RU2354702C2 - RECOMBINANT PLASMID DNA pHINS11 CODING HYBRID PROTEIN-HUMAN INSULIN PRECURSOR, Escherichia coli CELL, TRANSFORMED WITH RECOMBINANT PLASMID DNA pHINS11, Escherichia coli BACTERIA STRAIN JM109/pHINS11 - PRODUCER OF HYBRID PROTEIN-HUMAN INSULIN PRECURSOR, AND METHOD OF OBTAINING HUMAN INSULIN - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

Description

The invention relates to the field of biotechnology, in particular to the production of recombinant human insulin used for the preparation of drugs for the treatment of diabetes mellitus.

Insulin is a pancreatic hormone that regulates the processes of carbohydrate metabolism and maintaining normal blood sugar levels. The needs for this drug cannot be covered by insulin of animal origin due to the limited raw material base. In addition, there is a need for the use of various insulin preparations in the treatment of diabetes. Therefore, the development of methods for producing genetically engineered human insulin is an urgent medical task.

Currently, most of human insulin is obtained using the methodology for expressing the human proinsulin gene in Escherichia coli cells as part of fusion proteins in the form of insoluble inclusion bodies. A hybrid protein, the precursor of human insulin, consists of a leader peptide and the amino acid sequence of proinsulin, in which the A and B chains of insulin are connected by a C peptide. As leader sequences protecting the recombinant protein from proteolytic degradation, bovine protimosin [1], glutathione S-transferase [2], immunoglobulin binding (IgG) domain of protein A from S.aureus X [3], and others were used.

A known method of producing recombinant human insulin proposed by J. Nilsson et al. [4]. The method consists in cultivating a strain of E. coli that produces a hybrid protein containing human proinsulin and two synthetic IgG binding domains of staphylococcal protein A. The isolation scheme was to destroy bacterial cells, obtain inclusion bodies containing a hybrid protein, dissolve inclusion bodies, oxidative sulfitolysis of the hybrid protein , its renaturation, purification of the renatured fusion protein on IgG-Sepharose by affinity chromatography, its cleavage with proteolytic enzymes (trypsin and carbox peptidase B) and final purification of insulin by high performance reversed-phase liquid chromatography.

A significant disadvantage of this method is the high cost of the target product, due to the use for purification of the affinity sorbent, which is expensive to manufacture and short-lived in industrial use.

A known method of producing recombinant human insulin [5], including culturing a producer strain of Escherichia coli JM 109 / pPINS07, destroying cells by disintegration, washing inclusion bodies, dissolving the hybrid protein and restoring disulfide bonds in a buffer solution containing urea and dithiothreitol, renaturation of the recovered protein purification of the hybrid protein by ion exchange chromatography, cleavage of the hybrid protein by combined hydrolysis with trypsin and carboxypeptidase B, purification of insulin by a hydrophobic chromatog raffia using a Butyl-Toyopearl 650 sorbent followed by gel filtration.

The disadvantages of this method are as follows.

1. The use in the process of numerous stages of centrifugation: a) salting out insulin and centrifuging the precipitate obtained after hydrolysis of the fusion protein; b) clarification of the insulin solution by centrifugation before hydrophobic chromatography; c) reprecipitation of insulin and its centrifugation before gel filtration (paragraphs 5, 6 and 7 of the insulin purification scheme).

2. Under the stated conditions of the joint hydrolysis — the mass ratio of the hybrid protein, trypsin and carboxypeptidase B equal to 4000: 2: 1 (Example 1), a significant amount of difficultly separated impurity of B30-destreonininsulin is formed [6].

Known closest to the claimed method for producing recombinant human insulin [7], including culturing a producer strain of E. coli JM109 / pPINS07, destroying bacterial cells by disintegration, separating inclusion bodies containing a hybrid protein, dissolving them in a buffer containing urea and dithiothreitol, acid precipitation of impurity compounds, purification of the hybrid protein by chromatography on KM-Sepharose, cleavage of the hybrid protein with trypsin and carboxypeptidase B is carried out sequentially, while trypsinol products chromatography on SP-Sepharose, and the insulin fraction obtained after digestion with carboxypeptidase B was purified by reverse phase high performance liquid chromatography (RP-HPLC) followed by gel filtration.

