Method of producing recombinant plasmide dna encoding synthesis of bovine or human growth hormone

专利摘要:
The present invention provides a process for preparing selectable and autonomously replicating recombinant DNA expression vectors which comprise 1) a transcriptional and translational activating sequence which is in the reading frame of a nucleotide sequence that codes for a peptide or polypeptide; 2) a translational stop signal; 3) a translational start signal which is in the reading frame of a nucleotide sequence that codes for a functional polypeptide; and 4) an additional translational stop signal. The above-described nucleotide sequences are engineered to generate, upon transcription, a translatable polycistronic mRNA. The invention further provides a method of use.
公开号:SU1491346A3
申请号:SU853862498
申请日:1985-03-04
公开日:1989-06-30
发明作者:Джордж Шонер Рональд；Элизабет Шонер Бригитте
申请人:Эли Лилли Энд Компани (Фирма)；
IPC主号:

专利说明:

The invention relates to biotechnology and genetic engineering, and relates to a method for constructing recombinant plasmid DNA encoding the synthesis of bovine or human growth hormone.
The aim of the invention is to increase the yield of methionine growth hormone
The plasmid is constructed by combining the BamHI-Xbal or BamHl-EcoRI fragment of the pCZIOl plamid with the Xbal-BamHI fragment of the pIII78 plasmid with the BamHI-FnuII fragment of the plasmid pNM575. In addition to the obtained recombinant plasmid DNA, an EcoRI-Clal fragment of plasmid pNM608 with a synthetic linker is added.
The method is illustrated by the following.
examples
Example b As the starting material, a “5.1Kb fragment obtained by XLaT-VagN splitting of the plasmind pKEN021 is used.
Plasmid pKEN021 is obtained from pKENIII as follows: about 50 μg pKENIII burn 25 units. Hpall restriction enzyme in 300 μl of buffer containing 20 mM HCl (pH 7.4), 10 mM MgCl "and 6 mM p-mercaptozanol, at 37 ° C for 2 h. The resulting mixture was extracted twice with 300 μl of a mixture (50: 50) phenol and chloroform. The separated aqueous phase is then precipitated with 2.5 volumes of ethanol and volumes of ZM sodium acetate, the DNA is dissolved in 100 µl of electrophore
QD
WITH

about:
 cm
carved buffer and fractionated on a 5% polyacrylamide gel (acryl amide: bis ratio is 29: 1 in all gels except those that are specifically indicated). The gel is stained in a solution containing 0.5 µg / ml of ethidium bromide and exhibits bands with long-wave ultraviolet radiation. The 950Fr band is isolated and removed from α-nc by elution in a dialysis chamber. Extraction is then carried out with a phenol / CHCI3 mixture and precipitated with ethanol. The isolated DNA (approximately 2.5 µg) is dissolved in 25 µl of TEN (10 NaCl, 10 mM iris HCl, pH 7.4, and 1 1 sodium ethylenedinitrile tetrachloride CHEDTA, pH 8.0).
Approximately 2 μg of 950Lp Hpall fragment is burned with Alul restriction enzyme in 200 μl of buffer containing 50 KF1 NaCl, 6 mM Tris. HCl (pH 7.6), 6 mM MgClij and 6 mM p-mercaptoethanol, for 2 hours at 37 C. The DNA fractions are ionized on a 6% polyacrylamide gel. The resulting 462Lb Alul fragment was isolated and purified by the method described earlier. The Alul fragment (approximately 1 µg) was dissolved in 10 µl of T4 DNA ligase buffer (66 1 Tris HCl, pH 7.6; lOMMMgCl, 10 i dithiothrethiol, 0.4 mM ATP) containing 150 pmol of EcoRI-phosphorylated linker (5 GGAATTCC3) and 2 units, T4 DNA ligase. After incubation at 4 ° C for 16 h, the resulting mixture is heated at 65 ° C for 10 minutes and diluted to 1000 μl by adding EcoRI buffer (100 mM Tris HCl, pH 7.2; 50 mM NaCI, 10 mM, 6 mM p-mercapto-ethanol) and 40 units of EcoRI enzyme. After 2 hours, the sample was extracted with phenol / SSCC and precipitated with ethanol, DNA was dissolved in 20 µl of T4 DNA ligase buffer containing 0.1 units of T4 DNA ligase and 0.1 µg of PBR322 / 102, which was first linearized with EcoRI, and then treated with alkaline phosphatase. After ligation at 4 ° C for 16 v, the obtained DNA is used for the usual transformation of E. coli strain K12 HVAc.
Transformants are selected on agar plates containing 12 μg / ml of tetracycline, plasmids are isolated from resistant colonies by the rapid method
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Plasmid containing the 466Lr Xbal-BatnHI fragment is selected and used as a starting material in the following step about
About 2 μg of this plasmid, 2 units of Hindlll enzyme are burned out in 50 μl of Hindlll buffer (50 mM NaCl, 10 mM Tris HCl, pH 7.4; 10 mM MgCl and 6 mM p-mercapto ethanol) for one hour at 37 ° С, After extraction with phenol / CHC1 and ethanol precipitation of DNA is dissolved in 200 µl of buffer containing 300 mM NaCl, 30 mM sodium acetate (pH 4.25), 1 mM ZnClg and 200 units, I1 nuclei, After one hour at 15 C, the reaction is stopped by extraction with a mixture of feiol / CHCI3 and precipitation with ethanol, the obtained DNA is dissolved in 10 µl of T4 DNA of the ligase buffer containing 20 pmol of BamHI phosphorylated linkers (5 CCGGATCCGG3) and 2 units, T4 DNA l igazy. After 16 hours at 4 ° C., the reaction mixture was heated at 65 ° C. for 10 minutes to inactivate the ligase, and then diluted to 100 μl in BamHI buffer (150 mM NaCl, 20 mM Tris HCl, pH 8.0; 10 i MgCl (j 6 bM-mercaptoethanol) containing 20 units of WatNT enzyme After 2 hours at 37 ° C, the resulting mixture is purified on a 1% agarose gel. The gel is stained, a larger fragment (4.5 kb) is eluted after freezing, and then purified , extract with phenol / CHCl3 and precipitate with ethanol. The isolated fragment with BamHI sticky ends is dissolved in 20 µl of Q T4 DNA ligase buffer containing 0.1 units, T4 D To ligase. After 16 h with DNA used for transforming E, coli HBlO-lo Transformants are selected for ampicillin resistance — at 100 μg / ml and screened for sensitivity to 10 μg / ml of tetracyclino. Some plasmids that are Ap Tc, sequential processing yields a fragment of 466 and 305 bp, a plasmid with such characteristics is selected, and then modified to convert the EcoRI site of the 1pp promoter into the Hindlll restriction site.
2 μg of plasmid is hydrolyzed in 100 μl of EcoRI buffer with 0.2 units, EcoRI
for 10 min at 37 ° C, the reaction is stopped, natura for 10 min at h5 ° C, and after extraction with a phenol / CHCl1 mixture, DNA is precipitated with ethanol, dissolved in 200 µl of S1 nuclease buffer containing S1 nuclease at 1000 units / ml, and conduct the reaction at within one hour. PeaKUHXJ is stopped by extraction with phenol / CHCl1 and precipitated with ethanol o The resulting DNA is again suspended in 10 μl of T4 DNA ligase buffer containing 20 pmol of a phosphorylated Hindlll linker (5 CCAAGCTTGG 3) and 2 units. T4 DNA ligated After 16 hours with the mixture heated for 10 minutes at 65 ° C, diluted to 150 µl Hindlll with buffer containing 10 units, Hindlll enzyme, incubated for 2 hours at 37 ° C, and then fractionated into 1% - but agarose gel. The largest band (equivalent to a single piece of plasmid) is isolated and purified in the usual way, dissolved in 20 µl of T4 ligase buffer containing 0.2 units. T4 ligases are incubated for 16 hours at, and then used to transform E. coli HB101. Transformants are selected for ampicillin resistance, the isolated plasmids are analyzed in the usual manner by restriction enzyme analysis. A plasmid with an EcoRI-Hindlll ZOOR fragment is used as a cloning vector to add 3 sections of the 1pl gene.
About 2 μl of plasmid is burned out in 50 μl of Sail restriction buffer (150 mM NaCl, 6 mM Tris HC1, pH 7.9; 6 t MgClj, 6 i p-mercapto ethanol) with 2 units. Sail for one hour at 37 ° C and then diluted to 150 µl in BamHI buffer containing 2 units. BamHIo One hour later, 2.5 units of alkaline phosphatase were added, and then incubation was continued for one hour at 65 ° C. The material was extracted with a phenol / CHC1 mixture, precipitated with ethanol, a solution of sweat in TEN, and used as a cloning vector for fragment 1pp 3.
To obtain a fragment containing 1 pp 3 plot, 10 µg of pKENIII is burned out in 100 µl of Hpal buffer (20 mM KCli 10 mM Iris HCl, pH 7.4; 10 tM MgCl and 6 mM pz-mercaptoethanol) with 10 units. Hpal for 2 hours at
. Poslg; ext.11ChFS1VLIMP smgk phenol / CF.Cl t, and precipitated with tiolol DNA was dissolved in 10 µl of T4 DNA of ligase buffer, containing 20 pm1 phosphorylation of 1 Sail linker (5 GGTCGACC 3) and 2 units, 14 DNA ligases and then incubated for 16 hours at, inactivating the ligase, heating at 65 ° C for 10 minutes. The material obtained is diluted to 100 µl in Sail buffer containing 10 units. Sail, incubate for one hour at and then dilute.
up to 300 μl in PVUII buffer (60 mM NaGl, 6 mM Tris.HCl, pH 7.5; 6 mM MgClj ,, 6 mM b-mercaptoethanol), contains 10 units. PVUII restriction enzyme. After an hour
DNA was fractionated on a 5% polyacrylamide gel. Approximately 0.5 µg of 950bp fragment was isolated, purified and dissolved in TEN. 0.02 µg of the fragment is diluted in 20 µl of T4 DNA
ligase buffer containing 20 pmol of the phosphorylated BamHI linker (5 CCGGATCCGG3) and 2 units. THAT DNA ligase, and then incubated for 16 h at. The resulting DNA is heated for 10 minutes with, diluted to 100 µm with BamHI buffer, containing 20 units, BamHI, incubated for 2 hours at 37 ° C, and then fractionated on a 5% polyacrylamide gel to remove excess linker molecules. The obtained 950Lb fragment containing BamHI and Sail sticky ends is cleaned in the usual way and dissolved in 20 µg TA DNA of ligase buffer,
containing 0.2 μg of the cloning vector described previously, and 0.2 units T4 DNA ligated After incubation for 16 h at 4 ° C, the DNA is used to transform E. coli K12 HB101. Plasmids are obtained from ampicillin-resistant transformers and are routinely analyzed for the presence of a Sall-BamHI fragment. The target plasmid (-5,2kb) denote pKEN021o
10 μg of PKEN021 is burned out with 200 KCl of Xbal / BamHI buffer (150 NaCl, 10 mM Tris HCl, pH 8; 10 mM MgClgf 6 mM-mercaptoethanol) using 10 units. BamHI for an hour, and then 10 units, Xbal for another
hours at The target Xbal-BamHI scorched DNA is then treated with 2.5 units, alkaline phosphatase for 1.5 hours at 65 ° C, extracted
7
a mixture of phenol / sleep 1 in, precipitated with ethanol and a solution of 1T in 50 µl TEN for further use.
The plasmid ptrpED50chGH800 is used as a DNA source for a fermet that contains the coding sequence for a portion of the human growth hormone gene. The gene part of the human growth hormone ptrpED.SOchGHSOO plasmid contains a unique, Smal restriction site per bvr after the translational termination codon of the gene o This site is replaced with the BamHI site using the following method: 6 ug of plasmid is burned out 6 units. Sinal in 200 µl Smal restriction buffer (15 mM Tris-HCl, pH 8.0; 6 mM MgClii, .15 mM KC1 and 6 m P-mercaptoethanol) for 1.5 h at. After the burning is completed, extraction is carried out with a mixture of feola / CHCl3, DNA is isolated, precipitated with ethanol, and then dissolved in 24 m.


