RU2143492C1 - Recombinant plasmid encoding fused protein as precursor of human insulin (variants), strain of bacterium escherichia coli - producer of fused protein as precursor of human insulin (variants), method of human insulin preparing - Google Patents

Abstract

FIELD: biotechnology, molecular biology, genetic engineering. SUBSTANCE: invention relates to development of new recombinant plasmids expressing fused protein where amino acid sequences of lider peptide that is IgG-binding domain of protein A and human proinsulin are bound by two arginine residues (plasmid pRRproins) or lysine and arginine residues (plasmid pRRproins). Human insulin is prepared by a method that involves culturing the strain E. coli JM-109-pRRproins carrying plasmid pRRproins or the strain E. coli JM-109-pRRproins carrying plasmid pRRproins, isolation of inclusion bodies, solubilization, sulfitolysis and renaturation of fused protein by ion-exchange chromatography method, its cleavage with trypsin-like and carboxypeptidase B-like enzymes and insulin isolation. EFFECT: simplified technological process of human insulin preparing, increased yield of insulin. 6 cl, 4 dwg, 7 ex

Description

 The invention relates to the field of biotechnology and relates to means for producing recombinant human insulin.

 Insulin, a pancreatic hormone, is a globular protein consisting of two polypeptide chains (A and B) linked by disulfide bonds. Its lack of blood leads to a serious illness - insulin-dependent diabetes mellitus. The only therapy is regular insulin injections. Initially, only insulin of animal origin, isolated from the pancreas of farm animals, usually pigs, was used for these purposes. But this approach does not allow providing all those who need regular insulin injections. In this regard, methods have been developed for the production of the hormone by genetic engineering methods, based on the creation of strains producing hybrid proteins, the precursors of human insulin, based on E. coli cells, which accumulate in large quantities in cells in the form of inclusion bodies. A hybrid protein consists of a leader peptide and a peptide with the amino acid sequence of proinsulin, in which the A and B chains are connected by the so-called C-peptide. To obtain a biologically active hormone, it is necessary to cleave the leader peptide, renature proinsulin, forming three necessary disulfide bonds, and remove the connecting C-peptide.

 Known [FEBS Letters, 402, 1997, 124-130] is a method for producing genetically engineered human insulin based on the cultivation of a strain producing a hybrid protein in which the amino acid sequences of the leader peptide and proinsulin are connected via a methionine residue. The producing strain carries a plasmid obtained as follows: a commercially available cDNA library from the human pancreas was used as a matrix; The hybrid protein cDNA was obtained by polymerase chain reaction, and the primers were synthesized in such a way as to introduce the restriction enzyme sites BamH 1 and Sal I at the 5 ′ and 3 ′ ends, respectively, for cloning in the pQE-31 expression vector, into the amplified fragment. (Qiagene); an additional triplet encoding methionine was introduced immediately before the 5'-end of the proinsulin encoding sequence; the amplified fragment, after digestion with restriction enzymes BamH I and Sal I, was ligated into the same restrictase-treated and dephosphorylated vector using T4 DNA ligase. The resulting plasmid was transformed competent cells of E. coli strain XL2-Blue MRF '. A transformed producer strain of the hybrid protein was grown on LB medium containing 100 μg / ml ampicillin, inducing gene expression of isopropyl-β-D-thiogalactopyranoside (IPTG). At the end of cultivation, inclusion bodies were isolated from biomass, the hybrid protein was dissolved in 70% formic acid, and the leader peptide was cleaved by the action of bromine cyan. Then the peptide with the amino acid sequence of proinsulin was subjected to oxidative sulfitolysis, and the obtained proinsulin S-sulfonate was purified and renatured. Then, purified, renatured proinsulin was converted into a biologically active hormone by trypsin and carboxypeptidase B treatment according to the Kemler method. The described technological scheme is the most common.

 The disadvantages of the described method are, firstly, the multi-stage process, and, secondly, the use of a highly toxic reagent, bromine cyan, to cleave the leader peptide.

 It is known [Journal of Biotechnology, 48, 1996, 241-250] to use the plasmid pTrpZZ-R-proinsulin, which expresses a fusion protein, in which two IgG-binding domains of protein A and a peptide with a proinsulin sequence are connected via an arginine residue to produce human insulin. allows you to refuse in the technological scheme from the use of a highly toxic reagent - bromine cyan. In this case, E. coli strain O17 cells were used as the host for the plasmid and insulin production included the following stages: culturing the producer strain, isolating inclusion bodies, solubilizing the fusion protein, sulfitolysis and renaturation of the fusion protein, and purifying it by affinity chromatography on IgG-Sepharose and cleavage of the fusion protein with trypsin and carboxypeptidase-B to produce insulin, which was then chromatographed.

