RU2549710C2 - Method for purifying recombinant protein of type iii interferon-like factor - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is presented is a method for preparing a recombinant protein of type III interferon-like factor (ILF III) of the producing strain E. coli. The inclusion bodies E. coli are washed and dissolved with using 2% aqueous γ-cyclodextrin. That is followed by the sequential Ni-Sepharose, Q-Sepharose and SP-Sepharose chromatographic procedures. Refolding of a target protein is performed with using a mixture of cysteamine and cystamine at pH 10.5. The Amberchrome Profile XT20, Amberchrome Profile HPR10 and Kromasil 300-5C18 chromatographic procedures are sequentially performed.

EFFECT: invention enables optimising the ILF III purification environment at the stage of washing and dissolving the inclusion bodies Ecoli and provides 12% target protein yield.

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Description

The invention relates to biotechnology, and in particular to a technology for producing a recombinant protein of interferon-like factor III type (hereinafter, IPF III), in particular, to a method for its purification.

Known methods for purification of recombinant interferons (hereinafter, IFN), based on the selection of inclusion bodies, their washing and refolding of the target protein, followed by chromatography.

A known method of producing recombinant interleukin 29 (IL-29) in insoluble form in the form of inclusion bodies (US patent No. 7910313, 03/22/2011).

Compared to the soluble form, the expression of protein in the form of inclusion bodies has several advantages: insoluble protein aggregates do not exhibit toxic effects, are practically not attacked by proteases, and are released by centrifugation in high yield. At the same time, there are problems with a low yield of active, correctly folded protein after refolding and purification of IL-29, which is determined by the spatial structure of the latter.

A known method of purification of interleukin IL-29 and the use of detergent sodium dodecyl sulfate when it is received in insoluble form (LIS patent No. 7485700, 02/03/2009). However, with the relatively low toxicity of this drug, the use of sodium dodecyl sulfate in the process is fraught with a number of obstacles: this compound causes significant denaturation of the target protein, it cannot be removed from the solution, which in turn makes subsequent ion exchange chromatography necessary for the further IL purification process impossible -29.

There is also a known method for purifying IFN, which avoids the indicated drawbacks by using γ-cyclodextrin as a detergent, which does not have denaturing properties, does not form ionic bonds, is pH-independent, can be removed from solution, and ion-exchange chromatography is possible.

The closest selected as the closest analogue to the claimed one is a method for purifying interleukin IL-29 from inclusion bodies using affinity chromatography, cation exchange chromatography and gel filtration (US patent No. 7968315, 06/28/2011).

However, this method does not take into account the possibility of using reversed-phase chromatography to separate isomers with different spatial structures and does not characterize the features of its purification associated with this.

The present invention solves the problem of optimizing the purification conditions of IPF III at the stages of washing and dissolving inclusion bodies, for example, E. coli BL (21) IPF3 (7) cells producing recombinant IPF III protein, during the technological process of refolding the target protein and purification using highly sensitive protein chromatography (FPLC-Fast protein liquid chromatography) and high performance liquid chromatography (HPLC-High performance liquid chromatography).

