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    • 专利摘要:
    Novel upstream activation sites of the yeast PHO5 gene are used to produce inducible yeast hybrid promoters. The yeast hybrid promoters can be used to control transcription of a polypeptide coding region foreign to yeast in a yeast expression vector.
    公开号:SU1630616A3
    申请号:SU864028035
    申请日:1986-08-28
    公开日:1991-02-23
    发明作者:Хиннен Альберт；Мейхак Бернд
    申请人:Циба-Гейги Аг (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to biotechnology, in particular to the production of foreign polypeptides in yeast cells.
    The purpose of the invention is to increase the output-1 and the target product.
    The method consists in constructing a recombinant plasmid DNA containing the hybrid promoter PH05-GAPDH, consisting of Clo-SatI PH05, a promoter fragment of plasmid, p 31 / Y hole of size 545 bp, coupled with a synthetic Bglll linker with a Bglll-EcoRIGAPDH promoter fragment - - plasmids pGAI) H size 266 bp Sal I - Hind Ill fragment ppasmida pIDB207 / / 205-HIP size 6.3 kb and EcoRI-HfndITJ-frggment plasmid pJDB207 / pH05 (FC: 0) HTR size 643 bp (
    A 0.2 kb plasmid containing a disulfate-hogran Hgal-BamIII fragment of the plasmid p IL310L transforms the S. cerevisiae GRIM8 yeast strain obtained in the recombinant plasmid DNA and cultured the transformed cells in a liquid culture medium containing 0.03 g / l of potassium dehydrogen phosphate.

    Example 1. Construction PH05 promoter deletions.
    a) VAZZ splitting. Recombinant phage M13mp9 / HP05 Barn-Sal, containing the BNT-YAI fragment PH05, is used as a source of the PH05 promoter. 20 kg of phage DNA (RF: replicative form) digest pe05
    with about about
    CD

    with
    Sail striction endonuclease to obtain a linear DNA of approximately
    9kb. After extraction with phenol / chloroform, the DNA is precipitated with ethanol. DNA is re-suspended in
    10mK tris, pH 8.0, at a concentration of 0.5 μg / ml. 16 microns of Sail cleaved DNA digested 2 eg BA131 exonuclease (BRL) in 100 ml of 20 mM Tris, pH 8.0, 199 mm NaC, 12 mM MgCl,
    2 mM CaClЈ and 1 mM EDTA. Aliquots of 2 µg of DNA are each taken after 1, 2, 3, 4, 5, and 6 min of incubation at 3 ° C and mixed immediately with 50 µl of phenol and 60 µl of TNE. After extraction with phenol / chloroform and ethanol precipitation, the DNA was re-suspended in 10 ° Tris, pH 8.0, at a concentration of 100 µg / ml. To analyze the extent of exonucleolytic cleavage, Ba131 0, µ µg DNA for each time point was cleaved by endonuclease WatNT and analyzed on a 1.5% agarose gel in Tris-borate buffer, pH 8.3 (90 mM Tris-IICl, pK 9.3, 90 mM boric acid, 2.5 mG1 EDTA). On average, 100 lp is removed from each end of the fragment in 1 min of Ba131 cleavage.
    b) Adding EcoRI linkers to the treated Ba131 LNC. The day EcoRI linkers (5 -GGAATTCC-3, BRL) are resuspended in 250 μl of 10 mM Tris-HCl, pH 8, 1 mM EDTA. 2 μg of EcoRI linkers are treated with kinase in 75 μl of 60 mM Tris-HCl, pH 7.5, 10 mM MgClfc, 15 Mil DDT, 10 μM ATP, and 33 U of polynucleotide kinase. After 1 h at 37 ° C, the mixture is allowed to cool to room temperature and then stored at -20 ° C.
    Annealed double-stranded EcoRI linkers are blunt-ended with DNA fragments treated with Ba131, 0.5 μg of DNA treated with Ba131, incubated for 16 hours at room temperature with a 50-fold excess of EcoRI linkers treated with kinase and 20 μl of 60 mM Tris, pH 7.5, 10 mM MgClfe, .10 DTT, 4 mK ATP and 600 units of DTC ligase. After inactivation of T. ligase (10 min at), an excess of EcoRI linkers is split off 50 units, EcoRI in a volume of 50 µl. The DNA is extracted with phenol / chloroform, precipitated with ethanol and resuspended in 10 mM Tris-HCl, 1 mM EDTA (-TE). The DNA is then cleaved 5 units. BamHI and received
    the mixture is introduced into a 1.5% low melting point agarose gel i in tris-borate buffer. The bands are stained with ethidium bromide and visualized with long-wave UV-light at 366 nm. Wide diffuse bands between about 100 bp and 600 p. cut out from the gel, and the DNA is extracted.
