The method of producing oxytocin and recombinant plasmid dna pteilox4 and the escherichia coli strain bl21 (de3)/pteilox4 for its implementation

 

(57) Abstract:

The invention relates to biotechnology, in particular genetic and protein engineering. The method of producing oxytocin is based on the use of the new recombinant plasmid DNA pteilox4 that encodes a hybrid protein ILOX4, the amino acid sequence of which consists of N-terminal (63 amino acids) of the sequence of human interleukin-3 and oligomer oxytocin-containing Monomeric units, flanked lysine residues for specific enzymatic cleavage, and the producer strain E. coli BL21(DE3)/pteilox4, providing a high level of biosynthesis of hybrid protein (up to 30% of total cell protein). Recombinant plasmid pteilox4 consists of a HindIII/EcoR1 fragment of DNA plasmids pTE21L3 containing the promoter R8 8 gene of phage fd, the promoter and the amplifier broadcast of gene 10 of phage T7 transcription terminator of phage fd gene-lactamase and N-terminal part of the gene interleukin-3, and HindIII/EcoRI fragment representing a synthetic gene of the tetramer oxytocine-lysine. The Escherichia coli strain BL21 (DE3)/pteilox4 obtained by transformation of competent cells of E. coli BL21 (CARRIED) the corresponding plasmid pteilox4. Strain deposited under number VKPM B-7757. The Escherichia coli strain BL21 (DE3)/pteLOX4. Its enzymatic break down by subsequent or simultaneous recovery of disulfide bonds. Removal of C-terminal amino acid residue of the oxytocin of Decapeptide formed by splitting a hybrid protein is carried out either using carboxypeptidase Y (with simultaneous terminal amidation of glycine residue in the oxytocin) or using carboxypeptidase In subsequent chemical amidation terminal amino acids (glycine). The result is a stable recombinant protein ILOX4. The process is simpler, and the target product more sustainable. 3 S. p. f-crystals, 2 Il.

The present invention relates to biotechnology, in particular genetic and protein engineering. It includes a constructed in vitro recombinant plasmid DNA pteilox4 contributing to the biosynthesis of hybrid protein ILOX4, amino acid sequence which includes 63 N-terminal amino acids of human interleukin-3 and the tetramer oxytocine-lysine, the E. coli strain BL21(DE3)/pteilox4 producing a hybrid protein ILOX4 and a method of producing oxytocin on the basis of the above recombinant DNA and producer strain.

The peptide hormone oxytocin is secreted in human organismen smooth muscles of the uterus and the secretion of milk Breasts [3,4]. Thanks oxytocin is widely used in medicine and veterinary medicine [5].

The difficulties of isolation and purification of natural hormone make it inaccessible. Almost used oxytocin, obtained by chemical synthesis. The disadvantage of the chemical method is the complexity and the need for treatment of drug from other ones on the chemical nature of the impurities present in the drug due to inherent limitations of the synthetic process (non-quantitative outputs peptide synthesis and reactions of removal of the protective groups and others).

In this regard, a more promising is the method of obtaining hormone microbiological synthesis, which provides the ability to obtain the target product with a high yield from a relatively inexpensive starting material. It is known, however, that short peptides in bacterial cell quickly degrade. Therefore, to avoid degradation of the peptide, it must be obtained in the structure of the hybrid protein carrier and to allocate after chemical or enzymatic cleavage. The literature describes many such hybrids, in which the role of carrier protein performs-galactosidase, tumor necrosis factor (TNF), interferon and other is a small fraction of the peptide in the composition of the hybrid protein [6, 7, 8]. Oxytocin in the composition of the hybrid protein ILOX3 described by us in 1993, and the method of its production in 1997 issued a patent [9]. This method is the closest proposed in this invention and includes the cultivation of the producer strain obtained by transformation of Escherichia coli cells TG1 plasmid DNA TOTEilox3 with subsequent destruction of cells in the buffer solution, the selection of the hybrid protein ILOX3, its enzymatic cleavage by subsequent or simultaneous recovery of disulfide bonds and the replacement of the generated Decapeptide C-terminal amino acid residue on aminogroup. However, as shown by our further research, a hybrid protein ILOX3 and its higher homologues proved to be unstable and degraded in the process of biosynthesis, so that the destination hybrid protein contains on average at least three Monomeric units of oxytocin [10].

