gene encoding (tsp1) was then excluded from the strain
[00346] [00346] The deletion construct for the first protease gene, pep1 (TrelD74156), was designed as described above in Example 1. Generation of plasmids with tsp1 deletion
[00347] [00347] Plasmids with tsp1 alkaline trypsin-like serine protease deletion (TrelD71322 / TrelD73897, Dienes et al., 2007, Enz Microb Tech 40: 1087-1094), were constructed essentially as described for pep1 deletion plasmids in Example 1. 953 bp of the 5 'flanking region and 926 bp of the 3' flanking region were selected based on plasmids with sIp1 deletion. As for pep1, the first plasmid with tsp1 deletion (pTTv42) used bar as the selection marker. The fragments of the flanking region were produced by PCR using the primers listed in Table 3.1. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. Model DNA used in PCR of the flanking regions was the QM6a strain of T. reesei of wild type. The bar marker was obtained from pTTv41 (Example 1) through digestion with Notl. The vector backbone was pRS426 digested with EcoRV / Xhol, as in Example 1. The plasmid was constructed using the yeast homologous recombination method described in Example 1.
[00348] [00348] To clone the second plasmid with tsp1 deletion (PTTv72), the bar marker was removed from the plasmid with pTTv42 deletion through digestion with Notl. The p-yr4 disintegrating cassette was obtained from pTTv71 (Example 1) by digesting with Notl, bound to NotT cut pTTv42 and transformed into E. coli using conventional laboratory methods. Some transformants were cultured, the plasmid DNA isolated and digested for screening for correct binding and orientation of the pyr4 disintegrating cassette using conventional methods. A clone with the correct insert size and orientation was sequenced and stored. These sIp1 deletion plasmids (pTTv42 and pTTv72) result in a 1252 bp deletion at the sIp1 locus and cover the entire tsp1 coding sequence. Table 3.1: Primers to generate plasmids with deletion s- Ip1 pTTv42 deletion plasmid for TSP1 (TrelD71322 / TrelD73897), vector pRS426 backbone Primer Sequence T303 SF 71322 GTAACGCCAGGGTTTTCCCAGTCAC- ACGGTTTAAACTGCTGTTGCTG TTTGTTGATG (SEQ ID NO: 293) T304 71322 CCCGTCACCGAGATCTGATCCGTCACCG- 5r en GATCCACTTAAGCGGCCGC CTGTGGTGAGATCTCCAGACG (SEQ ID NO: 294)
[00349] [00349] To reuse pyr4 as the selection marker, removal of the pyr4 disintegrator cassette from the M195 strain with pep1 deletion was performed. The spores were spread on plates with minimal medium containing 20 g / l of glucose, 2 g / l of peptone protease, 1 ml / 1 of Triton X-100, 5 mM uridine and 1.5 g / l of 5-FOA , pH 4.8. Colonies resistant to 5-FOA were chosen after 5-7 days in 0.9% NaCl, completely suspended in vortex and filtered through a pipette with a cotton tip. To purify the clones in single cell clones, the filtrates were spread out again on the plates described above. The purified clones were sporulated on plates containing 39 g / l of agarose with potato dextrose. These clones were tested for auxotrophy for uridine by placing spores on plates with minimal medium (20 g / | glucose, 1 mL / | Triton X-100), where no growth was observed, indicating that the clones selected were pyr4-. All clones were subsequently tested by PCR (using the primers listed in Table 3.2) for removal of the disintegrating cassette and proved to be correct. The clone (9-35A-1A-a), used to generate the double protease deletion strain (M219), was designated with the strain number M196
[00350] [00350] To remove the vector sequence, plasmid pTTv72 (Atsp1-pyr4) was digested with Pmel and the correct fragment was purified from an agarose gel using a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the sIp1 deletion cassette was used to transform M196 (Apep1, pyr4-). Protoplast preparation and transformation were performed using pyr4 selection essentially as described for strains M181 and M195 with pep1 deletion in Example 1.
[00351] [00351] More than 100 colonies were chosen and 48 were selected by PCR using the primers listed in Table 3.2 for the correct integration of the deletion cassette and also deletion of tsp1 ORF using conventional laboratory methods. Four putative Atsp1 clones were purified into single cell clones. Deletion of slp1 was verified by Southern analyzes of these clones (Figure 18A), using conventional laboratory methods described in Example 1 for M181 and M195. Sourthern's analysis also indicated that only four transformants (two parallel clones of two transformants, clones 16-5AA, 16-5BA, 16-11AA, 16-11BA, Figures 18B and 18C) were individual members. The other clones were determined to have additional copies elsewhere in the genome and were discarded. To exclude the weak signal seen in Figure 18 for the sIlp1 ORF in transformants originating from the sIp1 gene, the tsp1 ORF deletion was confirmed by PCR using the primers in Table 3.2. No signal for ORF of tsp1 was obtained. The clone (16-5AA) used to remove the pyr4 disintegrating cassette (and to generate the M277 triple deletion strain) was designated with the M219 strain number (Apep1Atsp1). Generation of M252 strain with double deletions Apep1 Atsp1 that produces MABO1
[00352] [00352] To remove the vector sequence, plasmid pTTv42 (Atsp1-bar) was digested with Pmel and the correct fragment purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the sIip1 deletion cassette was used to transform the M181 strain (Apep1, Example 1). Preparation of protoplasts and transformation were performed using selection with bar essentially as described for the M182 strain with pep1i deletion in Example 1.
[00353] [00353] The colonies that grow on transformation plates were chosen as selective stripes. Rapidly growing clones as selective stripes were screened by PCR using the primers listed in Table 3.2 for correct integration using conventional laboratory methods. Putative disrupted entities were purified in single cell clones. Deletion of slp1 was verified by Southern analyzes of these clones (Figure 19A), using conventional laboratory methods described in Example 1 for M181 and M195. All clones were also verified as being unique members (Figures 19B and 19C). A double protease deletion clone (13-172D) was assigned the number M194. Table 3.2: Initiators for screening pyrá4 disintegrator cassette removal and for screening for tsp1i integration and strain purity
[00354] [00354] For screening pyr4 disintegrating cassette removal of M195 TO83 74156 5th seq GATCGACAAAGGTTCCAGCG (SEQ ID TO84 74156 3rd seq AATTGTATCATTCCGAGGCT (SEQ | D us a
[00355] [00355] The M194 strain with double protease deletion was used to generate M247 and M252 strains that express MABO1 antibody below. Construction of the M247 strain was performed by transforming M194 with MABO1 heavy and light chain constructs (pTTv1OI + pTTv102). Cepa M252 was built by transforming M194 with heavy and light chain constructs from MABO1 (pTTv99 + pTTv67). Both transformations were based on selection with hygromycin and
[00356] [00356] It has been shown that the double deletion strain (Apep1Atsp1) that produces MABO1 produces 261 mg / l of antibody, with 43% of full length antibody, when grown in a fermenter. The strain's protease activity was then tested by growing the strain in minimal medium for Trichoderma supplemented with g / l of consumed grain extract, 60 g / l of lactose and 9 g / l of bedrock acids at a pH of 5.5 and 22 "(C. The total protease activity against casein in this strain was determined to be 2.0 times less than the wild type M124 strain (Figure 20). 65 kD sIp1 serine
[00357] [00357] The protease that produces activity around 65 kDa was more difficult to identify due to its low level of expression and the proximity of size with several highly expressed proteins. Highly expressed proteins were previously identified as CBHI, CBHII, CIP2 and xylanase 4. Enhancements were made to better separate the 65 kD protease from the highly expressed proteins. Improvements included the use of a lower percentage of gel (7%) in the standard SDS PAGE gel zymogram and SDS PAGE gels to pass the samples a longer time, so that the molecular weight marker of 54 kD was on the bottom of the gel. In addition, the fermentation supernatant of a T. reesei transformant that produces rituximab antibody was also used to purify serine proteases. The rituximab antibody transformant is the M169 strain that produces rituximab and lacks protease deletions. The strain was grown in a minimal medium for Trichoderma supplemented with 20 g / l of grain extract consumed and 60 g / | lactose at a pH of 5.56 28 T. At
[00358] [00358] Peptide analysis showed that the protein with the second highest score was the protease tre51365. The protein with the highest score was xylanase 4, which was a contaminant in the sample. The protease subtilisin tre51365, now called sIp1, was found in three independent samples from three different purifications. In the sample with the best score, 6 peptides were found and sequenced by LC-MS / MS. The sequence coverage was 8%, since the gene encodes the native 882 amino acid protease that makes up a 93 kD protease. In zymography, a weak band at -90 kD can be seen along with blots up to 65 kDa, suggesting that the sIp1 protein itself is proteolysis, but maintains much of its activity. Generation of a plasmid with sIp1 deletion
[00359] [00359] The gene encoding SLP1 (slp1) was then deleted in the M244 strain that produces MABO1 antibody (Apep1).
[00360] [00360] The subtilisin sIp1 protease deletion plasmid (TrelD51365) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1. 1094 bp from the 5 'flanking region and 1247 bp from the flanking region 3 'were selected as the base of the sIp1 deletion plasmid. The fragments were produced by PCR using the initiators listed in Table 3.3. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods.
[00361] [00361] To generate the strain with double deletions of protease that MABO1 produces, the M181 strain with pep1 deletion (in Example 1) was transformed with the MABO1 heavy and light chain constructs (PTTv98 + pTTv67) using hygromycin selection and acetamide. The removal of the disintegrating cassette pyr4 from the pep1 site was performed essentially as described for M195 above (in the generation of the M219 strain with double protease deletion). This pyr4- strain was designated with the number M285 and used as a parent for subsequent protease deletion.
[00362] [00362] To remove the vector sequence, plasmid pTTv126 (AsIp1 -pyr4) was digested with Pmel and the correct fragment purified from an agarose gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the sIp1 deletion cassette was used to transform M285 (pyr4- of the M244 strain that produces MABO1 antibody, based on the M181 Apep1 strain). Preparation of protoplasts and transformation were performed using selection with pyr4 essentially as described for strains M181 and M195 with deletion of pep1 in Example 1.
[00363] [00363] The colonies that grow on transformation plates were chosen as selective stripes. Rapidly growing clones as selective stripes were screened by PCR using the primers listed in Table 3.4 for correct integration using conventional laboratory methods. Putative disrupted entities were purified in single cell clones.
[00364] [00364] Deletion of sIp1 in the M244 strain showed an expected improvement in the production of light and heavy chains (Figures 12 and 13). The sIp1 deletion strain (Apep1 Aslp1) was grown in a 2-liter shake flask culture containing 300 ml of minimum medium for Trichoderma supplemented with 40 g / I of lactose, 20 g / I of grain extract consumed and 9 g / I | of casamino acids and buffered at a pH of 5.5 with 100 mM PIPPS. As described in Example 2 above, the culture supernatants were separated on a 4-15% PAGE gel and immunoblot performed to detect MABO1 heavy and light chains. The heavy chain was produced at levels that were 2.8 times higher than the production levels of the M244 strain (Figure 13). The light chain was produced at levels that were 1.8 times higher than the production levels of the M244 strain (Figure 13). Identification of additional serine proteases
[00365] [00365] Serine proteases that degrade additional antibodies have been identified using other affinity linkers. The soy trypsin inhibitor (SBTI) effectively stabilizes antibody heavy and light chains. Therefore, it is able to inhibit proteases that are responsible for the cleavage of the antibody. Thus, in order to identify these proteases, affinity purification was performed with SBTI coupled to the agarose (Sigma% T0637).
[00366] [00366] The M44 strain of T. reesei was used to identify proteases. The M44 strain is a wild type strain with no expression of heterologous protein. The M44 strain was grown in minimum medium for Trichoderma supplemented with 20 g / | l of consumed grain extract and 60 g / | of lactose at a pH of 5.5 and 28 CC. A sample of 20 ml of M44 culture supernatant from a 217 hour sample was incubated with the SBAR-agarose affinity resin (1 ml) in 30 ml of binding buffer (50 mM Tris, NaCl a 0.5 M, pH 7.5) (pH 5.5; 28 O, 20 g / L of grain extract consumed, 60 g / L of lactose). The ligation buffer / supernatant mixture was combined in a 50 ml conical tube and stirred at room temperature for 1 hour. The mixture was then added to a glass column and washed with 200 ml of binding buffer. 50 ml of high salt buffer (1 M NaCl) was then used to further remove non-specific interactions. Finally, the column was washed again with 100 ml of binding / initial wash buffer. The column was then eluted with benzamidine. 0.8 M HCI in 50 mM Tris, pH 5.0. Fractions were collected in 0.5 ml volumes and subjected to a Bradford protein assay using BioRad's reagent with bovine immunoglobulin as a standard.
[00367] [00367] From all collected fractions, 190 µg of protein was purified from the SBTI affinity column. The peak fraction was washed in a Vivaspin Ultrafiltration centrifuge filter (Sartorius-Stedim) with a 10 kD molecular weight cut to remove the benzamidine inhibitor and the concentrated fraction. Concentrated fractions (CF3 and CF4) and non-concentrated fractions (F1-F4) were loaded onto a MABO2 zymogram gel (as described above) and a regular SDS PAGE gel for analysis. The results of the zymogram show that there are two visible proteolytic activities (Figure 22). The most prevalent band was visible at around 40 kDa and a weaker band was visible at around 26 kDa (Figure 22). In the zymogram gel, bands of darker colored proteins were on the white edges of the zymogram activity. Comparing them to concentrated fractions loaded on an SDS PAGE gel, these double bands could be seen around 38 kDa (Figure 23). The PAGE gel was a 4-15% gradient gel and the zymogram gel was 12%, so that the relative sizes may be slightly different. In the PAGE gel, a protein band could be clearly seen in the 26 kD area, which corresponded to the size of the second weakest activity in the zymogram.
[00368] [00368] To further analyze the proteolytic activity of purified CF3 protein, the fraction was tested for its ability to degrade the heavy chain of the rituximab antibody. A 5 µl sample of CF3 was incubated in sodium citrate buffer, pH 5.5 with 0.05 pg / ml rituximab. The incubated samples were then analyzed by immunoblotting using a specific anti-AP human IgG heavy chain antibody (Sigma tA3188) diluted 1: 30000 in TBST. The results of the immunoblot demonstrate that the proteases immediately degraded the heavy chain of the rituximab antibody. The full-length rituximab heavy chain migrated just over 50 kDa, while the initial degradation product was about 45 kD (Figure 24). In addition, overnight incubation generated an additional 38 kD product (Figure 24).
[00369] [00369] The proteases responsible for the activities in the zymogram were identified after peptide sequencing by LC-MS / MS. Gel sections containing proteins were cut from the SDS PA-GE gel shown in Figure 23 and submitted to trypsin digestion with trypsin modified for degree of sequencing (Promega ÉV5111). The resulting peptides were extracted from the gel and purified by C18 ZipTip (Millipore & ZTC18M096). The purified peptides were analyzed by LC-MS / MS in a TOF with quadrupole ESI-hybrid QSTAR Pulsar (AB Sciex).
[00370] [00370] The highest scoring protease was the subtilisin type protease, sIp2 (tre123244). Two sIlp2 peptides were found and sequenced, covering 6% of the full length sequence. The full length slIp2 protease is 58 kD, but it is usual for the active protease to be smaller in size.
[00371] [00371] Also other proteases have been found in adjacent regions. Analysis of the region below 26 kD identified the trypsin-like serine protease sIp1 (tre73897). This corresponded to the weak activity observed in the zymogram. As described above, this protease was identified through affinity purification with amino-benzamidine.
[00372] [00372] In addition, the entire SBTI affinity purified fraction was digested with trypsin in the solution to determine the entire protease content of the sample. Other proteases identified included the sIlp7 protease tre123865 (60 kD); the pep4 protease tre77579 (42 kD); and the sIp8 protease tre58698 (41 kD).
[00373] [00373] Trichoderma reesei subtilisin sIp5, sIp6 and sIip7 proteases were overproduced in Pichia supernatants for
[00374] [00374] Based on the results above, the s- Ip2 and sIp3 protease genes were each deleted from the M244 strain that produces MABO1 antibody.
[00375] [00375] Plasmids with subtilisin protease deletion sIp2 (tre1D 123244) and sIp3 (TrelD123234) were essentially constructed as described for plasmid pTTv41 with pep1 deletion in Example 1. 1000 bp from the region of 5 'and 1100 bp flanking of the 3' flanking region were selected based on the slp2 deletion plasmid. For slp3, 1000 bp from the 5 'flanking region and 1100 bp from the 3' flanking region were selected. The fragments were produced by PCR using the primers listed in Table 3.5. The products were separated by means of agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The model used in PCR of the flanking regions was the QM6a strain of T. reesei of wild type. The pyr4 disintegrating cassette was obtained from pTTv71 (Example
[00376] [00376] To generate the strain with double deletions of protease that produces MABO1 antibody, the strain M181 with pep1 deletion (in Example 1) was transformed with the MABO1 heavy and light chain constructs (pTTv98 + pTTv67) using selection with hygromycin and acetamide. The removal of the pyr4 disintegrating cassette from the pep1 locus was performed essentially as described for M195 above (in the generation of the M219 strain with double protease deletion). This pyr4- strain was assigned the number M285 and used as a parental for subsequent protease deletions.
[00377] [00377] To remove the vector sequence, plasmids pTTv115 (Asl | p2-pyr4) and pTTv116 (Aslp3-pyr4) were digested with Pmel and the correct fragments purified from an agar gel using QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of deletion cassette were used to transform M285 (pyr4- from the M244 strain that produces MABO1 antibody based on the M181 Apep1 strain) separately. Protoplast preparation and transformation were carried out using selection with pyr4 essentially as described for strains M181 and M195 with pep1 deletion in Example
[00378] [00378] Colonies that grow on transformation plates were chosen as selective stripes. Rapidly growing clones as selective stripes were screened by PCR using the primers listed in Table 3.6 for correct integration using conventional laboratory methods. Putative disrupted entities were purified in single cell clones. No pure clones were obtained, even after repeated purification steps. Table 3.6: Initiators for tracing the integration of slp2 (PTTvV115) and sIp3 (pTTv116) and strain purity For tracing the integration of pTTv115 Initiator Sequence TOS54 sIlp2 5tracking F. GATGCACCGCTGCGGCC (SEQ | DTACTAGGG2G2GG2GTGTGTGTGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTZDHXHPHHHP002H0 / 02 EPS D NO: 328) TO028 Pyr4 flang rev CATCCTCAAGGCCTCAGAC (SEQ ID NO: TO55 slp2 3trans R R GGCGTTGCTCCCCATGCG (SEQ ID NO: 330) T111 slp2 ORF F ATGCGGTCCGTTGTCGCC (SEQ ID NO: T1AG slag2) TOS56 sIp3 5translation F. GTGAATGGGTGGCAACATGA (SEQ ID NO: 333)
[00379] [00379] The M292 strain (Apep1Aslp2) and the M295 strain (Apep1AsIip3) were grown together with their sister transformers in a 2 liter shaking culture flask containing 300 ml of minimum medium for Trichoderma supplemented with 40 g / I of lactose, 20 g / l of extracted grains consumed and 9 g / l of casamino acids and buffered at a pH of 5.5 with 100 mM PIPPS. The culture supernatants were separated on a 4-15% SDS PAGE gel and an immunoblot was performed to detect the MABO1 heavy and light chain. The results show that both deletions improved the stability of MABO1 (Figures 12 and 13). The Aslp2 deletion improved the MABO1 heavy chain expression in culture in a shake flask about 2.4 times on day 7, compared to the parental M244 strain (Figure 13). Aslp3 improved MABO1 heavy chain expression in large shake flasks by about 1.5 times and MABO1 light chain expression by about 1.7 times compared to the parental M244 strain (Figure 13). In addition, when compared to AsIp3 and Agap1, AsIp2 showed the greatest increase in the expression of the MABO1 heavy chain compared to the expression of the heavy chain
[00380] [00380] When slp2 was deleted from the M306 strain with multiple deletion (Apep1Atsp1AsIp1), sIp2 deletion resulted in a reduction in sporulation and slower growth compared to the parent strain. Example 4 - TRICHODERMA STRETCHES WITH MULTI-DELETIONS PROTEASE PLAS
[00381] [00381] This example demonstrates increased antibody production and stability from Trichoderma reesei (T. reesei) strains containing multiple deletions of the protease genes identified above in Examples 1-3. Generation of Triple Deletion Strain Apep1 Atsp1 AsIp1
[00382] [00382] The T. reesei strain with a triple deletion Apep1 Atsp1 AsIp1 was generated and tested for improvement in antibody production. The strain was also used for new rounds of protean eliminations. Generation of triple protease deletion strain M277
[00383] [00383] To generate a triple deletion protease strain without a marker, the looping of the pyr4 marker was applied to the M219 strain, essentially as described above for the outpyr4 looping of the single Apep1 deletion protease strain. Three consecutive 5-FOA selection steps were performed to ensure that the selected clones came from single cells. Final clones were checked for the looping ofpyr4 by PCR (using the primers listed in Table 3.1); no specific signs were observed with annealing initiators with the part curled out of the pyr4. The looping was still verified by plating the clones on minimal medium plates with or without 5 mM uridine. The clone used to generate the triple protease strain with deletion was designated with strain number M228 (Apep1 Atsp1, pyr4 -).
[00384] [00384] The plasmid with pTTv126 deletion for the third protease gene, subtilisin as sIlp1 protease (TrelD51365) is described above (Table 3.3). This deletion plasmid results in 2951 bp deletion at the sIp1 locus and encompasses the complete sIp1 coding sequence