The main disadvantage of this method is the multistage process chain for the isolation of recombinant insulin. Sequential cleavage of the fusion protein with trypsin and carboxypeptidase B requires sequential chromatographic purifications of diarginin-insulin after tryptic hydrolysis of the fusion protein and insulin after cleavage with carboxypeptidase B, which leads to significant loss of the final product.

In addition, this method used a producer strain E. coli JM109 / pPINS07 carrying the recombinant plasmid pPINS07, which encodes a hybrid protein with a high proportion of leader peptide, comprising about 50% [8]. Therefore, the use of this strain in the production of insulin increases the cost of the production process due to increased costs of biosynthesis and reduces the yield of the target product.

The closest in technical essence to the invention in that part, which relates to the production of a producer strain of a hybrid protein with human proinsulin, are the producer strain E. coli JM109 / pHINS05 and recombinant plasmid DNA pHINS05 encoding a hybrid protein with human proinsulin, in which the N-terminal fragment of human gamma interferon was used as a leader sequence [9]. This plasmid construct directs the synthesis of a hybrid polypeptide with a higher proportion of human proinsulin. However, the disadvantage of the hybrid protein produced by E. coli strain JM109 / pHINS05 is its inefficient renaturation and, as a result, the unsatisfactory yield of the correctly folded polypeptide.

The present invention, in one part, is directed to obtaining a highly productive bacterial strain - a producer of human insulin precursor, and in the other - to simplify the process of obtaining highly purified recombinant human insulin and increase the yield of the final product.

To achieve this technical result, a method for producing human insulin is proposed, including cultivating a producer strain of Escherichia coli, destroying bacterial cells by disintegration, separating inclusion bodies, dissolving them in a buffer containing urea and dithiothreitol, renaturing and purifying the renatured fusion protein, cleaving it with trypsin and carboxypept B, followed by purification and obtaining the target product, while cultivating a new strain of E. coli JM109 / pHINS11 carrying the new plasmid pHINS11, purification of renata th e fusion protein is carried out by deposition of impurity compounds followed by chromatography on CM-Sepharose, cleavage of the fusion protein with trypsin and carboxypeptidase B are carried out simultaneously, as obtained after enzymatic cleavage of the insulin was purified by chromatography on SP-Sepharose, then insulin was purified by HPLC on reverse phase.

The new E. coli strain JM109 / pHINS11 carrying the plasmid pHINS11 is a producer of a fusion protein containing the amino acid sequence of human proinsulin.

The new recombinant plasmid pHINS11 determines the synthesis of a hybrid protein with a molecular weight of about 15.4 kDa, in which the N-terminal fragment of human gamma interferon is connected via the peptide linker HisProGlySerHisHisHisHisGlySerArg to the amino acid sequence of human proinsulin and ensures its high expression.

Recombinant plasmid pHINS11 was constructed on the basis of the known plasmid pHINS05 [9]. To improve the properties of the hybrid protein encoded by the pHINS05 plasmid, additional amino acid residues were introduced into its linker sequence to facilitate the correct folding of the polypeptide with human proinsulin, as well as its more efficient enzymatic cleavage. For this, the plasmid DNA pHINS05 was subjected to exhaustive hydrolysis with Bell and BamHI restriction enzymes, and the BelI-BamHI fragment obtained (4.2 kb) was ligated with the oligonucleotide duplex obtained by annealing the synthetic oligo-link24A oligonucleotides (SEQ ID NO : 4) and oligo-link24B (SEQ ID NO: 5). The ligation mixture was used to transform competent E. coli JM109 cells. The result was a recombinant plasmid pHINS09, the structure of which was confirmed by restriction analysis. The structure of the gene encoding the hybrid protein was confirmed by sequencing (figure 2).

To increase the copy number of the obtained plasmid pHINS09, it was delegated according to the rop gene (negative copy regulator) [10]. To this end, the DNA of plasmid pHINS09 was subjected to exhaustive hydrolysis with restriction enzymes Eco47III and SnaI, and the resulting Eco47III - SnaI (4.2 kb) was ligated. As a result, a 3.6 kb recombinant pHINS11 plasmid was obtained, the structure of which was confirmed by restriction analysis and sequencing.