The inventive method for purification of a recombinant protein of an interferon-like factor III type involves washing the inclusion bodies of the E. coli producer strain, occurring in the presence of a 2% aqueous solution of the basic detergent γ-cyclodextrin, dissolving the inclusion bodies in the presence of a 2% aqueous solution of γ-cyclodextrin, 0.1 M potassium sulfite and 0.01 M sodium tetrathionate, metal affinity chromatography on Ni-Sepharose with linear gradient elution with start buffer 2% γ-cyclodextrin, 6 M urea, 20 mM Tris-HCl with pH 8.0, 10 mM imidazole, and the final buffer 2% γ-cyclo dextrin, 6 M urea, 20 mM Tris-HCl with a pH of 8.0, 400 mM imidazole, with the release of the target protein in the range of imidazole concentrations from 75 to 125 mM, dialysis of fractions containing the target protein against 6 M urea, 20 mM Tris-HCl pH 8.0 and 2% γ-cyclodextrin, anion-exchange chromatography on Q-Sepharose with linear gradient elution, with 6 M urea, 20 mM Tris-HCl with pH 8.0, 10 mM NaCl, 2% γ-cyclodextria as starting buffer and the final buffer is 6M urea, 20 mM Tris-HCl with a pH of 8.0, 300 mM NaCl, 2% γ-cyclodextrin, with the yield of the target protein in the range of NaCl concentrations from 115 to 125 mM, dialysis of fractions containing the target protein against 6M urea, 20 mM Tris-HCl pH 8.0 and 2% γ-cyclodextrin, cation-exchange chromatography on SP-Sepharose with linear gradient elution with starting buffer 6M urea, 20 mM Tris-HCl with pH 8.0, 10 mM NaCl, 2% γ-cyclodextrin, and a final buffer of 6 M urea, 20 mM Tris-HCl with pH 8.0, 300 mM NaCl, 2% γ-cyclodextrin, with the yield of the target protein in the range concentrations of NaCl from 110 to 130 mm, refolding the target protein at a pH of 10.5 by adding a mixture of cysteamine and cystamine to a final concentration of 1 mm and 0.1 mm, followed by Conducting reversed phase chromatography on Amberchrome Profile XT20 columns at pH 10.5 with linear gradient elution with a start buffer of 10 mM NaCl, 2% acetonitrile, and a final buffer of 10 mM NaCl, 80% acetonitrile, pH 10.5 , with the yield of the target protein at a concentration of acetonitrile 54%, dialysis of fractions containing the target protein against a solution of 10 mm NaCl, pH 10.5, chromatography on Amberchrome Profile HPR10 at pH 10.5 with elution with a linear gradient, while the starting buffer is 10 mM NaCl, 2% acetonitrile, pH 10.5, and the final buffer is 10 mM NaCl, 80% acetonitrile, pH 10.5, with the yield of the target protein at a concentration of acetonitrile 58%, dialysis of fractions containing the target protein against a solution of 100 mm NaCl, the addition of trifluoroacetic acid to 0.1% and pH 1.0, chromatography on Kromasil 300-5C18 at pH 1.0 with linear gradient elution with a starting buffer of 0.1% trifluoroacetic acid, 2% acetonitrile, and a final buffer of 0.1% trifluoroacetic acid, 80% acetonitrile, with the yield of the target protein at an acetonitrile concentration of 35%.

The claimed invention is illustrated by drawings. Figure 1 shows the change in optical density OD (585) of the suspension of inclusion bodies in 6M urea with 20 mM TrisHCl pH 8.0 depending on the increase in the concentration of γ-cyclodextrin, and Figure 2 shows the chromatographic profile of elution of the refolded recombinant protein IPP III from the Kromasil 300 5C18, in Fig. 3 - electrophoresis of the refolded recombinant protein IDF III after all stages of HPLC chromatographic purification, in which track 1 - molecular weight standards, track 2 - recombinant protein IDF III.