[00385] [00385] To remove the vector sequence, plasmid pTTv126 (LOOPOUT Aslp1 -pyr4) was digested with Pmel and the correct fragment purified from an agarose gel using the QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the slp1 deletion cassette was used to transform M228 (Apep1 Atsp1, pyr4 -) above. Preparation of protoplasts and transformation were performed essentially as described in Example 1 for strains M181 and M195, using pyr4 selection.
[00386] [00386] 200 clones were chosen as first stripes. 48 of these stripes were screened by PCR using the primers listed in Table 4.1 for correct integration using conventional laboratory methods. Five putative triple protease disrupted entities (Apep1Atsp1 AsIp1) were purified in clones with a single cell. Deletion of sIp1 was verified by analyzes of the south of the five clones (Figure 26A). Southern analyzes were performed as described in Example 1. Southern analyzes also found that three of the clones were individual members (Figures 26B and 26C). The other two clones were shown to make additional copies elsewhere in the genome and were discarded. The clone used to remove the pyr4 disintegrating cassette (and to generate the quadratic deletion protein M307 below) was designated with the M277 strain number (Apep1 Atsp1 AsIip1). Table 4.1: Initiators for removing screening ofpyri Blaster cassette and for screening sIp1 strain integration and purity. For removing pyr4 disintegrating cassette screening from the
[00387] [00387] To generate the MABO1 antibody-producing strain for the third protease deletion, the pep11tsp1 double protease strain with M194 deletion (Example 3) was transformed with MABO1 heavy and light chain constructs (pTTv99 + pTTv67) using hygromycin and acetamide in the selection. This MABO1 strain with PEP1 / tspidouble, deletion was designated with the number M252. The removal of the disintegrator from the pyr4 PEP1 site cassette was performed essentially as described in Example 3 for M195 (in the M219 strain generation with
[00388] [00388] The third protease deletion for M284 was obtained using sIp1 deletion to construct pTTv128. This construct contains a KEX2 cassette native overexpression targeted to the sIp1 locus. Transformation was carried out essentially according to the protocol described in Example 1 for strains M181 and M195 using the pyr4 selection. The resulting strain is the one that produces MABO1 triple strain protects from the M304 deletion. Analysis that MABO1 Triple Protease Cepa produces with M304 deletion
[00389] [00389] The triple protease antibody (Apep1Atsp1 AsIlp1) deletion that produces MABO1 strain M304 has been shown to produce the MABO1 antibody with yields of up to 3.5 g / L culture (pH 5.5; 28 - 22 "CT, 60 g / L spent grain, 30 g / L glucose, 60 g / L lactose + lactose feed) and the quality of the product up to 84% of full-length IgG (see Example 6 below). The protease activity of the strain was also tested by the growth of the Trichoderma strain in minimal medium supplemented with 60 g / I of consumed grain, 30 g / | glucose and 60 g / I lactose at a pH of 5.5. The crop was grown at € and then transferred to 22 C during the production phase. Fedbatch cultivation was done with a lactose feed. The total protease activity against casein in this strain was determined to be about 3.2 times less compared to the wild type M124 strain (Figure 20). Comparison between single, double, triple and strain with deletions
[00390] [00390] The relative protease activity of culture supernatants from the single protease deletion fApep1) strain M181 (see Example 1), the double elimination of protease fApep1 Atsp1) strain M219 (see
[00391] [00391] Two proteolysis was seen in the M124 and M181 control Apep1 samples (Figure 27). The most predominant activity was observed between 65-90 kDa, which corresponds to sIlp1. The weakest activity was seen at about 28 kDa, which corresponds to sIp1. As expected, the M219 Apep1Atsp1i stock did not produce a zymogram band at 28 kD. Likewise, the M277 ApepiaAtspi Aslp1i strain produced no zymogram activity. The active size of sIip1 appears to be variable, since it was still active when it was cut up to 65 kD, although its adult size is 90 kD. The size variation can be seen in Figure 27.
[00392] [00392] The total protease activity against succinylated casein from cultures supernatant from M181, M219, M277 and elimination strains was also measured from day 3, day 5 and day 7 samples. The supernatants were first diluted to 2 mg / ml of total protein in sodium citrate at 50 MM, pH 5.5, before being tested. 50 ul of the diluted supernatant was loaded into a 96 well plate and 50 ul of succinylated casein was added to initiate the reaction. A buffered bottom control, instead of casein, was used for each sample. After the addition of casein, the protease reaction was allowed to proceed for 1 hour at 37 ° C (O. To develop the reaction of 50 µl TNBSA reagent was added to each well and the plate was incubated for 16 hours at 37 ° C (C The absorbance at 450 nm was measured for the plate set The nonspecific background signal is subtracted from the measurement of specific protease activity As shown in Figure 28, the supernatant samples from three Deletion protease strains contained less than the wild-type M124 protease activity.
[00393] [00393] The supernatant of the cultures of M277 and M124 (days 5 and 7), was diluted to 6 mg / ml in 50 mM sodium citrate buffer. For these diluted supernatants, MABO1 antibody was enriched to a final concentration of 0.05 µg / ul. These reactions were incubated at 37 CT overnight. The reactions were sampled at time zero, 1 hour and during overnight incubation. The 20 ul samples were loaded on a 4-15% SDS PAGE gel and run at 200 volts for 40 minutes. The gel was transferred at 100 volts for 1 hour to nitrocellulose for immunoblot. The membrane was blocked with 5% milk in TBST for one hour. The MABO1 heavy chain was detected with an anti-heavy chain of AP-conjugated antibody (Sigma t% A3188) diluted 1: 30000 in TBST. After washing the membrane with TBST, the blot was developed with the AP substrate (Pro-mega). Comparing the samples incubated overnight, it was clearly evident that the most degraded heavy chain in the
[00394] [00394] The M307 strain with an Apep1 Atsp1 AsSIp1 Agap1 quadruple deletion was generated and used for new rounds of protease eliminations. Generation of quadruple protease deletion strain M307
[00395] [00395] To generate a quadruple protease strain deletion without marker, removal of the pyr4 disintegrator cassette was applied to the M277 strain, essentially as described in Example 3, to remove thepyr4 disintegrator cassette from the single M195 fApep1 deletion protease strain ). Three consecutive 5-FOA selection steps were performed to ensure that the selected clones were from single cells. The final clones were checked for removal of the disintegrator cassette by PCR using the primers listed in Table 4.2, using normal laboratory methods. No specific signs were observed with annealing primers with the piece removed from pyr4. Removal was again verified by plating clones on plates with minimal medium with or without 5 mM uridine. There was no growth in the plaques without supplementation with uridine. The clone used to generate the quadruple protease deletion strain was designated with strain number M306 (Apep1 Atsp1 Aslp1, pyr4 -).
[00396] [00396] The plasmid with pTTv1 deletion 17 for the fourth protease gene, gap1 glutamic protease (TrelD69555) is described in Example 2 (Table 2.1). This deletion results in a plasmid pb deletion
[00397] [00397] To remove the vector sequence, plasmid pTTv1 17 (Agap1-pyr4) was digested with Pmel and the correct fragment was purified from an agarose gel using the QIAquick Gel Extraction Kit (Qigen). Approximately 5 µg of the gap1 deletion cassette was used to transform M306 (Apep1 Atsp1 AsIp1i, pyr4 -) above. Preparation of protoplasts and transformation were performed essentially as described in Example L To obtain strains M181 and M195 that use the pyr4 selection.
[00398] [00398] 150 clones were chosen as first stripes. 48 of these stripes were tracked by PCR using the primers listed in Table 4.2 for correct integration using conventional laboratory methods. Eight putative quadruple protease disrupted entities (Apep1Atspi Aslp1 Agap1) were purified in single cell clones. Gap1 deletion was verified by analyzes of the south of the eight clones (Figure 29a). Southern analyzes were performed as described in Example 1. Southern analyzes also found that three of the clones were individual members (Figures 29B and 29C). The other five clones were shown to make additional copies elsewhere in the genome and were discarded. The clone used to remove the disintegrating pyr4 cassette (and to generate the five-fold deletion protease strain M369 below) was designated with the strain number M307 (Apep1 Atsp1 Aslp1 Agap1). Table 4.2: Initiators for removing screening ofpyri Blaster cassette and for screening gap1 integration and strain purity. For removing pyr4 disintegrating cassette screening from the M277 Sequence Primer TO79 sIp1 scrn 5forw GCAGACAAACAGAGCAACGA
[00399] [00399] To generate quadruple protease strain with deletion with the production of MABO1 antibodies, removal of the pyr4, disintegrating cassette from the sIp1 locus of the M304 strain was performed essentially as described in Example 3 for M195 (in the M219 strain generation with deletion double protease). This pyr4 - strain was designated with the number M317 and used as a parent for subsequent protease deletion.
[00400] [00400] The fourth protease elimination for M317 was obtained using deletion gap1 to construct pTTv1 17. The transformation was carried out essentially in accordance with the protocol described in Example 1 for strains M181 and M195 using the pyr4 selection. The resulting strain is the quadruple that produces MABO1 protease strain deletion M371. Analysis of quadruple protease strain deletion
[00401] [00401] The total protease activity of culture supernatant of the M307 quadruple strain deletion was then measured and compared with culture supernatant of the M277 triple strain deletion and the wild-line M124 strain. Each strain was grown in 2 liters flasks with 300 ml TrMM containing 40 g / l lactose, 20 g / l spent grain extract and PIPPS at 100 mM pH 5.5. Day 7 samples of the supernatant were taken for the total protease assay. The total protein concentrations of the supernatants were measured using the BCA assay with bovine immunoglobulin as standard. The supernatants were serially diluted 1: 2 in sodium citrate buffer at a pH of 5.5. The diluted supernatants were added to the fluorescently labeled casein substrate and incubated at 37 CT. Fluorescence was measured after 1 hour at 485 nm excitation and 530 nm emission. The results showed that the protease activity rate of the M277 triple strain deletion was 3 times lower than that of the wild strain M124 and that the quadruple strain M307 was 8 times lower than that of the wild strain M124 (Figure 30).
[00402] [00402] In addition, Figure 20 summarizes the total protein activity against casein of the M188 single elimination strain, the M219 double elimination strain, the M277 triple strain, elimination, and the M307 quadruple deletion strain in relation to the wild type M124 strain. Thepep1 single deletion reduced protease activity by 1.7 times, PEP1 / tsp1 double deletion reduced protease activity by 2 times, PEP1 / tsp1i / tsp1 triple elimination reduced protease activity from
[00403] [00403] The antibody that produces MABO1 strain M371 contains a quadruple deletion Apep1Atsp1 AsIp1 Agap1. The strain was grown in the fermenter and compared to the MABO1 triple producer strain elimination under the same conditions. The batch cultivation was performed with the M371 strain that produced MABO1 and it was with PEP1, sip1, silph and deletions of the protease kex2 gap1 and overexpression. The strain was grown in minimal medium for Trichoderma, supplemented with 40 g / l solid grain consumed, 40 g / l glucose and 40 g / I | of lactose at a pH of 5.5. The crop was grown at 30 € T and then transferred to 22 C during the production phase. The batch cultivation was performed with the M304 strain that produced MABO1 and was with PEP1, sIp1 and deletions of the protease kex2 slp1 and overexpression. The strain was grown in minimum medium for Trichoderma, supplemented with 40 g / l solid grain consumed, 40 g / | glucose and 40 g / l lactose at a pH of 5.5. The crop was grown at € 30 and then transferred to € 22 during the production phase.
[00404] [00404] The calculated total length antibody yield was 20% higher in the gap1 strain eliminating from the day 6 sample. Under the same conditions, the quadruple deletion strain produced 1.9 g / L (897 mg / L of full length antibody) and the triple strain deletions produced 1.3 g / L (731 mg / L of full length antibody). From the fermenter supernatants, the total protease activity against casein was measured. The supernatant samples were diluted in sodium citrate buffer, pH 5.5, so that the total protein concentration was 0.15 mg / mL for all samples. For this diluted supernatant 10 µg / ml BODIPY casein was added to start the protease assay. The samples of each cultivation day were compared between the two different strains. The results show that it was not up to 30% less than the total protease activity in the gap1 strain deletion on day 5 (figure 31). On day 6, the protease activity was 20% lower, which correlates with the 20% increase in antibody yield on that day. Quintuple Strain Deletion
[00405] [00405] The M369 strain with a fivefold deletion Apep1 Atsp1 AsSIp1 Agap1 Agap 2 was generated and used for new rounds of protease elimination. Generation of quintuple protease deletion strain M369
[00406] [00406] To generate a fivefold strain protease deletion without marker, the removal of the disintegrating pyr4 cassette was applied to the M307 strain, essentially as described in Example 3, to remove the disintegrating cassette from the single M195 deletion protease strain (Apep1). Three consecutive 5-FOA selection steps were carried out to ensure that the selected clones came from single cells. The final clones were checked for removal of the disintegrator cassette by PCR using the primers listed in Table 4.3, with normal laboratory methods. No specific signs were observed with annealing primers with the part removed from the pyr4 Blaster cassette. Removal was again verified by plating clones on plates with minimal medium with or without 5 mM uridine. There was no growth in the plaques without supplementation with uridine. The clone used to generate the fivefold proteinase strain with deletion was designated with strain number M321 (Apep1 Atsp1 as IPL Agap1, pyr4 -).
[00407] [00407] The plasmid with pTTv145 deletion for the fifth protease gene, gap2 glutamic protease (TrelD106661) is described in Example 2 (Table 2.3). This plasmid deletion results in a 944 bp deletion at the gap2 locus and encompasses the complete GAP 2 coding sequence.
[00408] [00408] To remove the vector sequence, plasmid pTTv145 (LOOPOUT Agap 2-pyr4) was digested with Pmel and the correct fragment purified from an agarose gel using the QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the deletion gap2 cassette was used to transform M321 (Apep1 Atsp1 AsIp1 Agap1, pyr4 -) above. Preparation of protoplasts and transformation were performed essentially as described in Example 1 for strains M181 and M195 that use the pyr4 selection.
[00409] [00409] 100 clones were chosen as first stripes. All 20 growth bands were tested by PCR using the initiators listed in Table 4.3 for correct integration using conventional laboratory methods. 10 putative quintuple protease disrupted entities (Apep1 Atsp1 Aslp1 Agap1 Agap2) were purified in single cell clones and again screened by P-CR. Only one purified clone was negative for ORF gap2. The gap 2 deletion was verified by Southern analyzes of the clone (Figure 32A). Southern analyzes were performed as described in Example
[00410] [00410] The protease activity of the M369 strain was measured against the parental M307 strain. The protease gap2 deletion resulted in 23% less protease activity against casein (Figure 33). 6-Fold Strain Deletion
[00411] [00411] A 6 times protease deletion strain having eliminations
[00412] [00412] Apep1 Atsp1 As | Ip1 Agap1 Agap2Apep4 was generated and used for new protease rounds
[00413] [00413] exclusions. Pep4 Plasmids Generation of Elimination
[00414] [00414] The plasmid with pTTv181 deletion for the sixth protease gene, pep4 protease aspartic (TrelD77579) was essentially constructed as described for plasmid pTTv71 in Apep1 Example 1. 959 bp of the 5 'and 992 bp flanking region from the 3 'flanking region were selected on the basis of the plasmid, pept4 deletion. As forpep1, the first pep4 deletion plasmid (PTTv43, Table 4.4) carried out another selection marker, bar, which was replaced with the pyr4 Blaster cassette. The cassette was obtained from pTTv71 disintegrator through digestion with Notl, bound with cut Notl pTTv43, and then transformed into E. coli using standard methods. Some transformants were cultured, the plasmid DNA isolated and digested for screening for correct binding and orientation of the pyr4 disintegrating cassette using conventional laboratory methods. A clone with the correct insertion size and orientation was sequenced and stored (pTTv73, Table 4.4). The Blaster cassette was changed a little again: the direct repeat fragment used in removing ofpyr4 was changed from 308 bp ofpyr4 S "UTR of 300 bp direct repetition from the end of pep4 5 'flanking region (as in pTTv145, gap2-pyr4) .This was done by removing the existing pT4 disintegrating cassette from pTTv73 digestion with Notl. The pyr4 gene was amplified by PCR using pTTv73 as a template, using the primers in Table 4.4. of yeast homologous recombination used in cloning, the overlapping sequences for the vector were placed for the appropriate PCR initiators.
[00415] [00415] To generate a 6-fold protease strain with no marker deletion, removal of the pyr4 marker was applied to the M369 strain, essentially as described in Example 3, to remove the disinfecting cassette from the M195 strain (Apep1). Three consecutive 5-FOA selection steps were performed to ensure that the selected clones came from single cells. The final clones were verified by PCR using the primers listed in Table 4.5, using normal laboratory methods. Signs corresponding to the successful removal of the disintegrating cassette were obtained for all
[00416] [00416] To remove the vector sequence, plasmid pTTv181 (LOOPOUT Apep4-pyr4) was digested with Pmel and the correct fragment purified from an agarose gel using the QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the pep4 deletion cassette was used to transform M381 (Apep1 Atsp1i Aslp1i Agap1 Agap 2, pyr4 -). Preparation of protoplasts and transformation were performed essentially as described in Example 1 for strains M181 and M195 that use the pyr4 selection.
[00417] [00417] More than 200 transformants were chosen as first stripes. 32 growth bands were tested by PCR (using the primers listed in Table 4.5) for correct integration. Seven clones gave the expected signals and were purified for single cell clones and again screened by PCR using the primers listed in Table 4.5. Pep4 deletion was verified by Southern analyzes from five clones (Figures 34A and 34B), using conventional laboratory methods described in Example 3 for M181 and M195. Analisa Sul also indicated that all transformants (Figures 34C and 34D) were individual members. To exclude that the weak signal seen in the PCR screening for pep4 ORF in transformants originating from the PEPA4 gene, three clones were purified using more single-celled steps and reanalyzed by Southern and PCR hybridizations.
[00418] [00418] The quadruple-deletion protease strain M307, the five-fold deletion protease strain M369 and the 6-fold deformation-transforming deletion protease were grown in shaken flask cultures. Supernatant samples taken from large cultures in a shaking flask were grown in TrMM with 20 g / L of grain consumed and 40 g / L of lactose buffered with 100 mM PIPPS at a pH of 4.8. The pH was -4.25 on day 5. The 6 protein-elimination transformants tested were not the final strain, so there was some variation due to the purity of the spores. These were some of the best transformers, but spore purification was done later. Days 5 supernatants were diluted 1: 3 in 50 mM sodium citrate buffer pH 4.5. For this diluted supernatant BODIPY FL casein (10 µg / ml) and incubated together at 37 “C for 4 hours. For the protease activity of the assay was performed as described in the manufacturer's protocol (enzCheck protease kit assay% XE6638, Molecular Probes). The results of the protease activity can be seen in Figure 33.
[00419] [00419] There was a small reduction in protease activity when the five-fold deletion protease strain M369 was grown under acidic conditions. The gap2 deletion in the strain provided a 23% reduction in protease activity against casein. In the 6-fold deletion protease the pep4 protease aspartic strains were eliminated in the 5 studied transformants. The transforming enhancer showed a reduction of 35% in relation to the parental strain M369. Generation of 7-Fold Strain Deletion
[00420] [00420] The 7 times protease deletion strain has eliminations
[00421] [00421] ApepiAtsp1i Aslpb1i Agapi Agap2Apep44pep3 was generated and used for new rounds of protease exclusions.
[00422] [00422] The first pTTv188 deletion plasmid for the seventh protease gene, pep3 protease aspart (TrelD121133) was constructed essentially as described for the Apep1 plasmid pTTv41 in Example 1. 1,215 bp of the 10 'bp 5' flanking region of 3 'flanking region were selected as the basis of the, deleted plasmid pep3. As for gap2 (pTTv145) andpep4 (PTTv181) plasmids with the above deletion, in this plasmid the direct repeat fragment is an exception of 300 bp from the end of pep3 5 'flanking region. The fragments were produced by PCR using the primers listed in Table 4.6. As for pTTv181 (Apep4-pyr4) above, to allow changing the marker in the construct, Notl restriction sites were introduced on both sides of the selection marker and pyr4 for additional cloning the steps of an Ascl site were introduced between the and repeat pep3 5'diret 3'flanking. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. Mold used in PCR in the flanking regions of T. reesei was the wild type QM6a strain. The pyr4 marker gene was obtained from digestion with pTTv181 Notl. The vector layout was pRS426 digested with EcoRI / Xhol, as in Example 1. The plasmid was constructed using the yeast homologous recombination method described in Example 1.
[00423] [00423] The second elimination plasmid for aspartic proteasepep3 (TrelD121133), pTTv192, was constructed using the above plasmid pTTv188 as a skeleton. This second plasmid carries a native KEX2 (TrelD123156) overexpression cassette and uses the acetamidase gene (amdS) from Aspergillus nidulans as a selection marker. The Blaster pyr4 cassette was removed pTTv188 with Notl-Ascl double digest. The cDNA1 promoter fragments (model: PTHN3 plasmid DNA), native kex2 (model: genomic DNA QM6a T. reesei), trpC terminator (model: pHHO2 plasmid DNA) and AMDS marker (model: pHHOI plasmid DNA) were produced by PCR using the primers listed in Table 4.6. As for pTTv188 above, to allow altering the marker in the construct, Notl restriction sites were introduced on both sides of the amdS selection marker. The products were separated by means of agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The plasmid was constructed using the yeast homologous recombination method described in Example 1.