 The main disadvantages of this prototype method are the use of affinity chromatography, which is not technologically advanced for large-scale production, and a low yield at the stage of enzymatic conversion of the hybrid protein to insulin (44%).

 The invention is aimed at simplifying the process of obtaining human insulin while increasing the yield of the final product.

 To achieve this technical result, a method for producing human insulin is proposed, including culturing E. coli cells, isolating inclusion bodies, sequentially solubilizing, sulfitolizing and renaturing the hybrid protein, purifying the renatured hybrid protein, cleaving it with trypsin-like and carboxypeptidase-B-like enzymes and isolating insulin, while cultivating a new strain of E. coli JM-109-pRRproins bearing the new plasmid pRRproins, or a second new strain of E. coli JM-109-pKRproins bearing the other new plasmid pKRproins, a Rowan protein was purified by ion exchange chromatography. To increase the purity of the target product, the hybrid protein after sulfitolysis before renaturation, it is advisable to further purify by anion exchange chromatography.

 The E. coli strain JM-109-pRRproins, carrying the plasmid pRRproins, is a producer of a hybrid protein in which the amino acid sequences of the leader peptide, which is the IgG-binding domain of protein A, and human proinsulin are connected by two arginine residues.

 The E. coli strain JM-109-pKRproins, carrying the plasmid pKRproins, is a producer of a hybrid protein in which the amino acid sequences of the leader peptide, which is the IgG-binding domain of protein A, and human proinsulin are connected by lysine and arginine residues.

 The new recombinant plasmid pRRproins expressing a hybrid protein in which the amino acid sequences of the leader peptide, which is the IgG-binding domain of protein A, and human proinsulin are connected by two arginine residues, has a size of 5025 bp. and consists of the following elements: EcoR I - Hpa I fragment (212 bp) encoding a leader peptide, two binding arginine residues and the first two amino acids of the insulin B chain; Hpa I-Hind III fragment (257 bp) encoding the remainder of the B3-B30 insulin B chain, C-peptide and A-non-insulin; Hind III - EcoR I fragment (4556 bp) carrying the tac promoter and the ampicillin resistance gene.

 Another new recombinant plasmid pKRproins expressing a hybrid protein in which the amino acid sequences of the leader peptide, which is the IgG-binding domain of protein A, and human proinsulin, are connected by lysine and arginine residues, has a size of 5025 bp. and consists of the following elements: EcoR I - Hpa I fragment (212 bp) encoding a leader peptide, binding residues of lysine and arginine and the first two amino acids of the insulin B chain; Hpa I-Hind III fragment (257 bp) encoding the remainder of the B3-B30 insulin B chain, C peptide and insulin A chain; Hind III - EcoR I fragment (4556 bp) carrying the tac promoter and the ampicillin resistance gene.

 In FIG. Figure 1 shows the scheme of plasmid pNY1, which is the starting point for plasmids pRRproins and pKRproins. In FIG. 2 and 3 show construction schemes for plasmids pRRproins and pKRproins, respectively. Figure 4 shows a diagram of the technological process of obtaining human insulin.

 Plasmids pRRproins and pKRproins are obtained by targeted mutagenesis of the known plasmid pNY1 [Antibiotics and chemotherapy, 39, 1994, N4, 3-7; Bioorganic chemistry, 18, 1992, N12, 1478-1486], encoding a fusion protein in which the amino acid sequences of human proinsulin and the leader peptide, which is a single IgG - binding domain of protein A, are connected via a methionine residue. Plasmid pNY1 (Fig. 1) as a part of strain E. coli JM-109-I is stored in the culture collection of the State Scientific Center for Antibiotics (SSCA) under number 1864 and, if necessary, is isolated from it by the Birnboim-Doli method [Nucleic Acids Res., 7, 1979.1513-1523].