The inventive method of purification of a recombinant protein of an interferon-like type III factor from inclusion bodies of E. coli producer strain BL (21) IPF3 (7) involves dissolving and washing inclusion bodies in a solution containing 6 M urea, 20 mM Tris HCl pH 8.0 and 2% γ β-cyclodextrin followed by removal of insoluble material by centrifugation, oxidation of the target protein in a solution containing 6 M urea, 20 mM Tris HCl pH 8.0 and 2% γ-cyclodextrin with the addition of 0.1 M potassium sulfite and 0.01 M sodium tetrathionate, incubation 24 hours at a temperature of 20 C, dilution of the mixture five times, applying to a column of Ni-Sepharose, balanced with a solution containing 2% γ-cyclodextrin, 6 M urea, 20 mm Tris-HCl pH 8.0, washing with a solution containing 2% γ-cyclodextrin, 6 M urea, 20 mm Tris- HCl pH 8.0, 0.005 M NaCl, linear gradient elution with start buffer 2% γ-cyclodextrin, 6 M urea, 20 mM Tris-HCl with pH 8.0, 10 mM imidazole, final buffer 2% γ-cyclodextrin, 6 M urea , 20 mM Tris-HCl with pH 8.0, 400 mM imidazole, with the yield of the target protein in the range of imidazole concentrations from 75 to 125 mM, dialysis of fractions containing the target protein against 6 M urea, 20 mM Tris-HCl pH 8.0 and 2% γ -cyclo ectrina, application to a Q-Sepharose column, pre-equilibrated with 20 mM Tris-HCl with pH 8.0, elution with a linear gradient with a starting buffer of 6 M urea, 20 mM Tris-HCl with pH 8.0, 10 mM NaCl, 2% γ-cyclodextrin, the final buffer is 6 M urea, 20 mM Tris-HCl with a pH of 8.0, 300 mM NaCl, 2% γ-cyclodextrin, with the yield of the target protein in the range of NaCl concentrations from 115 to 125 mM, dialysis of fractions containing the target protein against 6 M urea, 20 mM Tris-HCl pH 8.0 and 2% γ-cyclodextrin, application to a column of SP-Sepharose, pre-equilibrated with 20 mM Tris-HCl pH 8.0, 2% γ-cyclodextrin, elution with linear gradient volume with starting buffer 6 M urea, 20 mM Tris-HCl with pH 8.0, 10 mM NaCl, 2% γ-cyclodextrin, final buffer 6 M urea, 20 mM Tris-HCl with pH 8.0, 300 mM NaCl, 2% γ- cyclodextrin, with the yield of the target protein in the range of NaCl concentrations from 110 to 130 mm, adjusting the pH of the 1M NaOH protein solution to pH 10.5, refolding by adding a mixture of cysteamine and cystamine to the final concentrations of 1 mm and 0.1 mm, respectively, incubation for 24 hours at a temperature of 20 ° C, application to a column of the Amberchrome Profile XT 20 series, elution with a linear gradient with a starting buffer of 10 mM NaCl, 2% acetonitrile, pH 10.5, and final m buffer 10 mM NaCl, 80% acetonitrile, pH 10.5, with the yield of the target protein at a concentration of acetonitrile 54%, dialysis of fractions containing the target protein against a solution of 10 mm NaCl, pH 10.5, application to the Amberchrome Profile HPR 10 column , linear gradient elution with a start buffer of 10 mM NaCl, 2% acetonitrile, pH 10.5, and a final buffer of 10 mM NaCl, 80% acetonitrile, pH 10.5, with the yield of the target protein at a concentration of acetonitrile 58%, dialysis of fractions containing the target protein against a solution of 100 mm NaCl, the addition of trifluoroacetic acid to 0.1% with a pH of 1.0, applying to a column of the Kromasil 300-5C18 series, linear gradient elution with a starting buffer of 0.1% trifluoroacetic acid, 2% acetonitrile, and a final buffer of 0.1% trifluoroacetic acid, 80% acetonitrile, with the yield of the target protein at an acetonitrile concentration of 35%.

The starting buffer: 10 mM NaCl, 2% acetonitrile, pH 10.5 was adjusted with 1 M NaOH, final buffer: 10 mM NaCl, 80% acetonitrile, pH 10.5 was adjusted with 1 M NaOH containing the target protein against a solution of 10 mM NaCl, pH 10.5 was adjusted with 1 M NaOH, application to a column of the Amberchrome Profile HPR 10 series, elution with linear gradient starting buffer: 10 mM NaCl, 2% acetonitrile, pH 10.5 was adjusted with 1 M NaOH, final buffer: 10 mM NaCl, 80% acetonitrile, pH 10.5 was adjusted with 1 M NaOH.

The control of the yield and activity of recombinant IPF III after purification showed compliance with the following criteria: yield 12%, multimers by gel filtration <2%, related proteins by reverse phase chromatography <3%, related proteins by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate ( SDS-PAGE) <5%, proteins of the producer strain by enzyme-linked immunosorbent assay (ELISA) <5 ng / mg, specific activity of IPF III corresponds to the standard preparation L-29 from R&D on the MDBK / VSV model. The specific activity of the standard R&D L-29 preparation on the MDBK / VSV model is ED50 0.2-1 million units / mg (known from the Internet at http://www.rndsystems.com/Products/1598-IL.