    . c) Subsidies in M13mp9. 3 µg Ml Smr split 15 units. EcoRI and 15 units. BamHI in a volume of 50 μl. After extraction with phenol and precipitation with ethanol, the DNA is resuspended in 50 µl of TE. 5 μl of vector DNA segments (about 200 ng) are mixed with 10 μl of these samples (DNA fragments obtained from different Ba131 digests, as described in Example 16) and ligated in full 20 μl in the presence of 60 mM Tris-HCl, pH 7 ,five,
    6 mM MgClii, 100 mM DTT, 1 mM ATP and 200 units of T DNA ligase transduce competent cells of E. coli strain M101 for 15 hours. Phages are grown and analyzed according to the size of their DNA inserts by cleavage with restriction enzymes EcoRI and BamHI.
    d) Determination of Ba131 end points of deletion by determining the sequence of amino acid residues by the Sangeru (deletion of the Sail site). Sequencing is carried out using a dideoxy DNA sequence determination system. The end points of the deletion are shown in Table 1.
    e) Determination of Ba131 deletion end points by determination of the sequence of amino acid residues by Sangeru (deletion from the BamHI site). Carry out a similar set of Ba13) deletions, as described in paragraphs a-c, except that M13mp9 PHH05 Barn-Sal is cut out using BamHI. Ba131 cleaved fragments are treated with EcoRI and Sail, and the resulting fragments are cloned into MlSmp9 cleaved with EcoRI and Sail. The end points of the deletion are shown in table 2,
    e) Construction of internal PH05 promoter deletions. The Ba13G deletion set, described under item g, results in left-hand PH05 promoter fragments ending with the EcoRI site, and the Ba131 deletion set, described in step e, results in the right-hand PHC5 pro 163061
    motor fragments ending with an EcoRJ site. The combination left and right in different positions create internal deletions that contain the EcoRJ linker segment at the deleted DNA site. Separate internal divisions are designed to cut out left and right of M13mp9 derivatives with restriction endonuclear-jo leases of EcoRI and BamHI (left) or EcoRI and Sail (right) and isolate the corresponding Fragments using soft agarose gel electrophoresis, as described in paragraph b. Equimol rs of left, right, and 200 ng BamHI and Sail cleaved M13mp9 vector DNAs are ligated, as described in paragraph c. After transduction in E. coli Ml01, white spots are selected, 20 are determined by RF and analyzed by restriction enzyme analysis (BamHI, Sail, EcoRI). The pieces shown in Table 3 are combined to create specific internal 25 deletions.
    Example 2. In vivo analysis of internal deletions of the PH05 promoter.
    The various deletions described in 30 of Example 1 are cloned into plasmid pJDB207 (PH05), replacing the wild-type PH05 Barn-Sal Fragment with a deleted version. After transformation of the yeast strain S.cerevisiae AH216. acid phosphatase activity is determined. The analysis indicates that the three zones essential for the PH05 expression are located in the following positions: 40
    1. between positions -349 and -383 (HAS1);
    2. between positions -225 and -263 (HAS2);
    3. Between -87 and -125 (TATA box).
    DNA fragments containing HAS or IA52 or HAS1 and IA52 PH05 can be obtained from recombinant bug M13mp9 (PH05) by cleavage with the appropriate endonucleases.
    Example 3. Designing PH05-GAPDH fusion hybrid promoters.
    In examples 1 and 2, the regions around the positions -365 IA51 (PH05) and another region around the -180 position of the IA82 (PH05) PH05 gene are designed - possible candidates for HAS with regulation 35
    55
    s 0 5
    0 0
    with
    0
    five
    five
    functions. HAS (PH05) is contained in 68 bp. The BamHI-Cla fragments, whereas both IA51 (PH05) and Sch52 (PH05) are contained in 368 bp. BamHI-BstEII fragment. Each two of these fragments are merged with two different GAPDHs, which include TATA box and GAPDH transcription initiation sites.
    a) Constructing a yeast gene library 30 μg of the full high molecular weight DNA of wild type yeast strain S288C is incubated for
    30.min with 2 units. EcoRI methylase in 250 µl EcoRI methylation buffer. The DNA is precipitated with ethanol, re-suspended in 500 microns of 25 mM Tris-HCl, pH 8.5, 2 mM MgCla (EcoRF buffer) and digested with EcoRI until a size distribution of the DNA fragments is obtained with a maximum in the range of 30-50 T.K. Yeast DNAs cleaved under EcoRI conditions are fractionally sized in a sucrose gradient (5-20% sucrose in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA). 30 fractions of 0.4 ml each are collected from the upper gradient value. Fraction 16 contains DNA fragments of 30–40 kb each. The DNA of this fraction, 3 μg, is precipitated with ethanol and ligated for 16 hours at 15 °, in full 15 μl with 15 μg of the cosmid vector pYcTf of the linearized EcoRI. Ligation was performed at 300 U of DNA ligase using the buffer system described. The DNA is packaged in vitro into bacteriophage / 1 / B, and the collected phage are used to transduce
     Q f
    E. coli strain HB101 (rk, mk, 1m, pro, gossa A). The transduction efficiency is about 5,000 ampicillin-resistant colonies per µg pVC of the vector. 300 amp colonies are selected and grown in LB medium.
    b) Isolation of the yeast GAPDH gene. The described gene library is screened with a synthetic oligonucleotide of the following structure: 5 - SSTSSATTTSSSSSSSSSS-31.