TEN, 40 "pmol of the BamHI phosphorylated adapter fragment is added to 0.5 µg (0.2 pmol, ends) of the plasmid burned using the method described above in 16 µl of l1ase buffer containing 2 units, T4 DNA ligase. The mixture is incubated for 2 h. at, 16 h - at, and then 10 minutes at 65 ° C. BamHI sticky end is obtained by conventional burning of BamH with a restriction enzyme. The enzyme cleaves the linker sequence in the same way. 1Sac BamHI site located at the beginning of the cloned human growth hormone cDNA sequence. This gives a 691Lp fragment with sticky BamHI ends, which are first isolated at 6% poly acrylamide gel, and then - in the usual way. The isolated DNA fragment is ligated (using 0.2 units of T4 DNA ligase in 20 µl of buffer under previously described conditions) with 0.2 µg of BamHI,
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scorched and alkaline phosphatase-treated pBR322. After 16 hours at 4 ° C, the resulting material was used to transform E. coli strain JA221 / NRRZD-15014). Transformants are selected on agar plates containing 100 µg / ml of ampicillin, then plasmids are isolated in the usual way and identified with a restriction enzyme and analyzed by gel electrophoresis. The target plasmids, designated .pNM575, contain the BamHI fragment (approximately 700 mg) and are propagated in the usual way for further use.
The DNA sequence of the mature human growth hormone contains one FnuDII site, which is 47F of the first nucleotide. 25 μg of pNM575 are burned out in 250 μl of BamHI buffer with 25 units of BamHI prn for an hour 691Lp fragment with BamHI sticky end is isolated from the 6% full acrylamide gel in the usual way and purified. After purification of the fragment, one third of it (equivalent to 8 μl of plasmid) is burned out in 100 μl of FnuDII buffer (6 NaCl, 6 mM Tris HCl, pH 7.4; 6 tiH MgCla, 6 mM p-mercaptoethanol) with 2.5 units of FnuDII in for 1.5 hours at 37 s. Electrophoresis on a 6% polyacrylamide gel and a standard isolation procedure make it possible to isolate a 538 bp DNA fragment containing the coding sequence for the last 175 amino acids of the gene followed by a translational stop signal.
A double-stranded DNA fragment is synthesized by the phosphothysphoric method to attach the 1pp promoter region to the coding region of human growth hormone.
5 СТА & АССВТАТТААТААТСТТСССАТТССАТ & АТОАСАТАААТТСССАА, rfffflll ll fflllT "flMlt ltflfI llftll lit
3 TSSSATAATATTATASASSSTAASSTASTASTASTATTSAAO & VTT-СSATTSSSTTATSSASSSSGGTTTSASAASOSTATSTSSO Z YSHOP
I I 1 1 I I I I I 1 I I f I t t f I I 11 I f And t I CH1 111J., El
CCTAACCCAATAGGTCC & AAAAACTGTTGC & ATAC & AG & C 5
Get the following segmenta:
f) CTAGAGGGTAT
2) TAATAATGTTCC
3) CATTGGATGAT
4) GAT GAT A A GTT С С
5) CAACCATTCCC
6) TTATCCAGGC
7) TTTTTGACAACG fi) CTATGCTCCG
9) SATTATTAATASSGT
fO) ATGGGAA
O) CTTATCATCATCCA
f2) GGTTGGGAA
f3) GGATAAGGGAAT
14) GTCAAAAAGCCT
/ 5) CGGAGCATAGCGTT
Using the segments obtained as described above, the addition reaction, catalyzed by TA ligase, is carried out in stages:
a) 5-unphosphorylated segment 1 is attached to 5-phosphorylated segment 2 in the presence of 5-phosphorus segment 9, using TA ligase to obtain duplex 1 DNA. Duplex is isolated by preparative gel electrophoresis on 15% polyacrylamide;
b) a 5-phosphorylated segment 3 is attached to a 5-phosphorylated segment A in the presence of a 5-phosphorylated segment 11 using TA ligase to obtain duplex 2 DNA, which is purified on a 15% polyacrylamide gel by electrophoresis;
c) a 5-phosphorylated segment is attached to a 5-phosphonated segment 6 in the presence of 5-phosphorylated segments 12 and 13 using a TA ligase to obtain duplex 3 DNA, which is purified by electrophoresis on a 15% polyacrylamide gel;
d) A 5-phosphorylated segment is attached to a 5-phosphorylated segment 8 in the presence of a 5-phosphorylated segment 1A and a 5-non-phosphorylated segment 15, using T4 liga-eu for oorlzonine DIC duplex 4, which is electrophoresis. ) (l polylkrplamydnom gel;
e) 2.3 nA duplex DNAs are then joined together by TA ligase to form duplex 5 DNA, which is purified by electrophoresis
0 15% polyacrylamide gel;
f) A 5-phosphorylated segment 10 and duplex 5 DNA are added in the presence of TA ligase to DNA duplex 1. The resulting duplex DNA is purified by electrophoresis on a 15% polyacrylamide gel. Then the duplex enzyme DNA is phosphorylated using TA polynucleotide kinase and (k) ATP according to -.
0 with the established methodology
The expression plasmid PNM702 was constructed by enzymatic addition of 0.1 pmol (O, A μg) of the Xbal-BAmHI fragment of the plasmid pKEN02l,
5 0.025 pmol of the synthetic DNA fragment and 0.3 pmol (0.08 μg) of the 538Lp fragment in 24 μl of the ligation buffer, using 1.5 units. THAT DNA is ligase; After incubation for
16 hours with the resulting mixture used to transform E. coli JA221, as described earlier, Transformants are selected on agar plates containing 100 µg / ml ampicilli
5 on, and cultured as a preferred source of the target expression plasmid in the usual manner.
The expression of human growth hormone is determined by the standard radioimmunoassay method. In determining, it is at a rate of at least 2 ppm molecules per cell.
Plaemid RSh2 expression of human growth hormone is used
5 as a starting material when constructing a plasmid expressing Me t-Phe-Pro-Zeu-Asp-Asp-Asp-Asp-Jys-bGH.
The plasmid rVCSAV is used as the source of two DNA fragments containing the coding sequence for a portion of the bovine growth hormone gene. The plasmid contains a bovine growth hormone 831b - the coding sequence cloned into the PstI PBR322 regression site.
10 µg of pNM702 partially burn 1 unit. PVU1I in 200 μl of PVUII restriction buffer (60 mM NaCl, 6 mM Tris HC1, pH 7.5; 6 mM MgClj, 6 mM p-mercaptoethanol) for 10 min at. After termination of the reaction by heating for 10 minutes with DNA, the material is treated with alkaline phosphatase and the fragments are isolated on a 1% agaroen gel. The linear DNA fragment of the size corresponding to the DNA without the A97Bp PVUlI fragment (goes a little earlier than the plasmid with one break) is isolated in the usual way, purified and used in the construction of the intermediate plasmid.
A fragment of the 494Lp PVUII plasmid pBR3A8 is obtained by burning 10 μg of plezmid in 200 μl of pVTJII buffer containing 10 units. pVUII, for an hour at. These fragments are scaled on a 6% polyacrylamide gel, the target 494Lp is shown and purified in the usual way.
The intermediate plasmid was constructed by reacting a 0.2 μg plasmid of the pNM702 PVUII fragment with 0.05 μg 494Fp fragment in 20 μl of T4 DNA ligase buffer containing 2 units of T4 DNA ligase for 16 hours at о After transformation and selection of transformants for resistance to ampicillin plasmids are analyzed in the usual way for the presence and appropriate orientation of the 494bp PVUII fragment, Plasmids with the 494bp PVUII fragment and 440bp of the Xbal-Smal fragment are selected for
Xbol
5 STASASSSTATATAATAAT & TTSSSATSTSATSAT & AG & ATAAC-, I I I I I I t g g I g (I I I t t I f g I I I t t I I t I t t t I g I I I f
3TCCCATAATTATTACAAC6CTAACCTACTACTACTATTCTTCCCAGCCAT & TCCTTCTC
M M I I M M I I t I I I I I I I
D A (; СТЕСТС && TASACAAACASSS
Upon receipt of the 63bp fragment, the following 9 segments are prepared:
CTACAGCGTAT
TAATAATGTTCC
CATTCGATCAT
GATGATAAGTTCC
CAGCCATGTCCTTGTC
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use in the further design.
10 µg of the intermediate plasmid are burned out with 1 eU of PVUII in 200 µl of PVUII buffer for 5 minutes at 37 ° C. After heating for 10 minutes at 65 ° C, the resulting mixture is distributed on a 1% agarose gel, and linear DNA is isolated, having only one A single PVUII is dispersed per molecule, and purified material (approximately 3 μg) is completely burned out with 5 eBo Xbal and treated with alkaline phosphatase. These fragments are distributed on a 1% agarose gel, the largest of them (the lost fragment between Xbal and the first PVUII site in human and bovine growth hormone) is isolated in the usual way.
The DNA sequence for the first 23 amino acids (69Bp) of bovine growth hormone up to the first PVUII site contains 2HpaII restriction sites, the first of which is located 23b from the first nucleotide of the coding sequence. The bbr fragment is synthesized by the phosphorometryphrine method. This first fragment corresponds to the 19Bp sequence from the Xbal site at the 1pp ribosomal binding site via the ATC-translational start signal followed by the coding sequence for the Phe-Pro-Zeu-Asp-Asp-Asp-Agp-Zys (24bp) and 20 nucleotides the sequence of bovine growth hormone (from Phe to the first Hpall site) and has the following sequence:
jr Hpaii
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6) ATGGGAACATTATTAATACCCT T) GTATSLTSATSAGSSA
8) ATG (JCTGGUAA (
9) CGG-ACAAGC AC
Using previously prepared segments, carry out a phased connection, catalyzing the process of T4 ligase:
13
a) a 5-unphosphorylated segment I is attached to a 5-phosphorylated segment 2 in the presence of a 5-phosphorylated segment 6 using TA ligase to form duplex 1 DNA, which is purified by electrophoresis on a 15% polyacrylamide gel;
b) 5-phosphorylated segments 3,4 and 5 are combined in the presence of a 5-phosphorylated segment 9 using T4 ligase to obtain duplex DNA, which is purified by electrophoresis on a 15% polyacrylamide gel. Then the duplex DNA is enzymatically phosphorylated, using
T4 polynucleotide kinase and () ATP followed by standard procedure.
The DNA fragment, which extends from the described Hpall site to the PVUII site, can be constructed synthetically or obtained from the starting pBH3 plasmid 48; 100 µl of plasmid pBR348 is burned out in 400 ml of PVUII buffer (50 units of PVUII) for 2 hours. After phenol extraction and precipitation with ethanol, DNA is dissolved in 400 µl of PstI buffer (50 NaCl, bM M Tris HCl, pH 7.4; 6 t MgCli, 6 mM (L-mercaptoethanol) with 50 units of PstI for 2 hours with DNA fragments distributed on a 6% polyacrypamide gel, 135 fragments containing the target 46L sequence, isolated and purified by standard methods. One third of the isolated DNA (equivalent to 33 μg) is subjected to limited burning of 1 unit of Hpall restriction enzyme in 100 μl of Hpall buffer (20 M Tris HCl, pH 7.4; 7 mM Mg ClQj 6 mM L-mercaptoethanol) for 40 min at 37 ° C. After heating at 65 ° C for 10 min, the DNA fragments are placed on a 5% acrnpamide gel (acrylamide: bis ratio 19: 1) with an appropriate size marker. The target 46F fragment, obtained by partial hpall of the 135B fragment, is purified by the standard method.
0.2 μg of the Xbal-PVUIl fragment of the plasmid vector, 3.2 pmol of the synthetic bzb fragment and 0.5 pmol of the 46bp fragment are incubated in 10 μl of a ligating yufer with 2 units of T4 DNA ligase for 16 hours. Leagues