[00424] [00424] The third aspartic proteasepep3 deletion plasmid (TrelD121133), pTTv205, was constructed using the plasmid pTTv192 above as a skeleton. The amdS marker was removed from pTTv192 digestion with Notl. Fragments of a new disintegrating pyr4 cassette (located after the KEX2 overexpression cassette) were produced by PCR using the primers listed in the Table
[00425] [00425] These plasmids with deletion for pep3 (pTTv188,
[00426] [00426] To generate a 7-fold protease strain with no marker deletion, the removal of the pyr4 marker was applied to the 6-fold deletion M396 strain, essentially as described in Example 3, to remove, pyr4 disintegrating cassette of the M195 strain (Apep1). Four consecutive 5-FOA selection steps were carried out to ensure that the selected clones came from single cells.
[00427] [00427] The final clones were verified by PCR using the initiators listed in Table 4.7, with normal laboratory methods. Signals corresponding to the successful removal of the disintegrating cassette were obtained. The removal of the disintegrating cassette was further verified by placing the clones on plates with minimal medium with or without 5 mM uridine. There was no growth in the plaques without uridine supplementation. Southern analyzes of four putative pyr4 clones - removal of the disintegrator cassette was verified for all clones (Figure 34E). The clone (25-120A-62) used to produce the 7-fold protease strain with deletion was designated with the strain number M402 (Apep1i Atspl Asipl Agap1 Agap2Apep4, pyr4 -).
[00428] [00428] Two parallel transformations were performed; one with the deletion to build pTTv188 (standardpep3 deletion) and the other with pTTv205 (KEX2 overexpression included). To remove vector sequence, plasmids pTTv188 and pTTv205 were digested with Pmel and the correct fragments purified from agarose gel using Q! - Aquick Gel Extraction Kit (Qiagen).
[00429] [00429] “Approximately 5 µg of cassette or deletion was used to transform M402 (Apep1 Atsp1 AsIp1 Agap1 Agap2Apep4, pyr4 -). Preparation of protoplasts and transformation were performed essentially as described in Example 1 for strains M181 and M195 using pyr4 selection.
[00430] [00430] Transformants were chosen as first stripes. Growing stripes were tested by PCR (using the primers listed in Table 4.7) for correct integration. Clones that provide the expected signals were purified into single cell clones and again screened by PCR using the primers listed in Table 4.7.
[00431] [00431] Pep3 deletion was verified by Southern analysis of selected clones using the methods described in Example 1. Selected clones were used to remove the disintegrating pyr4 cassette and to generate protease deletion strains 8 times (Figure 34E). Table 4.7: Initiators for screening removal ofpyrf M396 disintegrator cassette and for screening pep3 integration and strain purity. For blaster cassette screening removal of pyr4 T302 M396 Primer Sequence Sint GATTCATCACAGGGGCAGTOC 77 579 (SEQ ID NO: 403) T214 pep4 3f SEQ COGCTCTCAAACTGCCCAAA rl (SEQ ID NO: 404) for integrating screening pTTv188 Primer Sequence T625 -pep3 5intnew ACGTGAAGTTGCCCATCAA (SEQ ID NO: 405) TO26 Pyr4 orf 5rev2 CCATGAGCTTGAACAGGTAA (SEQ ID NO: 406) T626 pep3 3intnew GACCAATGGCTTCACGAAGT (SEQ ID NO: 407) TO61 pyr4 orTAGTCTTTTTTTG 2CTATTTGTTTTTTTF
[00432] [00432] The first deletion plasmid from the protease gene of the eighth, pep5 protease aspart (TrelD81004) was essentially constructed as described for the plasmid Apep1 pTTv41 in Example 1, but a second additional selection marker cassette (bar , Example 1) was placed after the pyr4 gene to create a plasmid with a deletion disintegrator
[00433] [00433] The second aspartic pep5 protease deletion plasmid (TrelD81004), pTTv229, was constructed using the above plasmid pTTv202 as a backbone. The double marker pyr4-bar was removed from pTTv202 by digestion with Notl. The pyr4 marker gene was obtained from 81 pTTv1 digestion with Notl. The cloning of the plasmid pTTv229 was done with standard ligation using T4 DNA ligase, at room temperature. Part of the ligation mixture was transformed into E. coli with electroporation. Some clones were cultured, the plasmid DNA was isolated and digested for screening for correct binding using conventional laboratory methods. Correct connection and orientation of the marker were also verified by sequencing. These pep5-deleted plasmids (pTTv202 and pTTv229, Table 4.8) result in a pb 1687 deletion at the pep5 locus and cover the complete Pep5 coding sequence.
[00434] [00434] 1348 bp of the 5 'flanking region and 1164 bp of the 3' flanking region were selected on the basis of the deletion pep5 plasmid. A 300 bp stretch from the edge of pep5 S'flanquamento was used as a direct repeat fragment. These fragments, as well as the second selection marker cassette, the bar (Example 1), were amplified by PCR using the primers listed in Table 4.8. The products were separated by means of agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. To allow removal of the complete double marker cassette, Notl restriction sites were introduced on both sides of the double marker cassette and an AsiS | between the two selection markers. An Ascl site was introduced between the direct pep5 repeat of the 5 'and 3' flanking regions. Vector skeleton was pRS426 digested with EcoRI / Xhol, as in Example 1. The selection marker pyr4 was obtained from pTTv181 (Apep4-pir above) by digesting with Notl. The plasmid was constructed using the yeast homologous recombination method described in Example 1. This pep5 deletion plasmid (pTTv202, Table 4.8) results in a 1687 bp deletion at the pep5 locus and covers the complete coding sequence of Pep5. Table 4.8: Primers for generating plasmid with deletion. A plasmid with deletion pTTv202 to pep5 (TrelD81004) backbone vector pRS426 Primer Sequence 5'pep5 T372 is GGTAACGCCAGGGTTTTCCCAGTCAC- GACGGTTTAAACGGAGG CTGCGACACCGTCTG (SEQ ID NO: 418) T373 pep5 rev 5'GCGCTGGCAACGAGAGCAGAGCAGCAG- TAGTCGATGCTAGGCG GCCECGCCeGGceTteAAACGACCTCCC (SEQ ID NO: 419) T376 5DR is pep5
[00435] [00435] To generate an 8-fold protease strain with no marker deletion, removal of the pyr4 marker was applied for stripping M486 strain 7 times (34-14A-one, pTTv205 in M402) essentially as described in the Example 3, to remove the pyr4, disintegrator from strain M195 (Apep1). Four consecutive
[00436] [00436] Selection steps with 5-FOA were carried out to ensure that the selected clones came from single cells.
[00437] [00437] The final clones were verified by PCR using the starters listed in Table 4.9, with normal laboratory methods. Signs corresponding to the successful removal of the disintegrating cassette were obtained for most of the clones. The removal of the disintegrating cassette was further verified by placing the clones on plates with minimal medium with or without 5 mM uridine. There was no growth in the plaques without uridine supplementation. Southern analyzes of the putative pyr4 - clones verified the removal of the Blaster cassette.
[00438] [00438] To remove the vector sequence, the plasmid was digested
[00439] [00439] The transformers were chosen as first stripes. Growing stripes are tested by PCR (using the primers listed in Table 4.9) for correct integration. Clones that provide the expected signals are purified into single cell clones and again screened by PCR using the primers listed in Table 4.9. Pep5 deletion is verified by Southern analyzes of selected clones using the methods described in Example 1. Table 4.9: Primers for removing 7 times ofpyri strain disintegrator cassette and for screening pep5 integration and strain purity. For removal of M486 pyr4 disintegrating cassette screening and strain purity Sequence Primer TO47 trpC term end FCCTATGAGTCGTTTACCCAGA (SEQ ID NO: 426) T854 pep3 3f r2 TGEGCCGAGTCTATGCGTA (SEQ ID NO: 427) TQTGGG44 SEQTGGG44 : 428) A 61 pyr4 orf 2F screening TTAGGCGACCTCTTTTTCCA (SEQ ID NO: 429) T855 pep3 orf f3 GTAAGACGCCCCGTCTC (SEQ ID NO: 430) T754 pep3 orf rev2 TGGATCATGTTGGCGACG (SEQ ID: 43)
[00440] [00440] This example demonstrates the ability of protease inhibitors to increase the production of full length antibodies in Trichoderma reesei production strains.
[00441] [00441] Based on the knowledge that the heavy chain is cleaved by trypsin and chymotryptic proteases, inhibitors of these two classes of enzymes have been tested against antibody degradation, both in vitro and in culture experiments, using an antibody from T. reesei producing strain. The soybean inhibitor trypsin inhibitor (SBTI) and chemostatin were tested, once they have been previously demonstrated in in vitro experiments, to stabilize heavy antibody chains. In vitro inhibitory treatment
[00442] [00442] Chemostatin and SBTI were analyzed in vitro with the
[00443] [00443] For this supernatant of 0.05 µg / ul diluted rituximab, 100 µM chemostatin, 1 mg / ml SBTI or a combination of both inhibitors were added to a total volume of 50 µl and shown at 0, 1 and 19 hours to assess the early and late degradation of the heavy chain of the rituximab antibody. The resulting heavy chain products were analyzed by immunoblot using and anti-heavy chain of AP-conjugated antibody (Sigma% A3188) diluted 1: 30000 in TBST (Figure 35). The initial degradation products generated from the heavy chain were approximately 42 kDa and 38 kDa, which were seen on the untreated control track for 1 hour (Figure 35). Additional fragments were generated after 19 hours, the two main products remained. Chemostatin treatment inhibited the initial production of the 42 kD fragment, while the SBTI treatment inhibited the 38 kD fragment from formation (Figure 35). The combination of the two compounds inhibited about 96% of the initial heavy chain degradation and about 75% of the degradation after 19 hours (Figure 35). These results demonstrate that the two inhibitors were able to effectively stabilize the heavy chain of the rituximab antibody. Treatment of T. reesei with Inhibitor cultures
[00444] [00444] The effectiveness of the inhibitors was also tested with the antibody producing strain rituximab, which contains a binding sequence and the VALEKR PEP1 deletion. This strain was grown in triplicate, in small flasks, both in the presence or absence of chemostatin, SBTI or pepstatin A. The small shake flasks contained 50 ml of TTMM plus lactose (40 9/1), of grains extract consumed ( 20 g / l |) and buffer with 100 mM PIPPS at a pH of 5.5. The SBTI inhibitor was added to the culture in final concentrations of 100, either ug / ml or 500 pg / ml. Chemostatin was used at 100 µM, and pepestatin A was used at 10 µM. Each of the three inhibitors was added to the cultures daily on day 2, 3,4 and 5.
[00445] [00445] The growth of the cultures was followed by pH daily from day 2 to day 7. For cultures grown with SBTI there were no significant differences in the pH of the cultures compared to untreated controls. In Pipps buffered cultures, the pH decreased from an initial pH of 5.5 to a pH of 4.8, after day 6. With chemostatin and pepstatin A, cultures were monitored until day 7. On day 7 , the mean pH for the control flasks was 4.6, while the mean pH of chemostatin treated cultures was 4.96 the mean pH of pepstatin The treated cultures was 5.0. Thus, for cultures treated with chemostatin and pepstatin A, there was a small reduction in growth.
[00446] [00446] Samples of culture supernatant (30 ul) were also collected on days 3, 4 and 5 for analysis of antibody production. The analysis was performed by immunoblots, using anti-AP heavy chain antibody conjugate (Sigma XA3188) and anti-AP anti-conjugate antibody light chain (Sigma tHtA3812). Each antibody was diluted 1: 30,000 in TBST. The total length rituximab heavy chain is about 51 kD, the CHBI fusion rituximab light chain is about 100 kD and the free rituximab light chain is about 28 kD.
[00447] [00447] The results of the immunoblot analysis of culture supernatant samples treated with SBTI are shown in Figure 36.
[00448] [00448] The results of the immunoblot analysis of culture supernatant samples treated with chemostatin and pepstatin A are shown in Figure 37. Chemostatin treated cultures showed results similar to those observed with SBTI (Figure 37). The rituximab heavy chain was stabilized on day 5 (Figure 37B). When compared to untreated control cultures, chytatin increased the amount of rituximab heavy chain produced total length, although the main degradation product at 38 kD was still seen.
[00449] [00449] Overall, it appears that the SBTI treatment was more effective in promoting higher protein production than the chemostatin treatment. However, the chemostatin treatment produced a heavy chain rituximab total length greater than the proportion of fragment. As can be seen in Figure 37B, the third sample of culture chemostatin showed approximately 90% of the full length heavy chain of rituximab compared to 10% of the heavy chain fragment. Thus, the combination of SBTI and chemotherapy treatment would be very beneficial to achieve higher yields of antibody production. Example 6 - Production of T. reesei antibodies
[00450] [00450] This example quantifies the amount of antibodies produced
[00451] [00451] Culture supernatants from each of the T. reesei protease deletion strains listed in Table 6.1 were filtered through 0.45 | J, with a syringe filter and adjusted to the binding buffer composition by adding 1/50 volume of 1 M sodium phosphate, pH 7, before purification. The affinity column was connected to an AKTA purifier and the purification was carried out according to the manufacturer's instructions. The following chromatography conditions were used: flow rate, 1 ml / min; detection at 280 nm; injection septum, ml; buffer A, 20 mM sodium phosphate, pH 7; buffer B, 0.1 M glycine-HCl, pH 2.7. Isocratic run with buffer A was conducted until the start of elution, which was performed with 5 ml of buffer B. The column was balanced with at least 5 ml of buffer A before each analysis. 1 ml of culture supernatant was injected for quantitative functioning. 0.5 ml fractions were collected in tubes containing 40 | V x | 0.5 M Tris, pH 9, during the elution step. The antibody was eluted markedly in two fractions, which were combined in a 1 ml sample. From the samples with the largest peak areas between each series of samples (fermentation), a run with 5-10 ml of injection volume was performed to obtain a more concentrated sample for analysis of gel filtration.
[00452] [00452] For quantification, a series of standard dilutions of antibody was prepared and run on 1 ml of similar Protein G HiTrap column volume for the analyzed samples. The standard curve (10-500 | J, g) was established from peak areas measured at 280 nm and used for the quantification of MABO1 or rituximab antibodies isolated from culture supernatants. The quality of the purified samples was verified by SDS PAGE.
[00453] [00453] At 250 V x | sample of each purified MABO1 and rituximab antibody was administered in Tris buffered saline (25 mM Tris, 140 mM NaCl and 3 mM KCl, pH 7.4) on a Superdex 200 10/300 GL gel filtration column (Amersham Biosciences), connected to an AKTA HPLC purifying system. The flow rate was 0.75 ml / min and the absorbance was measured at 280 nm. Fractions (0.75 ml) were collected over the entire period. The fractions that showed only one peak were concentrated and characterized on a standard SDS PAGE gel (Figure 38). The percentage of each eluted peak was calculated by dividing the peak area with the total sample area measured at 280 nm.
[00454] [00454] The antibody purification process is shown in Figure 38. Antibody Quantification
[00455] [00455] The amounts of antibodies produced by the protease deletion strains T. reesei are summarized in Table 6.1. Table 6.1: Synthesis of the quantity and quality of antibodies produced by cultures of the crude fermentor from the supernatant of T. reesei strains described in the Examples.
[00456] [00456] In Table 6.1, the total amount of antibody (mMAb) is the amount of protein that has been purified from the culture supernatant. After protein purification, the antibody was administered in size deletion chromatography to measure the amount of assembled full-length antibody. This amount was then referred to as "Full mAb".
[00457] [00457] As shown in Table 6.1, the M304 triple deletion strain (Apep1 Atsp1 Aslp1) obtained an antibody yield of 3500 mg / L of total IgG, with 2500 mg / L being correctly assembled at full length MABO1 antibody. This corresponds to 71% of the full length antibody. The improvement in the percentage of full length antibodies was a result of the sIp1 elimination. In contrast to the M304, M247 strain, the double elimination strain (Apep1Atsp1) achieved a 43% yield of full length antibody production (pH 5.5, 22 O; 9 gl casamino acids, 20 g / L of grain extract consumed, 60 g / L of lactose). Thus, it can be seen directly that the addition of the Aslp1 deletion significantly increases the quality of the product (25%). Improved quality of antibodies in M507 MABO1 production strain
[00458] [00458] Two MABO1 production strains were produced, M304 on a 3 protease deletion fund and M507 on a 7 protease deletion fund. The M304 strain was constructed with separate cassettes for the heavy and light chain (Figure 49). The heavy chain was integrated into the cbh1 locus and from the light chain to the EGLL locus. The M507 strain was made through the integration of a cassette containing the chain assembly within the heavy and light CBHI locus (Figure 49). MABO1 vector set guides heavy and light chains in opposite directions. The light chain uses the NVISKR cleavage site and the heavy chain uses the DGETVVKR cleavage site. The M304 strain has 3 excluded PEP1, sIp1 and slip1 proteases. The M507 has 7 proteases PEP1, sIp1, sIp1, gap1,
[00459] [00459] The bidirectional MABO1 vector set pTTv223 was transformed into the seven-fold deletion protease M486 strain with kex2 overexpression using standard protoplast transformation. The transformants were selected on acetamide-triton plates and the first stripes were screened by PCR for the 5 'and 3' integration of the amdS marker for the cbh1 locus. Double positive transformants were purified through individual spore cultures and spore actions were generated on DP plates supplemented with ampicillin.
[00460] [00460] Strains M304 and M507 were grown in fermenters with 30 g / | glucose, 60 g / I | of grain consumed, 60 g / l of lactose with lactose feed at 28 C and transferred to 22 “C at the end of the culture. The M507 strain was grown at a pH of 5.2 (cultivation bi-o000541b) and pH of 5.5 (cultivation bio00543b). The M304 strain was grown at a pH of 5.5 (cultivation bio00503b). The M304 fermentation sample bio00477b was included as a control in the immunoblot bio00503b. The bio00477b cultivation was carried out under the same conditions as described for bio00503b medium.
[00461] [00461] The M507 strain was grown at both pH 5.2 and pH 5.5 to study the effect of pH on antibody production. Samples of the fermentation supernatant of pH 5.2 were analyzed by Western blot and shown in Figure 50A and samples of 5.5 fermentation at pH, shown in Figure 50B. The antibody produced seemed quite similar in both M507 cultures. There was a little more light chain at pH 5.2 conditions. In both cultures were heavy chain fragments. The M304 strain was grown at a pH of 5.5 and the results can be seen in Figure 51. The amount of full-length heavy chain produced falls after day 7 in the harvest.
[00462] [00462] The concentrations of purified immunoglobulin in protein G of the three fermentation lanes can be seen in Table 6.2. The highest total antibody concentration for the M304 strain was 3.1 g / l on day 9. The highest concentrations for the M507 strain were on day 10, 3.0 gl at a pH of 5.2 and 2.8 g / l at a pH of 5.5. After size exclusion chromatography, the amount of full-length antibody was calculated for each sample (Table 6.3). The largest amount of full length antibody was 2.0 g / |! for both M507 fermentations on day 8 (pH 5.2) and day 9 (pH 5.5). M304 produced a similar level of 2.0 g / l of full-length antibodies on day 8. Table 6.2: Total antibody concentration determined after purification in protein G from culture samples and To Mr sm AA ea eme o ereess - rasa oncess Be po ea Boi rs ass po e ls po es Ts lr
[00463] [00463] The difference between strains M304 and M507 becomes evident when considering the percentage of antibody of full length produced during the course of cultures. The percentage of full-length antibodies was higher with the M507 strain compared to the M304 strain. The M507 strain grown at a pH of 5.5 produced the highest quality antibody, up to 78%, with total length on day 7. In M304 it was 68% on day 6, but then the product quality decreased compared to M507. The product of M507 was 73% of total length until day 9. Table 6.3: Concentration of full length antibody was calculated after chromatography by exclusion of star size Tatosete Tecoostas | Ass prey ass fo | E e er se E e mm PO e e Fm e e E graphs Table 6.4: Percentage of full-length antibody produced during the time span of COLO mm mA ess ess presses and E E E
[00464] [00464] The protease activity in the supernatant was compared between strains M304 and M507 grown under the same conditions. The Triab62 and Triab67 cultures were grown at a pH of 5.5, in 30 g / l glucose, 60 g / I | lactose, 20 g / l of whole grain consumed, 20 g / l of grain extract consumed with lactose feed at 28 C and changed to 22 C at the end of the culture.
[00465] [00465] Protein concentrations were determined from all supernatant samples from days 2-7. All supernatants were diluted in sodium citrate buffer, pH 5.5, so that all samples had a total protein concentration of 0.625 mg / ml. 100 µl of all diluted supernatants were added to a 96 well black plate using three repeated wells per sample. 100 ul diluted FL casein stock (10 pg / ml) made in sodium citrate buffer, pH 5.5, was added to each well containing supernatant. The plates were incubated covered with a plastic bag at 37 ° C. The fluorescence of the wells was measured after 2, 3 and 4 hours. Readings were made on the Varioskan fluorescent plate reader using excitation at 485 nm and emission at 530 nm.
[00466] [00466] The protease activity in the supernatant of the strain M507 with protease deletion 7 times was 2 to 2.5 times lower than M304 (protease deletion 3 times), see Table 6.5. The deleted acid proteases (gap1, gap2, pep4, pep3) contribute to this improvement. The protease activity in general of the 7-fold deleted strain is noticeably lower with the casein substrate. These data generally correlate with the results observed with full-length antibodies. Less protease activity leads to higher quality antibodies.
