RU2593172C2 - RECOMBINANT PLASMID DNA pER-TA1 GyrA-AcSer CODING SERINE ACETYLTRANSFERASE CAPABLE OF in vivo ACETYLATION OF N-TERMINAL SERINE DEACETYL-THYMOSIN α1 AND HYBRID PROTEIN CAPABLE OF AUTOCATALYTIC BREAKDOWN TO FORM HUMAN THYMOSIN α1, STRAIN OF Eschrichia coli C3030/pER-TA1GyrA-AcSer PRODUCER OF SAID PROTEINS AND METHOD OF PRODUCING GENETICALLY ENGINEERED HUMAN THYMOSIN - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to genetic and protein engineering and represents recombinant plasmid DNA pER-TA1GyrA-AcSer, intended for simultaneous biosynthesis of two proteins - hybrid polypeptide containing amino acid sequence of human thymosin α1 and intein, and enzyme E.coli - serine acetyl transferase, having molecular weight 5.04 MDa. Plasmid consists of BsaBI/NdeI DNA fragment of plasmid pTWTN-1, containing T7 promoter sequence and operator lacO; Ndei/PstI DNA fragment containing a sequence of hybrid protein, including adapted to said sites sequence of recombinant human thymosin α1, intein sequence Moss GyrA and sequence of chitin binding domain; Psti/NcoI DNA fragment coding reduced Shine-Dalgarno sequence and non-standard as codon-start amino acid valine; NcoI/XhoI DNA fragment containing a sequence of serine N-acetyl transferase; DNA fragment containing genetic marker of β-lactamase gene, determining resistance of transformed by plasmid pER-TA1GyrA-AcSer cells of E.coli to penicillin antibiotics; DNA fragment containing a sequence coding replication start point; DNA fragment coding sequence of lactose repressor LacI. Invention also relates to a strain Escherichia coli C3030/pER-TA1GyrA-AcSer, producing hybrid polypeptide, containing amino acid sequence of human thymosin α1 and intein, as well as serine acetyl transferase obtained by transformation of cells of Escherichia coli strain S3030 of recombinant plasmid DNA pER-TA1GyrA-AcSer.

EFFECT: invention enables to obtain recombinant human thymosin Alpha 1 with high output and simple production technology.

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Description

The invention relates to biotechnology, in particular to genetic and protein engineering. It can be used to produce recombinant human thymosin alpha 1.

Thymosin alpha 1 is a polypeptide produced by the thymus gland, which affects the cells of the immune system involved in the antiviral response, and also directly affects hepatocytes infected with the virus. The immunomodulating effect of thymosin alpha 1 is manifested in an increase in the number of CD4, CD8 lymphocytes and NK cells, as well as in bringing the immune response to the Th1 subtype. The antiviral effect is expressed in an increase in the expression of molecules of the main histocompatibility complex of the 1st class on infected cells and a direct suppression of virus reproduction.

Human thymosin alpha 1 is a 28-membered polypeptide acetylated at the N-terminal α-amino group (SEQ ID NO 2).

In world practice, thymosin alpha 1 is isolated from natural sources or obtained by chemical synthesis.

A known method for producing thymosin α1 from calf thymus using multistage chromatographic purification

(Goldstein, AL, T. LK Low, M. Mcadoo, J. Mcclure, G. B. Thurman, JL Rossro, CY Lai, D. Chang, SS Wang, C. Harvey, AH Ramel & J. Meienhofer. 1977. Proc. Nat. Acad. Sci. US 74: 725).

The disadvantage of this method is the difficulty of cleaning and the high cost of the resulting peptide.

An alternative way to obtain thymosin α1 is a biotechnological pathway using the expression of a recombinant gene in bacteria.

A known method of producing thymosin α1, in which the microbiological synthesis of a hybrid protein is carried out, including the amino acid sequence of thymosin alpha 1 and beta-galactosidase Escherichia coli

(Wetzel, R., Heyneker, HL, Goeddel, DV, Jhurani, P., Shapiro, J., Crea, R., Low, TLK, McClure, JE, Thurman, G. B., & Goldstein, AL Biochem. 1980. V. 19. P. 6096-6104).

The design described in this work has several disadvantages. Firstly, the hybrid protein β-galactosidase - thymosin α1 contains 24 methionines, which after treatment with bromine cyan leads to a complex mixture of products, the isolation of which target product is almost impossible. Secondly, the proportion of the target peptide in the hybrid protein is not more than 3%, which invalidates the high expression of the hybrid protein in bacterial cells.