The new recombinant plasmid pHINS11 encoding a fusion protein containing the amino acid sequence of human proinsulin is characterized by the following features:

has a size of 3640 bp .;

encodes a 139 aa fusion protein with a molecular weight of 15.4 kDa, in which the amino acid sequence of the leader peptide, which is 42 amino acid residues of the N-terminal fragment of human gamma interferon (SEQ ID NO: 1), is connected via the HisProGlySerHisHisHisHisGlySerArg peptide linker (SEQ ID NO: 2) to the amino acid human proinsulin sequence;

consists of: BamHI-EcoRI fragment of plasmid pKK223-3 [II] containing the tac transcription promoter; EcoRI-HindIII fragment comprising the fusion protein gene with the nucleotide sequence shown in FIG. 2 encoding the N-terminal fragment of human gamma interferon, the peptide linker HisProGlySerHisHisHisHisGlySerArg and the amino acid sequence of human proinsulin; HindIII-SnaI fragment of plasmid pKK223-3 containing the transcription terminator of the ribosome operon E. coli, β-lactamase gene (bla), replication initiation site (ori); Eco47III-EheI fragment of plasmid pKK223-3;

contains a tac transcription promoter, a gene encoding a fusion protein, in which the N-terminal sequence of gamma interferon is connected via the peptide linker HisProGlySerHisHisHisHisGlySerArg to the amino acid sequence of human proinsulin; as a genetic marker, the β-lactamase (bla) gene, which determines the resistance of ampicillin to bacterial cells transformed with the pHINS11 plasmid; unique recognition sites by restriction endonucleases located at the following distance to the right of the EcoRI site: BamHI - 159 bp, HindIII - 430 bp, Pvu I - 1370 bp

The advantage of the plasmid pHINS11 is that it provides a higher yield of the final insulin product by increasing the yield of a correctly folded fusion protein after its renaturation.

The E. coli JM109 / pHINS11 producing strain is obtained by transforming E. coli cells of the JM109 strain with plasmid pHINS11. After transformation, colonies grown on ampicillin medium are selected, plasmids are isolated from them and subjected to restriction analysis and sequencing. The cell line carrying the pHINS11 plasmid is plated several times on ampicillin agar medium and the resulting monoclonal culture is inoculated with 5 ml of ampicillin liquid medium. The culture is checked for the presence of inducible expression of the hybrid protein, packaged, glycerol is added and stored at minus 40 ° C.

The resulting producer strain Escherichia coli JM109 / pHINS11 is characterized by the following features.

Morphological features: small, rod-shaped cells, gram-negative, non-spore-bearing, 1 × 3.5 μm in size, motile, with well-defined inclusion bodies after induction of fusion protein synthesis.

Cultural traits: when growing on LB agar medium, the 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, minimal medium with glucose) is characterized by even turbidity.

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

Antibiotic resistance: cells of the producer strain show resistance to ampicillin (up to 500 mg / ml) due to the presence of the β-lactamase (bla) gene in the plasmid.

Stability of the plasmid in the strain. When maintaining the cells for several months on an agarized LB medium containing ampicillin, no plasmid loss or rearrangement affecting the expression of the fusion protein is observed.

In the new strain, the hybrid protein after induced expression accumulates in the form of inclusion bodies and its content is at least 30% of the total protein of the cell.

The new recombinant plasmid pHINS11 provides efficient biosynthesis of a hybrid protein containing human proinsulin in other bacterial strains of Escherichia coli, for example, the E. coli BL21 host strain (Example 3).

Figure 1 presents the physical map of the recombinant plasmid pHINS11; figure 2 is the nucleotide sequence of the gene of the hybrid protein with human proline in the plasmid pHINS11 and the amino acid sequence encoded by it.

The resulting producer strain E. coli JM109 / pHINS11 deposited in the collection of microorganisms of OJSC National Biotechnology under the number 11-05.

The method of obtaining human insulin is as follows.

The cultivation of the seed and main culture of the producer strain E. coli JM109 / pHINS11 is carried out on a nutrient medium containing, g / l: hydrochloric acid casein hydrolyzate - 30, baker's yeast extract - 14, disubstituted potassium phosphate three-water - 6, monosubstituted potassium phosphate - 3, magnesium sulfate - 0.5, glucose - up to 50, ampicillin sodium salt - 0.05, purified water - the rest. For inoculation of the fermenter, seed is used at a 1:10 dilution. To induce the synthesis of a hybrid protein, 1-isopropyl-β-D-1-thiogalactopyranoside (IPTG) is introduced in the middle of the logarithmic growth phase. During the growing process, the concentration of dissolved oxygen is maintained at 40 ± 15%, the amount of glucose introduced is regulated by the level of dissolved oxygen and pH. After cultivation, the culture is concentrated on a separator and the cells are destroyed on a Gaulin homogenizer in 0.1 M Tris buffer, pH 6.8-7, containing 1.5 M urea and 1 mM EDTA. Inclusion bodies are separated by centrifugation.