[00467] [00467] The protease stability of IGF1, hGH and IFNa2b model proteins was analyzed by placing them in supernatant of the M400 strain with protease deletion 6 times (Apep1Atsp1Asl | p1Agap1Agap2Apep4). The supernatant was collected from a large culture flask with CAH15 stirring. The undiluted supernatant from the CAH15 shaking culture flask was incubated with the purified model proteins with and without the pepstatin A (50 µM) and SBTI (0.2 mg / ml) inhibitors for 20 hours at 37 CT. The 5-day culture supernatant was at a pH of about 4.2. The reaction containing 0.05 upg / ul of model protein was collected after 20 hours. Sodium citrate at 50 mM, pH 4.0, enriched with model proteins (0.05 uvg9 / ul) was used as a buffer control.
[00468] [00468] From each reaction, 10 µl were loaded on 18% SDS PAGE gel and passed for 30 minutes at 200 V. The proteins in the gel were transferred to nitrocellulose for immunoblot. The nitrocellulose membrane was blocked for 1 hour at room temperature with 5% milk in TBST buffer. The individual blots were hybridized with the specific primary antibody to detect the appropriate model protein for 1 hour at room temperature on a shaker. The mouse anti-c | IGF1 antibody (R&D Systems tmab291) was used at 2 µg / ml diluted in TBST. The mouse anti-rhGH antibody (Abeam fttab51232) was used at 2 µg / ml diluted in TBST. The anti-mouse FNa2b antibody (Abeam ftab9386) was used at 1 µg / ml diluted in TBST. After washing the blot membranes quickly with TBST, the secondary antibody was added for 1 hour at room temperature with stirring. An AP-conjugated goat anti-mouse secondary antibody (Biorad ft170-6520) was diluted 1: 10,000 in TBST.
[00469] [00469] When incubated overnight in the supernatant, full-length proteins were observed for hGH, IFNa2b and IGF1, although most appeared to be degraded (Figure 42). There was a predominant degradation product of human growth hormone and IFNa2b of about 15 kD. However, these 3 model proteins were remarkably stabilized after treatment of the supernatant with the aspartic protease inhibitor pepstatin A. This inhibitor blocked the main proteases responsible for most of the protease activity. SBTI conferred only a small benefit to product stability. Although the optimal pH value for SBTI was higher than that used in the experiment (pH 4.2 versus optimal pH 8.0) and, therefore, the binding of these inhibitors to the target proteases may not be more efficient.
[00470] [00470] Pepstatin A effectively inhibits aspartic proteases. It is known from pepsin-A affinity purification studies that the aspartic proteases remaining in the supernatant are pep2, pep3 and pep5. Therefore, if the two or three remaining aspartic proteases are deleted from the supernatant, it will be practically free of aspartic protease activity. For the production of these model proteins, the aspartic proteases pep2, pep3 and pep5 would be considered the main proteases.
[00471] [00471] This same experiment was done with MABO1 to investigate its stability in the supernatant with the deletion of 6 proteases with and without inhibitors (Figure 43). Samples were collected as described above and immunoblot performed with an anti-AP heavy-chain antibody (Sigma t% A3188). After 20 hours of incubation, there was no significant degradation of the heavy chains. There was no obvious benefit from using inhibitors. The antibody was stable in this supernatant at pH 4.2. MABO1 production under more acidic conditions, such as a pH of 4.5 would likely improve production yield or at least reduce the amount of heavy chain cleavage that would occur.
[00472] [00472] To assess which inhibitors would best stabilize hGH production, cultures in 24 wells of these strains were performed. The M369 strain that produces human growth hormone (Apep1 Atsp1Aslp1Agap1Agap2) was grown with the individual components or with combinations of the following: trypsin and subtilisin SBTI inhibitor, SIP peptide acid protease inhibitor, LIP peptide acid protease inhibitor , pepstatin A free peptide aspartic protease inhibitor, pepstatin A immobilized on agarose beads, trypsin inhibitor and lime bean subtilisin BBI, chemostatin subtilisin inhibitor and BSA. Three independent wells were chosen for control wells, where no inhibitors or supplements were added. These two strains were grown in 3 ml of TTIMM with diamonium citrate without ammonium sulfate, PIPPS at 100 mM, 20 g / L of grain extract consumed, 40 g / L of lactose adjusted to a pH of 4.5. The 24-well plates were stirred at 800 rpm, 85% humidity and 28 CT. The cultures were grown for 6 days and covered with an air-permeable membrane.
[00473] [00473] Inhibitors were added first on day 1 and then daily starting on day 3. 100 µl samples were taken from the culture wells, starting on day 3. The mycelium was centrifuged for 5 minutes at 13k and the supernatant collected. Of the culture supernatant, 12 µl were loaded onto a 4-20% SDS PAGE gel and immunoblot made in nitrocellulose with the mouse anti-hGH antibody (2 pg / ml) and secondary goat anti-IgG antibody from AP-conjugated mouse diluted 1: 10,000 in TBST.
[00474] [00474] On day 4, human growth hormone can still be seen in the culture supernatant in all three control lanes (Figure 44). Two of the control lanes show a weak band and a control lane shows a clear band. The effect of inhibitors and supplements was immediately observed. Inhibitors / supplements that have had a great effect are highlighted in red and those with the best effect have an asterisk. Pepstatin A had a negative effect on the production of growth hormone. When used at 5 or 20 µM, hGH production appeared to be absent. It appears to have some toxic effect on production. Only when pepstatin was immobilized on agarose beads was this effect canceled out. One of the best treatments was pepstatin A beads plus 0.2 mg / ml SBTI (see the third asterisk in the blot in Figure 44). With only SBTI (0.2 mg / ml) there was no improvement in production, but there was a large degradation band present at 18 kD, which seems to be produced by the action of aspartic proteases.
[00475] [00475] Estimating expression levels in relation to the control sample at 200 ng, the control wells produced between 3-6 mg / L of hGH, treatment with BSA (0.25%) / SIP (50 uMy / SBTI (0.2 mg / ml) produced 24.5 mg / L, treatment with SIP (50 µM) / SBTI (0.2 mg / ml) produced 26.6 mg / L and the addition of pepstatin A / SBTI (0.2 mg / ml) produced 24.5 mg / L of hGH, so using a combination of inhibitors and additives worked best to increase production levels by at least four times. was to include an aspartic protease inhibitor in the mixture Example 8 - GENERATION OF T. REESEI DEFICIENT IN PEP7
[00476] [00476] The pep7 aspartic protease deletion plasmid pep7 (TrelD58669) is constructed essentially as described by plasmid pTTv41 with pep1 deletion in Example 1. 1062 bp from the 5 'flanking region and 1121 bp from the 3' flanking region are selected as the base of the pep7-deleted plasmid. The fragments are produced by PCR using the primers listed in Table 8.1. The products are separated by agarose gel electrophoresis and the correct fragments are isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. Model to be used in PCR of flanking and strain regions
[00477] [00477] The sIp5 aspartic protease deletion plasmid (TrelD64719) is constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1. 1044 bp from the 5 'flanking region and 1003 bp from the 3' flanking region are selected as the base of the sIp5 deletion plasmid. The fragments are produced by PCR using the primers listed in Table 9.1. The products are separated by electrophoresis on a guarose gel and the correct fragments are isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. Model to be used in PCR of the flanking regions is the QM6a strain of T. reesei of wild type. The pyr4 disintegrating cassette is obtained from pTTv71 (Example 1) through digestion with Notl. The vector backbone was pRS426 digested with EcoRI / Xhol, as in Example 1. The plasmid is constructed using the yeast homologous recombination method described in Example 1. This slp5 deletion plasmid results in deletion in the sIlp5 locus and covers the complete slp5 coding sequence.
[00478] [00478] Plasmid with sIp6 aspartic protease deletion
[00479] [00479] Plasmid pTTv269 with serine protease deletion sIp7 (tre123865) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1, except that the marker used for selection was pyr4-hgh from pTTv194.
[00480] [00480] 949 bp from the 5 'flanking region and 1025 bp from the 3' flanking region were selected based on plasmid pTTv269 with slp7 deletion. These fragments were amplified by PCR using the primers listed in Table 11.1. The model used in PCR of the flanking regions was the QM6a strain of T. reesei of wild type. The products were separated by means of electrophoresis
[00481] [00481] Plasmid pTTv330 with subtilisin sIp8 protease deletion (tre58698) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1, except that the marker used for selection was a double pyr4-hph marker.
[00482] [00482] 975 bp from the 5 'flanking region and 1038 bp from the 3' flanking region were selected based on the sIp8 deletion plasmid. A 298 bp stretch from the 5 'flanking end of sIp8 was used as a direct repeat fragment. These fragments were amplified by PCR using the primers listed in Table 12.1. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The pyr4-hph cassette was obtained from pTTv210 (Asepl-pyr4-hph) by digestion with Notl. To allow removal of the complete double marker cassette, Not! were inserted on both sides of the double marker cassette. An Ascl site was introduced between the direct repetition of the sIp8 5 'and 3' flanking regions. Vector skeleton was pRS426 digested with EcoRI / Xhol, as in Example 1. The plasmid was constructed using the yeast homologous recombination method described in Example 1. This sIp8 deletion plasmid (pTTv330, Table
[00483] [00483] Homologues of pep1, pep2, pep3, pep4, pep5 and pep7; tsp1; tsp1, sIp2, sIip3, sIp5, sIp6, sip7 and sIp8; gap1 and gap2 of T. reesei; and tpp1 counterparts have been identified from other organisms.
[00484] [00484] BLAST searches were conducted using the National Center for Biotechnology Information (NCBI) non-redundant amino acid database using Trichoderma reesei protease amino acid sequences as queries. Alternatively,
[00485] [00485] Figures 45 describes a phylogenetic tree of aspartic proteins from selected filamentous fungi.
[00486] [00486] Figure 46 shows a phylogenetic tree of subtilisin proteases from selected filamentous fungi.
[00487] [00487] Figure 47 shows a phylogenetic tree of glutamic proteases from selected filamentous fungi.
[00488] [00488] Figure 48 shows a phylogenetic tree of sedolisinA proteases from selected filamentous fungi. Example 14 - STRAIN GENERATION WITH PROTEIN DELETE 9 TIMES Generation of strain with protease deletion 9 times with deletions Apep1aAtspiAsIpiAgap1iAgap2Apep4Apep3Apep5Apep2 Generation of new plasmids with pep2 deletion
[00489] [00489] The first pep2 aspartic protease deletion plasmid pep2 (tre0053961), pTTv213, was constructed essentially as described for plasmid pTTv41 Apep1 in Example 1, but an additional second selection marker cassette carrying a hygromycin phosphotransferase gene (hph), was placed after the p-yr4 gene to create a deletion plasmid with a disintegrating cassette with double selection marker. The double marker system allows a) the use, for example, of hph as the initial resistance marker and faster selection; b) transformation of pyr4 + strains (without the need to generate pyr4- before transformation); and c) the removal of both transformant markers using 5-fluoro-orotic acid (as per the removal of the standard pyr4 disintegrating cassette) and simultaneous mutation of endogenous pyr4, resulting in the strain without pyr- marker. In addition to the double marker, the first deleted plasmid also contained a native kex2 overexpression cassette (tre123561; cDNA1 promoter, cbh2 terminator).
[00490] [00490] The second plasmid with pep2 aspartic protease deletion (tre0053961), pTTv232, was constructed using the above plasmid pTTv213 as a backbone. The kex2 overexpression cassette (pcDNA1-kex2-tcbh2) was removed by digesting pTTv213 with Ascl. The cloning of plasmid pTTv232 was done with standard ligation (self-ligation) using DNA ligase T4, at room temperature. Part of the ligation mixture was transformed into E. coli with electroporation. Some clones were cultured, the plasmid DNA was isolated and digested for screening for correct binding using conventional laboratory methods. Correct connection was also verified through sequencing.
[00491] [00491] The third plasmid with pep2 aspartic protease deletion (tre0053961), pTTv246, was constructed using the above plasmid pTTv232 as a backbone. The double marker pyr4-hph was removed from pTTv232 by digestion with Notl. The pyr4 marker gene was obtained from pTTv181 (Apep4-py4r above) by digestion with Notl. The cloning of the plasmid pTTv246 was done with standard ligation using DNA ligase T4, at room temperature. Part of the connection mixture was transformed into E. coli with electroporation. Some clones were cultured, the plasmid DNA was isolated and digested for screening for correct binding using conventional laboratory methods. Correct connection and orientation of the marker were also
[00492] [00492] 1000 bp flanking region 5 'and 1020 bp flanking region 3' were selected based on plasmids with pep2 deletion. A 300 bp stretch from the 5 'flanking end of pep2 was used as a direct repeat fragment. These fragments, as well as the second selection marker (hph) cassette, cCDNA1 promoter, native kex2 gene and c-bh2 terminator were amplified by PCR using the primers listed in Table 14.1. The products were separated by means of agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The selection marker pyr4 was obtained from pTTv181 (Apep4-pyr4 above) through digestion with Notl. To allow removal of the entire double marker cassette in pTTv213, Notl restriction sites were introduced on both sides of the marker marker cassette twice and a Swal site between the two selection markers. Ascl sites were introduced on both sides of the kex2 overexpression cassette (repetition between direct repetition in the 5 'and 3' pep2 flanking regions). Vector skeleton was pRS426 distributed with EcoRI / Xhol, as in Example 1. Plasmid pTTv213 was constructed using the yeast homologous recombination method described in Example 1. These pep2-deleted plasmids (pTTv213, pTTv232 and pTTv246, Table 14.1) result in a 1580 bp deletion at the pep2 locus and cover the complete PEP2 coding sequence.
[00493] [00493] To generate the strain with a protease deletion 9 times without a marker, removal of the pyr4 marker was applied to the strain M504 with a protease deletion 8 times (38-48A, pTTv229 in M496) essentially as described in Example 3, to remove the pyr4 disintegrator cassette from the M195 strain (Apep1). Consecutive 5-FOA selection steps were performed to ensure that the selected clones came from single cells.
[00494] [00494] The final clones were verified by PCR using the starters listed in Table 14.2 using conventional laboratory methods. Signs corresponding to the successful removal of the disintegrating shell were obtained for most of the clones. The removal of the disintegrating cassette was additionally verified by placing the clones on plates with minimal medium with or without 5 mM uridine. There was no growth in the plaques without uridine supplementation. The resulting strain used in the generation of a strain with a protease deletion 9 times was assigned a strain number M521.
[00495] [00495] To remove the vector sequence, plasmid pTTv246 (Apep2-pyr4) was digested with Mss! and the correct fragment purified from an agarose gel using a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the deletion cassette was used to transform a clone of the strain with a protease deletion 8 times M521 (Apep1 Atsp1Asl | p1Agap1Agap2Apep4Apep3Apep5, pyr4-). Preparation of protoplasts and transformation were carried out essentially as described in Example 1 for strains M181 and M195 that are selected with pyr4.
[00496] [00496] Transformants were chosen as first stripes. Growing stripes were tested by PCR (using the primers listed in Table 14.2) for correct integration. Clones that provide the expected signals were purified into single cell clones and again screened by PCR using the primers listed in Table 14.2. Pep 2 deletion was verified using Southern analyzes of selected clones (data not shown), using the methods described in Example 1. Clone 41-45G was assigned the strain number M574. Table 14.2: Initiators for screening pyr disintegrator cassette removal from the strain with protease deletion 8 times and for pTTv246 / Apep2-pyr4 integration screening and strain purity For screening of M504 pyr4 disintegrating cassette and strain purity Sequence Initiator | T755 peps 3f rev3 CTTCTGGTGACATTCCGAC
[00497] [00497] The first plasmid with aspartic protease deletion pep12 (tre119876), pTTv209, was constructed essentially as described for plasmid pTTv41 Apep1 in Example 1, but an additional second selection marker cassette, a synthetic construct bringing a phosphinothricin N-acetyl transferase (bar) gene from Streptomyces ssp., was placed after the pyr4 gene to create a deletion plasmid with a disintegrating cassette with double selection marker. The double marker system allows a) the use, for example, of bar as the marker of initial resistance and faster selection; b) transformation of pyr4 + strains (without the need to generate pyr4- before transformation); and c) the removal of both transformant markers using 5-fluoro-orotic acid (as in the removal of the standard pyr4 disintegrating cassette) and simultaneous mutagenesis of endogenous pyr4, resulting in the strain without pyr4- marker.
[00498] [00498] The second plasmid with pep12 aspartic protease deletion (trel 19876), pTTv245, was constructed using the plasmid pTTv209 above as a backbone. The double marker pyr4-bar was removed from pTTv209 by digestion with Notl. The new pyr4 marker gene was obtained from pTTv181 (Apep4-pyr4 above) by directing it with Notl. The cloning of the plasmid pTTv245 was done with | standard ligation using T4 DNA ligase, at room temperature. Part of the ligation mixture was transformed into E. coli with electroporation. Some clones were cultured, the plasmid DNA was isolated and distributed for screening for correct binding using conventional laboratory methods. Correct connection and marker orientation were also verified by sequencing.
[00499] [00499] 1019 bp of the 5 'flanking region and 895 bp of the 3' flanking region were selected based on plasmids with pep12 deletion. A 300 bp stretch from the 5 'flanking end of pep12 was used as a direct repeat fragment. These fragments were amplified by PCR using the primers listed in Table 14.3. The products were separated by means of agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The double marker (pyr4-bar) was digested from pTTv202 (Apep5-pyr4-bar) with Notl. To allow removal of the complete double marker cassette, Not! were inserted on both sides of the double marker cassette. An Ascl site was introduced between the direct repetition of the 5 'and 3' pep12 flanking regions. Vector skeleton was pRS426 digested with EcoRlI / Xhol, as in Example 1. Plasmid pTTv209 was constructed using the yeast homologous recombination method described in Example 1. These pep12-deleted plasmids (pPTTv209 and pTTv245, Table 14.3) result in a 2198 bp deletion at the pep12 locus and cover the complete coding sequence for PEP12.
[00500] [00500] To generate the strain with a protease deletion 9 times without a marker, removal of the pyr4 marker was applied to the strain M504 with a protease deletion 8 times (38-48A, pTTv229 in M496) essentially as described in Example 3, for removal of the pyr4 disintegrator cassette from the M195 strain (Apep1). Consecutive 5-FOA selection steps were performed to ensure that the selected clones came from single cells.
[00501] [00501] The final clones were verified by PCR using the starters listed in Table 14.4 using conventional laboratory methods. Signs corresponding to the successful removal of the disintegrating shell were obtained for most of the clones. The removal of the disintegrating cassette was additionally verified by placing the clones on plates with minimal medium with or without 5 mM uridine. There was no growth in the plaques without uridine supplementation. The resulting strain used to generate the strain with a protease deletion 9 times was assigned the strain number M521.
[00502] [00502] To remove the vector sequence, plasmid pTTv245 (Apep12-pyr4) was digested with Mssl and the correct fragment was purified from an agarose gel using a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the deletion cassette was used to transform a protease deletion M521 clone 8 times (Apep1 Atspi1Asl | p1iAgap1Agap2Apep44pep3Apep5, pyr4-). Preparation of protoplasts and transformation were carried out essentially as described in Example 1 for strains M181 and M195 that are selected with pyr4.
[00503] [00503] The transformers were chosen as first stripes. Growing stripes were tested by PCR (using the primers listed in Table 14.4) for correct integration. Clones that provide the expected signals were purified in single cell clones and again screened by PCR using the primers
[00504] [00504] Plasmid pTTv312 with aspartic protease deletion pep11 (tre121306) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1.
[00505] [00505] 956 bp from the 5 'flanking region and 943 bp from the 3' flanking region were selected on the basis of the plasmid with pep1 deletion. A 307 bp stretch from the 5 'flanking end of pep11 was used as a direct repeat fragment. These fragments were amplified by PCR using the primers listed in Table 15.1. The products were separated by means of agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The pyr4 cassette was obtained from pTTv181 (Apep4-pyr4 above) by digestion with Notl. To allow removal of the marker cassette, Notl restriction sites have been introduced on both sides of the cassette. An Ascl site was introduced between the direct repetition of the 5 'and 3' pep11 flanking regions. Vector skeleton was pRS426 digested with EcoRI / Xhol, as in Example 1. The plasmid was constructed using the yeast homologous recombination method described in Example 1. This pep11 deletion plasmid (pTTv312, Table 15.1) results in a 2624 deletion bp at the pep11 locus and covers the complete PEP11 coding sequence. Table 15.1: Primers for generating plasmids with pep11 deletion Plasmid with pTTv312 deletion (Apep11-pyr4), pRS426 vector skeleton
[00506] [00506] To generate a 10-fold protease deletion strain without a marker, removal of the pyr4 marker was applied to the MB574 strain with a protease deletion 9 times (41-45G, pTTv246 in M521) essentially as described in Example 3, for removing the pyr4 disintegrating cassette from the M195 strain (Apep1). Consecutive 5-FOA selection steps were performed to ensure that the selected clones came from single cells.
[00507] [00507] The final clones were verified by POR using the starters listed in Table 15.2 using conventional laboratory methods. Signs corresponding to the successful removal of the disintegrating shell were obtained for most of the clones. Removal of the disintegrating cassette was further verified by placing the clones on plates with minimal medium with or without 5 mM uridine. There was no growth in the plaques without uridine supplementation. The resulting strain used to generate the strain with the protease deletion was sometimes assigned the strain number M597.