The invention solves the problem of increasing the level of biosynthesis and stability of a new recombinant protein ILOX4, and simplifying the technology of production of recombinant oxytocin.

The problem is solved due to the fact that in the method of producing oxytocin, which includes the cultivation of the producer strain obtained by transformation of cells Escher is Elenium its enzymatic way with subsequent or simultaneous recovery of disulfide bonds and the replacement of the generated Decapeptide C-terminal amino acid residue on aminogroup, and characterized in that the quality of plasmid DNA using a specially designed DNA pteilox4, as a producer strain using strain Escherichia coli BL21(DE3)/pteilox4, which are cultivated in the rich environment and after the destruction of the cells produce a hybrid protein ILOX4, which is converted into the target product.

Use of recombinant plasmid DNA pteilox4,

- encoding the amino acid sequence of the hybrid protein ILOX4, including 63 N-terminal amino acids of human interleukin-3 and tetramer of oxytocin, flanked by lysine residues,

- having a molecular weight 2,19 Hmm,

- consisting of:

HindIII/EcoRI-fragment I DNA plasmids pTE2IL3 containing the promoter R8 8 gene of phage fd, the promoter and the amplifier broadcast of gene 10 of phage T7 transcription terminator of phage fd gene-lactamase and N-terminal part of the gene interleukin-3,

HindIII/EcoRI-fragment II, representing a synthetic gene of the tetramer oxytocine-lysine,

- contains:

as a genetic marker gene-lactamase determining the stability of the transformed plasmid pteilox4 of E. coli cells to penicillin antibiotics,

unique recognition sites of restriction endonucleases, located on the following p. O.

Use producing strains of Escherichia coli BL21(DE3) containing the recombinant plasmid DNA pteilox4, producing a hybrid protein ILOX4.

The present invention allows to obtain recombinant oxytocin on simple technology and high output.

Construction of recombinant plasmid DNA pteilox4 provides a high level of expression of a hybrid gene containing the gene of the tetramer oxytocine-lysine.

To construct plasmids used chemical approach to create optimal conditions for bacterial expression of variants of the structural gene and regulatory elements that control its expression.

Gene tetramer oxytocin, flanked by lysine residues, derived from appropriate synthetic oligonucleotides by enzymatic crosslinking of DNA-ligase vector fragments in three stages.

The first source vector fragment is known recombinant plasmid DNA pTE21L3 encoding human interleukin-3 [11]. The gene expression of interleukin-3 in this plasmid is controlled by the promoter R8 phage fd, as well as the promoter and the amplifier broadcast of gene 10 of phage T7. The E. coli strain BL21(DE3) containing plasmid ment is known recombinant plasmid DNA pte2il3g, modified by inserting in website restrictase EcoRI polylinker fragment (plasmid pte2il3ghll) [12]. At the end of the il3 gene of this plasmid lacks a termination codon and after site restrictase contains EcoRI site restrictase SalI.

Recombinant plasmid DNA pTE21L3 cleaved by endonucleases HindIII and EcoRI, the resulting fragment are ligated with the first synthetic HindIII/EcoRI fragment containing the gene dimer oxytocin (duplex A, Fig. 2), and obtain recombinant plasmid DNA pteilox2-1.

Recombinant plasmid DNA pte2il3gh11 cleaved by endonucleases EcoRI and SalI, the resulting fragment are ligated with the second synthetic EcoRI/SalI fragment containing the gene dimer oxytocin (duplex B, Fig. 2), and obtain recombinant plasmid DNA pteilox2-2.

Both recombinant plasmid DNA, pteilox2-1 and pteilox2-2, split by restrictase EcoRI and EcoRV and selected after electrophoresis in agarose gel fragments (plasmids pteilox2-1 - small, 255 p. O., plasmids pteilox2-2 - big, 3058 p. O.) are ligated and receive recombinant plasmid DNA pteilox4.

Offer producing strains of Escherichia coli BL21(DE3)/pteilox4 characterized by the following features:

Morphological features. Cells are rod-shaped form, gramotritsatelnymi on agar "Difco" - colonies are round, smooth, dull, shiny grey, smooth edge. With the growth in liquid medium (minimal medium with glucose or YT-broth) intensive form a smooth suspension.

Physical and biological characteristics. Cells grow at temperatures from 4 to 40oC at the optimum pH of from 6.8 to 7.5. As the source of nitrogen used as a mineral salt in ammonium form, and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. as a source of carbon use amino acids, glycerol, carbohydrates.