A known method in which instead of a "carrier" was used a small protein - tumor necrosis factor

(V.G. Korobko, E.F. Boldyreva, S.A. Filippov, N.P. Berkova, V.N. Dobrynin, V.A. Shmelev, S.G. Popov, S.I. Evsegneev, L. .U. Nosova. Bioorg. Chemistry 1992. V. 18. N 5 R. 646-659), (Shmelev V.A., Bunina Z.F., Kudryavtseva T.Yu. Molecular genetics, microbiology and virology. - 1995. V1. 9-14).

The disadvantage of this method was the decrease in the efficiency of cleavage of the hybrid protein compared with beta-galactosidase. The cleavage of the hybrid protein occurred with a small yield of the target peptide and was accompanied by the formation of difficult to separate products of nonspecific reactions.

A more promising design of a hybrid protein with a modified Sce VMA intein is known, capable of autocatalytic cleavage in the presence of a thiol reagent

(R.S. Esipov, A.I. Gurevich, V.N. Stepanenko, L.A. Chupova, D.V. Chuvikovsky, A.I. Miroshnikov. Bioorg. Chemistry 2004. V. 30 N5. P. 481 -486).

The disadvantage of this method was that the process of cleavage of the hybrid protein occurred uncontrollably - in the in vivo stage and subsequent stages of purification of the hybrid protein, without adding a thiol reagent.

The disadvantage of all the above methods was the need for chemical acetylation of the peptide to obtain the native structure of thymosin α1.

Closest to the claimed method for producing thymosin α1 is described in (YuantaoRen, Xueqin Yao, Hongmei Dai, Shulong Li, Hongqing Fang, Huipeng Chen and Changlin Zhou. Microbial Cell Factories 2011. 10:26). In this work, acetylation of the N-terminal serine of thymosin α1 was carried out in vivo by alanine N-acetyltransferase from E. coli. For this, the sequence of the modified intein Spl DnaX and thymosin α1 was cloned into the multi-copy plasmid pET22b (+), and the sequence of the alanine acetyltransferase (RimJ) was cloned into the low-copy plasmid pACYCDuet-1. Then E. coli strain BL21 (DE3) was transformed with the obtained plasmids.

The disadvantage of this method of producing acetylated peptide is that the presence of two plasmids in the producer strain requires the presence of two different antibiotics, which, when scaled, increases production costs. Another disadvantage of this method is the choice of alanine acetyltransferase as an acetylating enzyme that is not specific to serine, the N-terminal amino acid.

The invention solves the problem of obtaining a highly productive recombinant bacterial producer strain, allowing to obtain recombinant human thymosin α1 in high yield and by simplified technology.

The problem is solved due to the fact that:

1. The expression plasmid pER-TA1GyrA DNA was constructed by cloning the thymosin α1 gene into the vector plasmid pTWIN1. The plot encoding the altered Shine-Dalgarno sequence and the amino acid valine, which is non-standard as a start codon, is synthesized from two oligonucleotides and cloned into the vector pER-TA1GyrA. Next, using the artificially synthesized oligonucleotides, the portion of the Escherichia coli genome encoding the serine acetyltransferase is amplified and cloned into the vector plasmid pER-TA1GyrA-RBS. Thus, the vector pER-TA1GyrA-AcSer is obtained.

2. By transforming the plasmid DNA pER-TA1GyrA-AcSer of Escherichia coli C3030 cells, a producer strain is obtained,

3. In the induced cultivation of the obtained producer strain, the biosynthesis of the recombinant serine acetyltransferase and the accumulation in vivo of the TA1GyrA soluble fusion protein acetylated at the N-terminal α-amino group containing, along with thymosin alpha 1 human, the sequence of mini-intein Mhe Gyr A.

4. Cellular biomass is destroyed in the buffer solution and a clarified cell lysate containing the TA1GyrA fusion protein is isolated. TA1GyrA fusion protein is applied to a chitin sorbent balanced in a buffer containing 100 mM NaCl, washed with a buffer containing 2 M urea, and balanced in a buffer containing 100 mM DTT, thereby inducing autocatalytic cleavage of the fusion protein, then incubating for 24 hours at 25 ° C . The cleaved thymosin alpha 1 is then eluted in the same buffer. Further purification and analysis of recombinant human thymosin α1 is carried out by reverse phase chromatography. Identification of the resulting recombinant human thymosin α1 is carried out using mass spectrometry.