    10 μg of the oligonucleotide is treated with kinase using 10 μl of OG32p-ATP (3000 Curie / mmol, 10 μCi / μl of Tf by polynucleotide kinase. Positive cloning is detected autoradiographically. Plasmid DNA extraction results in a hybrid clone which
    20
    25
    2100 bp Hindi fragment encoding GAPDH. The cloned GAPDH gene has the same sequence as pgap 491.5
    c) Preparation of GAPDHs located below the promoter. 649 bp of the Tagl fragment, which includes positions from 27 to 675 from the ATG GAPDH gene, were cleaved, cleaved the 10 Tag hybrid plasmid, isolated the DNA fragment on a 1.2% soft soft agarose gel and extracted with hot phenol . Cloning of the Tagl fragment was carried out at the ClB site of pBR322. 1 μg pBR322 flake-g 15 are three units of Clal. 300 ng is ligated with approximately 300 ng of DNA insertion (649 Lp Tr 1 fragment), using 200 units of T DNA ligase in 20 µl. Transformation was carried out in E. coli HB101 for ampicillio resistance. Plasmid DNA is obtained and analyzed by restriction analysis. The orientation of the Tagl fragment is established using PgH restriction endonuclease in combination with BamHI. A plasmid is selected that contains the TagI site of position -675 near the HindIII site of pBR322. This plasmid, designated pBP322 / GAPDH, is linearized using BamHI, and Ba131 cleavage is performed as. described in example 1, except that use BglII-linkers. The size of Ba131 truncated Tagl fragment was determined by 35 restriction analysis (using Bglll and-HindIII).
    Example 4. Desulfatohirudin expression, controlled by PH05-GAPDH hybrid promoter. 40
    I. Regulation of the nucleotide sequence at the 5th end of the de-sulphathogular H, V, R gene.
    The nucleotide sequence encoding desulLatogirudin begins with a GTT codon, standing after the NH terminal valine in the final product. For convenient subcloning and expression in E. coli, the coding sequence is continued at the 5-terminus with eight nucleotidons, including the EcoRI-ori-ng site and the ATG-initiating codon. For an accurate skeletal i fusion of the hirudin coding sequence with the signal peptide coding sequence of PH05, these additional nucleotides are deleted
    0
    five
    0 5 0 5
    0

    by measuring the EcoRI restriction site to the growing end region, adding a synthetic oligonucleotide containing the HgAI recognition site in such a position that subsequent digestion with Hgal occurs directly upstream of the GTT codon,. Maintaining the Hgal restriction region in front of the desulfato-hudruin gene, 8 μg of the plasmid pML310 (EP 168342) was exhaustively digested with the restrictive EcoRI restriction enzyme DNA, extracted with the phenol / chloroform system and precipitated with ethanol. The protruding ends at position 5 are removed by nuclease S ,. The 4 μg pMb310 / EcqRI DNA was cleaved into 100 ml of a mixture consisting of 250 mM NaCl, 1 mM ZnSO, 30 mM sodium acetate at pH 4.6 with 20 units / ml of N nuclease S (Sigma) for 45 min at 37 ° C.
    The DNA is extracted with phonol / chloroform and precipitated with ethanol. DNA (pML310 / EcoRI / Si is resuspended in 100 μl of a solution containing 50 mM Tris-HCl, pH 8.0, and incubated in the presence of 2 units of alkaline calf intestinal phosphate for 1 h at 37 ° C. The enzyme is inactivated for 1.5 hours at 65 ° C. The concentration of NaCl in the incubation mixture is set equal to 150 mM. The dephosphorylated DNA (pMUS / i-EcoRI / S / CIAP) is purified by adsorption on the ion exchange column DN52 in a low salt content buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and then eluted with a high salt content buffer solution (1.5 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 M EDTA). The DNA was ethanol precipitated and resuspended in HgO in a concentration of 0.8 mg / ml ..
    An oligonucleotide of formula 5 — AGCGT.CGACCCT-3 is synthesized by the phospho-trifirne method. A self-complementary sequence of oligonucleotides is obtained containing the recognition site for the -GACCC restriction endonuclease HgAI. Annealing of two single strands results in the production of a double-strand DNA binding of 12 pairs of nitrogen-base bases.
    1.2 μg synthetic single-stranded oligodeoxynucleotide
    phosphorylated in 10 µl of the system consisting of 60 mM Tris-HCl with pH 7.5, 10 mM MgClfc, 5 M MDTT, 30.1 Ci (ft-32p ATP (30QO Ci mmol 1 and 6 units of T4 polynucleotide kinase) 30 min at 37 ° C, followed by substitution for 15 min in the presence of 0.5 mM ATP The resulting mixture is further incubated for 10 min at 75 ° C in order to inactivate the enzyme and then cooled to room temperature for annealing.
