RU2373286C2 - Method of producing recombinant protein, modified through attachment of human albumin from yeast culture fluid - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of producing recombinant protein human albumin-interleukin-2 or recombinant protein human albumin-alpha 16-interferon, modified by attachment of human albumin. The method involves technology of culturing yeast strain Pichia pastoris PS106/pPIC9HAbIL-2 or yeast strain Pichia pastoris PS106/pPIC9HAbIFNa-16 in modified culture medium BMGY, after which induction synthesis of target proteins is carried out at low temperature. Further, cells are removed and the medium is concentrated. Target proteins are then precipitated using ammonium sulphate or polyethyleneglycol 3350. Target proteins are then separated by gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents. Finally, affinity chromatography is then done on Cibacron F3GA sorbent.

EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.

3 cl, 1 tbl, 5 ex

Description

The invention relates to the field of biotechnology, medicine and pharmacology, relates to a method for producing recombinant proteins with therapeutic activity and modified by the addition of human albumin, including the technology of culturing producer strains and subsequent purification of the target proteins. The present invention is based in part on the discovery that hybrid proteins containing human plasma albumin coupled to the target protein have improved pharmacokinetic properties, such as longer half-lives, which improves the quality of life of patients (Yeh P et al. Proc. Natl Acad. Sci. 1992. V.89. P.1904-1908; Smith B. et al. Bioconjugate Chem. 2001. V.12. P.750-756; Halpem W. et al. Pharm. Res. 2002. V.19. P.1720-1729). The active part of the hybrid protein can be represented by proteins of various nature, which have biological activity that allows them to be used as drugs. For example, it can be enzymes, enzyme inhibitors, hormones, interferons and interleukins. Obtaining modified interferons and interleukins with a wide range of activities is especially important.

Known methods for producing recombinant proteins modified with human albumin from Kluyveromyces lactis yeast (US patent 6,686,179 published February 3, 2004), Saccharomyces cerevisiae yeast (US patent 7,045,318, published May 16, 2006), bacteria, Saccharomyces cerevisiae yeast and mammalian cells (US patent 6,946,134 published September 20, 2005; US patent 6,994,857 published February 7, 2006; US patent 7,238,667 published July 3, 2007). Bacteria as prokaryotic organisms are not able to carry out post-translational modification of proteins characteristic of eukaryotic organisms, and due to the conditional pathogenicity of prokaryotic producer strains, recombinant proteins may contain endotoxin impurities, so long-term use of recombinant proteins of a bacterial nature is accompanied by undesirable side effects (Primrose SBJol. 1986. V.61. P.99-116; Ershov F.I. Interferon system in norm and pathology, 1996). Expression systems based on mammalian cell culture allow the production of authentic heterologous proteins. A significant drawback of these systems is the complexity of working with cell cultures and the complexity of obtaining transgenic plants and animals, which increases the cost of the resulting recombinant proteins.

A drawback of the K.lactis and S. cerevisiae yeast expression system is the use of expression plasmids containing antibiotic resistance genes, which creates a risk of horizontal transfer. In addition, the proteins secreted by these yeasts are hyperglycosylated, which is accompanied by a change in their physicochemical properties (Schultz LD et al. // Gene. 1987. V.54. PI 13-123; Eckart MR, Bussineau CM // Curr. Opin Biotechnol. 1996. V.7. P.525-530).

Pichia pastoris yeast expression system is devoid of these deficiencies. The use of methylotrophic yeast Pichia pastoris as producers makes it possible to significantly increase the yield of recombinant proteins and to avoid hyperglycosylation of secreted heterologous proteins (Cereghino J.L., Cregg J.M. FEMS Microbiol.Rev. 2000. V.24. P.45-66). The accumulation of recombinant proteins in the culture fluid greatly simplifies the procedure for their purification. However, the yield of the final product largely depends on the technology used to cultivate the producer strain, isolation and purification of the recombinant hybrid protein.

A known method of producing recombinant proteins modified with human albumin from the yeast K. Lactis (patent US 6,686,179, published February 3, 2004), the closest to the claimed method, selected as a prototype. The known method consists in culturing at 30 ° C a recombinant yeast strain in a nutrient medium that provides cell growth and synthesis of the target protein, cell removal, ultrafiltration and subsequent dialysis of the culture medium, affinity chromatography on Blue-Trisacryl sorbents, two-stage ion-exchange chromatography on type S sorbents Sepharose FF or Q Sepharose FF, dialysis.