The technical result of autocatalytic cleavage of the hybrid protein is the formation of thymosin α1, the yield of which reaches 3% relative to the total protein of the cell with a drug purity of at least 98%.

To avoid the difficulties associated with the cleavage of the recombinant fusion protein using various proteases, such as enterokinase, factor X, etc., as well as to reduce the cost of this step, the mini-intein GyrA from Mycobacterium xenopi (Telenti, A., Southworth, M., Alcaide, F., Daugelat, S., Jacobs, WR Jr. and Perler, FB (1997). J. Bacteriol .. 179, 6378-6382.) For targeted autocatalytic cleavage of a hybrid into a target polypeptide and intein . For this purpose, the thymosin gene α1 (SEQ ID NO 1) is amplified by PCR using a plasmid with the artificial thymosin gene α1 as a template and cloned into the vector plasmid pTWIN1 containing the integin gene from Mycobacterium xenopi GyrA and the chitin binding site ( Chitin-Binding Domen) from Bacillus circulans (Telenti, A., Southworth, M., Alcaide, F., Daugelat, S., Jacobs, WR Jr. and Perler, FB (1997). J. Bacteriol .. 179, 6378 -6382; Evans, J, TC, Bermer, J., Xu, M.-Q., (1999) J. Biol. Chem., 274, 18359-18363), at restriction enzyme sites NdeI and SapI. Further, in order to obtain a polycistronic design, the region encoding the altered Shine-Dalgarno sequence, the amino acid valine nonstandard as the start codon, and the NcoI and XhoI restriction endonuclease sites are synthesized from two oligonucleotides and cloned into the pTWIN1 vector at the Pst I and BamHI restriction endonuclease sites. Then, using the artificially synthesized oligonucleotides, the portion of the Escherichia coli genome encoding the serine acetyltransferase is amplified and cloned into the previously obtained vector plasmid pER-TA1GyrA-RBS at the NcoI and XhoI restriction endonuclease sites. The resulting expression plasmid pER-TA1GyrA-AcSer, the structure of which is shown in FIG. 1, transform E. coli C3030 cells.

The TA1GyrA fusion protein resulting from expression of the recombinant gene is further processed in vivo with cleavage of N-formylmethionine and selectively acetylated at the N-terminal serine. This fusion protein is capable of autocatalytic cleavage into human thymosin α1 and intein. The yield of thymosin α1 of high purity (98%) after reverse phase chromatography reaches 3% relative to the total protein of the cell.

The proposed technical solution uses a producer strain Escherichia coli C3030 containing plasmid DNA pER-TA1GyrA-AcSer for superproduction and production of two proteins: a hybrid protein TA1GyrA containing a sequence of human thymosin α1 and intein Mxe GirA, and the enzyme, respectively, of E. coli serine acetyltransferase .

Use recombinant plasmid DNA pER-TA1GyrA-AcSer

- the coding amino acid sequence of recombinant human thymosin α1;

- consisting of the BsaBI / NdeI DNA fragment of the plasmid pTWIN-1, containing the sequence of the T7 promoter and the lacO operator; An NdeI / PstI DNA fragment comprising a fusion protein sequence comprising a recombinant human thymosin α1 gene sequence adapted to these sites, a Mxe GyrA intein sequence, and a chitin binding domain sequence; PstI / NcoI DNA fragment encoding a weakened Shine-Dalgarno sequence and the amino acid valine, which is non-standard as a start codon; An NcoI / XhoI DNA fragment containing the sequence of a serine N-acetyltransferase;

- containing a β-lactamase gene as a genetic marker, which determines the resistance of E. coli cells transformed with plasmid pER-TA1GyrA-AcSer to penicillin antibiotics; a sequence encoding a replication start point; DNA fragment encoding the LacI lactose repressor sequence.

The design of the recombinant plasmid DNA pER-TA1GyrA-AcSer provides a high level of expression of the TA1GyrA fusion protein gene cloned therein containing the thymosin alpha 1 gene connected to the Mxe GyrA gene at the 5 ′ end. To construct a plasmid, a chemical approach is used, which allows the use of optimal regulatory elements that control its expression for the expression of the cloned structural gene.