    The iZP-labeled DNA binding in the amount of 0.6 µg (170 lmol) is mixed with 2.4 µg (1.75 pmol ends) of pMl310 / EcoRI (S,) CIAP and ligated into 20 ml of a mixture consisting of from 60 mK Tris-HCl, pH 7.5, 10 mM MgClg, 5 mM DTT, 3.5 mM ATP 800 units. T4 DNA ligases, for 20 h at 150 ° C. The ligature is inactivated for 10 minutes at 85 ° C and the excess binder molecules are removed by precipitating DNA in the presence of 10 mM EDTA with pH 7.5, 300 mM sodium acetate with pH 6.0 and 0.54 volumes of isopropanol. After 30 minutes at room temperature, the DNA is suspended in 45 µl of the ligature mixture and lignified for 6 hours to form a circular DNA.
    Aliquots of a ligase mixture of 1 and 3 ml were added to 100 ml of calcium-treated transformation cells of E. coli HB101. The cells were allowed to cool in ice for 30 minutes, then incubated for 3 minutes at 4. ° C, cooled for 2 minutes in ice, and incubated for 1 hour at 37 ° C in 400 µl SOS medium. The resulting cells are concentrated to a volume of 100 µl and applied to plates with LB agar containing 50 µg / ml ampicillin.
    iо
    Twelve Transformed Agar
    colonies are individually grown in LB medium containing 100 µg / ml ampicillin. Plasmid DNA is prepared. The presence of a synthetic oligonucleotide linkage is confirmed by determining the sequence of amino acid residues using a single-stranded DNA seed fragment that hybridizes with the hirudin coding strand. The clone that contains the DNA binding in the correct position ahead of the hirudin gene is designated pML310L,
    50
    50
    five
    Q
    five
    Ii. The merging of PH05 signaling. sequences of the desulfatogirudine structural gene.
    a7 Isolation of the desulfatohirudin fragment. 12 µg of the plasmid pL310L DNA was fully digested with BatK and Pyul endonucleases. After DNA extraction, the two indicated restriction fragments were separated, in a 1, 2% agarose gel in a tris-borate-EDTA buffer, pH 8.3. The Pyul-BamHI fragment of 0.84 p, o, DNA was separated from the gel. eluted, the Pyu-BamHI fragment of pM1310L was further digested with Hgal endonuclease. The cleavage produces a 198 bp HgaI-BamHI fragment, which contains the complete coding sequence for mature ore desulphate. Additional cleavage with A1I1 does not affect this fragment, but eliminates another Hgal fragment of a similar size.
    The desired KgaI-BamHI fragment is separated from the other fragments on a 1.5% agarose gel in TBE buffer and eluted. The DNA is purified by ion exchange chromatography, precipitated with ethanol, the DNA is re-suspended in HgO at a concentration of 30 μg / ml (0.2 pmol / μl) ..
    Allocation of PK05 promoter region with part of the PH05 signal sequence. Plasmid p31 / PH05-TPA18 has a PH05 promoter and a PH05 signal sequence that is skeletally linked to a foreign structural gene (t-PA). Fragment 584 p. BamHI-Ball contains the PH05 promoter and the entire PH05 signal sequence, but with eight nucleotides on the 3rd terminus.
    p31 / PH05-TPA18 DNA in an amount of 8 µg was digested with Ball endonuclease (16 h at 37 ° C). The DNA is purified by extraction with phenol / chloroform and precipitation with ethanol; the DNA is resuspended at a concentration of 0.7 mg / ml.
    The correct connection between the PH05 signal sequence and the desulfatohirudin coding region is provided by a synthetic linkage of the formula:
    (1) 5 -CCAATCGA-3;
    (2) 3 -GGTTAGGT CAACA-5.
    The eight nucleotides at the 51 end of communication (S -CCAATCCA) are part of the PH05 signal sequence from the Ball region to the recession region. Five nucleotides protruding above the end of the 5 - oligonucleotide (2) are connected to the HgAI fragment of cleavage by 5. the termination of the desulfatohirudin auditing sequence.
    Individual single-stranded oligoacleotides (1) and (2) are synthesized by the JQ method using the photoether ether method. Oligonucleotides (1) and (2) in the amount of
    1.1 mcg and 1.8 mcg, respectively, are phosphorylated intravagually at their 5
    The ends are mixed in equimolar 15 quantities and annealed.