The disadvantages of this method are its multi-stage and low manufacturability, which limits the possibility of using the above scheme for the industrial production of recombinant hybrid proteins.

The technical result of the proposed method is to simplify and increase the efficiency of the technology for purification of the target protein due to a new method of culturing yeast-producers, providing the production of the target protein with biological activity comparable to unmodified proteins, which allows to obtain biologically active hybrid proteins suitable for creation on their basis drugs, and also creates the possibility of industrial production of recombinant fusion proteins.

The specified technical result is achieved by the fact that in the method for producing recombinant proteins modified by the addition of human albumin from yeast culture fluid, including culturing yeast strains in a nutrient medium that provides cell growth and synthesis of target proteins, cell removal, concentrated medium, precipitation of the target protein and its purification using gel filtration and affinity chromatography, in accordance with the claimed method cultivate strains of yeast Pichia pastoris, which are producer fusion proteins (human albumin-interleukin-2 and human albumin-alpha 16-interferon), cultivation is carried out in a modified medium, the synthesis of the target protein is carried out at 20-25 ° C, purification is carried out by precipitation of the target protein at 75% saturation of ammonium sulfate or 25% polyethylene glycol 3350, gel filtration on a Sephacryl HR 200 or BioRad P-300 type sorbent and affinity chromatography on Cibacron F3GA. In this case, the concentration of the culture medium is carried out using an AP-0.3-50PS membrane.

In addition, this technical result is achieved in that gel filtration on a Sephacryl HR 200 or BioRad P-300 sorbent is carried out using 20 mM sodium acetate buffer, pH 5.5, as a balancing and eluting solution.

In addition, this technical result is achieved by the fact that affinity chromatography on Cibacron F3GA is carried out using 20 mM sodium acetate buffer, pH 5.5, as a balancing solution; washing is carried out by sequential use of a balancing buffer solution and a balancing buffer solution containing 0.5 M sodium chloride, the target protein is eluted with a concentration gradient of potassium thiocyanate 0.5-1.0 M in 20 mM sodium acetate buffer, pH 5.5 or 50 mM Tris-HCl, pH 7.5-8.5.

In the claimed method, the technical result is achieved in this way by using the recombinant yeast strain Pichia pastoris PS106 (pPIC9HAbIL-2), a producer of the human albumin-interleukin-2 hybrid protein, previously obtained by the authors of the present invention (decision to grant a patent dated April 23, 2007 on the application RU 2006110098), and the strain of yeast P. pastoris PS106 (pPIC9HAbIFNa-16) obtained by the method previously claimed by the inventors of the invention (decision on the grant of a patent dated April 23, 2007 by application RU 2006110098) - producer of the albumin-alpha fusion protein and human 16-interferon, culturing the strains in a modified nutrient medium and inducing the synthesis of the target protein, removing cells, concentrating the culture medium, precipitating the target protein with ammonium sulfate or polyethylene glycol (PEG) 3350, gel filtration on sorbents like Sephacryl HR 200 or BioRad P- 300, affinity chromatography on sorbents of the type Cibacron F3GA.

The optimal conditions for the individual stages of obtaining the target hybrid proteins are as follows:

- cultivation of producer strains in a modified BMGY medium containing 0.05 M potassium phosphate buffer, pH 6.0;

- induction of the synthesis of the target protein at 22-25 ° C in a modified BMM medium containing 0.05 M potassium phosphate buffer, pH 6.0;

- concentration of the culture medium using an AP-0.3-50PS membrane;

- precipitation of the target protein at 75% saturation of ammonium sulfate or 25% polyethylene glycol (PEG) 3350;

- dissolving the obtained precipitate containing the target fusion protein in 20 mm sodium acetate buffer, pH 5.5;

- gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents in 20 mM sodium acetate buffer, pH 5.5;

- affinity chromatography on sorbents of the Cibacron F3GA type using 20 mM sodium acetate buffer, pH 5.5 as a balancing solution; removal of impurity proteins with 0.5 M sodium chloride in equilibration buffer, elution of the target protein with a concentration gradient of potassium thiocyanate 0.5-1.0 M in 20 mM sodium acetate buffer, pH 5.5 or 50 mM Tris-HCl, pH 7 5-8.5.