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I 4
This mixture is used to transform E. coli JA221. The resulting transformants, which contain the target plasmid pSh789, are selected for ampicillin resistance. The identity with the plasmid pNM789 was confirmed by conventional screening for the presence of 494 PNR and 109Lp Xbal-PVUII fragments.
Plasmma pŠ789 requires a single amino acid codon to be altered for complete conversion to bovine growth hormone, which is accomplished by removing the 28Bp PVUII to the BamHI fragment of pŠ789 and replacing it with a synthetic double-strand fragment having the following after-. conviction:
5 CTGTGCCTTCTAC 3
3 GACACGGAAGATCCTAG 5
10 μg of pNM789 are burned out with 1 unit, PVU1I in 200 μl of PVUII buffer for 5 minutes at 37 ° C. After heating for 10 minutes at 65 ° C, the mixture was diluted to 300 µl with the addition of BamHI buffer, burned to a finalization of 10 units. BamHI for 5 hours with 5 units. alkaline phosphatase and incubated for 65 h at 65 ° C. DNA fragments are separated on a 1% agarose gel; A DNA fragment of the size of a single-gap plasmid pNM789 is purified in the usual way. 0.02 μg of this fragment are ligated with 5 pmol of the synthetic fragment, using .2 udo T4 ligase in 20 μl of ligase buffer. Ligation was performed overnight at. After transformation, several plasmids are selected and screened for the corresponding PVUII (494bp) and Xbal-Bamll (628bp) fragments. Plasmids containing the above fragments constitute the target plasmid pNM789B.
Example 2. The E. coli K12 / pIM-l -A3 / NRRI.B-15733 bacterium is cultured in TV broth (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride; pH 7.4) with 30 μg / ml kanamycin at 25 ° C in accordance with the usual microbiological procedure. After that, the culture is diluted 1:10 in fresh broth, after three hours of incubation at 5 nl of culture, transferred to a 1.5 ml Eppendorf vial and centrifuged for s. Unless otherwise indicated, all manipulations are carried out at room temperature. The resulting over-.
sedimentary fluid is carefully removed with a sharp tip aspirator. The cell mass is resuspended in “MOO μl of freshly prepared lysozyme solution (2 μg / ml), which contains 2 μg / ml of lysozyme, 50 M glucose, 10 mM ETDA (diaminetetraacetate) and 25 1 Tris-HCl, pH 8. -200 µl of alkaline SDS (sodium dodecyl sulfate) solution (0.2 NaOH, 1% SDS) was added and the vial was gently inverted, and then kept at 0 ° C until the lysis was complete (approximately 5 minutes). Then, μl of 3 M sodium acetate is added and the contents of the ampoule are gently mixed, turning it over for a few seconds.
The ampule was kept at 0 ° C for at least 60 minutes and then centrifuged for 15 minutes to obtain an almost clear supernatant. The supernatant was transferred to a second ampoule of centrifuge, to which 3 volumes of cold 100% were added. ethanol. After keeping the ampoule on the dry ice-ethanol mixture for 5 minutes, the resulting precipitate was collected by centrifugation (5 minutes), the supernatant liquid was removed by suction. The collected mass was dissolved in 100 µl of TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA Obtain the target DNA of the pHS-X -A3 plasmid.
About 5 μl of plasmid pNM789B-DNA in 50 μl of HiSalt buffer is incubated with 10 units. each (BamHI and Xbal) recurrent enzyme at 37 ° C for 1 hour. After 5 µl of ZM sodium acetate (pH 8.0) was added, DNA was precipitated with 3 volumes of 100% ethanol. The target DNA digest is dissolved in 100 µl of TE buffer and stored at 0 ° C for further use.
5 STASA & 6 & TATTAATA ATC, GA6 CAT CAT TAA ATC TTC CCA CCC. m g and I I I I I I f f t I I III III trr HI (II III t ", fIT -
TCCCATAATTAT TAG AAC. GTC CTA CTA
Sh
TAG AAC- OGTi
cek
H
TCC CCC STS TTT CGC AAC CGT CTCCT III III I I I III III III III I ACC CCC CAG AAA CCC TTC CCA G
The target linker sequence is synthesized by the conventional modified phosphoether ester method.
About 200 pmol of the DNA linker of example 3, 1 μg of the plasmid pCZlOl
About 1 μg of the v-plasmid pIM-l -A3, 1 μg of the plasmid pMM789B Xbal-RamHI, AO μl of water, 5 μl (5 mM) ATP, 5 μl of the ligation mixture and 5 units. T4 DNA ligases are incubated at 20 ° C for an hour. After incubation at 65 ° C for 2 minutes, followed by cloning on ice, the resulting ligation mixture is used for dp transformation K12RV308 on TV plates (1% triptoton, 0.5% core extract, 0.5% sodium chloride, 1.5% agar, pH 7.4), containing 50 µg / ml canamycin.
Some of the resulting transformants (which can easily be shown by conventional agarose gel electrophoresis and other tests) contain only the target plasmid 10.8kb. The resulting cells are used to isolate the plasmid pCZlOl,
Example 3. The target fragment is constructed essentially according to the method of Example 2, except that the plasmid pCZlOl is used instead of the plasmid pNM789B. The target 10.2kb BamHI-Xbal restriction fragment is isolated and isolated by agarosy gel electrophoresis, then dissolved in 100 µl of TE buffer and stored for further use in dp.
The target fragment was constructed according to the method of Example 2, except that, instead of the plasmid rsH789B and Xbal restriction enzyme, the plasmid pCZlOl and HgiAI restriction enzyme were used. The target, 6kb BamHI-HgiAI restriction fragments are isolated in the usual manner by agarose gel electrophoresis, then dissolved in µl of TE buffer and stored for Further use.
ATS TTC CCA CCC III t ", fIT -
TAG AAC- OGTi
cek
H
3 5
55, 2kb BamHI-Xbal fragment n 0.5 µg of plasmid pCZlOl, 6kb BamHI-HgiAI fragment ligned, the resulting plasmid is used dp
171.
transforming E. coli KI2 RV308 according to the method of Example 2.
Some of the obtained transformants (which is easily shown by agarose gel electrophoresis and other tests) contain only the target plasmid, l-10.8kb. Such a transformant, designated E.coli K12RV308 / pCZnA, is selected, placed on TV agar containing appropriate antibiotics, and then cultured according to conventional microbiological techniques. As shown by SDS gelTACAGCCTATTAATA
I t I F I f I 1 t t 1 g
TSSSATAATTAT
& SS PBX Iff f I I CC & Tag
TCC TTG I 11 f f I AC & AAC
PBX f f I TAG
TCC f r r AG &
The target transformants, designated E. coli K12-RV308 / pCZ101,1, are placed on TV ara p, containing the appropriate antibiotics, and then cultured in the usual way. Plasmid pCZlOl was isolated. As shown by SDS gel electrophoresis, RLA, and other tests, transformants express met-bGH with high levels. Since the plasmid pCZlOl contains a thermally induced accelerated growth replicon, maximum expression of met-bGH occurs when cultured at temperature.
Example 5. The pGMI plasmid contains E. coli tryptophan operon containing the exception AZE1413. Consequently, it expresses a cast protein containing the first 6 amino acids of the trp leader and approximately the last third of the trpE polypeptide (hereinafter referred to for brevity as ZE), as well as the entire trpD polypeptide (all under the control of the trp promoter / operator system).
About 20 µg of the plasmid is burned with the restriction enzyme PNIIII, which splits the plasmid into 5 sites. The gene fragments are combined with EcoRI linkers consisting of self-complementary ligonucleotides of the sequence pCATGAATTCATG, providing an EcotlT cleavage site for subsequent cloning into a plasmid containing the EcoRI site.


