RU2729357C1 - Recombinant plasmid dna pf267 coding hybrid polypeptide containing proinsulin lisprum, and a strain of bacteria escherichia coli - producer of hybrid polypeptide containing proinsulin lispro - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to recombinant production of therapeutic proteins, and can be used to produce recombinant proinsulin lisprum human. Recombinant proinsulin is obtained in a hybrid polypeptide, in which the human HGS protein fragment sequence, substrate of tyrosine kinase is connected through peptide linker with sequence GlyHis6GlySerArg with amino acid sequence of proinsulin of lisprum human, with arginine as site of proteolysis between C-peptide and chain A. For this purpose recombinant plasmid DNA pF267 and strain-producer Escherichia coli BL21/pF267 are used.

EFFECT: invention provides synthesis of a hybrid polypeptide with an expression level of not less than 4_5 % of the crude weight of the cell biomass, enables increasing the proportion of insulin in the hybrid protein to 49_5 % and improving the purification efficiency of the end product by eliminating the closely related Arg0-A-lyspro admixture.

3 cl, 5 ex, 7 dwg

Description

Technology area

The invention relates to the field of biotechnology, in particular to genetic engineering. The invention provides a recombinant plasmid DNA for the production of recombinant insulin lispro and can be used for the preparation of drugs, in particular, insulins and their analogues for the treatment of diabetes mellitus.

Brief description of the invention

The invention relates to biotechnology. This invention can be used to obtain a recombinant proinsulin lispro, which contains a B chain, a C-peptide and a chain A. The proposed recombinant plasmid DNA containing a hybrid tac-promoter, the terminator of the ribosomal operon of E. coli , an artificial gene encoding a hybrid polypeptide, consisting of a short leader sequence of a fragment of the human HGS protein, a substrate tyrosine kinase, a peptide linker GlyHis6GlySerArg, an Arg proteolysis site, a B chain, an Arg proteolysis site, a C-peptide, an Arg proteolysis site and a chain A.

Also proposed is a strain of Escherichia coli - a producer of a hybrid polypeptide with a proinsulin lispro sequence, transformed with a recombinant plasmid DNA according to the invention, and a hybrid polypeptide consisting of a short leader sequence of a fragment of the human HGS protein, a substrate tyrosine kinase, a peptide linker GlyHis6 ArgGlySer B, Arg proteolysis site, C-peptide, Arg proteolysis site and chain A. In this case, the biosynthesis of the hybrid polypeptide is induced by isopropylthiogalactopyranoside, the level of its biosynthesis is not less than 4.5% of the wet sediment mass of the cell biomass.

The present invention makes it possible to obtain a hybrid polypeptide with a high proportion of insulin lispro (up to 49.5%) and to increase the purification efficiency of the target product due to the elimination of a closely related impurity Arg ₀ -A-lispro. The invention is aimed at obtaining a highly productive bacterial strain - the producer of the insulin analogue of lispro, the use of which can significantly reduce the cost of the production process of the analogue of insulin - insulin lispro.

Prior art

Diabetes mellitus is currently the most serious medical and socio-economic problem around the world. The number of patients with type 1 diabetes mellitus is constantly growing, and effective insulin replacement therapy, as the main method of therapy for insulin-dependent diabetes mellitus, is of particular importance in this regard (1). The economic losses associated with diabetes in Russia amount to about US $ 3.12 billion per year. By 2030, this amount may increase to 3.35 billion (2).

Effective insulin replacement therapy, as the main method of therapy for insulin-dependent diabetes mellitus, is of particular importance in this regard. Natural and recombinant insulins are no longer able to fully meet the needs of clinicians and patients. Modern medicine requires the use of more advanced antidiabetic drugs, such as genetically engineered analogues of human insulin.

Insulin Lizpro is the first insulin analogue developed and produced. The molecular structure of insulin lispro is identical to that of human insulin with the exception of positions 28 and 29 of the beta chain of the molecule, where lysine and proline are located in reverse order (Fig. 1).

In comparison with Lizpro, recombinant insulin has two main disadvantages - a relatively slow onset of action (usually an injection is required 30-40 minutes before a meal), and an increased duration of action, therefore, there is a risk of hyperinsulinemia. Natural and recombinant insulins are unable to normalize post-meal blood glucose peaks without the risk of hypoglycemia (3).