Combined use of reverse phase chromatography on columns with a matrix based on polystyrene / divinylbenzene, allowing the purification process at pH 10.5 and reverse phase chromatography on columns with a matrix based on spherical silica gel, modified with C18 groups, allowing the cleaning process at pH 1,0. This combination of reversed-phase chromatography at specific pH values makes it possible to obtain a homogeneous preparation with a fixed spatial structure.

Thus, the proposed method for purification of the recombinant protein IPF III (producer strain BL (21) IPF3 (7)) involves the optimization of the processes of washing the inclusion bodies, their dissolution and refolding of the target protein. Purification of the recombinant protein IPF III (producer strain BL (21) IPF3 (7)) is carried out using γ-cyclodextrin as the main detergent for washing and dissolving E. coli inclusion bodies. Chromatography is then carried out on Ni-Sepharose, on Q-Sepharose and on SP-Sepharose, followed by refolding of IPP III protein. It is then oxidized using a cysteamine / cystamine mixture at pH 10.5, followed by chromatography on Amberchrome Profile XT20, chromatography on Amberchrome Profile HPR10 and chromatography on Kromasil 300-5C18. The proposed purification method allows to provide a 12% yield of the target protein and to obtain the recombinant protein IPF III with specific activity corresponding to the standard R-D preparation L-29 on the MDBK / VSV model.

The invention can be illustrated by the following examples.

Example 1. Transformation of E. coli strain BL21 (DE3) and obtaining inclusion bodies

Competent cells of E. coli strain BL21 (DE3) were thawed in a water bath, 1.5 μl of plasmid solution was added, heated to 42 ° C for 2 minutes, then, adding 1 ml of LB growth medium, incubated for 1 hour at 37 ° C. As a result of transformation, the producer strain Bl (21) IPF3 (7) was obtained. The transformant containing the producer strain was transferred to 200 ml of LB medium with the addition of the antibiotic kanamycin at a concentration of 25 μg / ml and grown for 4 hours on a shaker at 37 ° C. It was further induced with a 0.5 mM IPTG solution and incubated on a shaker for another 2 hours at 37 ° C. During this time, E. coli cells produce the recombinant protein IAP III in inclusion bodies. Cells were besieged by centrifugation at 4000g for 10 minutes and lysed with 200 ml of a solution containing 2 mg / ml lysine and 1000 ED / ml DNAase for 30 minutes at 37 ° C. Next, the mixture was heated to 45 ° C. Inclusion bodies were precipitated by centrifugation at 8000 g for 120 minutes.

Example 2. Transformation of E. coli BLR strain (DE3) and preparation of inclusion bodies

Competent cells of E. coli strain BLR (DE3) were thawed in a water bath, 1.5 μl of plasmid solution was added, heated to 42 ° C for 2 minutes, then, adding 1 ml of EB culture medium, incubated for 1 hour at 37 ° C. The transformant containing the producer strain was transferred to 200 ml of EB medium with the addition of the antibiotic kanamycin at a concentration of 25 μg / ml and the antibiotic tetracycline 15 μg / ml. The culture was grown for 4 hours on a shaker at 37 ° C. It was further induced with a 0.5 mM IPTG solution and incubated on a shaker for another 2 hours at 37 ° C. During this time, E. coli cells produce the recombinant protein IAP III in inclusion bodies. Cells were pelleted by centrifugation at 4000 g for 10 minutes and lysed with 200 ml of a solution containing 2 mg / ml lysine and 1000 ED / ml DNAase for 30 minutes at 37 ° C. Next, the mixture was heated to 45 ° C. Inclusion bodies were precipitated by centrifugation at 8000 g for 120 minutes.