Isolation of the hybrid protein from inclusion bodies, its enzymatic cleavage and purification of insulin is carried out according to the following scheme:

- inclusion bodies are dissolved in 0.1 M Tris-HCl buffer, pH 8.0, containing 8 M urea, followed by the addition of dithiothreitol to a final concentration of 10 mM;

- a hybrid protein with human proinsulin is renatured in a 5-10-fold volume of 0.1 M glycine-NaOH buffer, pH 9-11 at a temperature of 10-14 ° C;

- acid deposition of impurity proteins is carried out by adjusting the pH to 4.0-5.5 with a HCl solution, and the precipitate formed is separated by microfiltration;

- purification of the hybrid protein is carried out by chromatography on KM-sepharose, balanced with 0.05 M Na-acetate buffer, pH 4.0 ÷ 5.5. The protein is eluted with a gradient of 0.1-0.6 M sodium chloride in 0.05 M Na-acetate buffer, pH 4.0 ÷ 5.5, containing 1.5 M urea;

- cleavage of the hybrid protein is carried out by the combined hydrolysis of trypsin and carboxypeptidase B with a mass ratio of hybrid protein: trypsin: carboxypeptidase B = 2000: 1: (1 ÷ 10) at pH = 6.9 ÷ 7.5 and a temperature of 4 ÷ 36 ° C. The reaction is stopped by the addition of a 10% hydrochloric acid solution to a pH of 3.6 ± 0.3;

- after enzymatic hydrolysis of the hybrid protein, insulin is purified by ion exchange chromatography on SP-Sepharose in 0.05 ÷ 0.2 M ammonium acetate buffer, pH 3.0 ÷ 6.0, containing 1 ÷ 6 M urea and 10 ÷ 50 mm KCl. Sorbed protein is eluted with a linear gradient of potassium chloride from 0 to 0.5 M in equilibration buffer. Fractions containing insulin with a purity of not less than 90% are combined and used to obtain a highly purified insulin substance;

- purification of insulin by preparative reverse-phase high-performance liquid chromatography is carried out using standard equipment "Armen" (France) or similar equipment from other companies.

The invention is illustrated by the following non-limiting examples.

Example 1. Construction of the plasmid pHINS11.

The plasmid pHINS11 is constructed on the basis of the known plasmid pHINS05 [9]. The pHINS05 plasmid DNA is subjected to exhaustive hydrolysis with restriction enzymes BelI and BamHI. For this, 5 μg of plasmid DNA pHINS05 in 20 μl of buffer containing 33 mM Tris-acetate, pH 7.9, 66 mM K-asetate, 10 mM Mg-acetate and 0.1 mg / ml BSA (buffer 1) and 10 units restriction enzymes BelI and BamHI are incubated for 1.0 hour at 37 ° C. A BclI-BamHI DNA fragment of about 4.2 kb was isolated from the hydrolyzate obtained. by electrophoresis in a 0.8% gel fusible agarose. Next, the DNA is deproteinized with phenol, a mixture of phenol with chloroform (1: 1), chloroform, precipitated with ethyl alcohol, and dissolved in 20 μl of water. The obtained BclI-BamHI fragment containing the hybrid protein gene and the vector part of the pHINS05 plasmid is ligated with a 20-fold molar excess of the oligonucleotide duplex obtained by annealing the synthetic oligo-link24A oligonucleotides (SEQ ID NO: 4) and oligo-lmk24B (SEQ ID NOQ 4B (SEQ ID NO: 4) (SEQ ID NO: 4) (SEQ ID NO: 4) :5):

oligo-link24A

5 'GATCACCCGGGATCTCATCATCAT 3'

3 'TGGGCCCTAGAGTAGTAGTAGTAC 5'

oligo-link24B

Competent cells of E. coli strain JM109 are transformed with a ligation mixture (10 μl) and plated on LB agar containing 100 μg / ml ampicillin. Plasmid DNA was isolated from the grown colonies and sequenced between the EcoRI and BamHI restriction enzyme sites to confirm insertion into the linker portion of the fusion protein gene. The result is the plasmid pHINS09.