    Phosphorylated double-stranded DNA binding in the amount of 1.3 µg (200 pmol), ligorogot with 7 mg (1.8 pmol) rZG / PH05-TPA18 cleaved with Ball J into 40 ml of a mixture consisting of 60 mM Tris-KCl, pK 7.5, 10 mM MgCl2, 3.5 mM ATP, 5 mM DTT and 1400 units of DNA ligase, for 16 hours. Ligase inactivation g for 10 min 25 at 85 ° C . The excess binding is removed by precipitating the DNA in the presence of 10 mM EDTA, 300 mM sodium acetate pH 6.0 and 0.54 volumes of isopropanol. The DNA was resuspended and an additional 30 were digested with the BamHI endonuclease. After extracting the DNA with a phenol / chloroform mixture and precipitating from ethanol, the two indicated restrictive fragments are separated in a 1.2% agarose gel in 35 Tris-borate-EDTA buffer, pH 8.3. 0.6 kb is recovered from the gel. fragment. DCC is eluted and further purified by DE52 ion exchange chromatography and ethanol precipitation. The RamHI-Hgal 40 DNA fragment, 0.6 kb in size, was resuspended at a concentration of 40 μg / ml.
    Selection pJDB207 yeast vector fragment. 9 μg of D5 pJDB207 PH5-TPA plasmid (12-2) are digested with BamHI endonuclease, DNA is extracted with phenol / chloroform and precipitated with ethanol, suspended in 50 mM Tris-HCl; pH 8.0, at a concentration of about 0.1 mg / ml and split 7 units. alkaline phosphate of calf intestine for 1 h 37 ° C. Phosphatase was inactivated for 1.5 hours at 65 ° C. DNA is purified by DE52 ion exchange chromatography and ethanol precipitation. A large BamHI fragment of 6.8 kb are separated in a 1.2% agarose gel in Gris-Gurat-EDTA buffer at pH 8.3. DNA is eluted and purified by DE52 ion exchange chromatography and ethanol precipitation. DNA was dissolved in water at a concentration of 0.4 mg / ml (0.1 pmol / µl).
    Ligating of the PH05 promoter fragment and the desulfatoguridine structural gene with pJDB207. The yeast vector pJDB207, PH05-primotor fragment with PH05 signal sequence and the desulfato-circulating structural gene are isolated as DNA fragments and are ligated to form an expression plasmid.
    For 20 hours at 15 ° C, 10 µl of a mixture consisting of 60 mM Tris-HCl, pH 7.5, 10 mM MgCLa, 5 mM DTT, 1 mM ATP, and 400 units of THz DNA ligase are ligated 0.3 pmol 0.6 kb, BaHI-Hoal fragment p31 / PH05-TPA18 and 0.3 pmol 0.2 kb Hoal-BaHI of the pM13101 fragment, 0.1 pmol 6.8 kb BamHI of the pJDB207P / PH05-TPA fragment (12 2).
    A 1 µl aliquot of the ligature mixture is added to 100 µl of the transformation competent E. coli HB101 cells. The cells are plated on LB-arapo plates containing 50 µg / ml ampicillin,
    R
    24 units of art colonies are individually grown in LB medium in the presence of 100 µg / ml ampicillin. Plasmid DNA is analyzed to determine the size and orientation of the insert by cleaving with the restrictive endonuclease Pstl. The clone with the correct orientation of the insert is designated as pJDB207 / PH05-HIR.
    Obtaining the plasmid pJDB207 / PH05 / (Eco) -HIR For the purpose of conveniently combining the elements of the YAS (PH05) GAPDH hybrid promoter with the desulfatohoirudin coding region, including the PH05 signal sequence (as in the plasmid pJDB207 / PH05-HIR) EcoRI restriction region 5 non-translated region between the original sections of the RHK and ATC coding region.
    15 µl of the pJDB207 / PHO-HRI plasmid was digested with Dral endonuclease. The resulting four fragments were separated on a 0.8% agarose gel in Tris-borate-EDTA buffer at pH 8.3. A 4.2 kb DNA fragment is regenerated from the gel, eluted and precipitated with ethanol. DNA is suspended in a concentration of 0.6 mg / ml.
    Each of two synthetic
    oligonucleotides corresponding to the formulas 5 -AATTCGATTACCAATGTTT-3 and 3 -GCTAATGGTTACAAA-5. (2.3 µg and 2.9 µg, respectively), are exposed to a kinase in 20 µl of a mixture consisting of 60 mM Tris, pH 7.5, 10 mM Mp.de, 5 mM DTT, 0.5 mM ATP and 20 units of T polynucleotide kinase. After 45 minutes at 37 ° C, the two reaction mixtures are combined, heated for 10 minutes at 75 ° C and cooled to room temperature. The annealed oligo-nucleotide linkage is stored at -20 ° C.
    Fragment size 4.2-tp. DrAT DNA in an amount of 6.5 µg (2.3 pmol) is incubated for 16 hours with a 70-fold excess of the named and annealed oligonucleotide binder in 50 µl of a mixture of 60 mM Tris, pH 7, 5, 10 mM MgClu, 5 mM DTT, 3.5 mM ATP, and 800 units 800 units of DNA ligase Tf. After inactivation of Tf, DNA ligases for 10 minutes with excess binding are removed by precipitating DNA in the presence of 10 mM EDTA, 300 mM sodium acetate pH 6.0 and 0.54 volume of isopropanol. The DNAs are cleaved with endonucleases EcoRI and Hindlll. The resulting fragments are separated in 1% agarose gel in Tris-borate-EDTA buffer, pH 8.3. A 643 fragment was eluted and precipitated with ethanol, resuspended at a concentration of 0.1 p mol / ml. The EcoRI-Hindlll fragment contains the PH05 signal sequence, the coding sequence of the desulfatoogirudin, and the PH05 transcription breaker.