[00508] [00508] To remove the vector sequence, plasmid pTTv312 (Apep11-pyr4) was digested with Mssl and the correct fragment purified from an agarose gel using a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the deletion cassette was used to transform a protease-deleted M597 clone 9 times (Apep1Atsp1AslpiAgap1Agap2Apep4Apep3Apep5Apep 2, pyr4-). Protoplast preparation and transformation were performed essentially as described in Example 1 for strains M181 and M195 that use pyr4 selection.
[00509] [00509] The transformants were chosen as first stripes. Growing stripes were tested by PCR (using the primers listed in Table 15.2) for correct integration. Clones that provide the expected signals were purified into single cell clones and again screened by PCR using the primers listed in Table 15.2. Pep11 deletion was verified by Southern analyzes of selected clones (data not shown), using the methods described in Example 1. Clone 47-62B was designated with the strain number M632. A purification step of a single additional cell was applied to the M632 strain to obtain the M658 strain with 10 times protease deletion. Table 15.2: Initiators for screening cassette removal should
[00510] [00510] Plasmid pTTv331 with tripeptidyl peptidase deletion tpp1 (tre82623) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1, except that the marker used for selection was a double pyr4-hph marker.
[00511] [00511] 1245 bp of 5 'flanking region and 1025 bp of 3' flanking region were selected based on the plasmid with tpp1 deletion. A 311 bp stretch from the 5 'flanking end of tpp1 was used as a direct repeat fragment
[00512] [00512] Another plasmid pTTv319 with aspartic protease deletion pep8 (tre122076) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1.
[00513] [00513] The second plasmid with pep8 aspartic protease deletion (tre122076), pTTv328, was constructed using the above plasmid pTTv319 as the backbone. The pyr4 marker was removed from pTTv319 by digestion with Notl. The pyr4-hph cassette was obtained from pTTv210 (Asepl-pyr4-hph) by digestion with Notl. Cloning of the plasmid pTTv328 was done with standard ligation using T4 DNA ligase, at room temperature. Part of the ligation mixture was transformed into E. coli with electroporation. Some clones were cultured, the plasmid DNA was isolated and digested for screening for correct binding using conventional laboratory methods. Correct connection and marker orientation were also verified by sequencing.
[00514] [00514] 1095 bp of the 5 'flanking region and 988 bp of the 3' flanking region were selected based on plasmids with pep8 deletion. A 324 bp section from the 5 'flanking end of pep8 was used as a direct repeat fragment. These fragments were amplified by PCR using the primers listed in Table 17.1. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The pyr4 selection marker used in pTTv319 was obtained from pTTv181 (Apep4-pyr4 above) by digesting with Notl. To allow removal of the p-yr4 marker cassette, Notl restriction sites were introduced on both sides of the cassette. An Ascl site was introduced between the direct repeat of the 5 'flanking region and 3' pep8 flanking region. Vector skeleton was pRS426 digested with EcoRI / Xhol, as in Example 1. Plasmid pTTv319 was constructed using the yeast homologous recombination method described in Example 1. These pep8-deleted plasmids (pTTv319 and pTTv328, Table 17.1) result in a 1543 bp deletion at the pep8 locus and span the complete PEP8 coding sequence. Table 17.1: Primers for generating plasmid with pep8 deletion Plasmid with pTTv319 deletion (Apep8-pyr4), vector skeleton pRS426 T1019 pep8 5flktiw / - | GTAACGCCAGGGTTTTCCCAGTCAC- vector GACGGTTTAAAC
[00515] [00515] The third pTTv266 deletion plasmid for pep8 aspartic protease (tre122076) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1, except that the marker used for selection was pyr4-hgh of pTTv194.
[00516] [00516] 1095 bp from the 5 'flanking region and 988 bp from the 3' flanking region were selected based on plasmid pTTv266 with pep8 deletion. These fragments were amplified by PCR using the primers listed in Table 17.2. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The selection marker pyr4-hgh used in pTTv266 was obtained from pTTv194 (Apep4-pir-HGH above) by digesting with Notl. To allow removal of the pyr4-hgh marker cassette, Notl restriction sites were introduced on both sides of the cassette. Vector skeleton was pRS426 digested with EcoRI / Xhol, as in Example 1. Plasmid pTTv266 was constructed with 3 'flanking region, 5' flanking region, pyr4-hgh marker and vector skeleton using the yeast homologous recombination method described in
[00517] [00517] To generate the five-fold protease deletion strain that produces IFN-a 2b, the M369 strain with five-fold protease deletion Apep1Atsp1 As / p1 Agap1Agap 2 a was transformed with the IFN-a expression cassette 2b (pTTv173) using hygromycin in the selection. This strain with a 5-fold protease deletion Apep1Atsp1 Aslp1 Agap1Agap2 that produces IFN-a 2b was designated with the number M401.
[00518] [00518] In order to study the level of expression of IFN-a 2b, the M401 strain with a 5-fold protease deletion (Apep1Atsp1 Asip1 Agap1Agap2) that produces IFN-a 2b was grown under cultivation conditions of pH 4.5 ; 28 - 22 ºC; 30 g / l glucose, 60 g / l lactose, 20 g / | of whole grains consumed and 20 g / Il of grain extract consumed. To analyze the level of expression of IFN-a 2b, the culture sample from day 3 was subjected to quantitative immunoblotting (Figure 54A). The samples were analyzed by comparison with a standard curve for IFN-a 2b and densitometric quantification was performed with the software Totallab Quant TL100. Immunoblot was performed with Abeam anti-| FN-a 2b antibody (ftab9386) diluted at 1 µg / ml in TBST. The secondary antibody was Bio-Rad AP-conjugated goat anti-IgG mouse secondary antibody (f4170-6520) diluted 1: 5000 in TBST. Protein patterns were loaded onto the gel corresponding to 50 ng, 100 ng, 200 ng and 400 ng of IFN-a 2b. The analysis showed that M401 produced IFN-a 2b with yields of up to 51.9 mg / l and 52% of the product was cleaved from the carrier molecule. Generation of M577 strain with 8 times protease deletion that produces IFN-a 2b
[00519] [00519] To generate the 8-fold protease deletion strain that produces IFN-a 2b, the M504 strain with 8-fold protease deletion Apep1Atsp1As | Ip1l Agap1Agap2Apep4Apep3Apep5 was transformed with an IFN-a 2b (pTTv254) expression cassette using acetamide na selection. This strain with a protease deletion 8 times Apep1Atsp1 Aslp1 Agap1Agap2Apep4Apep3Apep5 that produces IFN-a 2b was designated with the number M577. Analysis of strain M577 with 8-fold protease deletion that produces IFN-a 2b
[00520] [00520] To study the level of expression of IFN-a 2b, the M577 strain with protease deletion 8 times (Apepf1Atsp1 Aslp1 Agap1Agap2Apep4Apep3Apep5) that produces IFN-a 2b was grown under pH conditions of 4.5; 28 - 22 ºC; 2% yeast extract, 4% cellulose, 8% cellobiose and 4% sorbose. To study the expression of IFN-a 2b, the M577 fermentation samples were subjected to immunoblotting (Figure 54B). To analyze the level of expression of IFN-a 2b, the culture sample on day 4 was subjected to quantitative immunoblot (Figure 55). The sample was analyzed by comparison with a standard curve for IFN-a 2b and densitometric quantification was performed with the software Totallab Quant TL100. Immunoblot was performed with Abeam anti-IFN-a 2b antibody (fttab9386) diluted to 1 µg / ml in TBST. The secondary antibody was goat anti-IgG mouse secondary antibody from Bio-Rad (8170-6520) AP-conjugated diluted 1: 5000 in TBST. Protein patterns were loaded onto the gel corresponding to 50 ng, 100 ng and 200 ng of IFN-a 2b. The analysis showed that M577 produced IFN-a 2b with yields of up to 1780 mg / l and 66.5% of the product was cleaved from the carrier molecule. The 8-fold protease-deleted M577 produced 34 times more IFN-a 2b than the 5-fold protease-deleted M401 strain. Generation of the M652 strain with a 9-fold protease deletion that produces IFN-a 2b
[00521] [00521] To generate the 9 times protease deletion strain that produces IFN-a 2b, the M574 strain with 9 times protease deletion Apep1Atsp1As / Ip1 Agap1Agap2Apep4Apep3Apep5Apep2 has been transformed with an IFN-a 2b expression cassette (pTTv3) hygromycin in selection. This strain with a protease deletion 9 times Apep1Atsp1 Aslp1 Agap1Agap2Apep4Apep3Apep5Apep2 that produces IFN-a 2b was designated with the number M652.
[00522] [00522] To study the level of expression of IFN-a 2b, the M652 strain with protease deletion 8 times (Apep1Atsp1 Aslp1 Agap1Agap2Apep4Apep3Apep5Apep2) that produces IFN-a 1b was grown under pH conditions of 4.5; 28 - 22 ºC; 2% yeast extract, 4% cellulose, 8% cellobiose and 4% sorbose. To study the expression of IFN-a 2b, the M652 fermentation samples were subjected to immunoblotting (Figure 54B). To analyze the level of expression of IFN-a 2b, the culture sample on day 3 was subjected to quantitative immunoblotting (Figure 55). The sample was analyzed by comparison with a standard curve for IFN-a 2b and densitometric quantification was performed with the software Totallab Quant TL100. Immunoblot was performed with Abeam anti-IFN-a 2b antibody (ftab9386) diluted to 1 µg / ml in TBST. The secondary goat anti-IgG mouse AP-conjugated Bio-Rad (ft170-6520) diluted 1: 5000 in TBST. Protein patterns were loaded onto the gel corresponding to 50 ng, 100 ng and 200 ng of IFN-a 2b. The analysis showed that M652 produced IFN-a 2b with yields up to 1928 mg / l and 85% of the product was cleaved from the carrier molecule. The M652 strain with protease deletion 9 times produced slightly more than the MB577 strain with protease deletion 8 times and 37 times more IFN-a 2b than the M401 strain with protease deletion 5 times. Generation M670 strain with protease deletion 9 times with pep8 (tre122076) deleted from the M577 strain that produces interferon
[00523] [00523] To remove the deletion cassette, plasmid pTTv266 (Apep8-pyr4-hgh) was digested with Pmel and the correct fragment was purified using a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the deletion cassette was used to transform the M577 strain with a protease deletion 8 times
[00524] [00524] Transformants were collected and cultured on selection plates. Growing stripes were tested by PCR (using the primers listed in Table 18.1) for correct integration. Clones that provide the expected signals were purified into single cell clones and again screened by PCR using the primers listed in Table 18.1. Clone 82-9 was assigned the M670 strain number.
[00525] [00525] Plasmid pTTv268 with aspartic protease deletion pep11 (tre121306) was constructed essentially as described for plasmid pTTv41 with pep1 deletion in Example 1, except that the marker used for selection was pyr4-hgh from pTTv194.
[00526] [00526] 956 bp from the 5 'flanking region and 957 bp from the 3' flanking region were selected based on plasmid pTTv268 with pep71 deletion. These fragments were amplified by PCR using the primers listed in Table 18.2. The products were separated using agarose gel electrophoresis and the correct fragments were isolated from the gel with the gel extraction kit (Qiagen) using conventional laboratory methods. The pyr4-hgh cassette was obtained from pTTv194 (Apep-pyr4-hgh) by digestion with Notl. To allow removal of the marker cassette, Notl restriction sites were introduced on both sides of the cassette. Vector skeleton was pRS426 digested with EcoRI / Xhol, as in Example 1. The plasmid was constructed. Plasmid pTTv268 was constructed with 3 'flanking region, 5' flanking region, pyr4-hgh marker and vector skeleton using the yeast homologous recombination method described in Example 1. This pep11 deletion plasmid (pTTv268, Table 18.2) results in a deletion at the pep11 locus and covers the complete coding sequence of pep11. Table 18.2: Primers for generating plasmids with pep11 deletion Plasmid pTTv268 with deletion (Apep11-pyr4-hgh), vector skeleton pRS426 Primer —— Sequence T1009 pep 5flkiw vector GTAARCGCCAGGGTTTTCCCAGTCAC --—— 1 GACGGTATA
[00527] [00527] - To remove the deletion cassette, plasmid pTTv268 (Apep11-pyr4-hgh) was digested with Pmel and the correct fragment was purified using a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the deletion cassette was used to transform the M577 strain with a protease deletion 8 times (Apep1Atsp1Asl / p1Agap1Agap2Apep4Apep3Apep5). The M577 strain produces interferon alfa 2b. Protoplast preparation and transformation were performed essentially as described in Example 1, using hygromycin selection.
[00528] [00528] Transformants were collected and cultured on selection plates. Growing stripes were tested by PCR (using the primers listed in Table 18.3) for correct integration. Clones providing the expected signals were purified into single cell clones and again screened by PCR using the primers listed in Table 18.3. Clone 33-9 was assigned the M672 strain number. Table 18.3: Initiators for pTTv268 / Apep11-pyr4-hgh integration screening and strain purity For pTTv268 integration screening (Apep11-pyr4-hgh) Initiator —— Sequence T1013 pep1 | trace 5flk iwd 'TTACGACTCGATCCCTGTCE = T1084 trace 5flk pyrrev ——— TCTTGAGCACGACAATCGAC T1015 trace 3flk hygro ftivxd - GCATGGTTGCCTAGTGAATG = T1016 pep1 | 3flk rev tracking - GCCGCTAGGATCGTGATAAG == For ORFdepep1vT deletion screening T1017 peplit orffvxd - - GTGTCCCAGGACGACAACTT T1018 pep11 orfrev - - TGAAGGTTGCAGTGATCTCGE
[00529] [00529] To remove the deletion cassette, plasmid pTTv269 (Aslp7-pyr4-hgh) was digested with Pmel and the correct fragment was purified using a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the deletion cassette was used to transform the M577 strain with a protease deletion 8 times (Apep1Atsp1As / p1Agap1Agap2Apep4Apep3Apep5). The M577 strain produces interferon alfa 2b. Protoplast preparation and transformation were performed essentially as described in Example 1, using hygromycin selection.
[00530] [00530] Transformants were collected and placed on selection boards. Growing stripes were tracked by PCR (using the primers listed in Table 18.4) for correct integration. Clones that provide the expected signals were purified into single cell clones and again screened by PCR using the primers listed in Table 18.4. Clone 5-64 was designated with strain number M673. Table 18.4: Initiators for pTTv269 / As / p7-pyr4-hgh integration screening and strain purity. To pTTv269 integration screening (AS / LP7-pyr4-hgh) Primer Sequence T1092 slp7 screening 5flk ftiwd 'TTGGTTTGAACAGCTGCAAG T1084 screening 5flk pyrrev - TCTTGAGCACGACAATCGAC T1015 screening 3flk hygro ftivxd - GCATGGTTGCCTAGTGAATG = T1093 sip7 screening 3flk ev - ATGGTCAGCCAGAACCTGAC for deletion screening of ORFdes / pb7 T1094 slpr of fiwd - - TCTTGAGCCGTTTCTCGAAT = T1095 slp7 orfrev ———— CCGCTCTTAGATCGATGGTE Example 19 - M627 AND M629 CEPAS GENERATION WHICH PRODU- ZEM GO
[00531] [00531] Vectors pTTg156 and pTTg173 were constructed by adding the double marker selection cassette (hygromycin resistance marker gene (hph) between pkil promoter and cbh2 terminator, in addition to the pyr4 expression cassette) to the pTTg145 intermediate vectors and pTTg146. The intermediates were constructed by cloning through yeast recombination, marker cassettes were added by conventional cloning through digestion and binding with Not !.
[00532] [00532] The strategy for generating fragments for intermediate vectors is presented in Table 19.1 below. Primers used for fragment generation are listed in Table 19.2 below. Since the fragments for pTTg145 and pTTg146 were constructed according to the plan in Table 19.1, they were electroporated in the light Saccharomyces cerevisiae FY834 for plasmid assembly by means of homologous recombination. The yeast cells were grown in SC-ura for 2-3 days at 30 ° C (C. Colonies were then pooled from the plates and the pooled plasmid was purified using the phenol / chloroform extraction method. plasmids were transformed into competent E. coli cells by electroporation, electroporated cells were placed on selection plates with LB + amp, grown at 37 C overnight and colonies were screened by PCR. then, grown in fresh plates as pure cultures, the individual colonies were grown in liquid medium LB + amp and intermediate vectors pTTg145 and potential pTTg146 were purified according to standard protocols. restriction and the sequences were verified by sequencing.
[00533] [00533] Marker cassette was then added to the vectors by conventional NotT digestion of pTTg163 and binding to pTTg145 and pTTg146 intermediates linearized with Not !. Table 19.1: Strategy for the construction of intermediate vectors PTTg145-pTTg149. pTTv141 and pTTv11 are described in International Patent Application No. PCT / EP2011 / 070956. Plasmids pTTg124 and pTTv225 were used to construct the fragments specified in the table below.
[00534] [00534] The M507 strain of Trichoderma reesei that expresses MABO1 with a 7-fold protease deletion was transformed with the Pmel fragments of pTTg156 and pTTg173 that target the alg3 locus. Variable number of transformants (100-170 depending on the construct) were placed on selective plates. Based on PCR screening with the Phire Plant Direct PCR kit (Finnzymes F-130), clones with positive results referring to 5 'and 3' integration were selected for single spore placement and further screening for integration and deletion of a / g3 (5 pTTg156 transformation clones, 3 pTTg173 transformation clones). Initiators used for selection are listed in Table 19.3 below.
[00535] [00535] Strains traced by PCR were finally subjected to culture in a shaking flask and analysis of glycans. Final strains were named M629 (transformant pTTg173) and M627 (transformer
[00536] [00536] T. reesei strains M627 and M629 were fermented in 4% WSG, 2% glucose, 4% cellobiose, 6% lactose, pH 5.5 and sampling was carried out on days 3-6. Antibody titrations are shown in Table 19.4.
[00537] [00537] For analysis of N-glycans, MABO1 was purified from culture supernatants using filter plates with 96 HP MultiTrap Protein G wells (GE Healthcare), according to the manufacturer's instructions. Antibody concentrations were determined using UV absorbance against a standard curve for MABO1.
[00538] [00538] N-glycans were released from antibody precipitated with EtOH and denatured with SDS using PNGase F (Prozyme Inc.) in 20 mM sodium phosphate buffer, pH 7.3, in overnight reaction at 37 (C The released N-glycans were purified with Hypersep C-18 and Hypersep Hypercarb (Thermo Scientific) and analyzed with MALDI-TOF MS The results are shown in Table 19.6 In the M627 and M629 strains, GO levels varied between 24 , 3% to 41.7%, no GO was observed in the M507 strain, WSG culture in shake flasks of the M627 and M629 strains and glycan analysis
[00539] [00539] Strains M627 and M629 from 7. reesei were grown in shake flasks in TrMM, 4% lactose, 2% SGE, 100 mM PIPPS, pH 5.5, 28 CO. Sampling was done on day 5.
[00540] [00540] For analysis of N-glycans, MABO1 was purified from culture supernatants using filter plates with 96 HP MultiTrap Protein G wells (GE Healthcare), according to the manufacturer's instructions. Antibody concentrations in protein G eluents were determined by UV absorption against a standard curve for MABO1 (Table 19.5). The titrations in culture medium were not measured.
[00541] [00541] Release of N-glycans was carried out as above, the results are shown in Table 19.7. The GO levels were 21.1 and 56.9% for M627 and M629, respectively. Table 19.4: Antibody titers in supernatants from fermentation cultures of M627 and M629 strains fermented in medium
[00542] [00542] “Table 19.5: Antibody concentrations in Protein G eluents from M627 and M629 strains grown in WSG medium in EO as E shake flasks Table 19.6: Relative proportions of neutral N-glycans of purified renum earenaca antibody vans Jsssst 157 17o [1oel1nsf2s los [175122 erenac onvens rss 4000 Jos fo Jos Jon oz log Jor pssroNaa ros 199 5005 mr aan alste as aj af55a hunger or Tisstsar Jos foz foz [the Jos fox fox Rerenaca Has izassajao Joo Jos Jon oo Jos Jos Table 19.7: Relative proportions of neutral N-glycans of purified antibody on day 5 of strains M627 and M629 cultivated in WSG medium in shaking flasks err ques pose ea e nas o is pasa - vens isa oo na
[00543] [00543] The vectors for GnTI with different promoters are described in Table 20.1. The vectors were targeted to the T. reesei egl2 locus. Table 20.1: Description of human GNTI vectors with different promoters Materials and Methods
[00544] [00544] The strategy for generating vector fragments in Table 20.1 is presented in Table 20.2 and the initiators used for generating fragments are listed in Table 20.3. The fragments were amplified by PCR and the products were purified from the agarose gel. Digested pTTg152 vector was purified from the gel. All PCR amplifications were performed with high fidelity Phusion polymerase (Finnzymes). Fragments of pTTg153-pTTg171 were electroporated in the yeast Saccharomyces cerevisiae FY834 for plasmid assembly by homologous recombination. The yeast cells were cultured in SC-ura for 2-3 days culture at 30 O. The colonies were then pooled from the plates and the pooled plasmid was purified with the phenol / chloroform extraction method, as routine. The plasmid cluster was transformed into competent E. coli cells by electroporation. The electroporated cells were placed on selection plates with LB + amp, cultured 37
[00545] [00545] The M507 strain of Trichoderma reesei that expresses MABO1 with a 7-fold protease deletion was transformed with the Pmel fragments of pTTg153-pTTg171 vectors targeting the egl2 locus. Variable number of transformants were placed on selective plates. Based on POR screening with Phire Plant Direct PCR kit (Finnzymes F-130), clones with positive results regarding 5 'and 3' integration were selected for plates with single spores and new screening for egl2 integration and deletion. Strains selected by PCR were finally subjected to culture in a shaking flask and analysis of glycans. Shake flask cultures of M507 strains transformed with GnTI and promoter constructs
[00546] [00546] The M507 strain transformed with vectors from Tables 20.1 and 20.2 were grown in shaking flasks in TrMM, 4% lactose, 2% SGE, 100 mM PIPPS, pH 5.5, 28 CT and sampling was performed on day 5. An inactive GnTI construct was tested to determine possible effects of GICNAcMan5 glycans on the growth of T. reesei.
[00547] [00547] For analysis of N-glycans, MABO1 was purified, concentrations were determined and N-glycans analyzed with MALDI-TOF MS as described above. Analysis of MABO1 N-glycans showed that GnMan5 levels ranged from 8 to 79.2% of total glycans (Tables 20.4 and 20.5A and B). Inactive GnTI produced wild-type glycosylation, as expected. Table 20.4: GnTI constructs and antibody concentrations. Strain numbers for selected clones are provided in parentheses in the "Clones" column. Focus / vector Clones Antibody titration to gtomaemares | Cn ss a CO es a Bo a