Compared to recombinant insulin, Lispro exhibits a blood glucose profile much closer to the physiological profile (4). The reverse arrangement of lysine and proline allows the Lyspro molecule to dissociate twice as fast. As a result, the transition to the active form occurs two times faster than a similar process in recombinant insulins. When administered subcutaneously, the physiological effect of Lizpro occurs within 10-15 minutes after injection. The same features of the Lizpro molecule lead to an acceleration in reaching the peak of drug activity, which occurs approximately 1.5 hours after injection, while for ordinary insulins this time is 3-4 hours. The duration of action of Lizpro insulin is 3-4 hours (6-8 hours in the case of regular insulin). As a result, the curve of insulin activity of Lispro turns out to be almost identical to the curve of activity of endogenous insulin entering the bloodstream after a meal. Due to this, in patients, both the incidence of complications of diabetes mellitus itself and the risk of developing such complications of insulin therapy as hypoglycemia, which is observed with the use of insulin Lizpro 30% less frequently compared with recombinant insulins, decrease in patients (4).

In this regard, the development and implementation of modern methods of therapy for diabetes mellitus is one of the most priority areas of medicine and the pharmaceutical industry.

Currently, the most promising technology is the production of insulin and insulin analogs using methods of expression of the human proinsulin gene in Escherichia coli cells as part of fusion proteins in the form of insoluble inclusion bodies (5). The structure of a single-chain precursor (hybrid polypeptide) expressed in known human insulin-producing strains is a leader peptide, Arg or Lys or Lys Arg proteolysis site, B chain, Arg Arg proteolysis site, C-peptide , Lys Arg proteolysis site, chain A (Fig. 2).

The need to include leader peptides in the hybrid polypeptide is associated with the low stability of proinsulin in Escherichia coli cells , the half-life of which is 2 minutes (4). Glutathione transferase (6), immunoglobulin IgG, the binding domain of protein A from S. aureus (7), interleukin-2 (8), etc. are used as leader sequences that protect proinsulin from proteolytic degradation.

Technological schemes for obtaining insulin lispro and human insulin are identical and are characterized by the following stages: increasing the biomass of cells of the producer strain, isolation of inclusion bodies, denaturation of the hybrid polypeptide, renaturation of the hybrid polypeptide. The next stage of the technological process is enzymatic hydrolysis of the hybrid polypeptide using a mixture of two enzymes: trypsin and carboxypeptidase B. Under the action of trypsin, the peptide bond of the hybrid polypeptide is hydrolyzed mainly after arginine; under the action of carboxypeptidase B, positively charged amino acids are cleaved from the C-terminus of insulin or its analog (lysine and arginine). As a result of joint hydrolysis with trypsin and carboxypeptidase B, native insulin or insulin analogue lispro is formed.

The closest in technical essence to the proposed invention are recombinant DNA plasmids pPINS07 (9), pLP-3.1 (10), pHINS11 (11) and pHILP07 (12), providing the synthesis of hybrid polypeptides containing the IgG-binding domain of staphylococcal protein A (pPINS07, pLP-3.1) and the N-terminal fragment of human interferon gamma (pHINS11).

RF patent No. 2144957 describes the E. coli JM109 / pPINS07 strain, providing the synthesis of a hybrid polypeptide with an expression level of at least 25-30%. The recombinant plasmid DNA pPINS07 determines the synthesis of a hybrid polypeptide in which a single IgG-binding domain of staphylococcal protein A is linked via a peptide linker His ₆ GlySerArg to the amino acid sequence of human proinsulin. The advantages of the proposed construct are the high level of expression of the hybrid polypeptide and its efficient renaturation (refolding) and, as a consequence, the high yield of the correctly folded polypeptide. Recombinant plasmid DNA pLP-3.1, obtained by site-directed mutagenesis of plasmid pPINS07, expresses a fusion protein in which a single IgG-binding domain of staphylococcal protein A is linked via the His ₆ GlySerArg peptide linker to the amino acid sequence of proinsulin lispro (10).

A significant drawback of the recombinant plasmids pPINS07 and pLP-3.1 is that they encode a protein with a low proportion of insulin and insulin lispro, respectively, about 33%.

RF patent No. 2354702 describes the recombinant plasmid DNA pHINS11 and the E. coli strain JM109 / pHINS11. This recombinant plasmid DNA determines the synthesis of a hybrid polypeptide in which the N-terminal fragment of human interferon gamma is connected via a peptide linker HisProGlySerHisHisHisHisGlySerArg with the amino acid sequence of human proinsulin and ensures its high expression. The use of these structures in the technological process makes it possible to obtain highly purified human insulin with a purity of at least 98% and an activity of at least 27.5 IU / mg. This producer strain is characterized by a high level of biosynthesis of the hybrid polypeptide and a higher proportion of insulin in the hybrid polypeptide (up to 38%) compared to the recombinant plasmid DNA pPINS07 (up to 33%), while the stage of renaturation of the hybrid polypeptide produced is not efficient enough (11) ...