Significant differences of the proposed method from the prototype are:

- the use of hybrid proteins of yeast strains P. pastoris PS106 (pPIC9HAbIL-2) as a producer of the human albumin-interleukin-2 hybrid protein producer and PS106 (pPIC9HAbIFNa-16) as the producer of the human albumin-alpha 16 interferon hybrid protein;

- cultivation of yeast strains in a modified BMGY medium containing 0.05 M potassium phosphate buffer, pH 6.0;

- carrying out the stage of induction of the synthesis of the target protein at 20-25 ° C in a modified BMM medium containing 0.05 M potassium phosphate buffer, pH 6.0, which allows to obtain a higher yield of hybrid proteins;

- the consistent use of the stages of ultrafiltration and precipitation of the target protein with ammonium sulfate or polyethylene glycol (PEG) 3350, which leads to a significant reduction in the volume of the treated culture medium in the first stages of purification, which is of great importance when scaling the process of production of target proteins;

- two-stage chromatographic purification of hybrid proteins on sorbents such as Sephacryl HR 200 or BioRad P-300 and Cibacron F3GA, which allows to obtain biologically active hybrid proteins.

The essence and advantages of the claimed invention is illustrated by the following examples.

Example 1. The cultivation of strains of yeast P. pastoris - producers of the hybrid proteins albumin-interleukin-2 and albumin-alpha 16-interferon. After that, 1 ml of a suspension of P. pastoris yeast PS106 (pPIC9HAbIL-2), a producer of human albumin-interleukin-2 fusion protein or PS106 (pPIC9HAbIFNa-16), producer of a human albumin-alpha 16-interferon fusion protein, stored in 15% glycerol, was taken at -70 ° C, was introduced into 50 ml of modified BMGY medium (Table 1), in which, compared with the standard medium, the concentration of potassium phosphate buffer was 2 times lower and cultivated at 30 ° C on a rocking chair with stirring (200-220 rpm) within 24-36 hours until the optical density OD ₆₀₀ from 2.5 to 4.0, preferably 3.0. Then, 25 ml of the obtained inoculum is transferred into 1 liter of modified BMGY medium and cultured in 1.6 L Bioflo III fermenter (New Brunwick Sci. Co) at 30 ° C with stirring (230-450 rpm) for 68-72 hours to complete utilization of glycerin. Then the cells are sterilely harvested by centrifugation at 6-7 thousand rpm for 15 minutes, at + 4 ° C, and transferred to 1 l of modified BMM medium, which, unlike BMGY medium, contains methanol as the sole carbon source. When methanol is added, the synthesis of the target protein is induced. The methanol concentration is maintained in the range from 0.5% to 2.0%, preferably from 0.75 to 1.0%. Cultivation at the induction stage is carried out at 20-25 ° C, preferably at 22 ° C, for 96-120 hours, preferably 100-110 hours. The production of the target protein is tested by electrophoresis of the culture fluid proteins under reducing conditions. A decrease in the concentration of inorganic phosphate due to the use of a modified medium contributes to an increase in the production of hybrid proteins.

Table 1 The composition of the modified nutrient medium BMGY Environment component Quantity per liter MgSO ₄ × 7H ₂ O 0.5 g CaCl ₂ 0.4 g (NH ₄ ) ₂ SO ₄ 3.0 g yeast extract 10.0 g peptone 20.0 g Glycerol 10 ml Potassium phosphate buffer 1 M, pH 6.0 50 ml Biotin 1.8 mg CuSO ₄ × 5H ₂ O 16.6 mg Nal 0.35 mg MnSO ₄ 11.7 mg NaMoO ₄ × 2H ₂ O 0.74 mg H ₃ IN ₃ 0.1 mg CoCl ₂ 1.18 mg ZnSO ₄ × 7H ₂ O 0.2 mg FeSO ₄ × 7H ₂ O 0.3 mg

Analysis of proteins secreted by producer strains showed that lowering the temperature at the induction stage to 20–25 ° C prevents the proteolytic cleavage of the target proteins and thereby increase their yield.