    From the p31 / R plasmid, the 534 bp of the PH05 promoter fragment was isolated. 10 µg pZ1 / R are cleaved with the endonucleases EcoR and BamHI. Three fragments are separated on a 0.6% low melting agarose gel in Tris borate-EDTA buffer, pH 8.3, the BamHI-EcoRI fragment of 534 bp contains PH05 promoter comprising
    source sites of mRHK.
    i
    6 μg of plasmid pJDB207 / PH05-HIR
    digested with BamHI and Hindlll Coarse 6.5 kb, the fragment separated from other fragments in 0.6%. agarose gel in tris-borate-EDTA buffer, pH 8.3.
    The three described DNA fragments with the corresponding sticky ends are ligated with TU DNA ligase in a ratio of 534 bp. BamHI-EcoRI PH05-promoter fragment 0.2 pmol, 643 bp EcoRI-HindIII fragment (coding sequence of hirudin) 0.2 pmol, 6.5 tons, p.o, BamHI-Hindlll of the vector fragment 0.1 pmol.
    An aliquot of the ligase mixture in an amount of 1 µl was added to 100 µl of the calcium-treated transformational competent E. coli HB01 cells.
    s
    Twelve transformed amp colonies were individually grown in LB medium containing 10 µg / ml ampicillin. Plasmid DNA was analyzed by EcoRI and BamHI restriction digestion. One clone with the expected restriction fragments was isolated and designated pJDB207 / PH05 (Eco) -HIR.
    Iv. Ligating YAS1 (PH05) - GAPDH hybrid promoters with a desulfatohirudin region that encodes a protein.
    15 µg of the plasmid pJDB207 / FH05 (Eco) - HIR was digested with EcoRI and Hindlll. DNA fragments are separated in 1% agarose gel in tris-borate-EDTA buffer, pK 8.3. Fragment 643 p. eluted and precipitated with ethanol, suspended in HjO at a concentration of 0.1 pmop / μl. 6 μg of the plasmid pJDB207 / PH05-HlP are fully digested with endonuclease Hindlll and Sail. Large 6.3 t, p. the fragment / vector part) is isolated by electrophoresis, extracted with phenol, precipitated with ethanol, suspended in a concentration of 0.05 pmol / μl. 10 μl of the plasmid pGAPDH-EI is digested with Bglll and EcoRI, Fragment 266 bp. Bglll-EcoRI is separated on a 1.2% agarose gel in trisborate-EDTA buffer, pH 8.3, eluted, precipitated with ethanol, suspended in HgO at a concentration of 0.3 pmol / µg. 0.3 pmol 548 bp Sall-Bglll fragment containing UA51 (PH05), 0.3 pmol 266 bp BglII-EcoRI fragment of pGAPDHEI, 0.3 pmol 643 bp The EcoE1-H1pAH fragment of pL) B207 / PH05 (Eco) -HIR and 0.12 pmol 6.3 p.o. The SailHindIII vector fragment is ligated into 20 μl of a mixture consisting of 60; mM Tris, pH 7.5, 10 mM MgCle., 5 mM DTT, I mM ATP, and 400 units of DNA ligase 1 for 6 hours at Aliquots of a 1 µl and 3 µl ligand mixture were added to 100 µl of calcium-treated E. coli HB101 cells. Plasmid isolation from atomic colonies and restrictive analysis using Sail, Bglll, EcoRI and Hindlll were carried out as described. One positive clone is selected and assigned the designation pJDB207 / PAPEI-HIR (YAS1). A similar design is created using 201 p, o. The BgIII-EcoRI fragment isolated from pGAPDH-FI. One isolated plasmid is designated pJDB207 / PAPEI-HIR (YAS1). V. Ligation of YAS1 (PH05) -YAS2 (PHOS) -GAPDH Hybrid Promoters with the Protein Coding Region of Desulphatohoirudin. Plasmids pJDB207 / PAPEI10
    lony and analyze restrictive splitting. Single colonies are selected and their plasmid DNA is designated pJDB207 / PAPEI-HIR (YASl + YAS2) and pJDB207 / PAPEI-HIR (YASl + YAS2).
    Example 5. 31 p. The DNA sequence is sufficient to perform the functions of the phosphate control element.
    31 b.p. the sequence from the upper region of the PH05 promoter (position from -381 to -351), limited by two side deletions D1 0 and / M 3 (example 1e), can potentially contain a regulatory signal. This assumption can be verified by synthesizing two complementary oligonucleotides having the following structures: 5 HAATTCCAAATATATATTAAATATASS
    CGTTTTCGCAG-3 and 3 -GCTTATATATA ATTT AATCGTGCAAAA GCGTCTTAA-S.