Example 3. Transformation of E. coli Origami 2 strain (DE3) and preparation of inclusion bodies

Competent cells of E. coli strain Origami 2 (DE3) were thawed in a water bath, 1.5 μl of plasmid solution was added, heated to 42 ° C for 2 minutes, then, adding 1 ml of LB nutrient medium, incubated for 1 hour at 37 ° C. The transformant containing the producer strain was transferred to 200 ml of EB medium with the addition of kanamycin antibiotics at a concentration of 25 μg / ml, tetracycline 15 μg / ml and streptomycin at a concentration of 50 μg / ml. The culture was grown for 4 hours on a shaker at 37 ° C. It was further induced with a 0.5 mM IPTG solution and incubated on a shaker for another 2 hours at 37 ° C. During this time, E. coli cells produce the recombinant protein IAP III in inclusion bodies. Cells were pelleted by centrifugation at 4000 g for 10 minutes and lysed with 200 ml of a solution containing 2 mg / ml lysine and 1000 ED / ml DNAase for 30 minutes at 37 ° C. Next, the mixture was heated to 45 ° C. Inclusion bodies were precipitated by centrifugation at 8000g for 120 minutes.

Example 4. Transformation of E. coli Rosetta-gami 2 (DE3) strain and preparation of inclusion bodies

Competent cells of E. coli strain Rosetta-gami 2 (DE3) were thawed in a water bath, 1.5 μl of plasmid solution was added, heated to 42 ° C for 2 minutes, then, adding 1 ml of LB nutrient medium, incubated for 1 hour at 37 ° C. The transformant containing the producer strain was transferred to 200 ml of LB medium with the addition of kanamycin antibiotics at a concentration of 25 μg / ml, tetracycline 15 μg / ml, chloramphenicol at a concentration of 35 μg / ml and streptomycin at a concentration of 50 μg / ml. The culture was grown for 4 hours on a shaker at 37 ° C. It was further induced with a 0.5 mM IPTG solution and incubated on a shaker for another 2 hours at 37 ° C. During this time, E. coli cells produce the recombinant protein IAP III in inclusion bodies. Cells were pelleted by centrifugation at 4000 g for 10 minutes and lysed with 200 ml of a solution containing 2 mg / ml lysine and 1000 ED / ml DNAase for 30 minutes at 37 ° C. Next, the mixture was heated to 45 ° C. Inclusion bodies were precipitated by centrifugation at 8000 g for 120 minutes.

To implement the invention, the following operations were carried out.

1. Dissolution of inclusion bodies of E. coli BL (21) IPF3 (7) using γ-cyclodextrin

Inclusion bodies were resuspended in 6M urea with 20 mM Tris HCl pH 8.0, the optical density of this suspension was measured on a chromatograph at 585 nm, the optical density was OD (585) = 0.35 OE. The suspension was triturated with a 20% γ-cyclodextrin solution, measuring the optical density. At the time of a decrease in optical density by 10 times, i.e. to a value of OD (585) = 0.03OE, the concentration of γ-cyclodextrin was found to be sufficient. The results are shown in Figure 1. The optimal concentration of γ-cyclodextrin is 2%.

2. Monitoring the effectiveness of the method of purification and refolding of recombinant protein IPF III from inclusion bodies of E. coli BL (21) IPF3 (7)

After purification and refolding, the protein solution was dialyzed against a solution of 10 mM NaCl and 0.1% trifluoroacetic acid. 150 μl of the solution was taken and applied to a Kromasil 300-5C18 column with 4.6 mm ID x 150 mm. Analytical chromatography was performed. Eluted with a linear gradient. Starting buffer: 0.1% trifluoroacetic acid, 2% acetonitrile, final buffer: 0.1% trifluoroacetic acid, 80% acetonitrile. Protein is higher at 33% acetonitrile. The refolded recombinant protein IAP III was dialyzed against a solution of 100 mM NaCl. Protein purity was also determined by SDS page electrophoresis.