Example 2. Purification of the fusion protein albumin-alpha 16-interferon person. At the end of the cultivation process, yeast cells were removed by centrifugation at 6-7 thousand rpm for 15 minutes at + 4 ° C. All subsequent operations were carried out at a temperature of + 4 ° C. The supernatant was passed through a nitrocellulose filter with a pore diameter of 0.45 μm (Millpore Inc.).

The filtrate thus obtained was concentrated on an UPL-0.6 ultrafiltration unit using an AR-0.3-50PS membrane (NPK Biotest, Kirishi), cutting off compounds with a molecular weight of less than 50 kDa, at this stage low molecular weight components were removed and the initial volume up to 100-200 ml. Then, salt precipitation of the target protein was carried out, bringing the saturation of ammonium sulfate to 75%. The precipitate of the target protein was collected by centrifugation at 12-14 thousand rpm for 20 minutes. The resulting precipitate was dissolved in 20 mM sodium acetate buffer, pH 5.5, and applied to a column containing 280 ml of BioRad P-300 equilibrated with the same buffer. The first 75 ml was discarded, the fractions containing the target protein were combined. At this stage, there was a release from the bulk of the impurities.

The eluate thus obtained was applied to a column containing 2.5 ml of Cibacron F3GA affinity sorbent, equilibrated with 20 mM sodium acetate buffer, pH 5.5. The column was washed sequentially with 10 ml of equilibration buffer and 10 ml of equilibration buffer containing 0.5 M sodium chloride. The elution was carried out with a concentration gradient of potassium thiocyanate from 0.5 M to 1.0 M in 20 mM sodium acetate buffer, pH 5.5 or 50 mM Tris-HCl, pH 7.5-8.5. At this stage, residual impurity proteins, which were the products of proteolysis of albumin, which is part of the hybrid protein, were removed.

Electrophoresis under reducing conditions showed that the use of this purification scheme made it possible to obtain the human albumin-alpha 16-interferon hybrid protein preparation, the content of impurities in which was no more than 5%. The molecular weight of the human albumin alpha 16-interferon fusion protein was approximately 86 kDa. The average yield from a liter of culture medium was 7–9 mg.

Example 3. Purification of the fusion protein albumin-interleukin-2 person. At the end of the cultivation process, the yeast cells were removed by centrifugation at 6-7 thousand rpm for 15 minutes, at + 4 ° C. All subsequent operations were carried out at a temperature of + 4 ° C. The supernatant was passed through a nitrocellulose filter with a pore diameter of 0.45 μm (Millpore Inc.).

The filtrate thus obtained was concentrated on an UPL-0.6 ultrafiltration unit using an AR-0.3-50PS membrane (NPK Biotest, Kirishi), cutting off compounds with a molecular weight of less than 50 kDa, at this stage low molecular weight components were removed and the initial volume up to 100-200 ml. The target PEG 3350 protein was then precipitated at 25% saturation. The precipitate of the target protein was collected by centrifugation at 12-14 thousand rpm for 20 minutes. The resulting precipitate was dissolved in 20 mM sodium acetate buffer, pH 5.5, and applied to a column containing 200 ml of Sephacryl HR 200 equilibrated with the same buffer. The first 60 ml was discarded, the fractions containing the target protein were combined. At this stage, there was a release from the bulk of the impurities.

The eluate thus obtained was applied to a column containing 2.5 ml of Cibacron F3GA affinity sorbent, equilibrated with 20 mM sodium acetate buffer, pH 5.5. The column was washed sequentially with 10 ml of equilibration buffer and 10 ml of equilibration buffer containing 0.5 M sodium chloride. The elution was carried out with a concentration gradient of potassium thiocyanate from 0.5 M to 1.0 M in 20 mM sodium acetate buffer, pH 5.5 or 50 mM Tris-HCl, pH 7.5-8.5. At this stage, residual impurity proteins, which were the products of proteolysis of albumin, which is part of the hybrid protein, were removed.

Electrophoresis under reducing conditions showed that the use of this purification scheme made it possible to obtain a human albumin-interleukin-2 hybrid protein preparation with an impurity content of not more than 5%. The molecular weight of the human albumin-interleukin-2 fusion protein was approximately 82.0 kDa. The average yield from a liter of culture medium was 6-7 mg.

Example 4. The study of the biological activity of the hybrid protein albumin-alpha 16-human interferon.