    This sequence contains
    t5
    /
    31 p. O, sequence% to which
    HIR (YAs l)) HpJDB207 / PAPEl HIR (YASl) 25 pOI, they try EcoK restrictive
    in an amount of 3 µg each was digested with Bglll. After extraction with phenol and precipitation with ethanol 3, the recessive terminations of DNA are filled in with E.coli DNA polymerase (fragment of Klenow), the enzyme is inactivated for 10 minutes at. The DNA was further digested with Sail and 7.2 kb, the fragments were separated by electrophoresis on a soft agarose gel, 35 by extraction with phenol and precipitation with ethanol. Each fragment is re-sus. pend in HgO at a concentration of 0.05 pmol / μl. Such fragments contain the hirudin-coding region, 40 most vector sequences, and either of two different GAPDH promoter elements isolated from pGAPDH-EI or pGAPDH-FI,
    plots. Such EcoRI regions allow the sequence to be polymerized easily to form
    multimers.
    a) Cloning 31 p. element in vector LT98. Each of the two synthetic oligonucle otids, taken in an amount of 50 pmol, was treated with 20 units. PM polynucleotide kinases in 20 ml of a mixture consisting of 60 mM Tris, pH 7.5, 10 mK MgClfc, 5 mM DTT, 0.5 mM ATP, After 45 min of incubation at 37 ° C, the two reaction mixtures are heated, 10 minutes at 75 ° C and cooled to room temperature. Annealed oligonucleotides are stored at -200 ° C. The kinized and annealed oligonucleotides in coliPlasmid p31 / Y are cleaved with pmol and ligated for BstEII, incubated for 30 min in a volume equal to .15 μl. An EeoRI fragment of LT98 vector DNA (0.075 pmol) and is then added. IncubiE. coli DNA polymerase (Klenow fragment) as described and digested with Sail. Fragment 649 bp separated on a soft agarose gel and regenerated by extraction with phenol and precipitation with ethanol.
    O., 3 pmol L49 bp, fragment p31 / containing the YAS1-YAS2 (PH05) promoter element and 0.15 pmol each of 7.2 kb. fragments are ligated and transformed into E. coli HB101 cells. Plasmids are obtained from amp co50
    55
    After transformation into E. coli HB101 cells, the plasmids are isolated and analyzed by digestion with BamHI, individual plasmids with 1, 2, 3, 4 or 5 EcoRI fragments are selected and amino acid sequence determination is performed. lot-residues (according to the Sanger method), It was shown that the cultpletnye 31 p, o. elements are cloned in head-to-tail orientation.
    lony and analyze restrictive splitting. Single colonies are selected and their plasmid DNA is designated pJDB207 / PAPEI-HIR (YASl + YAS2) and pJDB207 / PAPEI-HIR (YASl + YAS2).
    Example 5. 31 p. The DNA sequence is sufficient to perform the functions of the phosphate control element.
    31 b.p. the sequence from the upper region of the PH05 promoter (position from -381 to -351), limited by two side deletions D1 0 and / M 3 (example 1e), can potentially contain a regulatory signal. This assumption can be verified by synthesizing two complementary oligonucleotides having the following structures: 5 HAATTCCAAATATATATAAATATASS
    CGTTTTCGCAG-3 and 3 -GCTTATATATA ATTT AATCGTGCAAAA GCGTCTTAA-S.
    This sequence contains
    five
    /
    31 p. O, sequence% to which
    5 perO prtashayut Esok restrictive
    plots. Such EcoRI regions allow the sequence to be polymerized easily to form
    multimers.
    a) Cloning 31 p. element in vector LT98. Each of the two synthetic oligonucle otids, taken in an amount of 50 pmol, was treated with 20 units. PM polynucleotide kinases in 20 ml of a mixture consisting of 60 mM Tris, pH 7.5, 10 mK MgClfc, 5 mM DTT, 0.5 mM ATP, After 45 min of incubation at 37 ° C, the two reaction mixtures are heated, 10 minutes at 75 ° C and cooled to room temperature. Annealed oligonucleotides are stored at -200 ° C. Kinated and annealed oligonucleotides in the number
    After transformation into E. coli HB101 cells, the plasmids are isolated and analyzed by digestion with BamHI, individual plasmids with 1, 2, 3, 4 or 5 EcoRI fragments are selected and amino acid sequence determination is performed. lot-residues (according to the Sanger method), It was shown that the cultpletnye 31 p, o. elements are cloned in head-to-tail orientation.