 The purpose of mutagenesis is: for plasmid pRRproins, to replace the ATG triplet encoding methionine with CGTCGC hexanucleotide encoding two arginines (Fig. 2), and for plasmid pKRproins, to replace the same triplet ATG with AAACGC hexanucleotide encoding arginine lysine pair Fig. 3). A fragment of the initial plasmid encoding the leader peptide is amplified by polymerase chain reaction using chemically synthesized oligonucleotides, one of which instead of the CAT triplet corresponding to methionine (meaning the complementary chain) contains GCGACG hexanucleotide corresponding to the arginine-arginine pair (for plasmid pRpro) GCGTTT hexanucleotide corresponding to the lysine-arginine pair (for plasmid pKRproins). The mutable sites in FIG. 2 and FIG. 3 are marked in bold. The amplified fragment was digested with restriction enzymes EcoR I and Hpa I and ligated into plasmid pNY1, from which the original sequence encoding the leader peptide was cut at the same sites. Each of the obtained recombinant plasmids contains a structural gene of the corresponding fusion protein expressed under the control of a strong tac promoter and an ampicillin resistance gene.

The producer strains of E. coli JM-109-pRRproins or E. coli JM-109-pKRproins are obtained by transforming E. coli cells of strain JM-109 with the plasmid pRRproins or pKRproins, respectively. After transformation, colonies grown on an ampicillin medium are randomly selected, plasmids are isolated from them and subjected to restriction analysis and sequencing according to the Sanger method. A cell line carrying a plasmid with a given mutation is subcultured several times onto ampicillin medium and the resulting monoclonal culture is inoculated with 5 ml of ampicillin liquid medium. Part of the culture is used to test the inducible expression of the fusion protein using SDS polyacrylamide gel electrophoresis (PAGE). Glycerin is added to the remainder and the cells are stored at minus 40 ^o C.

The resulting producer strains are characterized by the following features:
Morphological signs. The cells are straight, rod-shaped, about 2 μm in size, gram-negative, non-spore, with well distinguishable inclusion bodies after induction of fusion protein synthesis.

Cultural signs. Cells grow well on normal LB medium containing bactotryptone and yeast extract. On agarized LB medium after 24 hours of growth at 37 ^o C, large (3-4 mm) yellowish colonies form. When growing in a liquid medium, a uniform yellow haze is formed.

Physiological and biochemical characteristics. Cells grow at 15-39 ^o C with optimum at 37 ^o C at a pH of 6.8-7.0. The carbon source is glucose. Sources of nitrogen are bactotryptone, bactopeptone, yeast extract, etc.

 Antibiotic resistance. Cells are resistant to 50-100 μg / ml ampicillin.

 Stability of the plasmid in the strain. When cells are maintained for 6 months on an agar medium containing ampicillin, no plasmid loss or rearrangement is observed that affects the expression of the fusion protein.

 In the invented strains, the hybrid protein is synthesized endogenously in the form of inclusion bodies, and its content is 30-40% of the total number of cellular proteins.

The invented method for producing human insulin is as follows (figure 4):
- cultivate a strain of E. coli JM-109-pRRproins or E. coli JM-109-pKRproins on any suitable medium, for example rich LB medium. Superbroth, TY medium, etc .;
- isolate inclusion bodies, for example, by suspending the biomass of cells in a suitable buffer solution, disintegrating it, and centrifuging or microfiltering or ultrafiltration;
- solubilize the hybrid protein with any suitable solvent that does not violate its primary structure, for example, solutions of urea, guanidinium chloride, phosphoric or formic acids;
- known methods sequentially carry out sulfitolysis and renaturation of the hybrid protein;
- the renatured fusion protein is purified by ion exchange chromatography;
- purified renatured fusion protein is cleaved with trypsin-like and carboxypeptidase-B-like enzymes; enzymatic proteolysis can be carried out by sequential use of these enzymes or by the more commonly used one-step Kemler method [J. Biol. Chem., 246, 1971, 6786-6791];
- isolate insulin from the reaction solution by any known method, for example preparative reverse phase (RP) HPLC, cation exchange chromatography on S-Sepharose or Phospho-Biochrome K, anion exchange chromatography on TSK-DEAE-650M or Q-Biochrome A, gel chromatography on Sephadex G-50 or a combination of these methods.

 To obtain highly purified insulin that complies with pharmacopoeial requirements, it is advisable, after sulfitolysis, to render the fusion protein purified by anion exchange chromatography before renaturation, which can be performed, for example, on sorbents such as DEAE-Sepharose, DEAE-Sepharose FF, DEAE-Toyopearl, Mono Q HR, DEAE-Spheranit OH, Q-Biochrom.