The biological activity of the purified hybrid protein was tested for its ability to suppress the cytopathic activity of the vesicular stomatitis virus in the primary culture of L-41 tissue of human muscle tissue of human embryos sensitive to alpha-type interferons (Liu PT et al. // Protein Expres. Purif. 2001. V .22. P.381-387). Human leukocyte interferon OSO 42-28-90-87 (GISK, Moscow) was used as a standard.

A culture of L-41 cells of the musculoskeletal tissue of human embryos was grown in Nutrient medium MEM containing 2 mM L-glutamine, 10% serum fetal cows and antibiotics (penicillin and streptomycin, 100 units / ml) at (36.0 ± 1 , 0) ° С. After the monolayer was formed, the cells were removed from the glass with a 1: 1 Versen and trypsin mixture (FS 42-65 BC-93; FS 42-3321-96), the Versen and trypsin mixture was poured, the cells were suspended in a nutrient medium MEM needle containing L- glutamine and antibiotics (penicillin and streptomycin, 100 units / ml each), but without serum of cow fruits at a concentration of 150-300 × 10 ³ / ml. 200 μl of the prepared suspension of cells was introduced into 96-well plates and incubated at (36.0 ± 1.0) ° C in an atmosphere of (5.0 ± 0.5)% CO ₂ for 48-72 hours until the formation of a complete monolayer . The culture medium was removed, 100 μl of a series of two-fold dilutions of the test drug and standard were added to the wells and incubated at (36.0 ± 1.0) ° C in an atmosphere of (5.0 ± 0.5)% CO ₂ for 24 hours. Then, a dose of the Indiana strain of vesicular stomatitis virus (deposited in the GISC collection, per No. 11/82) corresponding to 100 TCD ₅₀ / 0.1 ml was added to each well with the test drug and standard and incubated at (36.0 ± 1 , 0) ° С in the atmosphere (5.0 ± 0.5)% СО ₂ within 24 hours. The antiviral activity of the test drug was calculated by the formula:

A _x = (A _std / a _std ) × a _x ,

where A _x is the antiviral activity of the test drug in IU / ml;

And _stand is the antiviral activity of the standard in IU / ml;

a _x is the titer of the test drug (the reciprocal of the dilution);

and _stand is the titer of the standard (inverse of dilution).

The specific activity of the purified preparation of the hybrid protein albumin-alpha 16-interferon person was 0.4-1.7 × 10 ⁷ IU / mg Given that the molecular weight of human alpha 16-interferon is 20 kDa and the molecular weight of albumin is 66 kDa, the proportion of the active component is human alpha 16-interferon in the fusion protein molecule is approximately 23%. Based on this, it can be concluded that the addition of albumin does not lead to a significant decrease in the biological activity of human alpha 16-interferon, which is 0.3-1.0 × 10 ⁸ IU / mg (RU 2203950, published May 10, 2003).

Example 5. The study of the biological activity of the hybrid protein albumin-interleukin-2 person.

The biological activity of the purified fusion protein was tested for its ability to proliferate mouse CTLL-2 interleukin-2-dependent T lymphocytes (Gearing AJH, Thorpe R. // J. Immunol. Meth. 1988. VI 14. P.3-9) . BRMP reference reagent human IL-2 (NCI, USA) was used as a standard.

CTLL-2 mouse mouse T lymphocytes were suspended in RPMI 1640 growth medium containing 2 mM L-glutamine and antibiotics (penicillin and streptomycin, 100 units / ml), 10% bovine serum at a concentration of 1 × 10 ⁶ / ml and introduced into 96-well plates, into which 100 μl of a series of two-fold dilutions of the test drug and standard were added before. The plates were incubated at (36.0 ± 1.0) ° C in an atmosphere of (5.0 ± 0.5)% CO ₂ for 42 hours. After that, 10 μl of a 0.005% solution of thiazoline blue (MTT) in RPMI 1640 medium without additives was added to each well and incubated for 6 hours at (36.0 ± 1.0) ° С in the atmosphere (5.0 ± 0.5)% CO ₂ . In viable cells stimulated by interleukin-2 as part of a fusion protein, navy-blue formazan crystals insoluble in aqueous solutions were formed. The plates were centrifuged for 5 minutes at 2000 rpm, supernatan was removed from the wells, 100-200 μl of dimethyl sulfoxide was added, shaken until the formazan crystals were completely dissolved, and absorbance was measured at wavelengths of 540 nm and 690 nm. The activity of the test drug was determined by comparing the absorbance values of the test drug and the standard.