The human thymosin gene α1 is obtained by amplification of the plasmid pIntTim3 (R.S. Esipov, A.I. Gurevich, V.N. Stepanenko, L.A. Chupova, D.V. Chuvikovsky, A.I. Miroshnikov. Bioorganic chemistry 2004 V. 30 N5. P. 481-486). Its end sections containing the restriction endonuclease sites corresponding to the vector are introduced by PCR with synthetic oligonucleotide primers A1 and B1 (SEQ ID NO 3) and then the gene is cloned into the vector plasmid pTWIN1.

The proposed producer strain Escherichia coli C3030 / pER-TA1GyrA-AcSer is characterized by the following features:

Morphological signs. The cells are rod-shaped, gram-negative, non-spore.

Cultural signs. Cells grow well on simple nutrient media. When growing on Difco agar, the colonies are round, smooth, cloudy, shiny gray, the edge is even. When growing on liquid media (on a minimal medium with glucose or LB broth) they form an intense smooth turbidity.

Physico-biological signs. Cells grow at temperatures from 4 ° C to 40 ° C with optimum pH from 6.8 to 7.5. As a source of nitrogen, both mineral salts in the ammonium form and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. are used. Amino acids, glycerin, carbohydrates are used as a carbon source.

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

The strain producing E. coli C3030 / pER-TA1GyrA-AcSer differs from the strain of the recipient E. coli C3030 only in the presence of recombinant plasmid DNA pER-TA1GyrA-AcSer, which gives it resistance to penicillin antibiotics.

Producer strains are obtained by transformation of competent E. coli C3030 cells with the corresponding recombinant plasmid DNA.

E. coli C3030 / pER-TA1GyrA-AcSer cells are both superproducer and producer. When isopropylthio-β-D-galactoside is induced, the biosynthesis of the TA1GyrA fusion protein is effective and accumulates in the cells in the amount of 15% of the total protein of the cell in soluble form. The production of the enzyme is 6% relative to the total proteins of the cell.

The invention is as follows. Recombinant plasmid DNA pER-TA1GyrA-AcSer was constructed, for which the recombinant human thymosin α1 gene was amplified by PCR with synthetic oligonucleotide primers (SEQ ID NO 3) containing NdeI restriction enzyme sites (gene N-terminus, primer A1I and S1). the end of the gene, primer B1), the obtained DNA is digested with the appropriate restriction enzymes and then ligated with the vector plasmid pTWIN1 cleaved at the same sites. Next, the region encoding the altered Shine-Dalgarno sequence, the amino acid valine non-standard as the start codon, and the restriction endonucleases PstI and BamHI, NcoI and XhoI (SEQ ID NO 6) are synthesized from two oligonucleotides and cloned into the resulting plasmid pER-TA1GyrA at the sites restriction of PstI and BamH1. The serine acetyltransferase gene is obtained by amplification of the E. coli genome by PCR with primers B3 and A3 (SEQ ID NO 5) containing the restriction endonuclease sites NcoI and XhoI. The resulting DNA was digested with the appropriate restriction enzymes and then ligated with the plasmid pER-TA1GyrA-RBS cleaved at the same sites. Competent E. coli C3030 cells are transformed with a ligase mixture and plated on LB agar containing 50 μg / ml ampicillin or another penicillin antibiotic. The resulting clones are analyzed by sequencing.

The strain producing E. coli C3030 / pER-TA1GyrA-AcSer is grown in a rich medium (YT-, LB-broth, etc.) (or isopropylthio-β-D-galactoside is induced and re-grown) to achieve maximum culture density.

(SEQ ID NO 1) shows the structure of the recombinant human thymosin α1 gene. The (SEQ ID NO 2) depicts the amino acid sequence of recombinant human thymosin alpha 1.

The invention is illustrated by the following figures.

Fig 1. Physical map of the plasmid pER-TA1GyrA-AcSer.

Fig 2. The electrogram of the lysate of the cells of the producer strain of E. coli C3030 / pER-TA1GyrA-AcSer,

The invention is illustrated by the following examples.

Example 1

Construction of recombinant plasmid DNA.