 Thus, the use of either the producer strain obtained on the basis of the new plasmid pRRproins or the producer strain obtained on the basis of the new plasmid pKRproins in the invented method makes it possible to exclude the use of the highly toxic bromine cyan reagent in the technological scheme and use ion-exchange chromatography instead of purifying the renatured protein affinity chromatography on IgG-sepharose, which simplifies the process and makes it more suitable for industrial use in large-scale production leadership. At the same time, this new method allows to increase the yield of the final product from a unit of dry cell biomass by reducing the molecular weight of the ballast portion of the hybrid protein — the leader peptide (mv hybrid protein in the prototype method — 25 kDa; mv hybrid protein in the inventive method - 16.8 kDa) and due to the greater level of expression (25% in the prototype method, up to 40% in the invented method). In the invented method for producing insulin, the yield at the enzymatic cleavage stage is 65% when using the E. coli strain JM-109-pRRproins and 80% when using the E. coli strain JM-109-pKRproins, which is significantly higher than in the prototype method (44 %).

 Example 1. Obtaining plasmids pRRproins.

 Plasmid pRRproins is obtained by targeted mutagenesis of the known plasmid pNY1 (FIG. 1) in accordance with the scheme shown in FIG. 2.

Plasmid pNY1 is isolated from the producer strain E. coli JM-109-I N1864, which is stored in the collection of GNCA strains. For this, the collection strain is plated on plates with LB agar containing 100 μg / ml ampicillin, and 5 ml of LB liquid medium containing 100 μg / ml ampicillin is inoculated with a free-standing colony. The culture is incubated at 37 ^o C and vigorous shaking overnight. 1 ml of the obtained overnight culture was added to 500 ml of LB medium with ampicillin and incubated at 37 ^° C until saturation (OD ₆₀₀ ≈ 4.0). Cells are pelleted by centrifugation at 6000g for 10 min and resuspended in 4 ml of 25 mM Tris-HCl (pH 8.0) containing 50 mM glucose and 10 mM EDTA. 1 ml of lysozyme solution (25 mg / ml) was added to the suspension and incubated for 10 min at room temperature. Then add 10 ml of a solution of 0.2 M NaOH with 1% SDS and mix until the suspension is completely clarified. Stand in ice for 10 minutes. 7.5 ml of 3M potassium acetate was added to the resulting solution and again kept in ice for 10 minutes. The formed precipitate was separated by centrifugation at 20,000 g, and the plasmid was planted with 0.6 volumes of isopropanol from the supernatant. The annular form of the plasmid is obtained by equilibrium centrifugation in cesium chloride in the presence of ethidium bromide (500000g, 14 hours) and is used for directed mutagenesis.

To introduce mutations into the nucleotide sequence of plasmid pNY1, oligonucleotides are synthesized:
I - 5'GAAACAGAATTCATGGCTGAC 3 'and
II - 5'TTGGTTAACAAAGCGACGGGATCCTTT 3 '.

100 μl of the reaction mixture for the polymerase chain reaction contains 1 μg of plasmid, 2.5 units Tag polymerase and the following components at the indicated concentrations: 1 μM primer I, 1 μM primer II, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl ₂ , 20 μg / ml gelatin, 0, 8 mM mixture of all four deoxynucleotide triphosphates. The reaction is carried out according to the following scheme: 94 ^° C. — denaturing (1 min), 45 ^° C. — annealing (2 minutes), 72 ^° C. — polymerization (1 minute). In total, 30 amplification cycles are carried out. At the end of the reaction, 1 μl of the mixture was analyzed by electrophoresis on a 1% agarose gel in 1xTBE (89 mM Tris base, 89 mM H ₃ BO ₃ 2 mM EDTA) in the presence of 0.5 μg / ml ethidium bromide. The amplified fragment was purified by ion-exchange HPLC on a DEAE-NPR column (2.6 x 150 mm) in a NaCl gradient from 0 to 1 M.

1 μg of plasmid pNY1 was digested with restriction enzymes EcoR I and Hind III under the following conditions: 6 mM Tris-HCl (pH 7.6), 6 mM MgCl ₂ , 6 mM 2-mercaptoethanol and 50 mM NaCl, 2 units. EcoR I, 2 units Hind iii. The volume of the reaction mixture is 30 μl. The reaction was carried out for 1 hour at 37 ^° C and stopped by heating at 65 ^° C. The EcoR I-Hind III fragment of plasmid pNY1 (4556 bp) was isolated by electroelution from a 1% agarose gel and dephosphorylated with bacterial alkaline phosphatase.