To obtain a rop deletion plasmid (negative copy regulator), 10 units of Eco47III and SnaI restriction enzymes generating blunt ends are added to 5 μg of pHINS09 plasmid DNA in 20 μl of buffer and the mixture is incubated for 1.0 hour at 37 ° C . Next, the DNA is deproteinized with phenol, a mixture of phenol with chloroform (1: 1), chloroform, precipitated with ethyl alcohol, and dissolved in 20 μl of water. Thus obtained DNA is ligated in 30 μl of ligation buffer in the presence of 5 units of T4 phage ligase DNA for 16 hours at 8 ° C. The competent cells of the E. coli strain JM109 are transformed with the ligation mixture (10 μl) and plated on LB agar containing 100 μg / ml ampicillin. Bacterial clones carrying plasmid DNA of 3.6 kbp were selected. The isolated plasmids are subjected to restriction analysis and sequenced according to the Sanger method. The result is the plasmid pHINS11.

Example 2. Obtaining a producer strain of E. coli JM109 / pHINS11.

Plasmid pHINS11 transform competent cells of strain E. coli JM109 and plated on LB agar containing 100 μg / ml ampicillin. Separately, a localized colony is reseed three times on plates with LB agar containing 100 μg / ml ampicillin. The resulting monoclonal culture was inoculated with 5 ml of liquid LB medium with ampicillin and incubated overnight with vigorous shaking at 37 ° C.

The resulting producer strain E. coli JM109 / pHINS11 is stored in 20% glycerol at minus 40 ° C.

To determine the level of inducible expression of the hybrid protein, the night culture was seeded at a dilution of 1:50 in 5 ml of LB liquid medium containing 100 μg / ml ampicillin and grown to a turbidity of 0.8 at 37 ° C on a rocking chair at 200 rpm. IPTG was added to the culture to a concentration of 1.0 mM and incubation was continued under the same conditions for 3 hours. Cells are collected by centrifugation, the pellet is suspended in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 3% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 10% glycerol and 0.01% bromophenol blue and heated for 3 minutes at boiling water bath. The obtained cell lysate was analyzed by electrophoresis in an 18% polyacrylamide gel with sodium dodecyl sulfate [12]. The gel is stained with Coomassie R-250, scanned and densitometric. According to densitometry, the content of the hybrid protein is at least 30% of the total protein of the cell.

Example 3. Obtaining a producer strain of E. coli BL21 / pHINS11.

Competent cells of E. coli strain BL21 transform plasmid pHINS11 DNA and plated on LB agar containing 100 μg / ml ampicillin. Separately, a localized colony is reseed three times on plates with LB agar 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.

The resulting producer strain E. coli BL21 / pHINS11 is stored in 20% glycerol at minus 40 ° C.

The productivity of the E. coli BL21 / pHINS11 strain was determined by the same method as for the E. coli JM109 / pHINS11 strain described in Example 2. According to densitometry, the content of the hybrid protein in the induced cells of the obtained strain was at least 30% of the total cell protein .

Example 4. Obtaining human insulin using a producer strain of E. coli JM109 / pHINS11.

Step 1. Cultivation of the biomass of the producer strain E. coli JM109 / pHINS11.

The cultivation of cells of the strain E. coli JM109 / pHINS11 is carried out under conditions that ensure the accumulation of the hybrid protein. At all stages of propagation of microbial cultures, a nutrient medium of the following composition is used (g / l): hydrochloric acid casein hydrolyzate - 30, baker's yeast extract - 14, disubstituted potassium phosphate three-water - 6, monosubstituted potassium phosphate - 3, magnesium sulfate - 0.5 glucose - up to 50, ampicillin sodium salt - 0.05, purified water - the rest.

Sowing crops are prepared in an amount of 1/10 of the volume of the sown nutrient medium and grown to optical density (OD) of 7-8 units (wavelength - 540 nm, optical path length -10 mm).

The conditions for growing crops are presented in table 1.

Table 1 pH 6.9 ± 0.2 pO ₂ 40 ± 15% Defoaming Sensor Signal Aeration and stirrer operation By level pO ₂ The amount of glucose and alkali administered According to the level of pO ₂ and pH Duration 4-6 hours Process end With OD 7-8

The cultivation of the main culture of the producer is carried out in a fermenter with a total capacity of 150 liters. The composition of the nutrient medium and the basic cultivation conditions are the same as for inoculum crops. To induce biosynthesis of the hybrid protein, an IPTG inducer is introduced in the middle of the logarithmic phase of the culture growth. Cultivation is continued until the formation of intracellular inclusions of the fusion protein (“inclusion body”) in 90-95% of the cells. Express control of the accumulation of inclusion bodies (TVs) is carried out using phase contrast microscopy of the preparations.