[00548] [00548] The strains M702, M704, M706, M710, M712, M716 and M507 from T. reesei were fermented in 4% WSG, 2% Glc, 4% cellobiose, 6% lactose, pH 5.5 and sampling was performed on days 3-6. Antibody titers are shown in Table 20.6. N-glycans were released and analyzed as described above using PNGase F.
[00549] [00549] Analysis of MABO1 N-glycans showed that the levels of GnMans5 varied from 1.8 to 68.5% of the total glycans (Tables 20.7 A, B and C). Inactive GnTI produced wild-type glycosylation, as expected, as did the control M507 strain. Table 20.6: MABO01 Antibody Concentrations Global Titration M704 0.299 0.399 0.415 0.479 Tables 20.7 A, B and C: Relative proportions of neutral N-glycans over MABO1 on 3.4, 5 and 6 Hex4HexNAc2 | 0.4 0.3 0.5 | o, 5 4 Hex5HexNAc2 | Man5 1257, | 36, 46, | 55, | 72, [9,7 [12, [16, | 20, [21, 45, | 63, | 70, 4 or 8 7 | 9 | 1 | 3 6 | 5 | 3 | 5 Hex6HexNAc2 1419, [11, | 12, 6.2 [9.2 | 7.2 | 5.6 | 10, | 13, 7.8 | 5.5 5 "1 jo Hex5HexNAc3 / [Gn- - | 1460, 40, | 29, 125, [14, | 67, | 68, | 64, [51, [45, [39, [22, [17, Mans | 5 | 5 | 7 | 1 | 8 | 5 | 5 | g | o | 7 2 2 | 1
[00550] [00550] The plasmids used in the generation of GNT1 strains with different Golgi objectification peptides (pTTv274, pTTv275, pTTv276, pTTv278, pTTv279, pTTv280) were all based on the parental plasmid in common pTTv265 with a GNT1 p5 (human) 38 amino acid N-terminal truncation. The lineage of pTTv265 is summarized in Table 21.1.
[00551] [00551] Plasmid pTTv77 contains 5 'and 3' flanking regions of egl2 (tre120312) for targeted integration into the T. reis genome and the cbh1 promoter for gene expression. Integration of the plasmid pTTv77 into the genome results in a deletion of 2456 bp at the egl2 locus. 1020 bp and 1024 bp regions of the egl2 locus were amplified for 5 'and 3' flanking. 2176 bp of the cbh1 locus were amplified for the promoter fragment. A model used in PCR reactions was T. reesei's genomic DNA. Primers used in PCR reactions are shown in Table 21.2. The disintegrating cassette of pyr4 was a Notl fragment of pTTv71 described above and the vector backbone was pRS426 digested with EcoRI / Xhol described above. All fragments were purified by standard laboratory methods and the plasmid was cloned by the yeast recombination method as described in the Examples. After recovering the E. coli plasmid, some clones were checked for correct recombination. Stored clone was verified by sequencing. Table 21.2: Primers used in cloning pTTv77 Initiator Name - - Initiator sequence T575 egl2 5flang F— GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT- - |
[00553] [00553] Plasmid pTTv264 is based on plasmid pTTv256. In pTTv264, the selection marker was changed from the disassembling cassette of pyr4 to the selection marker hygR. To clone pTTv264, plasmid pTTv256 was digested with Notl to release the pyr4 disintegrating cassette. HygR marker was amplified by
[00554] [00554] Plasmid pTTv265 is based on plasmid pTTv264 described above. In pTTv265, human GNT1 with N-terminal truncation of 114 nucleic acids (38 amino acids) was added under the gpdA promoter. To clone pTTv265, plasmid pTTv264 was linearized with Pacl. Human GNT1 was amplified by PCR from the synthetic plasmid pTTv1 carrying a full-length human GNT1 gene (P26572, pTTv11 is also described in International Patent Application No. POCT / EP2011 / 070956). The primers used for amplification are shown in Table 21.4. All fragments were purified using conventional laboratory methods. The plasmid was cloned by the yeast recombination method described above. After recovering the E. coli plasmid, some clones were checked for correct recombination and the stored clone was checked by sequencing.
[00555] [00555] - The truncated GnTI amino acid sequence of 38 amino acids in plasmid pTTv11 for construction of pTTv265. SVSALDGDPASLTREVIRLAQDAEV- LERQRGLLQQIGDALSSARGRVPTAAP PAQPRVPVTPAPAVIPIL- VIACDRSTVRRCLDKLLHYRPSAELFPIIVSQDCGHEETAQAIA SYGSAVTHIRQPDLSSIAVPPDHRK- QGYYKIARHYRWALGQVFRQFRFPAAVVVEDDL EVAPDFFEYFRATYPLLKADPSLWC- SAWNDNGKEQMVDASRPELLYRTDFFPGLGWL LLAELWAELEPKWPKAFWDDWMRRP- QRQGRACIRPEISRTMTFGRKGVSHGQFFDQH LKFIKLNQQFVHFTQLDLSYLQREA- DRDFLARVYGAPQLQVEKVRTNDRKELGEVRV QYTGRDSFKAFAKALGVMDDLKSGVY- RAGYRGIVTFQFRGRRVHLAPPPTWEGYDPSW N
[00556] [00556] Plasmids pTTv274, pTTv275, pTTv276, pTTv278, pPTTv279 and pTTv280 were all based on the plasmid pTTv265 described above. In these plasmids, different peptides that target Golgi were added to precede the N-terminally truncated human GNTI gene. To clone these plasmids, pTTv265 was linearized with Pacl. Different peptides that target Golgi were amplified by PCR using the primers shown in the Table
[00557] [00557] Fragments for transformations were released from plasmids pTTv274 (GNT2), pTTv275 (KRE2), pTTv276 (type KRE2), pTTv278 (OCH1), pTTv279 (ANP1) and pTTv280 (VAN1) (Tables 21.5A and 21.5A and 21.5A . All fragments were transformed individually into the M507 strain expressing MABO1 and transformations of protoplasts were performed essentially as described in the Examples for selection with hygromycin.
[00558] [00558] Clones that grew well on selective stripes were selected for 5 'and 3' integration in the egl2 locus. Positive clones for double integration were additionally screened for loss of egl2 ORF. The clones providing the desired results were purified by plating individual spores and clones derived from individual spores were checked by PCR as being pure integrating strains. Resulting strains are listed in Table 21.6 below. Table 21.6: Summary of strains with peptide that target Golgi for GnTI Strain Plasmid - Peptide of objectification -. TrelD = M607 pTTv274 - GNT2humana and the M685 pTTva7r5 - Ke2 215768. M610 pTTv276 - TipoKtel 6917 M615 pTTv278 Oct. 65646 M620 pTTv279 —AnDto 0 8251 M622 pTTv7ã0, M5 , M622, M685 and glycan analysis
[00560] [00560] The strains M507, M607, M610, M615, M620, M622 and M685 from T. reesei were fermented in 4% of consumed whole grains, 2% of glucose, 4% of cellobiose, 6% of lactose, pH 5 , 5 and sample-
[00561] [00561] Analysis of MABO1 N-glycans showed that GnMan5 levels varied between 4 and 66% of the total glycans (Tables 21.8 A, B and C). The control M507 strain showed wild-type glycosylation, as expected. Table 21.7: Concentrations of antibodies from strains with human GnTI with different targeting peptides Titration and as E asm e rse m e m e m e m e m e m e a s a ba e s e ba ez es e Bb Ps oras rg lg Tables 21.8 A, B and C: Relative proportions of MABO1 neutral N-glycans on days 5, 3, 4 and 6 of strains with human GnTI with different target peptides. NM [meo7 eto met US CSS PSPS PAU PO OS PS USE 2 4 Hex5HexNAc | Man5 1257, 79, [89, | 88, [93, [11, [24, [37, [42, [29, [44 , [68, | 74, sv a E Eb | ga Job SUS PA ES PS SO 3 Man4 | 5
[00562] [00562] Three silencing constructs were built to perform sip2 expression knockout (tre123244). These RNAi constructs contain a gpdA promoter, targeted integration for the pep2 protease site (tre53961) and a pyr4 loopout marker with direct 3 'pep2 repeat. Two short 19 pp target sequences and a large 448 bp sequence were inserted into this vector to create pTTv217, pTTv218 and pTTv263, respectively. These vectors were designed to knock down s / p2 expression and reduce its protease activity. The RNAi vectors were transformed into the pyr4 version of the M507 strain that produces MABO1. The vector pTTv204 is shown in Figure 52.
[00563] [00563] The RNAi expression vector pTTv204 was linearized with the restriction enzyme AsiS | I. The T846 and T847 primers were annealed together and integrated through yeast recombination in the pTTv204 vector. The initiators are shown in Table 22.1. The target sequence of 19 base pairs is contained in the resulting pTTv217 vector. Primers T848 and T849 were annealed together and integrated into the linearized pTTv204 vector to create the RNAi vector prTv218. This vector contains a target sequence of 19 base pairs. The initiators are shown in Table 22.1. The target sequences are shown in Table 22.2.
[00564] [00564] The vector pTTv263 was made in two parts and integrated in the vector pTTv204. Primers T965 and T967 were used to amplify a 506 base pair sense fragment, including the 58 base pair intronic sequence in the s / p2 gene. The vector pTTv204 was opened with the restriction enzyme AsiSI | and the 506 bp sense fragment was integrated into the yeast vector through recombination. The T1006 and T1007 primers were used to amplify a 448 base pair antisense fragment. The antisense fragment was digested with restriction enzymes Ascl and Fsel. The vector that includes the sense fragment was also digested with Fsel and Ascl. The vector fragment and antisense fragment were linked together to create the vector pTTv263. The initiators are listed in Table 22.1. The target sequence is shown in Table 22.2.
[00565] [00565] RNAi vectors pTTv217, pTTv218, pTTv263 were digested with Pmel to release the expression cassette. The fragments were separated by agarose gel electrophoresis and the correct fragments were isolated with a gel extraction kit (Qiagen) using conventional laboratory methods. Approximately 5 µg of the expression cassette was used to transform the M507 strain that expresses MABO1 antibody (version pyr4-). Preparation of protoplasts and transformation were performed essentially as described in Example 1 for strains M181 and M195 that use selection with bpyr4.
[00566] [00566] The short target sequence in the vector pTTv217 was designed to affect only specifically s / p2. The target sequence pTTv218 was homologous to sip3, slip5 and sIip6. The large 448 bp target sequence in the pTTv263 vector was to affect several subtilisins. The target sequences in these vectors are listed in Table 22.2. The resulting M665, M666 and M667 strains with knockdown were grown in small scale cultures.
[00567] [00567] Several pTTv217, pTTv218 and pTTv263 transformants were grown in cultures in 24 wells to compare their production of MABO1 against the control M507 strain. The strains were grown in TrMM with diamonium citrate without ammonium sulfate, PIPPS at 100 mM, 2% of grains extract consumed, 4% of lactose at a pH of 5.5. Duplicate wells were used for each transformer. Samples of cultures in 24 wells collected on day 6 were used for immunoblotting. The supernatant was diluted with sodium citrate buffer, pH 5.5, so that 0.5 µl of each supernatant could be loaded onto the 4-15% Criterion gel, mixed with LSB + BME and heated to 95 ° C for 5 minutes. Proteins were transferred to nitrocellulose with the Criterion blotter at 100 volts for 30 minutes. The nitrocellulose membrane was blocked with 5% milk in TBST for 1 hour. The heavy chain was detected with heavy anti-chain antibody AP -conjugate (Sigma t4A3188) diluted 1: 10000 in TBST After 1 hour of incubation with the detection antibody, the blot was washed with TBST and the membrane developed with AP substrate (Promega).
[00568] [00568] The results can be seen in Figure 53. Transformer 217.12G produced slightly higher amounts of heavy chain compared to M507 or the second transformant
[00569] [00569] Constructs that targeted several proteases were more successful in improving heavy chain expression. In general, pTTv218 transformants were consistently better than the control M507 strain. The lack of production observed in two of the pTTv263 transformers indicated that RNAi worked very well. When the s / p2 gene was deleted, the growth of the strain suffered and, thus, antibody expression also decreased. Transformants 263.36A and 263.124C grow very poorly and express very little sip2. This was confirmed by shaking flask and qPCR studies.
[00570] [00570] Dry weight measurements of cultures in a shake flask can be seen in Table 22.5. The strains were grown in TrMM with diamonium citrate without ammonium sulfate, PIPPS at 100 mM, 2% of grains extract consumed, 4% of lactose at a pH of 5.5. Duplicate flasks were used for each transformant. Transformer 263.124C had difficulty growing. In general, there was a small reduction in the growth rate of all strains expressing RNAi. This effect may be related to low levels of s / p2 expression.
[00571] [00571] To confirm that the expression s / p2 was effectively reduced by the expression of RNAi, qPCR studies were performed with the study mycelium in a shaking bottle. The RNA was purified from mycelium grown in a shake flask, the cDNA was synthesized and analysis by qPCR was performed. The expression of sip2, sip3, sIp5, sip6 and gpd1 was monitored with specific primers for the genes. Fold chances were measured against a control strain. The expression was normalized with gpd71.
[00572] [00572] Transformant 263.124C showed the highest negative regulation of s / p2 (Table 22.6). The large RNAi induced 36-fold negative regulation of the s / p2 gene, to the point where it was almost inactivated. The other transformants showed a much lighter knock-down activity that varied from 1.2 to 2.5 times. The lighter knockdown is more preferred because the strain grows better and can produce good levels of antibodies.
[00573] [00573] With two transformants, it was observed more closely
[00574] [00574] The M646 strain with s / p2 deletion was made by transforming the pTTv115 deletion cassette into M564 (version pyr4- of M507). The M564 pyr4- strain was created essentially as described in Example 3 to remove the pyr4 disintegrator cassette from the M195 strain (Apep1). Consecutive 5-FOA selection steps were carried out to ensure that the selected clones came from single cells.
[00575] [00575] The deletion cassette containing the flanking regions of s / p2 and pyr4 marker was removed from the vector by digestion with Pmel and the correct fragment was purified from an agarose gel using a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 µg of the deletion cassette was used to transform the M564 strain that produces MABO1 (Apep1Atsp1Asip1 Agap1Agap2Apep4Apep3, pyr4-). Preparation of protoplasts and transformation were performed essentially as described in Example 1 for strains M181 and M195 that use selection with pyr4.
[00576] [00576] The transformants were chosen as first stripes. Growing stripes were tested by PCR (using the primers listed in Table 22.3) for correct integration and loss of sip2 ORF. Clones giving the expected signals were purified into single cell clones and again screened by PCR using the primers listed in Table 22.3. The correct clone was designated as the M646 strain.
[00577] [00577] The M507 strain was grown in the Series FTR104 culture fermenter under the same conditions as M665, M666, M667 and M646. MG646 was the strain with a s / p2 deletion. M665, M666 and M667 were strains with RNAi silencing. The FTR104 cultures were grown in minimum medium for Trichoderma (TTIMM) plus 20 g / L yeast extract, 40 g / L cellulose, 80 g / L cellobiose and 40 g / L sorbose at a pH of 5.5 . The temperature was changed from 28 C to 22 C after 48 hours. The cultures were grown for 6 days. Minimum medium for Trichoderma contains 5 g / L of ammonium sulfate, 5 g / L of potassium dihydrogen phosphate, 1 ml / L of trace elements, 4.1 ml of calcium chloride at 1 M per L and 2.4 ml of 1 M magnesium sulfate per L of medium.
[00578] [00578] Total antibody concentrations were determined from days 3-6. On day 6, the M667 strain reached 3.81 g / L, see Table 22.8. After day 5, antibody expression dropped in the cultures of M507, M665 and M666. On day 6, the M507 strain produced 2.2 g / L, M665 reached 2.7 g / L and M666 produced 2.8 g / L. Thus, strains with the small RNAi target sequences produced slightly more antibody than M507, indicating that silencing works in these strains. The M646 strain with s / p2 deletion grew more slowly than the other strains. The s / p2 deletion strain produced slightly above 2 g / L on day 6. Fermentation of strains M507, M665, M666 and M667
[00579] [00579] Strains 217.12G (M665), 218.25F (M666) and 263,110F (M667) were grown in 1 L fermenters with 30 g / | glucose, 60 g / l lactose, 20 g / l WSG, 20 g / l SGE plus lactose feed at a pH of 5.5 starting at 28 “* C and going to 22 CT at the end of culture. The expression of MABO1 heavy and light chains was tested by immunoblot of samples of supernatant collected in
[00580] [00580] Total antibody expression was measured after protein G purification and the values are shown in Table 22.4, together with the results of two control strains. The M507 strain was grown with and without SBTI inhibitor under the same conditions. The levels of MABO1 expression in the M667 strain were higher than those measured in the M507 strain. On day 9, for example, the level of expression was twice as high for M667. The levels of expression observed with M667 were similar to the culture made with the addition of SBTI. Strains M665 and M666 produced levels slightly lower or similar to those of the control. There was an evident 2-fold increase in antibody expression compared to the standard MB507 strain.
[00581] [00581] The protease activity of the cultures listed in the Table
[00582] [00582] 100 µl of all diluted supernatants were added to the 96-well plate. Three replicate wells per sample were made. 100 µl of diluted FL casein stock (10 µg / ml) in sodium citrate buffer, pH 5.5 was added. The casein stock solution in the flask was 1000 µg / ml diluted in 200 µl of PBS. For each sample, a baseline control was included with 100 μl of the diluted supernatant and 100 μl of sodium citrate buffer, pH 5.5. The incubated plates containing supernatants and substrate were covered with a plastic bag at 37 ° C. Fluorescence was measured on the plates after 4 hours of incubation. Readings were taken on a fluorescence plate reader using excitation a 485 nm and emission at 530 nm.
[00583] [00583] The protease activity in the supernatant of the M665 strain was the lowest in general. Throughout culture, it was almost half that of M507. The activity of the large M667 hairpine vector was low as well, but started to decrease after 5 days, being lowest on day 10. This was where the production of antibodies to the M667 strain was highest on day 10. At the end of culture, both M665 and M667 culture supernatants had half of the protease activity compared to the M507 control. When the M507 culture was supplemented with protease inhibitor SBTI, the protease activity also dropped from day 6 to day 8 and remained lower than the M507 strain. The low protease activity at the end of the culture explains why the M667 strain produced twice the antibody compared to the M507 strain.
[00584] [00584] Seven strains of Trichoderma reesei were generated to express antibody fragments (Fab, antibodies with a single multimeric domain (sd-Ab's) and scFv) from different bases with protease deletion, as listed in Table 23. The architecture of the genetic expression chambers applied for this purpose was based on regulatory elements (promoter and terminator) of the cephalobiohydrolase gene | (cbh1). The catalytic domain of the CBHI protein has been modified to remove intronic sequences and used as a fusion partner to increase the expression and secretion of an antibody fragment. A reason for recognition for the Kex2 protease has been inserted among the fusion partners to promote cosecret release.
[00585] [00585] In order to prepare the flanking expression cassettes for transformation, the corresponding fragments were released from their respective vector skeletons by restriction digestion with Pmel and purified using Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).
[00586] [00586] As listed in Table 23, T. reesei strains with protease deletion were transformed with the purified expression cassettes using PEG-mediated protoplast transformation. The transformants were selected for resistance to hygromycin B or prototrophy for acetamidase by plating on culture medium containing hygromycin B as a selective agent or acetamide as the only source of nitrogen, respectively. Up to 48 transformants from each were screened by PCR for the homologous integration of the expression cassette at the cbh1 locus using a forward primer outside the 5 'flanking region fragment of the construct and the reverse primer within the modified CBH1 catalytic domain ( 5 'integration), as well as a direct primer inside the hygromycin B or acetamidase selection marker, respectively, and a reverse primer outside the 3' flanking region (3 'integration). From each transformation, five to seven independent transformants, for which PCR screening proved the correct integration of the construct to the cbh1 locus, were selected for purification of a single spore to obtain mononuclear clones. Proper integration of the rupture cassette was reconfirmed by PCR using the same combinations of primers as described above and the absence of the parental CBH1 locus was verified using a combination
[00587] [00587] The expression of antibody fragments is facilitated by the cellobiohydrolase | promoter. The strains were grown in batch fermentation for 7 days in medium containing 2% yeast extract, 4% cellulose, 8% cellobiose, 4% sorbose, 5 g / L KH, PO, and 5 g / L of (NH1) 2 / SO ,. The pH of the culture was controlled to a pH of 5.5 (adjusted with NH, OH) and the temperature was kept constant at 28 ºC. Fermentations were carried out in four reactors with a 2 L parallel glass vessel (DASGIP) with a culture volume of 1 L. Samples of culture supernatant were taken during the course of operations and stored at -20 CT. Samples were collected daily from the entire course of these cultures and production levels were analyzed by means of affinity liquid chromatography for all molecules. For each antibody fragment, the maximum titration, strain ID and proteinase deletion base are shown in Table 23.
[00588] [00588] The concentrations of Mab and sdAb were quantified by HPLC-Protein A chromatography, which is based on affinity chromatography with UV detection. The Fc domain of human class G immunoglobulins (class-subtype: I9G1, IgG2, IgG4, except I9G3) specifically binds to protein A, which is covalently bound to the stationary phase. The protein A binding affinity for the Fc domain is pH dependent. After binding at a pH of 7.5, the monoclonal antibody was eluted under acidic conditions at a pH of 2.0 and detected at 280 nm.
[00589] [00589] Fab concentrations were quantified by HPLC-antilambda chromatography, which is based on affinity chromatography with UV detection. The lambda chain of human Fab fragments specifically binds to an anti-lambda linker derived from camelid which is covalently linked to the stationary phase. After binding at a pH of 7.5, the monoclonal antibody was eluted under acidic conditions at pH 1.4 and detected at 280 nm. scFV2
[00590] [00590] ScFV2 concentrations were quantified by purification on Protein G HiTrap using an Avant system from Akta and subsequent UV detection. The part of the kappa light chain of scFV2 specifically binds to the protein G linker that is covalently bound to the stationary phase. After binding at a pH of 7.2, scFV2 was eluted under acidic conditions with 0.1 mM HCl and detected at 280 nm. scFV1-His
[00591] [00591] ScFV1-His concentrations were quantified by HisTrap HP purification using an Akant Avant system and subsequent UV detection. The protein's histidine marker binds specifically to Ni Sepharose. After ligation, the protein was eluted using 500 mM imidazole and detected at 280 nm.