The amplified fragment is similarly cleaved and ligated into the prepared plasmid using T4 DNA ligase at 11.8 ^° C. overnight. The mixture obtained after ligation transforms E. coli strain TG-1 cells in order to select a plasmid carrying the desired mutation.

The transformation is carried out as follows. The culture was grown to an optical density of 0.6 Units at 600 nm, centrifuged and the cells were gently suspended in 0.1 M ice-cold CaCl ₂ solution. The suspension is placed for 40 minutes in an ice bath, collected by centrifugation and resuspended in 1/20 of the initial volume of calcium chloride. To 100 μl of the obtained competent cells, 30 μl of the mixture was added after ligation, left for 40 minutes in an ice bath and heat shocked (42 ^° C., 2 minutes). Cells are diluted 10 times with LB medium, grown at 37 ^° C. for 1 hour and plated on plates with agarized LB medium containing 100 μg / ml ampicillin.

 Colonies capable of growing on a medium containing 100 μg / ml ampicillin were randomly selected. Plasmids isolated from individual colonies are subjected to restriction analysis and sequenced according to the Sanger method. As a result, the cell line carrying the pRRproins plasmid was selected.

 Example 2. Obtaining plasmids pKRproins.

Plasmid pKRproins is obtained by targeted mutagenesis of the known plasmid pNY in accordance with the scheme shown in figure 3. For this, oligonucleotides are synthesized:
I - 5'GAAACAGAATTCATGGCTGAC 3 '
II - 5'TTGGTTAACAAACGCTTTGCATCCTTT 3 '.

 The preparation is carried out by the method described in example 1. As a result, a cell line containing the pKRproins plasmid is selected.

 Example 3. Obtaining a strain of E. coli JM-109-pRRproins.

The plasmid pRRproins transform competent cells of E. coli strain JM-109 by the method described in example 1. Separately localized colony is reseeded three times on plates with LB medium containing 100 μg / ml ampicillin. The resulting monoclonal culture was inoculated with 5 ml of LB liquid medium and incubated overnight with vigorous shaking at 37 ^° C. To test the ability of the obtained strain to induced expression of the hybrid protein, 300 μl of the night culture was transferred to 2.7 ml of heated LB medium and grown at 37 ^o C until reaching the late logarithmic phase (≈1 h 50 min). IPTG was added to the culture to a concentration of 0.1 mM and incubated at 37 ^° C for another 4 hours. Cells were collected by centrifugation and washed with water to remove the medium, the pellet was dissolved by boiling for 5 minutes in the presence of SDS and 2-mercaptoethanol, and the presence of induced expression of the hybrid protein was checked by SDS-PAGE. According to the results of scanning stained Coomassie R-250 gel, the content of the hybrid protein is 35 ± 5% of the total protein of the cell.

The resulting strain of E. coli JM-109-pRRproins is stored in 20% glycerol at minus 40 ^o C.

 Example 4. The cultivation of the producer strain - E. coli JM-109-pRRproins.

The producer strain is grown in 15 ml of a liquid medium containing 10 g / l of bactotryptone (Difco), 5 g / l of yeast extract (Difco), 5 g / l of NaCl and 100 μg / ml of ampicillin, at 37 ^° C, overnight . The resulting overnight culture inoculated 10 flasks containing 100 ml of the same medium in a ratio of 1: 100. The cultures are incubated at 37 ^° C. and vigorously shaken until the late logarithmic phase is reached (1 hour 50 minutes). Fusion protein biosynthesis is induced by adding IPTG to a concentration of 0.1 mM. 4 hours after induction, 8.57 g of cell biomass of 70% moisture with a hybrid protein content of 35 ± 5% of the total protein in the cells was collected (according to SDS-PAGE).

 Example 5. Obtaining and culturing a strain of E. coli JM-109-pKRproins.

 The E. coli strain JM-109-pKRproins is obtained by transforming the E. coli strain JM-109 with the plasmid pKRproins. Obtaining, validation and cultivation of the strain is carried out according to the method described in examples 3 and 4.

 8.17 g of cell biomass of 70% moisture is obtained with a fusion protein content of 35 ± 5% of the total cell protein.

 Example 6. Obtaining human insulin using E. coli strain JM-109-pRRproins.

 The strain producing E. coli JM-109-pRRproins was cultured by the method described in example 4.