Resistance to antibiotics. Cells are resistant to penicillin antibiotics (up to 500 µg/ml).

Producing strains of E. coli BL21(DE3)/pteilox4 differs from strain-recipient E. coli BL21 (D3) only in the presence of recombinant plasmid DNA pteilox4, which gives it resistance to penicillin antibiotics.

Strains-producers obtained by transformation of competent cells of E. coli BL21(DE3) corresponding recombinant plasmid DNA.

Cells of E. coli BL21(DE3)/pteilox4 are producing a hybrid protein ILOX4. When induction of isopropylthio- -D-galactoside, and without induction with constitutive synthesis, there is an effective bio is 30% of the total protein of bacteria.

Producing strains deposited in Russian national collection of industrial microorganisms, N VKPM B-7757 dated March 16, 1999

The invention is as follows. Construct recombinant plasmid DNA pteilox4 in three stages, as described above. At the third stage are ligated EcoRI/EcoRV fragments of plasmids pteilox2-1 and pteilox2-2.

Ligase mixture transform competent cells of E. coli BL21(DE3) and plated on YT agar containing 50 µg/ml ampicillin or other penicillin antibiotic. The resulting clones analyzed by hybridization32P-labeled oligonucleotides C and D (Fig.2), and hybridization of clones secrete plasmid DNA, which is subjected to restriction analysis using restricted HindIII, EcoRI and BglII, and determination of nucleotide sequence between sites EcoRV and EcoRI. Producing strains of E. coli BL21(DE3)/pteilox4 grown in rich medium (YT-, LB-broth and others) (or induce isopropylthio-D-galactoside, and again is grown) to achieve maximum culture density.

The selection of the hybrid protein ILOX4 from cells of a producer includes the following stages:

the destruction of the grown cells one of the commonly used methods;

- washing buffer solutions Taurus enable from CBR 5-8 M urea solution, either a 5 M solution of guanidine hydrochloride, or another suitable solvent;

purification of the hybrid protein in the stepwise dilution of the solution in the urea or guanidine hydrochloride, or other methods.

The splitting of the hybrid protein is carried out by enzymatic followed by restoration of disulfide bonds for separation formed by the cleavage of peptide fragments. Enzymatic cleavage of the hybrid protein in which the Monomeric units of oxytocin flanked lysine residues using trypsin, and then restore the disulfide bonds using mercaptoethanol.

Removal of C-terminal amino acid residue of oxytocine-lysine formed by splitting a hybrid protein is carried out either using carboxypeptidase Y (with simultaneous terminal amidation of glycine residue in the oxytocin) or using carboxypeptidase In subsequent chemical amidation terminal amino acids (glycine) and receive oxytocin.

In Fig. 1 shows a partial structure of the plasmid pteilox4 in the gene region of the hybrid protein ILOX4 and corresponding amino acid sequence of the hybrid gene of oxytocin, and porosity of restricts that these duplexes was inserted into the vector plasmid pte2i13 (A duplex) and pte2il3gh11 (duplex B).

The invention is illustrated in the following examples.

Example 1. Construction of recombinant plasmid DNA pteilox4

Chemical synthesis of oligonucleotides perform solid-phase fostamatinib method on a DNA synthesizer ASM-102U (BIOSSET, Novosibirsk) with increasing oligonucleotide chain in the direction from the 3'end to the 5'-end using secure phosphamidon-5'-dimethoxytrityl-N-acyl-2'-deoxynucleoside-3'-O- ( -caitididmarie)-phosphites activated by tetrazole. The synthesis is carried out in the scale of 0.5-0.7 µm, using as a carrier of porous glass (pore size 500 A), to which 3'-succinate connection joining the first nucleoside link (load 20-30 µmol/g). Use synthetic cycle, described in [13].

For preparation of vector DNA plasmids pte2i13 (3 μg, 1 pmol) treated with 40 μl of buffer G (100 mm K-glutamate, pH 8,8, 25 mm Tris-acetate, pH of 7.6, 10 mm Mg-acetate, 1 mm mercaptoethanol) restrictase HindIII (10 units act. ) and EcoRI (10 units act.) for 1 h at 37oC. Vector fragment size 3,19 T. p. O. after electrophoresis in 1% agarose.