), к которому через 3′-сукцинатную связь присоединяют первое нуклеозидное звено (нагрузка 20-30 мкмоль/г). Используют синтетический цикл стандартного фосфоамидитного метода.Chemical synthesis of oligonucleotides is carried out by the solid-state phosphoamidite method on an ASM-102U DNA synthesizer (BIOSET, Novosibirsk) with the oligonucleotide chain being expanded from the 3′-end to the 5′-end using protected phosphamidites - 5′-dimethoxytrityl-N-acyl-2 T-deoxynucleoside-3′-O- (β-cyanethyl-diisopropylamino) phosphites activated by tetrazole. The synthesis is carried out on a scale of 0.5-0.7 μmol, using porous glass as a carrier (pore size 500

), to which, through a 3′-succinate bond, the first nucleoside unit is attached (load 20-30 μmol / g). The synthetic cycle of the standard phosphoamidite method is used.

To prepare the DNA vector of plasmid pTWTN1 (3 μg, 1 pmol) is treated with 40 μl of Y buffer (33 mm Tris-acetate, pH 7.9, 10 mm Mg-acetate, 66 mm K-acetate 1, 0.5 mm DTT, 0.1 mg / ml BSA) with the restriction enzyme SapI (10 units act.), And then in 40 μl of buffer O (50 mm Tris-HCl, pH 7.5, 10 mm MgCl ₂ , 100 mm NaCl, 0.1 mg / ml BSA) with NdeI restriction enzyme (10 units act.) for 1 h at 37 ° C. The vector fragment after electrophoresis in a 15% agarose gel was excised from the gel and transferred to 200 μl NT buffer, dissolved at 50 ° C for 5-10 min and applied to a NucleoSpin Extract II column. Wash with NT 3 buffer and elute with 50 μl of NE buffer.

To prepare the thymosin alpha 1 gene, amplification is carried out by PCR using a plasmid with the artificial thymosin gene α1 (0.01 μg in the sample) as a template, and synthetic oligonucleotides A1 and B1 (60 pmol each) as primers. PCR is carried out in a DNA amplifier, in a buffer solution consisting of each of four dNTPs at a concentration of 0.5 mM and 5 units. Act. Taq DNA polymerase, in the following mode: denaturation - 1 min at 94 ° С, annealing - 30 sec at 60 ° С, elongation - 40 sec at 72 ° С, 30 PCR cycles. The gene after electrophoresis in a 15% agarose gel is excised from the gel and transferred to 200 μl of NT buffer, dissolved at 50 ° C for 5-10 minutes and applied to a NucleoSpin Extract II column. Wash with NT 3 buffer and elute with 50 μl of NE buffer, then digest with the same restriction enzymes used in the preparation of the vector and isolate the target fragment from the agarose gel.

The resulting synthetic fragment with the recombinant human thymosin α1 gene in an amount of 2 pmol is added to a solution of 1 μg of the above vector fragment in 10 μl of buffer (20 mM Tris-HCl, pH 7.56, 10 mM MgCl ₂ , 0.2 mM rATP, 10 mM dithiothreitol) and are ligated with 10 units. Act. T4 DNA ligases for 12 hours at 10 ° C.

DNA containing the altered Shine-Dalgarno sequence, the amino acid valine, which is non-standard as a start codon, as well as restriction endonucleases sites are obtained from two synthetic oligonucleotides A2 and B2 (SEQ ID NO 4). The oligonucleotides are mixed in an equimolar amount (600 pmol each), denatured at 90 ° C and annealed at a slow decrease in temperature to 30 ° C.

Plasmid pER-TA1GyrA (3 μg, 1 pmol) was treated in 40 μl of buffer O (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 100 mM NaCl, 0.1 mg / ml BSA) with PstI restriction enzyme (10 units act.), and then in 40 μl of buffer R (10 mM Tris-HCl, pH 8.5, 10 mM MgCl ₂ , 100 mM KCl, 0.1 mg / ml BSA) with BamHII restriction enzyme (10 units act .) for 1 h at 37 ° C. The vector fragment after electrophoresis in a 15% agarose gel was excised from the gel and transferred to 200 μl NT buffer, dissolved at 50 ° C for 5-10 min and applied to a NucleoSpin Extract II column. Wash with NT 3 buffer and elute with 50 μl of NE buffer.

The resulting synthetic fragment with a modified Shine-Dalgarno sequence (SEQ ID NO 6) in an amount of 2 pmol is added to a solution of 1 μg of the above vector fragment in 10 μl of buffer (20 mm Tris-HCl, pH 7.56, 10 mm MgCl ₂ , 0 , 2 mM rATP, 10 mM dithiothreitol) and are ligated with 10 units. Act. T4 DNA ligases for 12 hours at 10 ° C. The result is the plasmid pER-TA1GyrA-RBS.