 To isolate inclusion bodies, cells are suspended in 70 ml of an aqueous buffer solution containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl and 0.1 M Trilon B, and then destroyed by ultrasonic disintegrator. In the resulting suspension, the total protein content determined by the Lowry method is 850 mg. Inclusion bodies are separated from water-soluble impurities by centrifugation (15000 g, 30 min).

The fusion protein is solubilized by dissolving inclusion bodies for 6 hours at 30 ^° C. in 60 ml of 8 M urea containing 0.1 M NaCl, 0.01 M dithiothreitol, 0.01 M Trilon B, 50 mM Tris-HCl (pH 7.5). Insoluble particles are removed by centrifugation (15,000 g, 30 min).

The hybrid protein is subjected to sulfitolysis to obtain the hybrid protein S-sulfonate, for which 1.5 g of sodium sulfite and 120 mg of sodium tetrathionate are added to the solution, the pH is adjusted to 9.0 with 2 M NaOH and the solution is allowed to mix for 6 hours. The hybrid protein S-sulfonate is precipitated by adjusting the pH of the solution to 5.2 with 25% acetic acid and diluting the solution 3 times with distilled water. The precipitate formed is separated by centrifugation (5000 g, 10 min), dissolved in 25 ml of 30 mM Tris-HCl (pH 7.5) and chromatographed in the same buffer on a column with DEAE-Sepharose FF (1 x 25 cm) in a concentration gradient from 0 to 1 M NaCl for 4 hours. The elution rate is 40 ml / hour. Fractions were analyzed for the presence of S-sulfonate fusion protein by ion-exchange HPLC (Protein PAK DEAE 5 PW column (7.5 mm x 7.5 cm) (Waters), 30 mM Tris-HCl (pH 7.5), 1 ml / min, NaCl gradient from 0 to 1 M) using obviously pure S-sulfonate. Fractions with S-sulfonate fusion protein are combined, desalted on a 2.5 x 20 cm column with Sephadex G-25 F in 50 mM NH ₄ HCO ₃ and lyophilized. The result is 177 mg of S-sulfonate fusion protein of 90% purity, the yield of which at this stage is 53% of the fusion protein in biomass.

Next, the hybrid protein S-sulfonate is renatured: it is dissolved in 265 ml of 50 mM glycine buffer (pH 10.5) cooled to + 4 ^° C, 12.7 mg of cystine is added and after dissolution 7.54 μl of 2-mercaptoethanol is added. The solution was stirred for 24 hours while maintaining a pH of 10.5 by the addition of 0.2 M NaOH. The increase in the concentration of the renatured fusion protein in the solution is monitored by reverse phase (RP) HPLC using a knownly pure sample of the renatured fusion protein. At the end of the reaction, the solution was concentrated on a Minitan apparatus with a PTGC membrane (Millipore, USA) to 20 ml, desalted as described above, and lyophilized. The result is 169 mg of a product containing 48 mg of a renatured hybrid protein (yield at the 30% stage; yield at this stage with respect to the hybrid protein in biomass -15.9%).

Purification of the renatured fusion protein is carried out on a 1 x 11 cm column with S-Sepharose. 167 mg of the sample was dissolved in 15 ml of 50 mM CH ₃ COONa (pH 4.2) containing 6 M urea and chromatographed in the same buffer in a concentration gradient from 0 to 0.5 M NaCl for 4 hours at a flow rate of 40 ml /hour. Fractions with a renatured hybrid protein (according to RP-HPLC) are combined, desalted and lyophilized. The result is 48 mg of a renatured fusion protein of 90% purity. (The yield at this stage is 90%, the yield at the stage relative to the hybrid protein in biomass is 14.34%).

Enzymatic proteolysis is carried out as follows: 45 mg of the obtained sample is dissolved in 2 ml of 50 mM Tris-HCl (pH 7.8), heated to 37 ^° C in a thermostat, 9 μl of freshly prepared trypsin solution (5 mg / ml, 35 U / mg ) and 9 μl of a solution of carboxypeptidase B (5 mg / ml, 135 U / mg). After 30 minutes, the reaction was stopped by adding 100 μl of glacial acetic acid. The two main reaction products are identified by comparison with standard samples by RP HPLC as human insulin and C-peptide. The insulin content in the reaction mixture is 8.8 mg, which corresponds to 65% yield at this enzymatic stage.