The main parameters of the cultivation process of the producer strain E. coli JM109 / pHINS11 are shown in table 2.

table 2 Full fermenter capacity 150 l Fermenter working capacity 100 l The volume of the nutrient medium 90 l The volume of sowing crops 10 l Working temperature 36.5 ± 0.5 ° C pH 6.9 ± 0.2 pO ₂ 40 ± 15% Defoaming Sensor Signal Aeration and stirrer operation By level pO ₂ The amount of glucose and alkali administered According to the level of pO ₂ and pH IPTG Introduction With OD 7-9 Process end With the formation of TB in 90-95% of cells

Stage 2. Isolation of inclusion bodies.

The growth of the culture in the fermenter is stopped by a sharp reduction in the intensity of mixing, the culture is cooled to 10-14 ° C and concentrated on the separator 8-10 times. Tris-basic is added to the resulting suspension to a concentration of 0.1 M, urea - up to 1.5 M, EDTA - up to 1 mM. Buffered to a pH of 6.8-7.0, the suspension is passed three times through a Gaulin homogenizer at a pressure of 700-800 atm and a temperature of 15-20 ° C. Cell homogenate is passed through a flow centrifuge (g = 18000). The bulk of inclusion bodies (at least 90%) is deposited in the centrifuge rotor. The wet sediment of inclusion bodies, 2.6 kg, is discharged from the centrifuge rotor, frozen and stored at minus 40 ° C. Losses of the hybrid protein during isolation of inclusion bodies do not exceed 15%.

Step 3. Dissolution of inclusion bodies containing the fusion protein, and renaturation of the fusion protein.

In a 30 L reactor filled with 18 L of a buffer solution containing 0.1 M Tris-HCl, pH 8.0 and 8 M urea, 1.8 kg of inclusion Taurus paste was charged and a stirrer was turned on. After dissolution of the inclusion bodies, dithiothreitol is added to the reactor to a final concentration of 10 mM and stirring is continued for 10-12 hours at 15 ° C. The amount of fusion protein in solution was 174 g.

160 l of glycine-NaOH buffer, pH 9-11, are poured into a cooling reactor with a volume of 250 l, pH 9-11 and cooled to a temperature of 10-14 ° C. After cooling the buffer solution, a hybrid protein solution with reduced disulfide bonds is fed to the reactor. The hybrid protein solution is incubated for 20-24 hours, mixing and maintaining the temperature in the reactor 10-14 ° C. After renaturation of the hybrid protein with the formation of correctly closed S-S bonds (control by RP-HPLC), acid deposition of impurity proteins is carried out by acidifying the reaction medium in the reactor to pH 4.0-5.5 with a hydrochloric acid solution. Stirring is stopped and after 4-5 hours the supernatant is clarified on a microfiltration unit.

The content of the renatured hybrid protein in the supernatant is 85 g.

Stage 4. Purification of the renatured hybrid protein on KM-Sepharose.

A correctly folded fusion protein is sorbed from the filtrate onto a 5 L ion exchange column filled with KM-Sepharose, previously equilibrated with 0.05 M Na-acetate buffer, pH 4.0 ÷ 5.5. Sorbed protein is eluted from the column with a gradient of 0.1-0.6 M sodium chloride in 0.05 M Na-acetate buffer, pH 4.0 ÷ 5.5, containing 1.5 M urea. Fractions containing a hybrid protein with a purity of not less than 95% (control by RP-HPLC) are combined and used for further work.

The result is 63 g of renatured hybrid protein with a purity of at least 90%.

Step 5. Joint hydrolysis of the hybrid protein with trypsin and carboxypeptidase B and purification of insulin on SP-Sepharose.