Also described is a recombinant plasmid DNA pHILP07, obtained by site-directed mutagenesis of plasmid pHINS11, with the expression of a hybrid polypeptide, in which the N-terminal fragment of human interferon gamma is connected through a peptide linker HisProGlySerHisHisHisHisGlySerArg with the amino acid sequence proprogram 12.

A common disadvantage of the hybrid polypeptides in the described patents is the "Lys Arg" proteolysis site between the C-peptide and the A-chain, allowing the formation of a closely related impurity Arg0-A-lispro.

In general, the disadvantages of previously used genetic constructs for the expression of a hybrid polypeptide include:

1. Low yield of the final product - insulin lispro or insulin itself. This is due to the fact that leader peptides are rather extended amino acid sequences and make up from 40 to 60% of the mass of the hybrid polypeptide.

2. Formation of a closely related impurity Arg ₀ -A-lispro as a result of trypsin hydrolysis of the peptide bond in the position after lysine between the C-peptide and the A-chain (Fig. 2).

The objective of the present invention is to construct a recombinant plasmid DNA that provides a high level of inducible expression of a hybrid polypeptide with a high proportion of insulin lispro and a modified proteolysis site between the C-peptide and the A-chain in order to prevent the formation of a closely related impurity Arg0-A-lispro during enzymatic hydrolysis of the hybrid polypeptide ...

The problem is solved by constructing the recombinant plasmid DNA pF267 and the Escherichia coli BL21 / pF267 strain, transformed with it and providing the synthesis of a hybrid polypeptide with an expression level of at least 4.5% of the wet weight of the cell biomass.

Description of figures

FIG. 1 shows the molecular structure of Lispro (source: US FDA, NDA 21-017).

FIG. 2 shows the structure of a single-chain precursor (hybrid polypeptide) in insulin-producing strains.

FIG. 3 shows the structure of a DNA sequence obtained by chemical synthesis.

FIG. 4 shows the arrangement of primers Pr237 and Pr238 on plasmid pF173 for site-directed mutagenesis of the proteolysis site between the C-peptide and the A-chain.

FIG. 5 shows the arrangement of primers Pr072 and Pr073 on plasmid pF265 for site-directed mutagenesis of the insulin B chain sequence.

FIG. 6 shows the primary structure of the EcoRI / HindIII fragment of plasmid pF267 encoding the fusion polypeptide. Initiating and terminating codons are underlined, restriction endonuclease recognition sites are indicated.

FIG. 7 shows a physical map of the recombinant plasmid pF265. Restriction endonuclease sites are indicated, pBR322 ori - plasmid replication initiation site, Ptac - hybrid transcription promoter, rrnB - transcription terminator of E. coli ribosomal operon, Leader peptide - MetLeuTyrTyrGluGlyTyrTyrGluGlyTyrTyrGluGlyTyrTyrGluGlyTyrTyrGluGlyLeuGlnAspyr , Prolizpro is proinsulin lispro, LacI is a transcriptional repressor that regulates transcription of the Tac promoter.

Detailed description of the invention

According to the invention, there is provided a recombinant plasmid DNA pF267 encoding a hybrid polypeptide in which the sequence of a fragment of the human protein HGS, a substrate of tyrosine kinase, is connected via a peptide linker GlyHis6GlySerArg to the amino acid sequence of proinsulin lispro,

with arginine as a proteolysis site between C-peptide and chain A, with a molecular weight of 1.4 MDa (4537 bp),

containing

- BglII / XhoI fragment of plasmid pBR322, including the β-lactamase gene (bla), which determines the resistance of bacterial cells to ampicillin;

- replication initiation site (ori);

- ROP gene regulating the copy number of the plasmid;

- XhoI / EcoRI fragment, which is a hybrid Tac transcription promoter;

- EcoRI / HindIII fragment containing an artificial gene, in which the sequence of the short leader peptide MetLeuTyrTyr GluGlyLeuGlnAsp is connected via a peptide linker GlyHis6GlySerArg to the amino acid sequence of proinsulin lispro;

- HindIII / BglII fragment containing the terminator of the ribosomal RNA operon rrnB, transcriptional repressor LacI, which regulates transcription of the Tac promoter, unique recognition sites by restriction endonucleases with the following coordinates: EcoRI - 115, BamHI - 176, SpeI - 438, HindlII - 444, 1872, NdeI - 2476, AatII - 4468.

Moreover, this recombinant plasmid DNA can have a size of 4537 bp.

In addition, the proposed strain of Escherichia coli BL21 / pF267 - the producer of a hybrid polypeptide with a proinsulin lispro sequence, transformed with the recombinant plasmid DNA according to the invention.