 The hydrolyzate is subjected to preparative RP HPLC in two cycles on a RAD PAK micro Bondapak C 18 cartridge in a Z-module holder from Waters (USA). As the mobile phase, a concentration gradient of acetonitrile from 29.6% to 35.2% for 1 hour in 0.25 M ammonium acetate buffer (pH 4.5) is used. The elution rate is 1 ml / min. Fractions with an insulin content of more than 97% were collected and combined (according to RP HPLC). After lyophilization of the combined fractions, 4.4 mg of highly purified human insulin are obtained (basic substance content - 98%). The output at the stage of purification is 50%, the total yield is 4.65%.

 Additionally, the authenticity of the obtained product with human insulin is confirmed by the determination of the N-terminal amino acid sequences of A- and B-chains; a comparison of the peptide maps of the obtained preparation and the standard sample after treatment with protease V8 from S.aureus and mass spectrometry (mm 5807).

 Example 7. Obtaining human insulin using E. coli strain JM-109-pKRproins.

 To obtain human insulin, use the biomass obtained by culturing the E. coli strain JM-109-pKRproins in example 5.

 The method is carried out according to the method described in example 6. The protein content in the suspension obtained after ultrasonic destruction of the cells is 817 mg. S-sulfonate obtained after purification (169 mg) has a purity of 90%. The output at the stage is 53%.

 After renaturation, 152 mg of a product containing 45.6 mg of a renatured hybrid protein of 90% purity is obtained (yield at the stage is 30%, yield at this stage relative to the hybrid protein in biomass is 15.9%).

 As a result of purification, 45 mg of a renatured fusion protein of 90% purity is obtained. The output at the stage is 90%, the total yield is 14.3%.

 The output of insulin at the stage of enzymatic proteolysis is 80%, which corresponds to 10.8 mg of insulin in the reaction mixture.

 After purification, 5.4 mg of highly purified human insulin with a basic substance content of 97% is obtained. The output at the stage of purification is 50%, the total yield is 5.72%.

Claims (6)

 1. Recombinant plasmid pRRproins expressing a hybrid protein in which the amino acid sequences of the leader peptide, which is the IgG-binding domain of protein A, and human proinsulin are connected by two arginine residues, has a size of 5025 bp. and consists of the following elements: EcoR I - Hpa I fragment (212 bp) encoding a leader peptide, two binding arginine residues and the first two amino acids of the insulin B chain; Hpa I-Hind III fragment (257 bp) encoding the remainder of the B3-B30 insulin B chain, C peptide and insulin A chain; Hind III - EcoR I fragment (4556 bp) carrying the tac promoter and the ampicillin resistance gene.  2. Recombinant plasmid pKRproins expressing a hybrid protein in which the amino acid sequences of the leader peptide, which is the IgG-binding domain of protein A, and human proinsulin, are connected by lysine and arginine residues, has a size of 5025 bp. and consists of the following elements: EcoR I - Hpa I fragment (212 bp) encoding a leader peptide, binding residues of lysine and arginine and the first two amino acids of the insulin B chain; Hpa I - Hind III fragment (257 bp) encoding the remainder of the B3-B30 insulin B chain, C peptide and insulin A chain; Hind III - EcoR I fragment (4556 bp) carrying the tac promoter and the ampicillin resistance gene.  3. The strain Escherichia coli JM-109-pRRproins, carrying the plasmid pRRproins according to claim 1, is a producer of a hybrid protein in which the amino acid sequences of the leader peptide, which is the IgG-binding domain of protein A, and human proinsulin are connected by two arginine residues.  4. The strain Escherichia coli JM-109-pKRproins, carrying the plasmid pKRproins according to claim 2, is a producer of a hybrid protein in which the amino acid sequences of the leader peptide, which is the IgG-binding domain of protein A, and human proinsulin are connected by lysine and arginine residues.  5. A method of producing human insulin, including culturing E. coli cells, isolating inclusion bodies, solubilizing, sulfitolysis and renaturation of the fusion protein, purifying the renatured fusion protein, cleaving it with trypsin-like and carboxypeptidase-like enzymes, and isolating insulin, characterized in that it is cultivated E. coli strain JM-109-pRRproins according to claim 3 or E. coli JM-109-pKRproins according to claim 4, and the renatured hybrid protein is purified by ion exchange chromatography.  6. The method according to claim 5, characterized in that the hybrid protein is further purified by anion exchange chromatography after sulfitolysis before renaturation.

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