3. Chromatographic purification of the recombinant IPF III protein on reverse phase sorbents

Chromatography was performed on an Amberchrome Profile XT20 22 mm ID x 250 mm column. The reaction mixture after refoding was applied to a chromatographic column. Eluted with a linear gradient. Starting buffer: 10 mM NaCl, 2% acetonitrile, pH 10.5 (adjusted with 1 M NaOH), final buffer: 10 mM NaCl, 80% acetonitrile, pH 10.5 (adjusted with 1 M NaOH). Gradient volume 400 ml. The eluate was fractionated according to the chromatograph, collecting individual peaks. The target is a peak converging at a concentration of acetonitrile of 54%. The volume of fractions that included this peak was 92 ml. Fractions were dialyzed against a solution of 10 mM NaCl, pH 10.5 (adjusted with 1 M NaOH) and applied onto a Amberchrome Profile HPR10 preparative column 22 mm ID × 250 mm. Amberchrom Profile HPR10 22 mm ID × 250 mm column was chromatographed on a target peak coming off the Amberchrome Profile XT 20 22 mm ID × 250 mm column. Eluted with a linear gradient. Starting buffer: 10 mM NaCl, 2% acetonitrile, pH 10.5 (adjusted with 1 M NaOH), final buffer: 10 mM NaCl, 80% acetonitrile, pH 10.5 (adjusted with 1 M NaOH). Gradient volume 400 ml. The target is a peak converging at a concentration of acetonitrile of 58%. The volume of fractions including this peak was 63 ml. Fractions were dialyzed against a solution of 100 mM NaCl, then trifluoroacetic acid was added thereto to 0.1%. The sample was then applied to a Kromasil 300-5C18 21.2 mm ID x 250 mm column. Eluted with a linear gradient. Starting buffer: 0.1% trifluoroacetic acid, 2% acetonitrile, final buffer: 0.1% trifluoroacetic acid, 80% acetonitrile. Gradient Volume 500 ml. The target is a peak converging at a concentration of acetonitrile of 35%. The volume of fractions including this peak was 59 ml.

4. Oxidation and refolding of the recombinant protein IPF III.

0.1 M potassium sulfite and 0.01 M sodium tetrathionate were added to the dissolved E. coli BL (21) IPF3 (7) inclusion bodies in 6 M urea with 20 mM Tris HCl pH 8.0 and 2% γ-cyclodextrin. Incubated 24 hours at a temperature of 20C °.

A solution of the recombinant protein IPF III in 6 M urea, 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2% γ-cyclodextrin. This solution was adjusted with 1 M NaOH to a pH of 10.5 and a mixture of cysteamine / cystamine was added to a final concentration of 1 mM / 0.1 mM, respectively. We incubate for 24 hours at a temperature of 20C °. The reaction mixture is applied to a column with a reversed-phase sorbent of the Amberchrome Profile XT 20 series.

5. Chromatographic purification of the recombinant protein IAP III on Ni-Sepharose, Q-Sepharose and SP-Sepharose in the presence of 2% γ-cyclodextrin.

The solution of oxidized inclusion bodies was diluted 5 times with 2% γ-cyclodextrin, 6 M urea and 20 mM Tris HCl pH 8.0. Centrifuged for 120 minutes at 17,000 g. The supernatant was applied to a Ni-Sepharose column pre-equilibrated with a solution: 2% γ-cyclodextrin, 6 M urea, 20 mM Tris-HCl pH 8.0. Washed with a solution: 2% γ-cyclodextrin, 6 M urea, 20 mM Tris-HCl pH 8.0, 0.005 M NaCl, to get rid of proteins not bound to the sorbent. The target protein was eluted with a linear gradient. Starting buffer: 2% γ-cyclodextrin, 6 M urea, 20 mM Tris-HCl (pH 8.0), 10 mM imidazole, final buffer: 2% γ-cyclodextrin, 6 M urea, 20 mM Tris-HCl (pH 8.0), 400 mM imidazole. The target protein goes in the range of imidazole concentrations from 75 to 125 mm. Fractions containing the target protein were combined and dialyzed against 6 M urea, 20 mM Tris-HCl pH 8.0 and 2% γ-cyclodextrin. The resulting protein solution was applied to a Q-Sepharose. Column pre-equilibrated with 20 mM Tris-HCl (pH 8.0). Eluted with a linear gradient. Starting buffer: 6 M urea, 20 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2% γ-cyclodextrin, final buffer: 6 M urea, 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2% γ-cyclodextrin. The target protein comes in a range of NaCl concentrations from 115 to 125 mm. Fractions containing recombinant IPF III protein were combined and dialyzed against 6 M urea, 20 mM Tris-HCl pH 8.0 and 2% γ-cyclodextrin.