Synthetic fragment with the gene dimer of oxytocin (A) (Fig. 2) in an amount of 2 pmol, was added to a solution of 1 μg of the above described vector fragment in 10 μl of buffer L (20 mm Tris-HCl, pH 7,56, 10 mm MgCl2, 0.2 mm rATP, 10mm dithiotreitol) and are ligated with 10 units of the act. T4-DNA ligase for 6 h with 10oC.

An aliquot of the reaction mixture used to transform competent cells of E. coli BL21(DE3). Transformants plated on plates with YT-agar containing 50 μg/ml ampicillin. Screening of recombinants carried out by using hybridization of colonies in situ with32P-labeled oligonucleotide D (Fig.2). From hybridization of clones secrete DNA plasmids pteilox2-1 and analyzed by endonucleases HindIII and EcoRI, and determining the nucleotide sequence of the regulatory area, the beginning of the structural gene and the cloned synthetic fragment.

Similarly, DNA plasmids pte2i13gh11 (3 μg, 1 pmol) using restricted EcoRI (10 units act.) and SalI (10% act.) prepare EcoRI/SalI-vector value of 3.3, etc., of O., 1 µg of which are ligated with 2 pmol of the synthetic fragment with the gene dimer oxytocin (B) (Fig.2), and after transformation of competent cells of E. coli BL21(DE3) get clones of recombinants, of which there are DNA plasmids pteilox2-2.

32P-labeled oligonucleotides C and D (Fig.2). From the hybridization of two oligonucleotides clones secrete plasmid DNA pteilox4, which is analyzed by endonuclease XbaI, HindIII, EcoRI and SalI, and determining the nucleotide sequence of the regulatory area, the beginning of the structural gene and the cloned synthetic fragment.

Example 2. Obtaining E. coli strain BL21(DE3)/pteilox4 (PMBC 7757) - producer of the hybrid protein ILOX4 and determine its productivity.

Cells of E. coli BL21(DE3) carrying plasmid pteilox4, the structure of which is confirmed by the data analysis (see example 2), are producing a hybrid protein ILOX4.

The producer strain E. coli BL21(DE3)pteilox4 grown at 37oC in 100 ml of YT-broth (pH 7.0) with 50 mcg/ml ampicillin for 2 h on a rocking chair with speed 190 rpm to the turbidity of A550of 0.7-0.8, add isopropylthio- -D-galactoside to a concentration of 0.2 mm and continue the process for another 6 hours Every hour take a sample of 2 ml, define A550and the amount of culture, soota with dye bromophenol blue handle 20 sec ultrasound, heated 3 min at 100oC and the samples, 1 μl used for electrophoresis in 15% SDS-PAG. Gel stain, Kumasi R-250 according to standard methods and scan to determine the relative amount of protein in the band of the hybrid protein.

Example 3. Receiving oxytocin.

1) Isolation of the hybrid protein ILOX4.

Wet cells (100 g) is suspended in 200 ml of buffer (50 mm Na-phosphate, 1 mm EDTA, pH 7.5), add lysozyme (100 µg/ml), incubated for 30 min at 20oC, add a solution of MgCl2(10 mm) and Tenkasu (10 μg/ml), incubated at 20oC to viscosity loss (30 min), diluted with 2 l of buffer (50 mm Na-phosphate, 10 mm EDTA, 4 M urea, 1% Triton X100, pH 7.5) and centrifuged 20 min at 10000 g. The residue is suspended in 2 l of buffer (50 mm Na-phosphate, 10 mm EDTA, 4 M urea, 1% Triton X100, pH 7.5) using an ultrasonic disintegrator Sonifier 240 (Branson) and centrifuged 20 min at 10000 g. Washing buffer I repeat, then similarly washed with buffer (50 mm Na-phosphate, 1 mm EDTA, pH 7.5). Sediment resuspended in 2 l of buffer (50 mm Na-phosphate, 1 mm EDTA, 5 M guanidine hydrochloride, pH 7.5) and incubated at 20oC. the resulting solution was diluted with stirring 8 l of buffer (50 mm Na-phosphate, 1 mm EDTA, pH 7.5) and centrifuged.

2) Enzymatic hydrolysis of hibri money-carbonate buffer, pH 8.5, treated with 2 mg of trypsin 6 h at 37oC to dissolve the precipitate. Complete hydrolysis of the protein is controlled by HPLC using a resin Sepharon C-18 in the concentration gradient of 20-80% acetonitrile in 0.1% triperoxonane acid.