权利要求:
Claims (30)
[1]
1. Filamentous fungal cell comprising at least three endogenous proteases having reduced activity and a recombinant polynucleotide that encodes a heterologous polypeptide, characterized by the fact that the polypeptide is produced at a level at least twice the level of production of the polypeptide in a corresponding parental filamentous fungal cell in which the proteases have no reduced activity.
[2]
2. Filamentous fungal cell, according to claim 1, characterized by the fact that, when the cell is an Aspergillus cell, the total protease activity is reduced to 50% or less of the total protease activity of the corresponding parental Aspergillus cell in which the proteases have no reduced activity.
[3]
3. Filamentous fungal cell according to claim 1 or 2, characterized by the fact that the total protease activity is reduced to 49% or less, preferably 31% or less, of the total protease activity of the corresponding parental filamentous fungal cell in which the proteases have no reduced activity.
[4]
4. Filamentous fungal cell according to any one of claims 1-3, characterized by the fact that at least three genes encoding endogenous proteases each comprise a mutation that reduces or eliminates the corresponding protease activity.
[5]
5. Filamentous fungal cell according to any of claims 1-4, characterized by the fact that at least four genes encoding endogenous proteases each comprise a mutation that reduces or eliminates the corresponding protease activity.
[6]
6. Filamentous fungal cell, according to any of the preceding claims, characterized by the fact that the cell
fungal cell is a Trichoderma fungal cell, a Myceliophthora fungal cell, an Aspergillus fungal cell, a Neurospora fungal cell, a Penicillium fungal cell or a Chrysosporium fungal cell. anti
[7]
7. Filamentous fungal cell, according to any of the preceding claims, characterized by the fact that the fungal cell is from Trichoderma reesei.
[8]
8. Filamentous fungal cell, according to claim 6 or 7, characterized by the fact that said cell is of the wild type for pep4 protease.
[9]
9. Filamentous fungal cell, according to claim 6, 7 or 8, characterized by the fact that the cell has reduced or no protease activity in at least three of the following proteases: pep1, tsp1 and sip1 or gap1, sip1 and pep1.
[10]
10. Filamentous fungal cell, according to one of claims 6 or 7, characterized by the fact that the cell has reduced or no detectable protease activity in at least three or four proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, sip1, sip2, sIp3, sIp7, gap1 and gap 2.
[11]
11. Filamentous fungal cell, according to claim 10, characterized by the fact that each of the genes encoding the three or four proteases with reduced or non-existent protease activity comprises a mutation that reduces or eliminates the corresponding protease activity.
[12]
12. Filamentous fungal cell according to claim 6 or 7, characterized by the fact that the cell has reduced or no protease activity in at least eight proteases, each of the genes encoding the eight proteases comprising a mutation that reduces or eliminates the corresponding protease activity and the eight proteases are pep1, tsp1, sip1, gap1, gap2, pep4, pep3 and pepo.
[13]
13. Filamentous fungal cell according to claim 6 or 7, characterized by the fact that the cell comprises three to six proteases having reduced or no detectable activity and each of the three to six proteases is selected from the group consisting of pep1, pep2, pep3, pep4, pep5, tsp1, sIp1, sip2, slp3, gap1 and gap2.
[14]
14. Filamentous fungal cell according to any one of claims 6-13, characterized in that the cell comprises a gene that encodes an additional protease that comprises a mutation that reduces the corresponding protease activity and the protease additional is selected from the group consisting of pep7, pep8, pep11, pep12, top1, gap2, sIp3, sIp5, sIp6, sip7 and sip8.
[15]
15. Filamentous fungal cell according to any of the preceding claims, characterized by the fact that the heterologous polypeptide is a mammalian polypeptide.
[16]
16. Filamentous fungal cell, according to claim 15, characterized by the fact that the mammalian polypeptide is glycosylated.
[17]
17. Filamentous fungal cell, according to claim 15, characterized by the fact that the mammalian polypeptide is selected from the group consisting of an antibody and antigen-binding fragments thereof, a growth factor, an interferon, a cytokine and an interleukin.
[18]
18. Filamentous fungal cell, according to claim 15, characterized by the fact that the mammalian polypeptide is selected from the group consisting of insulin-like growth factor 1 (IGF1), human growth hormone (hGH) and interferon alpha 2b (IFNa2b).
[19]
19. Filamentous fungal cell, according to any of the preceding claims, characterized by the fact that the fungal cell further comprises ALG3 having reduced activity.
[20]
20. Filamentous fungal cell, according to claim 19, characterized by the fact that the gene encoding ALG3 comprises a mutation that reduces or eliminates the corresponding activity.
[21]
21. Filamentous fungal cell according to any one of claims 1-20, characterized in that the fungal cell further comprises a polynucleotide encoding an α-1,2-mannosidase.
[22]
22. Filamentous fungal cell according to any of the preceding claims, characterized by the fact that it still comprises a catalytic domain of N-acetylglucosaminyl transferase | and, optionally, a catalytic domain of N-acetylglucosamini! transfer | l.
[23]
23. Filamentous fungal cell according to claim 22, characterized by the fact that it also comprises a first polynucleotide that encodes the catalytic domain of N-acetylglucosaminyl transferase | and, optionally, a second polynucleotide that encodes the catalytic domain of N-acetylglucosaminyl transferase | 1.
[24]
24. Filamentous fungal cell, according to any of the preceding claims, characterized by the fact that it still comprises a polynucleotide that encodes a mannosidase | and / or a galactosyl transferase.
[25]
25. A method for improving the stability of a polypeptide comprising: a) supply of the filamentous fungal cell, as defined in any of the preceding claims; and b) cell culture so that the heterologous polypeptide
Be expressed, characterized by the fact that the heterologous polypeptide exhibits increased stability compared to the heterologous polypeptide produced in a corresponding parental fungal filamentous cell in which the proteases have no reduced activity.
[26]
26. Method of producing a heterologous polypeptide, characterized by the fact that it comprises: a) supply of the filamentous fungal cell, as defined in any of claims 1-24, b) culture of the cell so that the heterologous polypeptide be expressed; and c) purification of the heterologous polypeptide.
[27]
27. Method according to claim 25 or claim 26, characterized in that the filamentous fungal cell further comprises a carrier protein.
[28]
28. Method according to claim 27, characterized by the fact that the carrier protein is CBH1.
[29]
29. Method according to any of claims 26-28, characterized by the fact that the culture is in a medium comprising a protease inhibitor.
[30]
30. Method according to any one of claims 26-29, characterized by the fact that the culture is in a medium comprising one or two protease inhibitors selected from SBT1 and chemostatin.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US10731168B2|2020-08-04|Protease deficient filamentous fungal cells and methods of use thereof