The cleavage of the hybrid protein with trypsin and carboxypeptidase B is carried out in a stainless steel reactor with a cooling volume of 30 l, equipped with a mixing device. 13 l of a solution of the hybrid protein are introduced into the reactor, the solution is cooled to 4-7 ° C and its pH is adjusted to 7.2-7.3 by the addition of a 1 M tris-base solution. Then, a solution of trypsin and carboxypeptidase B in a weight ratio of hybrid protein: trypsin: carboxypeptidase B equal to 2000: 1: 1.25 was introduced into the reactor. The hydrolysis reaction is carried out for 10-12 hours, monitoring by the method of RP HPLC the accumulation of insulin in the hydrolyzate. The cleavage reaction is stopped by acidifying the hydrolyzate to a pH of 3.4 ± 0.2 with a 10% hydrochloric acid solution to a pH of 3.6 ± 0.3. Potassium chloride was added to the reactor to a concentration of 40 mM. The resulting solution was applied to a 5 L chromatographic column filled with SP-Sepharose, previously equilibrated with buffer: 2 M urea, 0.1 M ammonium acetate, pH 3.6. The column was washed with the same buffer. Sorption of insulin is eluted by a linear gradient of potassium chloride from 0 to 0.5 M in a balancing buffer. Combine fractions containing 13.2 g of insulin with a purity of at least 95% (control by RP-HPLC).

Step 6. Obtaining highly purified insulin by preparative reverse phase high performance liquid chromatography (RP HPLC)

Purification of insulin by RP HPLC was carried out on standard equipment "Armen" (France). An integrated system for HPLC includes the following elements:

- chromatograph "Armen" (productivity 100 ml / min, pressure up to 150 atm),

- Control block,

- double-head high pressure diaphragm pump,

- high-pressure pump for pumping samples for separation,

- ultraviolet detector with variable wavelength "Knauer",

- chromatographic column, stainless steel., V = 4 l,

- A computer for managing and processing data.

The preparation of the chromatographic unit for operation is carried out according to the manufacturer's instructions for individual units and the unit as a whole.

An insulin solution from the previous purification step in the amount of 13.2 g of protein is fed into the prepared column using a pump for supplying a sample from the container. Turn on the programming device and carry out the separation according to the following program (see table 3).

Table 3 Time min The rate of elution, cm ³ % solution B 0 350 26 5 350 27.5 fifteen 350 28.5 twenty 350 thirty 25 350 70 thirty 350 26

The separation process is recorded at 220 nm on a flow spectrophotometer with a sensitivity of 2.0. Protein fractions are collected using a chromatograph collector or manually. The collected fractions of the main peak of insulin are analyzed for impurities by RP-HPLC.

After purification, 11.4 g of highly purified human insulin with a basic substance content of 98% are obtained.

Example 5. Joint hydrolysis of the hybrid protein with trypsin and carboxypeptidase B and purification of insulin on SP-Sepharose.

The hydrolysis conditions of the hybrid protein with trypsin and carboxypeptidase B and the subsequent purification of insulin on SP-Sepharose are shown in table 4.

Table 4 Conditions for hydrolysis of the hybrid protein with trypsin and carboxypeptidase B Chromatography conditions on SP-Sepharose The mass ratio of the hybrid protein: trypsin: carboxypeptidase B t ° C pH Urea concentration, M The concentration of NH ₄ Ac, mm pH The concentration of KCl, mm in the sample 2000: 1: 4 15 ± 1 7.5 ± 0.1 1,5 130 3.3 45 2000: 1: 1.5 4 ± 1 6.9 ± 0.1 1,5 130 3.3 45 2000: 1: 2 5 ± 1 7.0 ± 0.1 2 one hundred 3.6 40 2000: 1: 10 36 ± 1 7.2 ± 0.1 3 80 4.2 thirty 2000: 1: 10 36 ± 1 7.5 ± 0.1 6 fifty 5.5 10

Thus, the proposed method allows to obtain highly purified human insulin with a purity of not less than 98% and an activity of not less than 27.5 U / mg.

The authenticity of the obtained target product is confirmed by the following parameters:

- by coincidence of the retention time of the main peak of the target product and the international reference standard USP (Pharmacopoeia USP 23, USA);

- by definition of the biological activity of the target product (VFS 42-3045-98), which is at least 27 U / mg;

- the coincidence of the peptide maps of the target product and the reference standard USP.

- by determining the molecular weight of the target product by mass spectroscopy.

The purity of the obtained target product is confirmed by pharmacopoeia USP 23, USA and amounted to:

- the content of the target product, determined by HPLC, at least 98%;

- the content of desamidoinsulin is less than 2%.