Also proposed is a hybrid polypeptide, in which the sequence of a fragment of the human protein HGS, a substrate of tyrosine kinase, is connected via a peptide linker with the sequence GlyHis6GlySerArg with the amino acid sequence of human proinsulin, with arginine as a proteolysis site between the C-peptide and the A chain, obtained using a recombinant plasmid DNA according to the invention.

A common disadvantage of hybrid polypeptides in the patents described in the prior art is that the resulting hybrid polypeptide includes a "Lys Arg" proteolysis site between the C-peptide and the A-chain, allowing for the formation of a closely related impurity Arg0-A-lispro.

The present inventors have found that by significantly reducing the size of the hybrid polypeptide by using a short leader sequence of 31 amino acids and changing the proteolysis site between the C-peptide and the A-chain to "Arg", high yields of the target hybrid polypeptide can be achieved.

The leader peptide contains a short sequence of a fragment of the human protein HGS, a substrate of tyrosine kinase, which forms a pronounced spatial structure of the "alpha-helix" type. The presence of the N-terminal alpha-helical region in the leader peptide has a positive effect on the level of renaturation of the hybrid polypeptide.

The present invention makes it possible to obtain a hybrid polypeptide with a high proportion of insulin lispro (up to 49.5%) and to increase the purification efficiency of the target product due to the elimination of a closely related impurity Arg ₀ -A-lispro. The invention is aimed at obtaining a highly productive bacterial strain - producer of the insulin analogue of lispro, the use of which can significantly simplify the production process of the insulin analogue of insulin lispro and reduce the cost of purifying the target product.

The initial plasmid for the creation of the pF267 expression vector was the pF019 plasmid, which is a recombinant pBR322 vector (13), in which the tetracycline resistance gene was completely removed by molecular cloning (example 1).

At the next stage, a 1.6 kb DNA fragment was synthesized containing the following elements and restriction sites (Fig. 3):

- Tac promoter and Lac operator to control the expression of a hybrid polypeptide (14), flanked by XhoI and EcoRI restriction sites.

- The transcription terminator of the rnnB operon of the strain K-12 ER3413 (REGION: 4143199..4143361), in front of which is a HindIII restriction site.

- LacI gene of strain K-12 ER3413 (REGION: 365804 to 367006) with a BglII restriction site at the 3 'end. The introduction of the LacI gene into an expression construct solves the problem of controlling the expression of a hybrid polypeptide using a wide range of host strains, including BL21, which is characterized by increased stability of recombinant polypeptides due to the removal of cell proteases lon and ompT.

The synthesized DNA fragment and plasmid pF019 were incubated with restriction endonucleases XhoI and BglII for 3 hours at 37 ° C. The corresponding DNA fragments were isolated from the agarose gel using the Quiagen kit and ligated with T4 DNA ligase. After electroporation of the XL1-Blue strain of E. coli, plasmid DNA isolated from the grown colonies were tested by PCR and sequencing.

Thus, an expression vector pF20 was obtained based on the plasmid pBR322, containing the Tac promoter, the rnnB terminator and the LacI gene encoding the transcriptional repressor Tac promoter, as well as EcoRI and HindIII restriction sites for subsequent insertion of the gene encoding the hybrid polypeptide.

The DNA sequence of the hybrid polypeptide containing the IgG-binding domain of protein A from Staphylococcus aureus , the peptide linker His6GlySerArg and human proinsulin as a leader peptide, was excised from the plasmid pPIN07 (9) using EcoRI / HindIII restriction endonucleases and inserted at the appropriate restriction sites into the restriction vector pF20 in the manner described above, wherein a BamHI restriction site formed by the coding sequence of two amino acids of the peptide linker GlySer (GGATCC) appeared in the resulting vector. As a result, a recombinant plasmid DNA pF100 was obtained.

At the next stage, recombinant plasmid DNA pF173 was obtained after replacing the DNA sequence of the IgG-binding domain of protein A from Staphylococcus aureus with a DNA sequence encoding a shorter leader peptide MetLeuTyrTyrGluGlyLeuGlnAsp, which is a fragment of the human protein HGS, a substrate (TKS) kinase example 2).

The amino acid sequence of LeuTyrTyrGluGlyLeuGlnAsp is located in the alpha-helical region of the TKS protein (3D structure no. In the PDB database: 3F1I). The presence of an alpha-helical region in the leader peptide facilitates efficient renaturation of the hybrid polypeptide.

By the method of site-directed mutagenesis in the plasmid pF173, the codon "aag" at position 373-375 corresponding to the amino acid lysine of the proteolysis site "Lys Arg" between the C-peptide and the A-chain of proinsulin was removed. As a result, a recombinant plasmid DNA pF265 was obtained expressing human proinsulin as part of a hybrid polypeptide (example 3).