    1716
    b) Cloning in pJDB207. Oligomers 3 b.p. they are tested for their promoter-controlling function by embedding them upwards from the F element of the GAPD promoter. Plasmid pJDB207 / PAPEI-EGI (YASl) is shortened to obtain a plasmid with the crossed out element YAS1. Such a plasmid was digested with Sail and Bglll, purified on a gel and a large vector fragment was isolated. In an independent reaction mixture, the same plasmid was digested with BamHI. Recessive S1 terminations are filled with Klein's DNA polymerase, using all four NTFs for this purpose. The blunt-ended sites are expanded with phosphorplated BglII binder and, after cleavage with Sail and Bglll, a DNA fragment having an approximate length of 400 bp is harvested on a gel. A large vector fragment is ligated with a Sall-Bglll fragment approximately 400 bp long. using TCs DNA ligase. After transformation of E. coli HB101 and isolation of the plasmid, a plasmid not containing PH05 YAS is obtained. This plasmid is designated as pJDB207 / GAPFI-EGI. This plamid was digested with Bglll and serves as a vector for cloning 31 bp of oligomers. 1,198, containing 1, 2, 3, 4, or 5 oligonucleotide inserts, are digested with BamHI. Fragments of different sizes are isolated by gel purification and are independently ligated with pJDB207 / GAPFI-EGi; cut endonuclease Bglll. The mixture was cleaved with Bglll to remove the undesired re-ligated vector without the DNA insert and then used to transform E. coli HB101. The resulting plasmids were analyzed by restriction analysis using Sail and Dral (region within the GAPDH promoter part).
    Example 6 Expression of Polypeptides
    The CPF18 strain of Saccha-romyces cerevisiae is transformed with the plasmids obtained.
    S. cerevisiae CPF18 transformants were grown in 10 ml of Difco medium without amino acids, in which 2% glucose and 20 mg / l L-histidine were added, in a 50 mm Erlenmeyer flask, with shaking at 5 0 about 5
    0
    five
    24 hours at 30 ° C to a density of 3x10 cells / ml. These cells are washed with 0.9% Nad solution and used to inoculate 50 ml of minimal medium with low P:. Obtained according to the recipe for Difco Jeast Nitrogen Base medium (without amino acids, but containing 0.03 g / l, I g / l of KCl and 10 g / l of L-asparagine instead of (2% glucose and 1 g / l of L-histidine). Cultures are inoculated until a density of about 4x10 cells / ml is reached and stirred at 200 rpm for 42 h at 30 ° C.
    Yeast secretes desulfato-urein compounds into culture broth. After fermentation for 22 hours, a 10 ml sample is taken from the culture medium and is enriched with proteins by desalting and concentration on a column. The column is washed twice with 1.5 ml of a water-acetonitrile (9: 1) system with 0.1% trifluoroacetic acid. Desulfatokirudine compounds are eluted from the column with a water-acetonitrile-0.1% trifluoroacetic acid system (3: 2 v / v,). 2 ml of eltoate is concentrated to a final volume of 400 ml, Desulh-atogirudine is identified by analysis using high-pressure liquid chromatography, a comparison with authentic desulfatohirudine, and using a thrombin inhibition test.
    权利要求:
    Claims (1)
    [1]
    The results obtained are presented in Table 4, Formula of the invention.
    A method for producing desulfatohovirudin, involving the isolation and purification of the target product, characterized in that, in order to increase the yield of the target product and simplify the method, a recombinant plasmid DNA containing the promoter PH05-GAPDH consisting of the Clal-Sall PH05 promoter is constructed - a 545 Lp p31 / Y plasmid fragment linked by the Bglll linker to the Bglll-EcoRI GAPDH promoter fragment of the pGADHs plasmid-size 266 Gbp; The EcoRI-HindIII fragment of the plasmid pJDB207 / PH05 (F, co) -HIR 643 Lp in size, encoding desulfatogiru21
    1630616
    ; 22
    Continuation of table 3
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    同族专利:
    公开号 | 公开日
    AT62510T|1991-04-15|
    EP0213593B1|1991-04-10|
    NO863458D0|1986-08-28|
    DD254212A5|1988-02-17|
    DK175093B1|2004-05-24|
    GR862209B|1986-12-31|
    ES2006383A6|1989-04-16|
    NO175871C|1994-12-21|
    GB8521496D0|1985-10-02|
    IL79837D0|1986-11-30|
    IE58844B1|1993-11-17|
    NO175871B|1994-09-12|
    IE862307L|1987-02-28|
    NO863458L|1987-03-02|
    PH24715A|1990-10-01|
    JPH07110233B2|1995-11-29|
    IL79837A|1992-06-21|
    AU6203286A|1987-03-05|
    EP0213593A1|1987-03-11|
    PT83273B|1989-05-12|
    ES2006382A6|1989-04-16|
    HUT42525A|1987-07-28|
    JPS6251995A|1987-03-06|
    CA1318616C|1993-06-01|
    US5436136A|1995-07-25|
    FI91280B|1994-02-28|
    ES2001618A6|1988-06-01|
    FI91280C|1994-06-10|
    DK409686D0|1986-08-28|
    FI863430A0|1986-08-25|
    HU211207B|1995-11-28|
    ZA866535B|1987-04-29|
    DK409686A|1987-03-01|
    PT83273A|1986-09-01|
    FI863430A|1987-03-01|
    AU599329B2|1990-07-19|
    DD267739A5|1989-05-10|
    DE3678647D1|1991-05-16|
    NZ217388A|1989-02-24|
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