---

US10988791B2|2021-04-27|Multiple proteases deficient filamentous fungal cells and methods of use thereof

---

US9701970B2|2017-07-11|Promoters for expressing genes in a fungal cell

---

US20180215797A1|2018-08-02|Regulatory Protein Deficient Trichoderma Cells and Methods of Use Thereof

---

WO2020127198A1|2020-06-25|Tandem protein expression

---

TW202031898A|2020-09-01|Carbon-source regulated protein production in a recombinant host cell

---

TN et al.0|gk kkk kMMkkk S kkSkk BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM

同族专利:

---

公开号 | 公开日

---

JP6245528B2|2017-12-13|

---

RU2645252C2|2018-02-19|

---

EP3358000A2|2018-08-08|

---

MX355226B|2018-03-23|

---

US11180767B2|2021-11-23|

---

CN107868758A|2018-04-03|

---

SG11201403818XA|2014-08-28|

---

DK2800809T3|2018-05-22|

---

MX2014008272A|2015-02-24|

---

JP2018038419A|2018-03-15|

---

AU2013207165B2|2018-10-04|

---

EP3358000A3|2018-12-19|

---

US9567596B2|2017-02-14|

---

US20170275634A1|2017-09-28|

---

EP2800809A2|2014-11-12|

---

KR20140114860A|2014-09-29|

---

CA2861697A1|2013-07-11|

---

US20140370546A1|2014-12-18|

---

CN104245919B|2017-07-14|

---

EP2800809B1|2018-03-07|

---

WO2013102674A2|2013-07-11|

---

WO2013102674A3|2013-11-14|

---

US20190233831A1|2019-08-01|

---

IL233052D0|2014-07-31|

---

JP2015512611A|2015-04-30|

---

RU2014132178A|2016-02-20|

---

CN104245919A|2014-12-24|

---

AU2013207165A1|2014-07-03|

---

US20210139922A1|2021-05-13|

---

US10240159B2|2019-03-26|

---

US10731168B2|2020-08-04|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US4500707A|1980-02-29|1985-02-19|University Patents, Inc.|Nucleosides useful in the preparation of polynucleotides|

---

US4475196A|1981-03-06|1984-10-02|Zor Clair G|Instrument for locating faults in aircraft passenger reading light and attendant call control system|

---

US4447233A|1981-04-10|1984-05-08|Parker-Hannifin Corporation|Medication infusion pump|

---

US4439196A|1982-03-18|1984-03-27|Merck & Co., Inc.|Osmotic drug delivery system|

---

US4447224A|1982-09-20|1984-05-08|Infusaid Corporation|Variable flow implantable infusion apparatus|

---

US4487603A|1982-11-26|1984-12-11|Cordis Corporation|Implantable microinfusion pump system|

---

US4816567A|1983-04-08|1989-03-28|Genentech, Inc.|Recombinant immunoglobin preparations|

---

US4486194A|1983-06-08|1984-12-04|James Ferrara|Therapeutic device for administering medicaments through the skin|

---

US4758512A|1984-03-06|1988-07-19|President And Fellows Of Harvard College|Hosts and methods for producing recombinant products in high yields|

---

US4596556A|1985-03-25|1986-06-24|Bioject, Inc.|Hypodermic injection apparatus|

---

US4683195B1|1986-01-30|1990-11-27|Cetus Corp|

---

US4790824A|1987-06-19|1988-12-13|Bioject, Inc.|Non-invasive hypodermic injection device|

---

US4941880A|1987-06-19|1990-07-17|Bioject, Inc.|Pre-filled ampule and non-invasive hypodermic injection device assembly|

---

US5700637A|1988-05-03|1997-12-23|Isis Innovation Limited|Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays|

---

CA1333777C|1988-07-01|1995-01-03|Randy M. Berka|Aspartic proteinase deficient filamentous fungi|

---

GB8822228D0|1988-09-21|1988-10-26|Southern E M|Support-bound oligonucleotides|

---

US5312335A|1989-11-09|1994-05-17|Bioject Inc.|Needleless hypodermic injection device|

---

US5064413A|1989-11-09|1991-11-12|Bioject, Inc.|Needleless hypodermic injection device|

---

GB2276169A|1990-07-05|1994-09-21|Celltech Ltd|Antibodies specific for carcinoembryonic antigen|

---

US5846802A|1992-04-15|1998-12-08|Buxton; Frank|Fungal Protease|

---

AU680816B2|1992-05-15|1997-08-14|Quest International B.V.|Cloning and expression of DNA encoding a ripening form of a polypeptide having phamnogalacturonase activity|

---

US5383851A|1992-07-24|1995-01-24|Bioject Inc.|Needleless hypodermic injection device|

---

DK52393D0|1993-05-05|1993-05-05|Novo Nordisk As|

---

US5674728A|1993-11-03|1997-10-07|Novartis Corporation|Aspergillus niger vacuolar aspartyl protease|

---

AT256191T|1994-06-17|2003-12-15|Novozymes As|A MUSHROOM IN WHICH THE AREA GENE IS CHANGED AND AN AREA GENE FROM ASPERGILLUS ORYZAE|

---

US6060305A|1994-06-30|2000-05-09|Novo Nordisk Biotech, Inc.|Non-toxic, non-toxigenic, non-pathogenic Fusarium expression system|

---

CN1164868A|1994-11-08|1997-11-12|诺沃挪第克公司|Tripeptidyl aminopeptidase|

---

CN1225550C|1995-03-20|2005-11-02|诺沃奇梅兹有限公司|Host cell expressing reduced levels of metalloprotease and methods using host cell in protein production|

---

AU7112796A|1995-09-27|1997-04-17|Chiron Corporation|Method of making a protease deficient host|

---

US5776730A|1995-12-15|1998-07-07|University Of Hawaii|Neurospora hosts for the production of recombinant proteins, and methods for producing same|

---

EP0866872A1|1995-12-15|1998-09-30|Novo Nordisk A/S|A FUNGUNS WHEREIN THE areA, pepC AND/OR pepE GENES HAVE BEEN INACTIVATED|

---

WO1997026330A2|1996-01-19|1997-07-24|Novo Nordisk Biotech, Inc.|Morphological mutants of filamentous fungi|

---

CN1120235C|1996-03-27|2003-09-03|诺沃奇梅兹有限公司|Alkaline protease deficient filamentous fungi|

---

WO1997046689A1|1996-06-05|1997-12-11|Gist-Brocades B.V.|Fungal metallo protease genes|

---

ES2256895T3|1996-09-19|2006-07-16|Novozymes A/S|GUEST CELLS AND PROTEIN PRODUCTION METHODS.|

---

US7883872B2|1996-10-10|2011-02-08|Dyadic International , Inc.|Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose|

---

US6806062B1|1998-10-05|2004-10-19|Novozymes A/S|Fungal transcriptional activator useful in methods for producing polypeptides|

---

AU2755300A|1999-02-02|2000-08-25|Novozymes Biotech, Inc.|Methods for producing polypeptides in fungal cells|

---

AU2658200A|1999-02-22|2000-09-14|Novozymes A/S|Oxaloacetate hydrolase deficient fungal host cells|

---

DE60138947D1|2000-04-13|2009-07-23|Dyadic Internat Usa Inc|SCREENING OF LIBRARIES OF EXPRESSED DNA IN FILAMENTOUS MUSHROOMS AT HIGH PERFORMANCE RATE|

---

US7122330B2|2000-04-13|2006-10-17|Mark Aaron Emalfarb|High-throughput screening of expressed DNA libraries in filamentous fungi|

---

WO2001068864A1|2000-03-14|2001-09-20|Novozymes A/S|Fungal transcriptional activator useful in methods for producing polypeptides|

---

DE60103174T2|2000-12-07|2005-05-12|Dsm Ip Assets B.V.|PROCESS FOR PREVENTING OR REDUCING TURBIDITY IN BEVERAGES|

---

CA2438680A1|2001-02-23|2002-09-06|Dsm Ip Assets B.V.|Genes encoding proteolytic enzymes from aspargilli|

---

WO2003054186A1|2001-12-21|2003-07-03|Dsm Ip Assets B.V.|New rennets|

---

US20040018573A1|2002-04-18|2004-01-29|Power Scott D|Production of functional antibodies in filamentous fungi|

---

WO2004067709A2|2003-01-17|2004-08-12|Elitra Pharmaceuticals, Inc.|Identification of essential genes of aspergillus fumigatus and methods of use|

---

CN100547078C|2003-03-31|2009-10-07|诺维信股份有限公司|In the mutants of aspergillus that lacks enzyme, produce the method for biological substance|

---

US8716023B2|2003-12-09|2014-05-06|Novozymes, Inc.|Methods for eliminating or reducing the expression of a genes in a filamentous fungal strains|

---

GB0405659D0|2004-03-12|2004-04-21|Horticulture Res Internat|Altered expression in filamentous fungi|

---

DK1756145T3|2004-06-16|2014-09-29|Dsm Ip Assets Bv|PRODUCTION OF POLYPEPTIDES THROUGH IMPROVED SECRETION|

---

WO2006040312A2|2004-10-12|2006-04-20|Dsm Ip Assets B.V.|Fungal transcriptional activators useful in methods for producing a polypeptide|

---

GB0424940D0|2004-11-11|2004-12-15|Danisco|Transcription factors|

---

MX2007007862A|2004-12-30|2007-08-17|Genencor Int|Acid fungal proteases.|

---

DK2333045T3|2005-04-12|2017-02-20|Danisco Us Inc|GEN-INACTIVATED MUTANTS WITH CHANGED PROTEIN PRODUCTION|

---

JP5131457B2|2005-08-03|2013-01-30|旭硝子株式会社|Yeast host, transformant and method for producing heterologous protein|

---

US7858360B2|2005-10-17|2010-12-28|Novozymes A/S|Use of fungal mutants for expression of antibodies|

---

EA014783B1|2005-11-29|2011-02-28|ДСМ АйПи АССЕТС Б.В.|Dna binding site of a transcriptional activator useful in gene expression|

---

CA2657975A1|2006-06-29|2008-01-03|Dsm Ip Assets B.V.|A method for achieving improved polypeptide expression|

---

EP2082047A2|2006-08-29|2009-07-29|Danisco US, INC., Genencor Division|Compositions and methods for improved protein production|

---

WO2008053018A2|2006-11-02|2008-05-08|Dsm Ip Assets B.V.|Improved production of secreted proteins by filamentous fungi|

---

US9862956B2|2006-12-10|2018-01-09|Danisco Us Inc.|Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi|

---

US8680252B2|2006-12-10|2014-03-25|Dyadic International , Inc.|Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi|

---

CN105420268A|2006-12-21|2016-03-23|诺维信股份有限公司|Methods of reducing or eliminating expression of genes in filamentous fungal strains by transitive RNA interference|

---

EP2508612B1|2007-04-03|2017-11-29|Oxyrane UK Limited|Glycosylation of molecules|

---

WO2008153712A2|2007-05-21|2008-12-18|Danisco Us, Inc., Genencor Division|Method for introducing nucleic acids into fungal cells|

---

CA2704548A1|2007-11-01|2009-05-07|Danisco Us Inc.|Signal sequences and co-expressed chaperones for improving protein production in a host cell|

---

US8633010B2|2007-12-06|2014-01-21|Novozymes A/S|Fungal PepC inhibitor|

---

US20090253173A1|2008-04-08|2009-10-08|Danisco Us Inc., Genencor Division|Filamentous fungi with inactivated protease genes for altered protein production|

---

AU2009266989B2|2008-07-03|2013-05-02|Pfenex, Inc.|High throughput screening method and use thereof to identify a production platform for a multifunctional binding protein|

---

CA2732104A1|2008-07-29|2010-02-04|Danisco Us Inc.|Increased production of aspartic proteases in filamentous fungal cells|

---

CN102227502B|2008-09-30|2016-01-13|诺维信股份有限公司|The method of polypeptide is produced in the enzyme defect mutant of empiecement sickle spore|

---

JP2012509082A|2008-11-18|2012-04-19|ダニスコ・ユーエス・インク|Filamentous fungi with reduced ptrB activity for modified protein production|

---

WO2010068800A1|2008-12-12|2010-06-17|Novozymes, Inc.|Polypeptides having aspartic endopeptidase activity and polynucleotides encoding same|

---

ES2845200T3|2009-03-16|2021-07-26|Danisco Us Inc|Chrysosporium lucknowense protein production system|

---

ES2589655T3|2009-10-16|2016-11-15|Merck Sharp & Dohme Corp.|Method for producing proteins in Pichia pastoris that lack detectable cross-binding activity to antibodies against host cell antigens|

---

US20120213728A1|2009-10-30|2012-08-23|Merck|Granulocyte-colony stimulating factor produced in glycoengineered pichia pastoris|

---

MX2012004993A|2009-10-30|2012-06-12|Merck Sharp & Dohme|Methods for the production of recombinant proteins with improved secretion efficiencies.|

---

WO2011053612A1|2009-10-30|2011-05-05|Merck Sharp & Dohme Corp.|Method for producing therapeutic proteins in pichia pastoris lacking dipeptidyl aminopeptidase activity|

---

EP2501805B1|2009-11-19|2019-02-20|Oxyrane UK Limited|Yeast strains producing mammalian-like complex n-glycans|

---

US8986974B2|2009-12-18|2015-03-24|Novozymes, Inc.|Methods for producing polypeptidies in protease-deficient mutants of trichoderma|

---

AU2010334383B2|2009-12-21|2014-10-02|Centre Hospitalier Universitaire Vaudois |Synergic action of a prolyl protease and tripeptidyl proteases|

---

CA2788992A1|2010-02-24|2011-09-01|Merck Sharp & Dohme Corp.|Method for increasing n-glycosylation site occupancy on therapeutic glycoproteins produced in pichia pastoris|

---

WO2012021883A2|2010-08-13|2012-02-16|Dyadic International, Inc.|Novel fungal enzymes|

---

US9322027B2|2010-08-20|2016-04-26|Shell Oil Company|Expression constructs comprising fungal promoters|

---

WO2012048334A2|2010-10-08|2012-04-12|Dyadic International Inc.|Novel fungal proteases|

---

US9068235B2|2010-11-02|2015-06-30|Codexis, Inc.|Fungal strains|

---

BR112014004190A2|2011-08-24|2017-03-01|Novozymes Inc|method for constructing a filamentous fungal strain, filamentous fungal strain, method for producing multiple recombinant polypeptides, and tandem construct|

---

EP2760997A4|2011-09-30|2015-02-11|Codexis Inc|Fungal proteases|

---

AU2013207165B2|2012-01-05|2018-10-04|Glykos Finland Oy|Protease deficient filamentous fungal cells and methods of use thereof|

---

AU2013348178A1|2012-11-20|2015-05-14|Shell Internationale Research Maatschappij B.V.|Recombinant fungal polypeptides|AU2013207165B2|2012-01-05|2018-10-04|Glykos Finland Oy|Protease deficient filamentous fungal cells and methods of use thereof|

---

US20140273163A1|2013-03-14|2014-09-18|Glycobia, Inc.|Oligosaccharide compositions, glycoproteins and methods to produce the same in prokaryotes|

---

MX2016000306A|2013-07-10|2016-08-08|Novartis Ag|Multiple proteases deficient filamentous fungal cells and methods of use thereof.|

---

CA2954974A1|2014-07-21|2016-01-28|Glykos Finland Oy|Production of glycoproteins with mammalian-like n-glycans in filamentous fungi|

---

BR112017007949A2|2014-10-24|2018-01-23|Danisco Us Inc|method for alcohol production by the use of a tripeptidyl peptidase|

---

MX2017005268A|2014-10-24|2017-07-26|Dupont Nutrition Biosci Aps|Proline tolerant tripeptidyl peptidases and uses thereof.|

---

EP3259364B1|2015-02-20|2020-07-01|Roal Oy|Methods for controlling protease production|

---

CN106190874B|2015-05-06|2020-06-26|中国科学院天津工业生物技术研究所|Method for enhancing production of filamentous fungal protein|

---

CN104990881A|2015-07-09|2015-10-21|辽宁省农业科学院蔬菜研究所|Method for quickly detecting vigor of morchella esculenta L. hyphae|

---

EP3380609B1|2015-11-24|2020-11-11|Novozymes A/S|Polypeptides having protease activity and polynucleotides encoding same|

---

WO2017115005A1|2015-12-29|2017-07-06|Teknologian Tutkimuskeskus Vtt Oy|Production of fusion proteins in trichoderma|

---

EP3523415A1|2016-10-04|2019-08-14|Danisco US Inc.|Protein production in filamentous fungal cells in the absence of inducing substrates|

---

CN108588060B|2017-03-07|2020-12-01|武汉康复得生物科技股份有限公司|Recombinant oxalate decarboxylase expressed by filamentous fungus host cell|

---

US20200299742A1|2017-07-31|2020-09-24|Biopetrolia Ab|Fungal cell with improved protein production capacity|

---

WO2019046232A1|2017-08-30|2019-03-07|Novozymes A/S|Combined use of an endo-protease of the m35 family and an exo-protease of the s53 family in the fermentation of starch|

---

EP3696274A4|2017-10-10|2021-07-28|Ajinomoto Co., Inc.|Method for manufacturing protein|

---

WO2019175477A1|2018-03-12|2019-09-19|Teknologian Tutkimuskeskus Vtt Oy|A subunit vaccine against porcine post-weaning diarrhoea|

---

US10787656B2|2018-05-04|2020-09-29|Fomia BioSolutions, Inc.|Variant alkaline protease enzyme compositions and methods|

---

CA3111168A1|2018-08-29|2020-03-05|Toray Industries, Inc.|Mutant strain of trichoderma reesei and method for producing protein using same|

---

CN111088244A|2018-10-23|2020-05-01|中国科学院天津工业生物技术研究所|Application of protease gene in promotion of cellulase production and complex nitrogen source utilization|

---

WO2021007630A1|2019-07-16|2021-01-21|Centro Nacional De Pesquisa Em Energia E Materiais|Modified trichoderma fungal strain for the production of an enzyme cocktail|

---

CN110724644A|2019-12-05|2020-01-24|西北农林科技大学|Trichoderma reesei engineering bacterium for producing recombinant human proinsulin and application thereof|

---

WO2022023584A1|2020-07-31|2022-02-03|Biotalys NV|Methods of increasing recombinant protein yields|

法律状态:
2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2020-11-24| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2021-03-09| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|

---

2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

---

申请号 | 申请日 | 专利标题

---

US201261583559P| true| 2012-01-05|2012-01-05|

---

US61/583,559|2012-01-05|

---

PCT/EP2013/050126|WO2013102674A2|2012-01-05|2013-01-04|Protease deficient filamentous fungal cells and methods of use thereof|

---