Recombinant plasmid DNA pF267 was obtained by site-directed mutagenesis of recombinant plasmid DNA pF265, in which the sequence "ccgaag" encoding the amino acids proline and lysine was replaced by the sequence "aagccg" encoding the amino acids lysine and proline (example 4).

The recombinant plasmid DNA pF267 obtained as a result of the described molecular genetic manipulations, encoding a hybrid polypeptide with the amino acid sequence of proinsulin lispro, is characterized by the following features:

has a molecular weight of 1.4 MDa (4537 bp);

consists of the following structural elements (Fig. 7):

- BglII / XhoI fragment of plasmid pBR322, including the β-lactamase gene (bla), which determines the resistance of bacterial cells to ampicillin;

- replication initiation site (ori);

- ROP gene regulating the copy number of the plasmid;

- XhoI / EcoRI fragment, which is a hybrid Tac transcription promoter;

- EcoRI / HindIII fragment containing an artificial gene, in which the sequence of the short leader peptide MetLeuTyrTyrGluGlyLeuGlnAsp is connected via a peptide linker GlyHis6GlySerArg to the amino acid sequence of lispro proinsulin;

- HindIII / BglII fragment containing the ribosomal RNA operon terminator rrnB, and

- transcriptional repressor LacI, which regulates the transcription of the Tac promoter;

contains unique recognition sites by restriction endonucleases with the following coordinates: EcoRI - 115, BamHI - 176, SpeI - 438, HindIII - 444, BglII - 1872, NdeI - 2476, AatII - 4468.

The advantages of the proposed recombinant plasmid DNA construct are the high proportion of insulin lispro in the hybrid polypeptide (up to 49.5%), the presence in the leader peptide of a sequence that forms a spatial structure of the "alpha-helix" type to improve the efficiency of renaturation of the hybrid polypeptide, modification of the proteolysis site between C -peptide and A-chain, excluding the formation of a closely related impurity Arg0-A-lispro during enzymatic hydrolysis of the hybrid polypeptide.

To obtain a producer strain of a hybrid polypeptide with proinsulin lispro, electrocompetent cells of the recipient strain Escherichia coli BL21 were transformed with recombinant plasmid DNA pF267 by electroporation according to the described method (15) and plated on LB agar containing 100 μg / ml ampicillin.

The resulting Escherichia coli BL21 / pF267 producer strain is characterized by the following features:

Morphological features: Cells are small, rod-shaped, gram-negative, non-spore-bearing, 1x3.5 µm in size, motile, with well-distinguishable inclusion bodies after induction of hybrid polypeptide synthesis.

Cultural traits: when growing on agar medium LB colonies are round, smooth, translucent, shiny, gray; the edge is even, the diameter of the colonies is 1-3 mm, the consistency is pasty. Growth in liquid media (LB, glucose minimal media) is characterized by even haze.

Physiological and biochemical signs: cells grow at a temperature of 4-42 ° C, the optimum pH is 6.8-7.6. As a source of nitrogen, both mineral ammonium salts and organic compounds are used: amino acids, peptone, tryptone, yeast extract. Glycerin, carbohydrates, amino acids are used as a carbon source for growth on a minimal medium.

Antibiotic resistance: cells of the producer strain are resistant to ampicillin (up to 300 mg / ml).

The invention is illustrated by the following examples.

Example 1. Construction of an intermediate genetic construct pF019

The PCR product was 2.8 kb in size, the resulting plasmid pBR322 amplification with primers Pr033 (5'-ccaacgtaac agatct (BglII ) aacagctcgaa ctcgag (XhoI) ttgaagacgaaagggcctcg- 3 ') and Pr039 (5'-ccaaccgtaac agatct (BglII ) ctgtggaacacctacatctg- 3' ), excised from the gel, purified using the Qiagen kit. After incubation with restriction endonuclease Bgl II for 3 hours at 37 ° C, the purified DNA fragment was ligated using T4 DNA ligase and used to transform XL1-Blue E. coli strain (Agilent Technology, 200249) by electroporation (15). The correctness of plasmid DNA isolated from colonies grown on LB medium with ampicillin (100 μg / ml) was confirmed by sequencing. As described above, plasmid pF019 was obtained, which is a fragment of plasmid pBR322 with complete deletion of the tetracycline resistance gene.

Example 2. Construction of an intermediate recombinant plasmid DNA pF173

To introduce a short leader peptide into proinsulin, primers pr096 (tcaacgtaac gaattc t atg ctg tat tat gaa ggc ctg cag gac ggc agc agc cac cac catcatcatcatcatggatcccg ) and pr130 (tgcctggcggcagtagtag) were synthesized Primer pr096 contains an EcoRI restriction endonuclease recognition site; the region coding for the amino acid sequence of the leader peptide; sequence corresponding to the peptide linker His6GlySerArg construct pF100.