Information sources

1. Tang J., Xue Y., Fan X., Fu Y. // Clin. J. BiotechnoL, 1993. v. 9. p. 71-78.

2. Berg H., Walter M., Mauch L., Seissler J., Northemann W.J. // Immunol. Methods, 1993, v. 164, p. 211-231.

3. Wei G., Hu M. H., Tang L. G. // Biochem. Mol. Biol. Int., 1995, v. 35. p. 37-46.

4. Nilson J., Jonasson P., Samuelsson E., Stahl S., Uhlen M. // Journal of biotechnology, 1996, v. 48, p. 241-250.

5. RF patent RU 2208637 C1, 20.07.2003.

6. USSR patent SU 1829939 A3, 07.23.93. Bull. Number 27.

7. RF patent RU 2232813 C1, 20.07.2004. Bull. No. 20.

8. RF patent RU 2144957 C1, 01/27/2000.

9. RF patent RU 2263147 C1, 10.27.2005. Bull. No. 30.

10. Twigg A.J. and Sherrat D. // Nature, 1980, v. 283, p. 216-218.

11. Brosius J., Dull T. J., Sleeter D.D., Noller H.F. // J. Mol. Biol., 1981, v. 148, p. 107-127.

12. Laemmli U.K. // Nature, 1970, v. 227, p. 680-687.

Claims (6)

1. The hybrid protein is the precursor of human insulin of the formula (I) [leader] - [linker] - [proinsulin] (I),
where leader is a leader peptide sequence for expression of a fusion protein containing 42 amino acid residues of the N-terminal fragment of human gamma interferon (SEQ ID NO: 1);
linker is the linker sequence of HisProGlySerHisHisHisHisGlySerArg (SEQ ID NO: 2);
proinsulin is the amino acid sequence of human proinsulin (SEQ ID NO: 3). 2. DNA encoding a hybrid human insulin precursor protein and characterized by a nucleotide sequence (SEQ ID NO: 6) that determines the amino acid sequence of the hybrid protein according to claim 1. 3. Recombinant plasmid pHINS11 (figure 1), containing DNA encoding a hybrid protein according to claim 2, size 139 aa, in which the N-terminal fragment of human gamma interferon is connected via the peptide linker HisProGlySerHisHisHisHlyGerSerArg with the amino acid sequence of human proinsulin having molecular weight 2.36 MDa (3534 bp) containing the BamHI-EcoRI fragment of the pKK223-3 vector plasmid, including the tac transcription promoter, the EcoRI-HindIII fragment with the gene having the nucleotide sequence shown in FIG. 2 and encoding N end fragment of gamma interfer per person, HisProGlySerHisHisHisHisGlySerArg peptide linker and the amino acid sequence of human proinsulin, HindIII-SnaI fragment of plasmid pKK223-3, including transcription terminators of the E. coli ribosomal operon, β-lactamase gene (bla), site of replication of the I-fragment III or pKK223-3, including unique recognition sites of restriction endonucleases located at the following distance to the right of the EcoRI site: BamHI - 159 bp, HindIII - 430 bp, PvuI - 1370 bp 4. An Escherichia coli cell transformed with pHINS11 recombinant plasmid DNA according to claim 3 is a producer of a hybrid protein containing human proinsulin. 5. The bacterial strain Escherichia coli JM109 / pHINS11 is a producer of a hybrid protein containing human proinsulin. 6. A method for producing recombinant human insulin, including culturing a strain of Escherichia coli, a producer of a hybrid protein with human proinsulin, disrupting cell disintegration, separating inclusion bodies containing the hybrid protein, dissolving them in a buffer containing urea and dithiothreitol, renaturation of the hybrid protein, acid deposition of impurities compounds, and purification of the hybrid protein, its enzymatic digestion with trypsin and carboxypeptidase B, followed by purification and isolation of insulin, characterized in that lead cultivation of the strain Escherichia coli JM109 / pHINS11 - producer of a hybrid protein containing human proinsulin; purification of the hybrid protein is carried out by chromatography on KM-sepharose, balanced with 0.05 M Na-acetate buffer, pH 4.0 ÷ 5.5, while the protein is eluted with a gradient of 0.1-0.6 M sodium chloride in 0.05 M Na -acetate buffer, pH 4.0 ÷ 5.5, containing 1.5 M urea; cleavage of the hybrid protein is carried out by combined hydrolysis with trypsin and carboxypeptidase B, and the resulting insulin is purified by ion exchange chromatography on sorbents with sulfopropyl groups in 0.05–0.2 M ammonium acetate buffer, pH 3.0–6.0, containing 1–6 M urea and 10 ÷ 50 mm KCl, and then by reverse phase high performance liquid chromatography.

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