To 10 ml trypsinogen of the hydrolyzate ILOX4 added 1 mg of carboxypeptidase B and incubated for 1 h at 37oC. the Solution is frozen, lyophilized and produce dezaminooxytocin reversed-phase chromatography on resin Sepharon C-18 in the concentration gradient of 20-80% acetonitrile in 0.1% triperoxonane acid.

3) the Transformation of oxytocin acids in oxytocin

50 mg oxytocin acid dissolved in 2 ml of 0.25 M HCl in methanol and incubated for 1.5 hours at room temperature. To the obtained solution of methyl ester oxytocin acid was added 2 ml of 10 M methanol NH3the mixture was incubated for 18 h at room temperature and evaporated. Receive 40 mg of oxytocin.

LITERATURE

1. Lederis K.//Gen.Compar. Endocrinol. 1961. V. 1. N 1. p. 80-86.

2. Du Vigneaud V.//Science. 1956. V. 123. p.956.

3. Du Vigneaud V, Ressler Ch., Trippet S. // J. Biol.Chem. 1953. V. 205. p.949-957.

4. Dale H. H./Biochem.J. 1909. V. 4. p.427-447.

5. Mashkovsky, M. D.// "Drugs". M, Medicine. So 2. C. 72.

6. Black 21063, priority from 14.11.85, CL C 12 N 15/00.

8. USSR author's certificate N 1724691, priority from 20.06.90, CL C 12 N 15/42.

9. RF patent N 20085585, priority from 18.06.93, CL C 12 N 15/16.

10. A. I. Gurevich, I. A. Kachalina, A. L. Cousin, M. D. Korosteleva, K. C. Maltsev, O. A. Mirgorodsky, A. I. Miroshnikov. Bioorgan, chemistry, I. 22, No. 1, 1996, S. 14-19.

11. A. I. Gurevich, N. In.Kaptsova, S. C. Lutsenko, V. A. Smirnov, A. N. Kurkin, A. C. Agaev. Bioorgan, chemistry, I. 17, No. 5, 1991, S. 647-653.

12. A. I. Gurevich, P. S. Esipov, T. A. Kachalina, A. L. Cousin. Bioorgan, chemistry, I. 21, N 4, 1995, S. 282-288.

13. Atkinson T. , Smith M.//in: Oligonucleotide synthesis; apractical approach. 1984. Ed. Gait, M. J. p. 35-81. IRL Press, Oxford.

1. The method of producing oxytocin, which includes the cultivation of the producer strain obtained by transformation of Escherichia coli cells plasmid DNA with subsequent destruction of cells in the buffer solution, the selection of the hybrid protein, its enzymatic cleavage by subsequent or simultaneous recovery of disulfide bonds and the replacement of the generated Decapeptide C-terminal amino acid residue on aminogroup, characterized in that the quality of plasmid DNA using a specially designed DNA pteilox4, as a producer strain using strain Escherichia coli BL converted into the target product.

2. Recombinant plasmid DNA pteilox4 encoding the amino acid sequence of the hybrid protein ILOX4, including 63 N-terminal amino acids of human interleukin-3 and tetramer of oxytocin, flanked by lysine residues, having a molecular weight 2,19 Hmm, consisting of: HindIII/EcoRI-fragment I DNA plasmids pTE2IL3 containing the promoter R8 8 gene of phage fd, the promoter and the amplifier broadcast of gene 10 of phage T7 transcription terminator of phage fd gene-lactamase and N-terminal part of the gene interleukin-3 and Hindlll/EcoRI-fragment II, representing a synthetic gene of the tetramer oxytocine-lysine, containing as a genetic marker gene-lactamase determining the stability of the transformed plasmid Pteilox4 of E. coli cells to penicillin antibiotics, a unique recognition sites of restriction endonucleases that are located in the following distance to the right of the NdeI site: HpaI - 46 p. O., EcoRI - 251 p. O., BgIII - 674 p. O., PST - 2073 p. O., BamHI - 3215 p. O., XbaI - 3273 p. O., EcoRV - 3309 p. O.

3. The Escherichia coli strain BL21(DE3), VKPM B-7757 containing recombinant plasmid DNA pteilox4, producing a hybrid protein ILOX4.6

 

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