The reaction mixture (100 μl) for polymerase chain reaction (PCR) consisted of the following components:

- 10 μl of 10-fold buffer for PFU DNA polymerase,

- 0.1 ng of plasmid DNA pF100,

- 1 μl of 10 mM dNTP mixture,

- 0.5 μl of each primer (10 μM),

- 2 units Pfu DNA Polymerase,

- 2 units Taq DNA polymerase.

PCR was carried out using a DNA amplifier T100 Thermal Cycler, Bio-Rad under the following conditions: 96 ° C (5 min), 30 cycles - 96 ° C (15 sec), 55 ° C (15 sec), 72 ° C (30 sec ).

The amplification products were analyzed by agarose gel electrophoresis using the intercalating dye GelRed, Biotium. The PCR product corresponding to 600 bp was excised from the gel and purified using the Zymoclean ™ Gel DNA Recovery Kit. Electrophoresis was performed in standard TBE (Tris-borate-EDTA) buffer using 1% agarose. The resulting DNA was visualized using the Fusion-SL2-400 gel documenting system.

The PCR product cut from the gel and plasmid pF100 were incubated with restriction endonucleases EcoRI and HindIII for 3 hours at 37 ° C. The corresponding DNA fragments (330 bp and 4.2 kb) were isolated from the agarose gel using the Quiagen kit and ligated with T4 DNA ligase. After electroporation of the XL1-Blue strain of E. coli, plasmid DNA isolated from the grown colonies was analyzed by PCR and sequencing.

Example 3. Construction of intermediate recombinant plasmid DNA pF265 using site-directed mutagenesis of plasmid pF173

To modify the Lys Arg proteolysis site between the C-peptide and the A-chain of proinsulin, oligonucleotides Pr237 (cgtggtatcgttgaacagtg) and Pr238 (ctgcagagaaccttccagag) containing a phosphate group from the 5'-end were synthesized (Fig. 3).

The reaction mixture (100 μl) for polymerase chain reaction (PCR) consisted of the following components:

- 10 μl of 10-fold buffer for Pfu DNA polymerase,

- 0.1 ng of plasmid DNA pF173,

- 1 μl of 10 mM dNTP mixture,

- 0.5 μl of each primer (10 μM),

- 2 units Pfu DNA Polymerase,

- 2 units Taq DNA polymerase.

PCR was carried out using a DNA amplifier T100 Thermal Cycler, Bio-Rad under the following conditions: 96 ° C (5 min), 30 cycles - 96 ° C (15 sec), 55 ° C (15 sec), 72 ° C (3 min ).

The amplification products were analyzed by agarose gel electrophoresis using the intercalating dye GelRed, Biotium. The PCR product corresponding in size to the plasmid pF173 (4.5 kb) was excised from the gel and purified using the Zymoclean ™ Gel DNA Recovery Kit. Electrophoresis was performed in standard TBE buffer using 1% agarose. DNA was visualized using the Fusion-SL2-400 gel documenting system.

To obtain plasmid DNA pF265, the ends of the PCR product isolated from the gel were ligated with T4 DNA ligase (NEB). The ligation mixture (10 μL) was used to transform the BL21 strain by electroporation (15). After transformation, the colonies grown on a medium with ampicillin were selected, plasmids were isolated from them and analyzed by sequencing the hybrid polypeptide sequence. As a result, we obtained a recombinant plasmid DNA pF265 expressing proinsulin as part of a hybrid polypeptide.

Example 4. Obtaining the final genetic construct of the recombinant plasmid DNA pF267, expressing the hybrid proinsulin polypeptide Lispro

Recombinant plasmid DNA pF267 was obtained by site-directed mutagenesis of recombinant plasmid DNA pF265. For this, oligonucleotides (primers) Pr072 ( aagccg acccgtcgtgaagctgaag) and Pr073 (ggtgtagaagaaaccacgttc) containing a phosphate group from the 5'-end were synthesized. Primer Pr072 contained the aagccg sequence encoding the amino acids lysine and proline (Fig. 5).

The reaction mixture (100 μl) for polymerase chain reaction (PCR) consisted of the following components:

- 10 μl of 10-fold buffer for PFU DNA polymerase,

- 0.1 ng of plasmid DNA pF265,

- 1 μl of 10 mM dNTP mixture,

- 0.5 μl of each primer (10 μM),

- 2 units Pfu DNA Polymerase,

- 2 units Taq DNA polymerase.