13) 13
electrophoresis., KIA and drupgh tsslishsh, the resulting cells exprgsg.iropali met-bCH in bol yum number. Since plasmapd p (; 7.114 contains a thermoconductive replicon of accelerated growth, maximum expression of met-HTH is observed at a culture growing temperature of about 37 C.
dExample A. Purposeful construction was carried out in accordance with the procedure of Example 3 with the difference that instead of the linker sequence of the example, the sequence
CCA TTO CAT CAT CAT TAA PBX TTC ..., (t r t f f t f I tit f t r. I I f GGT AAC CTA CTA CTA ATT TAG AA &
CTC TTT CCC AAC CCT CTCCT 3
I I r I t f t I f f f 1 r r f I f f t.
GAC AAA CCC TTC CCA C 5
20 μg of DNA fragments obtained from pGMI are treated with 10 units, T4 DNA ligase in the presence of 200 pmol 5 -phosphorylated synthetic ligonucleotide pCATGAATTCATC and 20 μg T4 DNA ligase buffer
(20 mM Tris, pH 7.6; 0.5 mM ATP,
10 mM MgClI, 5 mM dithiothretiol) overnight. The solution is then heated for 10 min while stopping ligation. Linkers
they were digested with EcoRI by burning, fragments (now with EcoRI ends) were isolated using 5% polyacrylamide gel electrophoresis. The three largest fragments were extracted from
gel, first staining with etidocumbromide, then determining the position of the fragments under the action of ultraviolet radiation and expressing hegel segments that are of interest.
Each of the gel fragments with 300 μl of 0.1 x TBE was placed in a dialysis chamber and subjected to electrophoresis at 100 F. For one hour in 0.1 x HTE buffer (TBE buffer contains
10.8 g Tris base, 5.5 g boric acid, 0.09 g NajEATA in I l). The aqueous solution is selected from the chamber for dialysis, extracted with phenol, chloroform and adjusted to 0.2 M
related to sodium chloride. The DNA is then isolated in water after precipitation with ethanol. Fragments containing the trp promoter / operator with EcoRI sticky ends are identified by incorporating into a tetracycline-sensitive plasmid, which, after switching on, the promoter / operator becomes resistant to tetracycline.
All DNA isolations of the fragment described hereinafter, in this example, are performed using PAGE followed by electroelution as described above.
The pBRHI plasmid expresses ampicillin resistance and contains the tetracycline resistance gene, but since it does not have an associated promoter, it does not express this resistance. The cells in which the plasmid is found, respectively, are susceptible to tetracycline. If you enter the promoter / operator system in the EcoRI site, the plasmid will express tetracycline resistance.
Plaemid pBRHI digested with EcoRI. The enzyme is removed, extracting with phenol, and then chloroform. The DNA is resuspended in water after precipitation with ethanol. The obtained DNA in separate reaction mixtures was combined with each of the three DNA fragments obtained in Example 5, and ligated with the TA DNA ligase, as described previously. The DNA present in the reaction mixture is used in dp transformation of competent E. coli K12294 / NPPZB-15625 / using a standard procedure, then the bacteria are placed on ZB plates containing 20 μg / ml ampicillin and 5. μg / ml tetracycline.
Several tetracycline-resistant colonies are selected, plasmid DNA is isolated and designated pBRHtrp. The presence of the target fragment is confirmed by restriction enzyme analysis. Plasmid pBRHtrp expressing fo-lac tamazu, confers resistance to ampicillin and contains a DNA fragment that includes the trp promoter / operator. The DNA fragment also encodes the first protein (designated ZE), containing the stitching of the first six amino acids of the trp leader and approximately the last third of the trpE polypeptide; a second protein (designated D), corresponding to approximately the first half of the trpD polypeptide, and a third protein that encodes a tetracycline resistance gene.
Plasmid pBRHt-.rp is burned with EcoRI
restriction enzyme. The resulting fragment, isolated by PAGE and electroelution, was combined with EcoRI, burned with plasmid pSOMII. The resulting mixture is pulsed with T4 DNA.
ligase, the obtained DNA is transformed into E. coli K12 294, as described previously. Transformant bacteria are selected on plates containing ampicillin. The resulting colonies
ampicillin resistant are screened by hybridization. The fragment containing the trp promoter / operator isolated from pBRHtrp, and then labeled radioactive P, use
in the above procedure as a probe. Some positive hybridization colonies are selected. Plasmid DNA was isolated. The orientation of the inserted fragments is determined by
restriction analysis using
Bglll and BamHI enzymes in double burning Colonies containing the target plasmid with a trp promoter / operator fragment in the appropriate orientation are grown on ZB medium containing 10 μg / ml ampicillin. The target plasmid is designated pSOM7A2 and is used for the subsequent constructs described below.
Plasmid pSOM7 & 2 is burned with Hindlll, and then lambda exonuclease (5 to 3 exonuclease) under conditions chosen so that burning occurs behind the Bglll restriction site
inside the ZE coding region. About 20 µg of Hindlll scorched pSOM742 is dissolved in buffer (20 mM glycine buffer, pH 9.6; 1 mM MgCli, 1 mM p-mercaptoethanol). Received
the mixture is treated with 5 units. lambda exonucleases for 60 min at room temperature. The reaction mixture obtained in this way was extracted with phenol, chloroform and precipitated with ethanol,
In order to obtain the EcoRI residue at the distal end of the ZE gene enzyme, primer pCSTSTSSATSAT is synthesized by an improved phosphotriester method and hybridized with the single-strand ZE end of the gene fragment obtained by burning with lambda exogenase. Hydrogenation is carried out, a solution of 20 µg of treated lMb-
yes ecogenesis dt solution
Hybridization of a 20 μg linda-exogenase-treated Hindlll product of the plasmid pSOM742 in 20 μl of HjiO and combined with f μl of a solution containing 80 pmol of the 5-phosphorylated oligonucleotide described previously About the synthetic fragment is hybridized with the ZE end of the coding sequence, a the rest of the single stranded portion of the ZE fragment is filled with Klenow polynerase 1 using dATP, dTTP, dCTP and dCTP. Klenowa I polymerase is a fragment obtained by proteolytic cleavage of DNA polymerase I. It contains 5-3 polymerizing activity, 3 - 5 exonucleotic activity, but not 5 -3 exo nucleotic activity of the original enzyme.
The reaction mixture is then heated to and allowed to cool slowly, after which 4 µl of Klenow enzyme is added. After incubation for 15 minutes at room temperature, then for 30 minutes, the reaction is stopped by adding 5 μl of 0.25 M EDTA. The reaction mixture is extracted with phenol, chloroform and precipitated with ethanol. DICs are sequentially digested with Bglll restriction enzyme. The resulting fragments are separated by PAGE. The autoradiogram obtained for the gel reveals a labeled P fragment of expected length of approximately 470F, which is isolated by electroelution. The ZE / d fragment has a Bglll terminus and a blunt end encoding the beginning of the primer.
The plasmid pTY1 is constructed by inserting the synthetic gene for thymosin-CU 1 into plasmid pBR322. Synthesis of thymosin-0 1 coding DNA includes the synthesis and subsequent ligation of 16 oligonucleotides (T -T, b) raet. ATCs include N-thermal modules; the 5 ends provide a single-stranded adhesive end to allow for the attachment of plasmids of cleaved EcoRI and BamHI. As is easily seen, the Bglll site in the center of the pus helps the analysis of recombinant plasmids.
Oligodeoxyribonucleotides T synthesized by the modified phosphotriester method are presented in Table. one.
Fully treated trideoxy0
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ribonucleotides D (8b mg, 0.05 mmol) and 2 (180 mg, 0.1 mmol) unblock in 5 hydroxyls by treating with 2% BSA at 7: 3 (v / v) chloroform / methanol (10 and 20 MP respectively) for 10 min at. The reaction is stopped by adding saturated aqueous ammonium bicarbonate (2 ml), the material is extracted with the form (25 ml) and washed with water (ml). The organic layers are dried (over magnesium sulphate), concentrated to small volumes (about 5 ml) and precipitated by adding petroleum ether (fraction ZZ-BO C). Colorless precipitates were collected by centrifugation and dried in a desiccator under vacuum to obtain 6 and 8, respectively (each homogeneous) using TLC on silica gel.
Trimers 1 and 3 (270 mg, 0.15 mmol; 1A5 mg, 0.075 mmol) are converted to 5 their phosphodiesters (5 and 7) by treatment with a mixture of triethylamine / pyridine / water (1: 3: v / v, 10 ml) in for 25 min at room temperature. The reagents are removed in a rotary non-evaporator. The residual residues are rewired with anhydrous pyridine (ZiO ml) o Trimer 8 (0.05 mmol) and trimer 7 are combined with TPST (50 mg, 0.15 mmol) in anhydrous pyridine (3 ml). The reaction mixture is left under vacuum at room temperature for 2 hours. TZC analysis shows that 95% of make-up artist 8 is converted into a hexamer product (visualized by definition of the DMT group by spraying with 10% aqueous sulfuric acid and heating at 70 ° C) . The reaction is quenched by adding water (1 ml). The solvent is evaporated in vacuo under reduced pressure. After pyridine is removed by co-evaporation with toluene, the hexamer is unblocked at position 5 with 2% BSA (8 ml), as described previously for trimers 4 and 2. The product (10) is purified on a column of silica gel (Merkc 60 N, 3.55 cm) by step gradient elution with chloroform / methanol (98: 2 - 95: 5, v / v). The fractions which contain product 10 are evaporated to dryness. In a similar way, trimer 3 is combined with 6. The fully protected product is directly purified on silica gel. Last connection unlocked5
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231491346
at the H end with a mixture of triethylamine / pyridine / water, as previously described prior to the preparation of fragment 9.
Hexamers 9 and 10 are combined in anhydrous pyridine (2 ml) with TPSTe (75 mg, 0.225 mmol) as a condensing agent. After completion of the reaction (4 hours, room temperature), the resulting mixture was spinned in a rotary evaporator, and the residue was chromatographed on silica gel. Product II (160 mg) obtained by precipitating petroleum with ether was homogeneous according to TZC, Part 15 of compound 11 (20 mg ) in pyridine (0.5 ml), is fully deblocked by treating with concentrated ammonium hydroxide (7 ml, 8 h), and then treated with 80% acetic acid (15 min, room temperature). After evaporation of the acetic acid, the solid residue is dissolved in 4% aqueous hydroxide
24
T4 DNA ligase. The reaction product is purified by gel electrophoresis on a 15% polyacrylamide gel containing 7M urea. The four isolated products are ligated together, the reaction mixture is separated by electrophoresis on a 10% polyacrylamide gel. Electroelution of DNA in a tinosine-ovale size sequence (1 gene (90-10 base pairs)).
Plasmid pBR322 (0.5 µg) is treated with BamHI and EcoRI with restriction endonucleases, the fragments are separated by polyacrylamide gel electrophoresis. A larger fragment is removed from the gel by electroelution and sequentially ligated with the collected synthetic DNA. This mixture is used to transform E.co-11K12294. A 5% transformation mixture is placed on ZB plates containing 20 µg / ml ampicillin. Four received colonies resistant to
thirty
ammonium (volume / volume, 4 ml) and extra- 25 ampicillin, sensitive to tetraging with ethyl ether (3 2 2 ml). The aqueous phase is concentrated to 1-2 ml, part is treated with HPZC to purify 12c. The fractions corresponding to the main peak are collected (approx. 2 A554 U) and concentrated to 5 ml. The final product 12 is desalted on Biogel P-2 (1 cm), eluting with 20% aqueous ethanol, dried to dryness and resuspended in water (200 µl) until solution AQSI Y is obtained. Sequence 12 is confirmed by two-dimensional sequence analysis
The complete thymosin-o / 1 gene is assembled from 16 synthetic oligonucleotides. Microgram amounts of oligonucleotides from Te to quantitatively phosphorylate () - ATF in the presence of T4 polynucleotide kiase to obtain specific activities of approximately 1 Ci / mol. Radiolabeled fragments are purified by gel electrophoresis (20% polyacrylamide / 7M urea) T Sequences in the eluted fragments are identified by two-dimensional electrophoresis (homochromatography). The T fragments are left unphosphorylated to reduce undesired polymerization during subsequent ligation reactions. These oligonucleotides (2 μg each) are assembled into four groups of four fragments.
cyclin, which suggests the inclusion of the tetracycline resistance gene. Analysis of the plasmids from these four colonies showed that in each case. The plasmid, burned out with pTh 1, contains the third site, which is not in pBR322 itself, which indicates the presence of the thymosin-Ct1 gene, and a fragment (approximately 105 base pairs) obtained by cleavage of the BamHI / EcoRI. Plasmid pThcA I contains a gene that determines resistance to ampicillin, and structural gay, which determines thymosin-o 1, cloned
40 on its 5 end-coding strand in the EcoRI site and on its H end in the BamHI site of the Gene Thymosin also contains the Bglll site. To create a plasmid capable of priming the ZE / d / fragment,
d5 previously prepared, pThedl was burned with EcoRI followed by Klenow 1 polymerase reaction with dTTP and dATP to blunt EcoRI residues. EgIII burning the resulting product creates a linear DNA fragment containing the ampicillin resistance gene, and its opposite ends are the sticky Bgllll residue and the blunt end. The resulting product can be recycled by reacting with a ZE / d / fragment containing a Bdllll sticky end and a blunt end, in the presence of T4 ligase to obtain the plasmid pTr24. At the same time EcoRI site revos50
55
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T4 DNA ligase. The reaction product is purified by gel electrophoresis on a 15% polyacrylamide gel containing 7M urea. The four isolated products are ligated together, the reaction mixture is separated by electrophoresis on a 10% polyacrylamide gel. Electroelution of DNA in a tinosine-ovale size sequence (1 gene (90-10 base pairs)).
Plasmid pBR322 (0.5 µg) is treated with BamHI and EcoRI with restriction endonucleases, the fragments are separated by polyacrylamide gel electrophoresis. A larger fragment is removed from the gel by electroelution and sequentially ligated with the collected synthetic DNA. This mixture is used to transform E.co-11K12294. A 5% transformation mixture is placed on ZB plates containing 20 µg / ml ampicillin. Four received colonies resistant to
ampicillin sensitive to tetra
cyclin, which suggests the inclusion of the tetracycline resistance gene. Analysis of the plasmids from these four colonies showed that in each case. The plasmid burned out by pTh 1 contains the III site that is not in pBR322 itself, which indicates the presence of the thymosin-Ct1 gene, and a fragment (approximately 105 base pairs) obtained by splitting BamHI / EcoRI. Plasmid pThcA I contains a gene that determines resistance to ampicillin, and structural gay, which determines thymosin-o 1, cloned
on its 5 end-coding strand in the EcoRI site and on its H end in the BamHI site of the gene for thymosin also contains the Bglll site. To create a plasmid capable of priming the ZE / d / fragment,
previously prepared, pThedl was burned with EcoRI followed by Klenow 1 polymerase reaction with dTTP and dATP to blunt EcoRI residues. EgIII burning the resulting product creates a linear DNA fragment containing the ampicillin resistance gene, and its opposite ends are the sticky Bgllll residue and the blunt end. The resulting product can be recycled by reacting with a ZE / d / fragment containing a Bdllll sticky end and a blunt end, in the presence of T4 ligase to obtain the plasmid pTr24. At the same time, the EcoRI site is recreated
is given in a position where ligation of the blunt end takes place.
Sequential vorsiganie pTrpZABglll and EcoRI, followed by PAGE and electroelution yields a fragment having codons for ZE / d / polypeptide Bgllll sticky end and EcoRI sticky end adjacent to the GO 3 encoding terminusuo ZE / d / fragment was cloned into Bgllll join r50M7D2 plasmid to production of ZE plippeptid / samotostatin cross-linking protein, expressed under the control of tryptophan promoter / operator.
16 µg of pSOM7u2 plasmid is diluted in 200, µl of buffer containing 20 mM Tris (pH 7.5), 5 mM MgClI, 0.02% detergent RAO and 100 mM NaCl, and 0.5 units are processed. EcnRI. After 15 minutes, the reaction mixture is extracted with phenol, chloroform, precipitated with ethanol, and Bgllll are subsequently burned. The larger of the obtained fragments is isolated using PAGE and then electroelution. This fragment contains the ZE / P / codons for the proximal end of the ZE polypeptide, that is, the one that is located earlier than the BgLIII site. This fragment is then ligated with the above ZE / d / fragments in the presence of. T4 DNA ligase to obtain plasmid P50M7L2 / 14, which, after transformation into E. coli K12 294, efficiently produces a cross-linking protein consisting of a completely reconstructed ZE polypeptide and samotostatin under the control of tryptophan promoter / operator.