The plasmid pER-TA1GyrA-RBS (3 μg, 1 pmol) is treated in 40 μl of Y buffer (33 mm Tris acetate, pH 7.9, 10 mm Mg acetate, 66 mm K-acetate 1, 0.5 mm DTT, 0.1 mg / ml BSA) with restriction enzyme NcoI (10 units act.), And then in 40 μl of buffer R (10 mm Tris-HCl, pH 8.5, 10 mm MgCl ₂ , 100 mm KCl, 0.1 mg / ml BSA) with restriction enzyme XhoI (10 units act.) for 1 h at 37 ° C. The vector fragment after electrophoresis in a 15% agarose gel was excised from the gel and transferred to 200 μl NT buffer, dissolved at 50 ° C for 5-10 min and applied to a NucleoSpin Extract II column. Wash with NT 3 buffer and elute with 50 μl of NE buffer.

To prepare the serine acetyltransferase gene, PCR amplification is carried out using E. coli genomic DNA (0.01 μg per sample) as a template, and synthetic oligonucleotides A3 and B3 (60 pmol each) as primers. PCR is carried out in a DNA amplifier, in a buffer solution consisting of each of four dNTPs at a concentration of 0.5 mM and 5 units. Act. Taq DNA polymerase, in the following mode: denaturation - 1 min at 94 ° С, annealing - 30 sec at 60 ° С, elongation - 40 sec at 72 ° С, 30 PCR cycles. A fragment of 564 bp in length after electrophoresis in a 15% agarose gel, they are excised from the gel and transferred to 200 μl NT buffer, dissolved at 50 ° C for 5-10 minutes and applied to a NucleoSpin Extract II column. Wash with NT 3 buffer and elute with 50 μl of NE buffer, then digest with restriction enzymes, which are used to prepare the vector.

The resulting synthetic fragment with a sequence of serine acetyltransferase in an amount of 2 pmol is added to a solution of 1 μg of the above vector fragment in 10 μl of buffer (20 mm Tris-HCl, pH 7.56, 10 mm MgCl ₂ , 0.2 mm rATP, 10 mm dithiothreitol ) and are ligated with 10 units. Act. T4 DNA ligases for 12 hours at 10 ° C.

An aliquot of the reaction mixture is used to transform competent E. coli C3030 cells. Transformants are plated on plates with LB agar containing 50 μg / ml ampicillin. The plasmid pER-TA1GyrA-AcSer DNA was isolated from the clones and analyzed using NdeI, SapI, PstI, BamHI, NcoI and XhoI endonucleases. Screening of recombinants is carried out using sequencing. The physical map of the plasmid pER-TA1GyrA-AcSer is shown in FIG. 1. Indicated restriction endonuclease sites. Ori is the site of initiation of replication of the tsamid. Ap ^R - gene for resistance to ampicillin. LacI - lactose operon repressor gene. TA1GyrA is a recombinant protein gene encoding human thymosin alpha 1 and GyrA intein. serAc is a gene encoding a serine acetyltransferase.

Example 2

Obtaining a producer strain of E. coli C3030 / pER-TA1GyrA-AcSer and determining its productivity.

The E. coli C3030 / pER-TA1GyrA-AcSer producing strain is obtained by transforming competent E. coli C3030 cells with the pER-TA1GyrA-AcSer plasmid as described in Example 1. E. coli C3030 cells carrying the plasmid, the structure of which is confirmed by analysis data (see Example 1) are producers of two proteins.

The strain producing E. coli C3030 / pER-TA1GyrA-AcSer is grown at 37 ° C in 100 ml of LB-broth (pH 7.0) with 50 μg / ml ampicillin for 2 hours on a shaker at a speed of 190 rpm until turbidity A ₅₅₀ 0.7-0.8, add isopropylthio-β-D-galactoside to a concentration of 0.2 mm and continue the process for another 6 hours, or continue growing in the absence of an inducer for 6 hours. Each hour, a 2 ml sample is taken, A ₅₅₀ is determined and the amount of culture corresponding to 1 ml with A ₅₅₀ 1.0, centrifuged for 5 minutes at 6000 rpm. The precipitated cells in 100 μl of lysing buffer with dye bromphenol blue are treated with ultrasound for 20 seconds, heated for 3 min at 100 ° C and 4 μl samples are used for electrophoresis in 15% SDS-PAGE. The gel was stained with Coomassie R-250 according to a standard technique and scanned using a Shimadzu CS-930 densitometer. In FIG. 2. presents an electrogram of the cell lysate of the producer strain E. coli C3030 / pER-TA1GyrA-AcSer, where M is the molecular weight standard, L is the cell lysate, 1 is a hybrid protein containing thymosin alpha 1, 2 is serine acetyltransferase.