PCR was carried out using a DNA amplifier T100 Thermal Cycler, Bio-Rad under the following conditions: 96 ° C (5 min), 30 cycles - 96 ° C (15 sec), 55 ° C (15 sec), 72 ° C (3 min ).

The amplification products were analyzed by agarose gel electrophoresis using the intercalating dye GelRed, Biotium. The PCR product corresponding in size to the recombinant plasmid DNA pF265 (4.5 kb) was excised from the gel and purified using the Zymoclean ™ Gel DNA Recovery Kit. Electrophoresis was performed in standard TBE buffer using 1% agarose. The resulting recombinant plasmid DNA was visualized using the Fusion-SL2-400 gel documenting system.

To obtain recombinant plasmid DNA, the ends of the PCR product isolated from the gel were ligated with T4 DNA ligase (NEB). A ligation mixture (10 μL) was used to transform the BL21 strain by electroporation (15).

After transformation, the colonies grown on a medium with ampicillin were selected, plasmids were isolated from them and analyzed by sequencing the hybrid polypeptide sequence. As a result, a recombinant plasmid DNA pF267 was obtained, expressing proinsulin Lizpro as part of a hybrid polypeptide.

Example 5. Determination of the productivity of the hybrid polypeptide producer strain

An individual colony of E. coli BL21 cells containing recombinant plasmid DNA pF265 was inoculated with 2 ml of LB medium containing ampicillin at a concentration of 100 μg / ml, grown in a thermal shaker at 37 ° C for 22 hours with stirring (170 rpm) ... After measuring the optical density OD _{600 of the} overnight culture, 40 ml of liquid LB medium containing 100 μg / ml ampicillin (OD starting ₆₀₀ = 0.1) were inoculated and grown for 1-2 hours at 37 ° C on an incubator shaker with stirring (180 rpm ) until the optical density reaches OD ₆₀₀ = (0.8 ± 0.2). 20 ml of culture was transferred to another flask (control without induction), 10 μl of 1M IPTG (isopropyl-beta-galactopyranoside) (final concentration of IPTG - 0.5 mM) was added to the remaining 20 ml. After 4 hours of incubation on an incubator shaker with stirring (180 rpm), the cell cultures were centrifuged (15 min, 3000 rpm), the mass of the wet cell pellet was determined, and frozen. The content of the hybrid polypeptide in% of the wet sediment weight was measured by capillary electrophoresis.

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14.de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proceedings of the National Academy of Sciences of the United States of America. 1983 Jan; 80 (1): 21-5. PubMed PMID: 6337371.

15. Lessard JC. Transformation of E. coli via electroporation. Methods in enzymology. 2013; 529: 321-7. PubMed PMID: 24011058. Epub 2013/09/10.

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LIST OF SEQUENCES

<110> ZakrytoeAktsionernoeObshchestvo "FARM-Kholding"

<120> Recombinant Plasmid DNA pF267 encoding Hybrid polypeptide comprising lispro proinsulin, and lispro proinsulin- hybrid polypeptide producer bacterial strain Escherichia coli