The pBR322 plasmid is burned with Hinglll, the protruding Hindlll ends are burned with SI nuclease. Burning SI with a nucleotide involves treating 10 µg of Hindlll with split pBR322 in 30 µl of SI buffer (0.3 M NaCl, 1 mM ZnCLj, 25 mM sodium acetate; pH 4.5), with 300 units. SI nuclease for 30 min at. The reaction is stopped by adding 1 µl of a 30vl nuclease stop solution (0.8 M Tris base, 50 EDTA). The resulting mixture was extracted with phenol, chloroform, precipitated with ethanol, and then burned with EcoRI, as previously described. The fragment obtained by PAGE followed by electroelution, has EcoRI sticky and blunt ends, encoding
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the latter strand starts with nucleotide thymidine. The scorched Hindlll SI residue, starting with thymidine, can be attached to the Bgllll residue treated with polymerase, therefore, the plasmid p50M7D2, obtained in Example 5, was burned by theglll, and the obtained Bgllll sticky samples were treated twice with Klenov 1 polymerase using all 4 deoxynucleotide tri-phosphates, and the cryptochemicals were purified by cryptocid mixtures. followed by PAGE treatment and electroelution of a small fragment gives a linear DNA region containing the tryptophan promoter / operator and the ZE codons of the proximal sequence located earlier than the Bglll site (ZE / P /). The resulting product has an EcoRI end and a blunt end, obtained when filling the Bglll site. However, the Bglll site is reconstructed, the ligir is the blunt end with the blunt end of the above fragment of the burned Hindlll SI fragment. Thus, two fragments are ligated in the presence of T4 DNA ligase before the formation of the recrystallized plasmid ИКRIKU 10, which is propagated by transformation into competent E. coli K12294 cells. Tetracycline-resistant cells containing recombinant plasmid pHU are selected, and plasmid DNA is extracted. After burning Bglll and PStI, followed by isolation of PAGE and electroelution of a large fragment, the target linear piece of DNA with PStI and Bglll is obtained with sticky ends. This DNA fragment derived from rnAc contains a source of replication and is therefore suitable for use as a component in the construction of plasmid pH17D4d1, in which both genes encoding trpZE (polypeptide cross-linking protein and tetracycline resistant) are controlled by the trp promoter / operator.
Plasmid.RVOM7D2D4, obtained in example 5, is subjected to partial digestion of EcnRI followed by digestion of PStI. The resulting fragment containing the trp promoter / operator is selected by PAGE followed by electroelution. Partial EcoRI burning is necessary to obtain a fragment that is cleaved by the number 3 of the end of the gene.
samotostatina, but was not split on the EcoRI site, which takes place between the ampicillin resistance gene and the trp promoter / operator.
Ampicillin resistance, lost by Pstl cleavage in the ampicillin resistance gene, is restored by ligation with the final RNA DNA linear derivative previously obtained in Example 5.
As a first step in the design of the gene encoding the first 32 amino acids of proinsulin, a series of 18 oligonucleotides are presented, presented in Table. 2
Some nucleotides are used to construct the human insulin B chain. Two nucleotides (B5 and B10) include Hpall and the terminal BamHl and EcoRI restriction sites. Terminal sites are useful for cloning purposes.
The 8 H1-H8 oligonucleotides previously used to construct the left half of the human insulin B chain contain the coding sequence for 1-13 amino acids of the B chain gene and additional N-terminal methionino. The right half of the B chain is constructed from oligonucleotides B, B, B B4 . BS, Wb, B7, B, BO and ligation using T4 DNA ligase. The resulting gene fragment encodes the human insulin chain and the first arginine of the bridging chain Hpall restriction site is inserted into the gene sequence into the same reading fragment, similar to the Hpall site, in the human insulin gene. After purification of the ligated gene fragment by polyacrylamide gel electrophoresis and the largest eluted band in the duck fragment, the cleaved plasmid pBR322 is inserted into HindIII-BamHI. The resulting plasmid, designated pVZ, is included in E. coli K12 294 by transformation. The plasmid confers resistance to antibiotics (ampicillin and tetracycline) and, as found, contains the target nucleotide sequence
Two 58 base-pair fragments of the Hindi11-VaHa1 pV3 fragment and 46 base pairs of the EcoRI-Hindlll pBHI fragment are ligated to obtain a fragment having EcoRI and BamHI ends.
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This fragment is ligated in the usual manner into EcoRI and BamHI, the restriction plasmid pBR322. The resulting plasmid, designated p1B3, is then cloned in E. coli K12 294. After the usual amplification and isolation of the p1B3 plasmid, EcoRI and Hpall are burned to produce a synthetic gene fragment that encodes N-terminal proinsulin amino acids led by methionine. The synthetic gene is isolated by a conventional method using polyacrylamide gel electrophoresis.
Decanucleotide 18, which contains both the BamHI recognition sequence and the 3 portion of poly-imidylic acid, containing about 20 residues, is synthesized and used to stimulate AMV reverse transcriptase to synthesize cDNA. A primer is obtained using deoxynucleotidyl transferase with 1 μm BamHI decanucleotides in a reaction volume of 0.6 ml containing 1.5 “10 10 μm TTPo. The reaction is carried out at 37 ° C for one hour in a buffer system. PolyAmRNA (2.5 µg) of human insulinoma was isolated and transformed into double-stranded cDNA (in almost the same way; “Sa”, the reaction volume (80 µl) containing 15 1 Tris HCl (pH 8.3 at), 21 mM KS1, 8 mM MgClg, 30 mM mercaptoethanol, 2 mM dCCGGATCCGGTT primer, g and 1 mM dNTPS, preincubated with after addition of AMV reverse transcriptase, are incubated for 15 min. cDNA is synthesized in a reaction volume of 150 μl, containing 25 Tris HCl (pH 8.3), 33 KCl, 4 mM MgCL, 15 1 L-mercaptozanol, 1 mM dNTPS, and 9 units of Klenow polymerase I. The resulting mixture is incubated at 15 ° C for 90 minutes, and then 15 hours at. Carry out nuclease burning. 37 ° C, using 1000 units of S / nuclease / .The double cDIC (0.37 mcc) is treated by electrophoresis on an 8% polyacrylamide gel. DNA fragments larger than 500 base pairs are eluted. To the 3 ends of the fragments oligodeoxycytidyl acid residues are added using terminal deoxy-nucleotidyl transferase, cLNA with dC ends are annealed to pPjR322, then Ps is burned tl restriction enzyme and sew with deoxyguanidilic acid using terminal deoxynucleotidyltransferase. The resulting plasmids are used to transform E. coli K 12 294. The obtained cells are placed on ZB and TET medium. Tetracycline resistant colonies, but sensitive to ampicillin, are isolated and screened for plasmids that contain three PstI restriction sites. One plasmid, designated pHPOA, contains an insert of 600 base pairs, gives the expected PstI restriction type, and contains a BamHI site between the 3 polyA and polyCC, introduced during cDNA production.
The synthetic gene segment encoding the first 31 amino acids of pro-insulin was isolated from 50 µg of the p1B3 plasmid using the restriction endonuclease EcoRI and Hpall, as previously described. The first fragment also contains the ATC sequence for methionine instead of the proinsulin presequence
The cDNA gene segment, encoding amino acids 32-86, as well as the translational stop signal and the 3 untranslated region of mRNA, is isolated from 40 µg of the plasmid pH1104, which is processed first by BamHI and then by Hpall restriction enzymes. These fragments were electrophoresed on a polyacrylamide gel, followed by electroelution. The gene fragments were combined by treating T4 DNA with ligase in 20 µl of ligase buffer at 4 ° C for 24 hours. The mixture was diluted with 50 µl, extracted in the usual manner with phenol and chloroform, and then precipitated with ethanol.
The resulting DNA is treated with BamHI and EcoRI restriction enzymes to regenerate these and remove the gene polymers. The collected insulin gene was isolated by polyacrylamide gel electrophoresis and ligated (using T4 DNA ligases) for EcoRI and BamHI digestion of plasmid pBR322. The obtained DNA is used to transform E. coli K12 294, and then the resulting colonies are screened for tetracycline resistance and
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ampicillin. Plaemid pH13, isolated from one of these colonies, contains the target proinsulin gene, which is then characterized using the analysis of nucleotide sequences.
The complete human proinsulin gene, including the N-terminal codon that encodes methionine, is isolated from the plasmid pH13 by treatment with EcoRI and BamHI with restriction enzymes. The target fragment is purified by gel electrophoresis, and then ligated (using T4 DNA ligase) with partial digest of the plasmid pSOM7u2 & 4 Pstl-EcoRI (prepared in Example 5) and large from Pstl-Bglll fragments of the plasmid RNA (obtained in Example 5). Then, μg of the complete human proinsulin gene with EcoRI and BamHI ends, 4 μg of the Pstl-EcoRI (partial) pSOM7A2u4 fragment and about 1 μg of the Pstl-Bglll fragment of RNAA are ligated at 4 C for 5 24 h, using T4 DNA ligase in the ligation buffer . The ligated DNA mixture is used to transform E. coli K12 294. Colonies that grow on both ampicillin and tetracycline are selected. They were found to contain the target plasmid pHI7u4JSl and express the molecular weight protein expected from fusion trpZE-proinsulin, Plasmid pH1 A1, which express the above-mentioned protein, is fully characterized both by the DNA sequence and by restriction sites of both included genes, and also by vector.
Example 6. About 5 μg of DNA plasmid pH 17D4D1, 10 μl (10 X) of reaction buffer, 80 μl of water and 5 μl (5 units) of the Hpal restriction enzyme are incubated at 37 ° C for J h. Then the reaction is quenched by incubating 70 ° C e for 5 min. The mixture is cooled on ice, about 12 µl of Ø Tris HC1 (pH), 7 µl of water and 1 µl (10 units) of ERoRI restriction enzyme are added. The resulting mixture was first incubated at 37 ° C for one hour, and then for 5 minutes. After cooling with ice, the obtained DNA was extracted with phenol and chloroform: isoamyl alcohol (24: 1), and then precipitated with ethanol. Target - 253b EcoRI-Hpal fragment in the usual way you 5
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divided by electrophoresis on a polyacrylamide gel with electroelution, precipitated with EOH, then dissolved in µl of TE buffer and stored until further use.
- 20 μg of DNA plasmid pH17DAA l in 32 μl (51) of EcoRI reaction buffer, 198 μl of water and A μl of EcpRI restriction enzyme (20 units) are incubated for about an hour. The reaction is then terminated by incubation for 5 min. The resulting 862Lp EcoRI fragment is isolated by electrophoresis on a 6% polyacrylamide gel, followed by electroelution and precipitation with ethanol. The fragment is then dissolved in MOO µl Hpal buffer containing 16 units. Hpal restriction enzyme. After 1.5 hours of incubation, with 7.5 units of TagI restriction enzyme added, incubated for one more hour, the resulting fragments are made in the usual manner on a 7.5% polyacrylamide gel. The target Hpal-Tagl fragment was isolated by electroelution and precipitation with ethanol, and then dissolved in 5 µl of TE buffer.
5 μg of plasmid pBR322 DNA, 10 μ ClaC of the reaction mixture, 77.5 μl of water and 7.5 μl (15 units) of the Clal restriction enzyme are incubated for an hour. Then the reaction is completed by incubating the mixture for 5 minutes. The mixture is cooled on ice and about 12.5 µl of W Tris HC1 (pH. 7.2), 6.25 µl of W NaCl, 2.5 µl of O, 1 M MgCl and 1 µl (10 u.) Of EcoRI restriction enzyme are added. enzyme. The resulting mixture was in5; CCASA AT & TTC CCA TT & GAG GAT CAT TAA AT & TTC CCA CCC ATS
rrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrаrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrr
3 TCT TAC AAC C & T AAC СТА СТА СТА ATT TAG AA & BCT C & G TAG
Tcc ttco tcc- ccc era ttt ccc AAC eat STST
r 11 f f f f m r f f r r f I f f f I f I M I r ACG AAC AG-C CC & CAC AAA CG & TTB CGA C
The target linker sequence is synthesized by the modified phosphotriester method (this synthesis is specifically illustrated in Example 1).
About 20 pmol of the DNA linker of example 7; 1 μg pNC608 digest Example 7; 0.5 µl of the plasmapc pCZlOl-U.Zi RamHI-EcoRI fragment (incubate first with
1 h and then for
5 minutes. After cooling on ice, the obtained DNA is processed by electrophoresis on a 1% agarose gel. The coarse fragment is isolated by elution and precipitated with ethanol, then dissolved in (-20 µl TE buffer) and stored until further use.
About 0.2 MKr v-253bp of the EcoRI-Hpal fragment and 0.5 μg of the Hpal-TagI fragment of Example 6 and 0.1 μg of the EcoRIGlal digest of Example 6 are ligated and used to transform E. coli K12 RV308.
Part of the obtained transformants, as shown by electrophoresis
on agarose gel and other tests, contains only target, 6kb plasmidoo. Such a transformant, designated E. coli K12PV308 / PNM608, is selected, placed on TV agar, containing
appropriate antibiotics, and then cultured using conventional microbiological techniques. The plasmid rMMBOV is the preferred source — 287βp EcoRI-Clal / TagI /
fragment of plasmid pH7J.
Example 7 o About 5 μg of the DNA of the plasmid pNM608 in 50 μl of Medium Salt buffer is incubated with 10 units. each of the restriction enzymes EcoRI and Clal at 37 ° C for 1 hour. After adding 5 µl of 3 M sodium acetate (pH 7.0), the DNA is precipitated with 3 volumes of 100% ethanol. The target didster DNA is dissolved in 100 µl of TE buffer and stored at 0 ° C until further
use
I
3
obtained in accordance with the procedure of Example 2, except that EcoRI and the corresponding buffer are used instead of the Xbal restriction enzyme and buffer), I μg of the plasmid pCZlOl 0.6kb of the BaraHI-HgiAI fragment of Example 2 is ligated, the resulting plasmid is used for transformation E. coli K12KU308 „
Part of the obtained transform} {comrade, which is convenient to show using agarose gel electrophoresis and other tests, contains only the target .Skb plasmid. Such a transformant, designated E. coli K12PV308 / pCZ105.1, is selected on TV agar, contains appropriate antibiotics, and then cultured using conventional microbiological techniques. The resulting cells, as shown by SDS by gel electrophoresis, RIA and other tests, express met - in GH in large
5 SBAA AT & TTS SSA TT & GAT rdt & AH TAA ATG TTS SSA & SS AT &
, f m I I f g г г г f f г г f г г г t f г г г f fir г г г г г I Mл м г
3TDT TAS AAG C & T AAS CTA CTA CTA ATT TAS AA & && T C & C TAS
TCC TTG TCC CGC CT & TTT GCC AAC SST CTGCT 5
r g f f I f I I r I f r t f r r r f r I I r r t.
AG6 AAC ACC CCG GAG AAA CGG TTG CGA C5
The above specific linker sequence is constructed in accordance with the usual method.
Target transformants designated E. coli K12RV308 / pCZ105.2 are placed on TV agar containing the appropriate antibiotics and then cultured for the production and isolation of the plasmid pCZ105.2o in the usual way as shown by SDS electrophoresis.
5 CGACC PBX GAT CAG-GAG TAA AT & TTC CCG GCT ATG TCC TTG
.rrr rrr rrt rrt rtf fir rrr rrr iri rrr tri rii irr
at TGG TAC STA GTC STO ATT TAC AAG CGC CGA TAC AGG AAC
TCC GCC CTC TTT CCC AAC GCT CTCCT 3 t I 11 I r r I I r I r I I I r I r I I, 1
AGG CGG GAG AAA CGG TTG CGA C5
Construct the specified linker sequence. Target transformants, designated E. coli K12RV308 / pCZ106, l, are placed on TV agar containing the appropriate antibiotics, and then cultivated by ordinary means for subsequent production and isolation of the plasmid, l. These transformants, as observed using SDS gel electrophoresis, PIA, and other tests, express met-bGH at high levels.
kolchrstnlh Since CPC plasmipl pC7.1P5, 1 contains a rapid-growth thermo-inducible roplicone, maximum expression net-bGH occurs at cultivation temperatures of about
Example 8; Pelar constructions are obtained in accordance with the method of Example 7 with the difference that instead of the linker sequence of Example 7, the following DNA linker sequence is used:
RTA and other tests express net-bGH with high levels. Since the plasmid pCZ105.2 contains a heat-induced fast-growth replicon, maximum expression of met-bGH is observed when cultured at temperatures around.
Example 9. The target design was carried out as described in Example 7, but instead of the linker sequence of Example 7, the DNA sequence is used.
Since the plasmid pCZ106.1 contains a thermo-induced rapid growth replicon, maximum expression of roet-bGH is observed during cultivation during temperature analysis near.
Example 10. The target design was carried out substantially in accordance with the method of Example 7, but instead of the linker sequence of Example 7, the DNA sequence was used.
5 SODSS AT & GAT CAT AAC TAA AT & TTT CC & CCT ATG TCC TTG
, tif ftr Iff III III III III III III III ifi irr irr
3 TCC TAG STA STA TTC ATT TAG AAA GCC CGA TAG AGC AAG
TCC CCC CTC TTT CCC AAG CCT irr r rI tit IrI r r r ftr r r I ABS CCC & AA AAA CCC TTC CCA
The above linker sequence is constructed according to a conventional procedure.
Target transformants, designated here by E. coli, K12RV 308 / pCZn2, are placed on TV agar containing the appropriate antibiotics, and then cultured in the usual way for subsequent production and isolation of the plasmid pCZ112 ,. These transformants, as shown by SDS gel electrophoresis, RIA and other tests,
R
five
CCAGG ATG GAT CAT AAC CAD -IMl f f I r I r III irr fir
J TC & TAC STA CTA TTC GTG
TCC f f r
ACC