The resulting protein solution was applied to a column of SP-Sepharose, previously equilibrated with 20 mM Tris-HCl (pH 8.0). Eluted with a linear gradient. Starting buffer: 6 M urea, 20 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2% γ-cyclodextrin, final buffer: 6 M urea, 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2% γ-cyclodextrin. The target protein comes in a range of NaCl concentrations from 110 to 130 mm.

Claims (1)

A method of purification of a recombinant protein of an interferon-like type III factor, including washing the inclusion bodies of the E. coli producer strain, occurring in the presence of a 2% aqueous solution of the basic detergent γ-cyclodextrin, dissolving the inclusion bodies in the presence of a 2% aqueous solution of γ-cyclodextrin, 0.1 M potassium sulfite and 0.01 M sodium tetrathionate, metal affinity chromatography on Ni-Sepharose with linear gradient elution with start buffer: 2% γ-cyclodextrin, 6 M urea, 20 mM Tris-HCl with pH 8.0, 10 mM imidazole, and final buffer: 2% γ-cyclodext in, 6 M urea, 20 mM Tris-HCl with a pH of 8.0, 400 mM imidazole, with the yield of the target protein in the range of imidazole concentrations from 75 to 125 mm, dialysis of fractions containing the target protein against 6 M urea, 20 mm Tris-HCl pH 8.0 and 2% γ-cyclodextrin, anion exchange chromatography on Q-Sepharose with linear gradient elution, with 6 M urea, 20 mM Tris-HCl with pH 8.0, 10 mM NaCl as starting buffer, 2% γ-cyclodextrin, and the final buffer is 6 M urea, 20 mM Tris-HCl with a pH of 8.0, 300 mM NaCl, 2% γ-cyclodextrin, with the yield of the target protein in the range of NaCl concentrations from 1 15 to 125 mM, dialysis of fractions containing the target protein against 6 M urea, 20 mM Tris-HCl pH 8.0 and 2% γ-cyclodextrin, cation exchange chromatography on SP-Sepharose with linear gradient elution with start buffer: 6 M urea, 20 mM Tris-HCl with pH 8.0, 10 mM NaCl, 2% γ-cyclodextrin, and final buffer: 6 M urea, 20 mM Tris-HCl with pH 8.0, 300 mM NaCl, 2% γ -cyclodextrin, with the yield of the target protein in the range of NaCl concentrations from 110 to 130 mm, refolding of the target protein at pH 10.5 by adding a mixture of cysteamine and cystamine to a final concentration of 1 mm and 0.1 mm, the following its reverse phase chromatography on Amberchrome Profile XT20 columns at pH 10.5 with linear gradient elution with a start buffer: 10 mM NaCl, 2% acetonitrile, pH 10.5, and final buffer: 10 mM NaCl, 80% acetonitrile, pH 10.5, with the yield of the target protein at a concentration of acetonitrile 54%, dialysis of fractions containing the target protein against a solution of 10 mM NaCl, pH 10.5, chromatography on Amberchrome Profile HPR10 at pH 10.5 with linear gradient elution, this starting buffer is 10 mM NaCl, 2% acetonitrile, pH 10.5, and the final buffer is 10 mM NaCl, 80% acetonitrile, pH 10.5, with the yield of the target protein at a concentration of acetonitrile 58%, dialysis of fractions containing the target protein against a solution of 100 mm NaCl, the addition of trifluoroacetic acid to 0.1% and pH 1.0, chromatography on Kromasil 300 -5C18 at pH 1.0 with linear gradient elution with a start buffer: 0.1% trifluoroacetic acid, 2% acetonitrile, and a final buffer: 0.1% trifluoroacetic acid, 80% acetonitrile, with the yield of the target protein at a concentration of acetonitrile 35% .

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