<140> RU 2019116104

<141> 2019-05-23

<160> 1

<210> 1

<211> 10

<212> PRT

<213> E. coli

<400> 1

GlyHisHisHisHisHisHisGlySerArg

<210> 2

<211> 11

<212> PRT

<213> E. coli

<400> 2

HisProGlySerHisHisHisHisGlySerArg

<210> 3

<211> 9

<212> PRT

<213> E. coli

<400> 3

MetLeuTyrTyrGluGlyLeuGlnAsp

<210> 4

<211> 9

<212> PRT

<213> E. coli

<400> 4

HisHisHisHisHisHisGlySerArg

<210> 5

<211> 8

<212> PRT

<213> E. coli

<400> 5

LeuTyrTyrGluGlyLeuGlnAsp

<210> 6

<211> 53

<212> DNA

<213> E. coli

<400> 6

ccaacgtaacagatct 16

aacagctcgaactcgag 17

ttgaagacgaaagggcctcg 20

<210> 7

<211> 37

<212> DNA

<213> E. coli

<400> 7

ccaaccgtaacagatct 17

ctgtggaacacctacatctg 20

<210> 8

<211> 79

<212> DNA

<213> E. coli

<400> 8

tcaacgtaacgaattct 17

atgctgtattatgaaggcctgcaggacggcagcagccaccac 42

catcatcatcatggatcccg 20

<210> 9

<211> 20

<212> DNA

<213> E. coli

<400> 9

cgtggtatcgttgaacagtg

<210> 10

<211> 20

<212> DNA

<213> E. coli

<400> 10

ctgcagagaaccttccagag

<210> 11

<211> 25

<212> DNA

<213> E. coli

<400> 11

aagccgacccgtcgtgaagctgaag

<210> 12

<211> 21

<212> DNA

<213> E. coli

<400> 12

ggtgtagaagaaaccacgttc

<210> 13

<211> 720

<212> DNA

<213> E. coli

<400> 13

tttgtttaactttaagaaggagagaattct atg ctgtattatgaa 45

aaacaaattgaaattcttcctctcttaagatacgacataatactt 45

ggcctgcaggacggcagcagccaccaccatcatcatcatggatcc 45

ccggacgtcctgccgtcgtcggtggtggtagtagtagtacctagg 45

cgttttgttaaccaacacctgtgcggttctcacctggttgaagct 45

gcaaaacaattggttgtggacacgccaagagtggaccaacttcga 45

ctgtacctggtttgcggtgaacgtggtttcttctacaccaagccg 45

gacatggaccaaacgccacttgcaccaaagaagatgtggttcggc 45

acccgtcgtgaagctgaagacctgcaggttggtcaggttgaactg 45

tgggcagcacttcgacttctggacgtccaaccagtccaacttgac 45

ggtggtggtccgggtgctggtagcctgcaaccgctggctctggaa 45

ccaccaccaggcccacgaccatcggacgttggcgaccgagacctt 45

ggttctctgcagcgtggtatcgttgaacagtgctgcacctctatc 45

ccaagagacgtcgcaccatagcaacttgtcacgacgtggagatag 45

tgctctctgtaccagctggaaaactactgcaac tagtaa gcttag 45

acgagagacatggtcgaccttttgatgacgttgatcattcgaatc 45

<210> 14

<211> 106

<212> PRT

<213> E. coli

<400> 14

MetLeuTyrTyrGlu5

GlyLeuGlnAspGlySerSerHisHisHisHisHisHisGlySer15

ArgPheValAsnGlnHisLeuCysGlySerHisLeuValGluAla15

LeuTyrLeuValCysGlyGluArgGlyPhePheTyrThrLysPro 15

ThrArgArgGluAlaGluAspLeuGlnValGlyGlnValGluLeu 15

GlyGlyGlyProGlyAlaGlySerLeuGlnProLeuAlaLeuGlu 15

GlySerLeuGlnArgGlyIleValGluGlnCysCysThrSerIle 15

CysSerLeuTyrGlnLeuGluAsnTyrCysAsn11

<---

Claims (11)

1. Recombinant plasmid DNA pF267 for expression in Escherichia coli of human lispro proinsulin as part of a hybrid polypeptide having a plasmid map as shown in FIG. 7, with a molecular weight of 1.4 MDa (4537 bp), encoding a hybrid polypeptide, in which the sequence of a fragment of the human protein HGS, a substrate of tyrosine kinase, is connected through a peptide linker GlyHis6GlySerArg with the amino acid sequence of proinsulin lispro, with arginine as a proteolysis site between the C-peptide and chain A, comprising the amino acid sequence SEQ ID NO: 14, while plasmid DNA pF267 contains: - BglII / XhoI fragment of plasmid pBR322, including the β-lactamase gene (bla), which determines the resistance of bacterial cells to ampicillin; - replication initiation site (ori); - ROP gene regulating the copy number of the plasmid; - XhoI / EcoRI fragment, which is a hybrid Tac transcription promoter; - EcoRI / HindIII fragment containing an artificial gene, in which the nucleotide sequence corresponds to SEQ ID NO: 13; - HindIII / BglII fragment containing the terminator of the ribosomal RNA operon rrnB, transcriptional repressor LacI, which regulates transcription of the Tac promoter, unique recognition sites by restriction endonucleases with the following coordinates: EcoRI-115, BamHI-176, SpeI-438, HindlII-444, 1872, NdeI - 2476, AatII - 4468. 2. The Escherichia coli BL21 / pF267 strain is a producer of a hybrid polypeptide with the proinsulin lispro sequence, the strain obtained by transforming the cells of the E. coli BL21 strain with the recombinant plasmid DNA pF267 according to claim 1. 3. Hybrid polypeptide of human proinsulin lispro, in which the sequence MetLeuTyrTyrGluGlyLeuGlnAsp of the fragment of human HGS protein, a substrate of tyrosine kinase, is connected through a peptide linker with the sequence GlyHis6GlySerArg with the amino acid sequence of the proinsulin lyspro site with the amino acid sequence of the proinsulin lyspro site and the amino acid sequence of the site with arulinine lyspro as a peptide chain, including the proinsulin lyspro peptide chain as a peptide amino acid sequence of SEQ ID NO: 14.

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