CCC CTC TTT
f I I r r r Ml
CCC CAC AAA
Gcg
AAC r r t
C6C TT & CCA
CCT f r f
Design the specified linker sequence in accordance with the usual method.
Target transformants, designated E. coli K12RV309 / pCZl12.2, are placed on TV agar containing the appropriate antibiotics, and then cultured until the production and isolation of the plasmid pCZll.2.2. These transformants, as indicated by gel electrophoresis, RIA, and other tests, express met-bGH with you
CTACAGGGTATTAATA ATC DAT t: f f I I g I f f g f I f f f I r f I TCCCATAATTAT TAC GTA
ATB TCC TTC TCC CCC CTC TTT
"II Ml I If III Ml t f I Iff
TAC ACC AAC ACC CCC GAC AAA
Construct the specified linker sequence in accordance with the common method with
The target transformants, designated E. coli K12RV308 / pCZ1000, are placed on TV agar containing the appropriate antibiotics and then cultured in the usual way to obtain and isolate the plasmid pCZlOOO.
CTGCT 3 i5
high levels of met-bGH are expressed. Since the plasmid pCZl12.1 contains a thermally induced rapid growth replicon, the maximum expression of met-bGH is observed when cultured at temperatures of about 27 ° C.
Example 11, Target con (trouting is carried out in accordance with the method of Example 7, but instead of the linker sequence of Example 7, the DNA sequence is used
TAA PBX TTT CCC CDT PBX TGC
Iff itl ffi rrr f f frr fff
ACC TAG AAA CCC CCA TAG ACC
CTCCT I
WITH
J
five
sochi levels. Since the plasmid pCZ112,2 contains a thermally induced rapid growth replicon, the maximum expression of met-bGH occurs at a culture temperature of about.
Example 12o Purposeful construction was carried out in accordance with the procedure of Example 3, replacing the linker sequence of Example 2 with a DNA linker sequence.
CAC GAC. GAT TAA PBX TTT CCT GCT
Iff If I f fff Iff TIL I M
GTC iCTC; CTA ATT TAC AAA GGA SSH
AAC I r r TTC
CCT 111
CCA
CTOCT t
with
3 5
five
These transformants, as shown by gel electrophoresis, RIA, and other tests, express raet-bGH with high levels. Since the plasmid pCZlOOO contains a thermally induced rapid growth replicon, maximum expression occurs when cultured at temperatures around.
Example 13, Target design was carried out practically in accordance with the method of Example 3, except that instead of 5 STA & ASSSTATATA ATC CAT CAT CAC TAA AT & TTT SSS SST ATS TSS
, f M f f f 1 I t f t g I g I t g I III lit Iff lit I I f r I r
3 TCCCATAATTAT TAG СТА & TC
ATT TAC AAA C & C
CCA
t I r TAC ACC
TTC TCC CGC CTC TTT CCC AAC CCT BTSC
Iff lir lir | (t fri lit 111 III 1
AAC ACC CC & GAC ADA CCC CCC TTG CCA C
The indicated linker sequence is constructed in accordance with the usual procedure.
Target transformants, designated here by E. coli KI 2PV308 / pr, Zl 000.1, are placed on TV agar containing the appropriate antibiotics, and then 20 are cultured in the usual way for subsequent preparation and isolation of the plasmid pCZlOOO. , RIA and other tests, express 25
5 CTACAGGGTATTAATA PBX CAT GAT
g f I I I I I I I I I f lit r I r III
jTCCCATAATTAT TAC CTA CTA
TCC TTC TCC CCC CTC TTT CCC AAC CCT CTCCT J
, t I f I I I I I I I I I I I I I I (I (f I I f r I.
AG & AAC AGG CCG CAC AAA CCG TTG CCA C 5
The above link levels are constructed. Since the plasmid core sequence in accordance with pCZ 1000 contains thermally induced with the usual method, 35 replicon of rapid growth, maximum
Target transformants, designated for expression, occur in cultured here E, coli K12RV308 / pCZ1060, are placed on i-TV agar containing appropriate antibiotics, and conventional
40
tivirovaya at a temperature of about 37 ° C
Example 15 The target construct was cultured to obtain and isolate plasmid pCZ1OfiO. These transformants, as shown by gel electrophoresis, RIA, and other tests, express met-bGH with you CTACAGGGTATTAATA ATC & AT CAT AAG TAA ATG TTT CCG CCT ATC
, .ffirilllfllt “If MI Ml Ml Ml MI IMIIIflllM
3 TCCCATAATTAT TAC СТА СТА ТТС ATT TAC AAA GGC CCA TAC
TCC TT & TCC GGC CT & TTT CCC
lit Mr ml III ml lit i m
AGC AAC ACG CCC CAC AAA CCC
The above linker sequence is constructed in the usual manner. .
Target transformants, designated here by E, coli K12RV308 / PCZ1120 are placed on TV agar containing the corresponding antibiotics, and then, in the usual way, the cultivar sequence of Example 3 is used with the DNA liquera sequence
lit Iff lit I I r r I r
TAC AAA C & C
CCA
t I r TAC ACC
3
high levels of met-bGH. Since the plasmid pCZlOOO contains a thermally induced rapid growth replicon, maximum expression of raet-bGH occurs when cultivated at a temperature of about
Example lAg Target construction is carried out in accordance with the method of Example 3, using instead of the linker sequence of Example 3 DNA linker sequence
TAA ATG TTS
I f 1 I f III
ATT TAC AAG
CCA GCC PBX
III I t I Ml
CCT CCC TAC
on expression occurs during culture
0
tivirovaya at a temperature of about 37 ° C
Example 15, Target design was carried out in accordance with the method of Example 3, but instead of the linker sequence of Example 3, the DNA linker sequence was used
AAC CST GTCCT 3
I M I I I I ..I
TTC CCA C 5
viral to obtain and isolate plasmid pCZ1120. These transformants, as shown by gel electrophoresis, RTA and other tests, express high levels of met-bGH. Since plamemi and pCZn20 contain a thermoinduced replicon of rapid growth,
39
Maximum expression of met-bGH occurs at temperatures around.
Example 16, About 5 μg of the pNM575 plamid (prepared in Example 1) in 50 μl of Medium Salt buffer is incubated with 10 units of each (BamHI and FnuDII) from restriction enzymes 5 CTA & ACG-CTATTAATA ATC TTC
, f I g f I I g I 1 f f t I g f t I
3 TCCCATAATTAT TAG AA &