Example 3

Obtaining a hybrid protein TA1GyrA, its autocatalytic cleavage and isolation of recombinant human thymosin α1.

After fermentation, the cells of the TA1GyrA hybrid protein producer (biomass) are separated by centrifugation (5000 g, 20 min, 4 ° C), destroyed on an ultrasonic disintegrator in buffer (50 mM Tris / HCl, 10 mM EDTA, 1 mM PMSF) and isolated by centrifugation ( 15000 g, 45 min) clarified cell lysate. Put on a chitin sorbent balanced in a buffer containing 50 mm Tris / HCl, 100 mm NaCl, washed with a buffer containing 50 mm Tris / HCl, 100 mm NaCl, 2 M urea, and balance in a buffer containing 50 mm Tris / HCl, 100 mM NaCl, 100 mM DTT, thereby inducing autocatalytic cleavage of the fusion protein, then incubated for 24 hours at 25 ° C. Further purification of recombinant human thymosin α1 is carried out by reverse phase liquid chromatography. The analysis of the obtained product is carried out using RP HPLC. Fractions with a TA1 protein content of at least 98% are pooled and lyophilized. The yield of recombinant thymosin α1 is 3 mg per 100 g of cells or 3% of the total protein of cells.

The resulting recombinant human thymosin α1 is identified using TOF-ESI mass spectrometry using an Agilent technologies 6224 mass spectrometer. The resulting recombinant thymosin α1 signal corresponds to a calculated mass value of 3108.31 Da.

Claims (3)

1. Recombinant plasmid DNA pER-TA1GyrA-AcSer, designed for the simultaneous biosynthesis of two proteins - a hybrid polypeptide containing the amino acid sequence of human thymosin α1 and intein, and the enzyme E. coli - serine acetyltransferase having a molecular weight of 5.04 MDa; consisting of:
BsaBI / NdeI DNA fragment of plasmid pTWTN-1 containing the sequence of the T7 promoter and the lacO operator; An NdeI / PstI DNA fragment comprising a fusion protein sequence comprising a recombinant human thymosin α1 gene sequence adapted to these sites, a Mxe GyrA intein sequence, and a chitin binding domain sequence; PstI / NcoI DNA fragment encoding a weakened Shine-Dalgarno sequence and the amino acid valine, which is non-standard as a start codon; An NcoI / XhoI DNA fragment containing the sequence of a serine N-acetyltransferase; a DNA fragment containing, as a genetic marker, a β-lactamase gene that determines the resistance of E. coli cells transformed with pER-TA1GyrA-AcSer plasmid to penicillin antibiotics; DNA of a fragment containing a sequence encoding a replication origin; DNA fragment encoding the sequence of the lactose repressor LacI. 2. The strain Escherichia coli C3030 / pER-TA1GyrA-AcSer, producing a hybrid polypeptide containing the amino acid sequence of human thymosin α1 and intein, as well as serine acetyltransferase obtained by transformation of Escherichia coli C3030 strain cells with recombinant plasmid DNA pERA AcSy pG-AcGy. one. 3. A method for producing recombinant human thymosin α1, comprising transforming the Escherichia coli strain C3030 of the obtained plasmid DNA pER-TA1GyrA-AcSer according to claim 1, culturing the resulting producer strain Escherichia coli C3030 / pER-TA1GyrA-AcSer according to claim 2, separation TA1GyrA in soluble form after destruction of cells using an ultrasonic disintegrator in a buffer solution (50 mM Tris / HCl, 10 mM EDTA, 1 mM PMSF), adsorption of the TA1GyrA fusion protein on a column with chitin sorbent, inducing autocatalytic cleavage of the protein TA1GyrA with buffer containing dithiotreitol, incubation on a sorbent for 24 hours at 25 ° C, elution of the target peptide and purification of the target product by reverse phase chromatography.

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