The specific activity of the purified preparation of the human albumin-interleukin-2 hybrid protein was 1.4-2.2 × 10 ⁵ IU / mg. Given that the molecular weight of 0-glycosylated recombinant human interleukin-2 is 16.0 kDa (Vita N. et al. Lymphokine Res. 1990. V.9. P.67-79), and the molecular weight of albumin is 66 kDa, fraction the active component — alpha 16 human interferon — in the fusion protein molecule is approximately 20%. Based on this, it can be concluded that the addition of albumin does not reduce the biological activity of alpha 16 human interferon, which is 1.0 × 10 ⁶ IU / mg (RU 2140283, published October 27, 1999).

Thus, the claimed method for producing recombinant proteins modified by the addition of human albumin, including the technology of culturing producer strains and subsequent purification of the target proteins, allows to obtain target proteins with biological activity comparable to unmodified proteins. Taking into account the fact that hybrid proteins containing human albumin attached to the target protein have improved pharmacokinetic properties, it can be concluded that, based on purified hybrid proteins: human albumin-interleukin-2 and human albumin-alpha 16-interferon can be effective drugs have been obtained for the treatment of various viral diseases (Guo JT et al. J. Virol. 2001. V.75. P.8516-8523; Popovich AM Interleukin-2: immunobiology and immunotherapy. 2004; Siebeck N. et al. Am. J. Vet. Res. 2006. V.67. P.1406-1411; Human Genome Sciences Reports. 2007), Oncology their diseases (Borden EC Oncologist. 1998. V.3. P.198-203; Bukowski RM Semin. Oncol. 2000. V.27. P.204-212; Molchanov O.E. et al. Current trends in immunotherapy of malignant tumors . 2001) and human autoimmune diseases (Piskova J. et al. Invest. Ophtalmol. Vis. Sci. 2006. V.47. P.3946-3950).

The claimed method in comparison with world analogues has a simpler scheme for the isolation of target proteins and higher efficiency due to the prevention of proteolysis of target proteins, which allows to obtain biologically active hybrid proteins suitable for creating drugs based on them with improved pharmacokinetic properties for the treatment of diseases of various etiologies , as well as the possibility of obtaining target proteins on an industrial scale.

Claims (3)

1. A method of producing a recombinant protein modified by the addition of human albumin from a yeast culture fluid, comprising culturing yeast strains in a growth medium that provides cell growth and synthesis of the target protein, removing cells, concentrating the medium, precipitating the target protein and purifying it using gel filtration and affinity chromatography, characterized in that the cultivar of the yeast strain Pichia pastoris PS106 / pPIC9HAbIL-2 is a producer of the hybrid protein albumin-interleukin-2 or strain of the yeast Pichia past oris PS106 / pPIC9HAbIFNa-16 - producer of the human albumin-alpha 16-interferon fusion protein in a modified BMGY medium with a 2-fold reduced concentration of potassium phosphate buffer, the synthesis of the target protein is carried out at 20-25 ° C, albumin-interleukin-2 human is purified by precipitation with 25% polyethylene glycol 3350 and isolated by gel filtration on a Sephacryl HR 200 sorbent, and human albumin-alpha 16-interferon is purified by precipitation at 75% saturation with ammonium sulfate and isolated by gel filtration on a BioRad P-type sorbent 300, in conclusion pr drive target protein affinity chromatography on Cibacron F3GA. 2. The method according to claim 1, characterized in that gel filtration on a Sephacryl HR 200 or BioRad R-300 sorbent is carried out using 20 mM sodium acetate buffer, pH 5.5, as a balancing and eluting solution. 3. The method according to claim 1, characterized in that the affinity chromatography on Cibacron F3GA is carried out using 20 mM sodium acetate buffer, pH 5.5, as a balancing solution, while washing is carried out sequentially using a balancing buffer solution and a balancing buffer solution, containing 0.5 M sodium chloride, and the elution of the target protein is carried out with a concentration gradient of potassium thiocyanate 0.5-1.0 M in 20 mm sodium acetate buffer, pH 5.5 or 50 mm Tris-HCl, pH 7.5-8 ,5.