SSA forest ATT SSS TTL TSS ASS
rrr rif f l TG1 tif llf irr
CCT TC & TAA GCC AAT ACC TCC
I
The above linker sequence is constructed in accordance with the synthesis method specifically illustrated in Example 1 "
Ligation and transformation are carried out as follows.
About 20 pmol of the linker DNA of Example 16, 1 μg of the BamHI-FnuDII digest of Example 16 and 0.5, 2kb of the BamHI-Xbal fragment of Example 3 are ligated and the resulting plasmid is used to transform E. coli K12RV308 according to the method of Example 2.
Part of the obtained transformants, as shown by agarose gel electrophoresis, contains only the target plasmid 10.8kb. The transformants designated here by E. coli K12RV308 / pCZ2II40 are selected, placed on TV agar containing the appropriate antibiotics, and then cultured using conventional microbiological techniques. The resulting cells, as shown by SDS gel electrophoresis, RIA and other tests, express met-bGH with high levels. Since the plasmid pCZ1140 contains thermally induced replica
is maximal
fast growth
5 CTAGACGGTATTAATA PBX TTG GAG GAT GATTAL PBX TTC SSA
I I I I I I I I f (f I I til III r I I II II III I M III
TCCCATAATTAT TAG AAC CTC STA STATT TAG AAG GGT
CCG ATG JGG TTG TCG GGC CTG TTT CCC AACGCT GTGCT G
r tI f f f r r r Iff III fir Iff I I r r rrt r I r
CCC TAC ACC AAC ACG GCC CAC AAA CCC TTGCCA C5, OR
J CTACACGCTATTAATA ATG TTG CCA TTG GATGAT CAT TAA PBX TTC
I t r f r I I I I I r r III III III III III r I t III I I I I I I
TGCCATAATTAT TAC AAO CCT AAG BATTERY STA ATT TAC AAG
io
TOB at 1 hour. After adding 5 µl of ZM sodium acetate (pH 7.0), the DNA is precipitated with 2 volumes of 100% ethanol and recovered. The target DNA digest is dissolved in 1UO µl TE buffer and stored until further use.
TTG & AG SAG CAT TAA ATS TTS
III 111 g t g I t g Ml f g I g f t
AAC CTC STA CTA ATT TAS AA &
CAC AAS
f g I I I
CIC TTC
eat ATC CTC CO 3 fit g t I g I I g. CCA TAG CAG & C 5
five
0
0 5 0
H
Compression met-bGH occurs when cultivated at temperatures around.
I
F o r mula inventions

权利要求:
Claims (1)
[1]
1 "Method for producing recombinant plasmid DNA encoding the synthesis of bovine or human growth hormone by combining DNA fragments encoding a lipoprotein promoter, a transcription initiation region, a translation start codon, a growth hormone and a stop signal of translation, differing in that, in order to increase the output of methionine growth hormone, BamHI-Xbal or BamHI-EcoRI fragments of the plasmid pCZlOl, about 10.2 kb in size, including reclicon, which lose the copy number control, and the lipoprotein promoter of E.coli, and the plasmid in pCZlOl, ligating the Xbal-BamHI fragment of the pIM-l A3 plasmid with a size of about 10.2kb with the Xbal-BamHI fragment of the plasmid pNM789b and the synthetic linker of the following nucleotide sequence is obtained:
With the BamHl-FnuIl fragment of the pNM575 plasmid with a translation initiation codon, a start codon, a peptide and ribosome binding site, a translation stop signal and the first 14 amysyacid residues of bovine growth hormone or the first 15
human amino acid residues - growth hormone,
2, the method according to claim 1, is also distinguished by the fact that an EcoRI-Clal fragment of the pYMbOb plasmid of 0.29 kb in size is added to the recombinant DNA, including)
g Z1A913A644
E. coli tryptophan promoter, which gives the nucleotide sequence a synthetic linker has the following usefulness:
5 ССАА АТС ТТС SSA TTG GAC CAT GAT ТАА АТС ТТС SSA GCC
I I I I I g III lit III III III III III III III III
TOT TAG AA & CCT AAC CTC CTA STA ATT TAC AAC GCT CG &
AT & TCC TTC TCC CCC CTG TTT GCC AAC GCT GTGCT 3 III III III III til III III III til 111 I
TAG AG & AAC AGG CCC GAC AAA CGG TTG CCA C 5, OR
 CGACA ATG TTC
111 r I I r I I
TGT TAG AAC
TG TCC TTC TCC
II n r I I I III
AC AGG AAC AGG
CCA I r r
CGT
GCC I I I
TTG GAT III lit
AAC CTA
STO-TTT 111 III
CAT I I
Cta
GCC I t I
GAT TAA A
fit III t
STA ATT T
AAC I I I
GCT I I t
GT
I
CCG GAC AAA CGG TTG CGA C
C GAC C I I I
TGG
TG TCC t r III
ATG I t t
Tac
GCC f I I
GAT f t I
Cta
CTC I I I
CAG t t t
GTC
GAG TAA ATG TTC CCC GCT
Ml III III III III til
CTC ATT TAC AAG GGC CCA
TTT I I I
GCC 1 11
AC AGG CGG GAG AAA CGG
AAC GCT GTGCT
III lit t
TTD CGA C5,
5 CCACC ATG CAT CAT AAG TAA ATG TTT CCG GCT ATG TCC
t t I til III tit "Mill III III III I t I t I I III
TGC TAC CTA CTA TTC ATT TAC AAA GGC CGA TAC AGD
TTG TCC GG-C CTC TTT GCC AAC GCT GTGCT W lit III III lit "11 t t I t t t t t
AAC ACC CCG GAC AAA CGb TT & CGA S S, OR
5 CGACC AT & GAT GAT AAG GAG TAA ATG TTT CCG GCT ATG
til lit III lib III I-I I t I I 1 I I I I I t I t lit III
TGG TAC CTA CTA TTC CTC AGC TAD AAA GGC CGA TAC
TCC TTO TCC GGC CTC TTT GCC AAC GCT GTGCT
Ml r I I til lit Ml f t t t I t t I f III t
AGG AAC forest CCG GAG AAA CCG TTG CGA.C
CAT I I
Cta
CC I t I
GAT TAA ATG TTC CCA GCC
fit III til lit tit Ml
CTA ATT TAC AAG G & T CGC
AAC I I I
GCT I I t
GTGCT
I
GG TTG CGA C
ATG TTC CCC GCT
III III III til
TAC AAG GGC CCA
J
5 ,, AND / 1И
AT & TCC III 111
TAC AGG
OR
L 5
Table 1

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同族专利:

---

公开号 | 公开日

---

DOP1985004337A|1990-09-17|

---

ES540936A0|1988-05-01|

---

ES8702495A1|1987-01-01|

---

IL74507D0|1985-06-30|

---

AU589355B2|1989-10-12|

---

GR850571B|1985-07-08|

---

EP0154539A2|1985-09-11|

---

JPS611387A|1986-01-07|

---

DK99985A|1985-09-07|

---

HUT40162A|1986-11-28|

---

NZ211313A|1988-05-30|

---

DK99985D0|1985-03-05|

---

ES550012A0|1987-06-01|

---

EP0154539A3|1986-12-30|

---

PT80051B|1987-03-24|

---

CA1283374C|1991-04-23|

---

ES550013A0|1987-01-01|

---

CN85101555A|1987-01-24|

---

AU3950285A|1985-09-12|

---

KR850006703A|1985-10-16|

---

ZA851626B|1986-10-29|

---

ES8802322A1|1988-05-01|

---

PT80051A|1985-04-01|

---

KR900001015B1|1990-02-24|

---

ES8706207A1|1987-06-01|

---

EG17786A|1990-10-30|

---

HU202587B|1991-03-28|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US58659284A| true| 1984-03-06|1984-03-06|

---