RU2123010C1 - Method of preparing recombinant interferon-alpha-2 from insoluble inclusion bodies - Google Patents

Abstract

FIELD: biotechnology, biochemistry. SUBSTANCE: cells are disrupted by ultrasonic oscillation and solubilization of inclusion bodies and destruction of interferon oligomers are carried out by reaction of oxidative sulfitolysis at protein concentration 5-10 mg/ml in solution of 6-7 M Gdn-HCl. Low-molecular impurities are separated by dialysis against 7-8 M urea. Renaturation of sulfitized derivative is carried out in the presence of 3-4 M urea in a mixture of oxidized and reduced glutathione at ratio 1:10. Method ensures to obtain 400 mg native protein from 30 g biomass and can be used for large-scale preparing. Method is used in medicinal and microbiological industry for production of human alpha-2-interferon. EFFECT: improved method of preparing, increased yield of native protein. 2 tbl, 5 ex

Description

 The proposed solution relates to biotechnology and is intended for use in the medical and microbiological industry for the production of human alpha-2-interferon, which is necessary for the treatment of various viral and oncological diseases.

 The production of recombinant proteins in microbial cells is often accompanied by the formation of insoluble aggregates localized in the cytoplasm and called inclusion bodies (TBs). To isolate the target protein, contaminating cellular proteins are removed, the protein is solubilized in the detergent and its conformation is restored, obtaining a biologically active protein.

 Several methods are known for isolating alpha interferon from inclusion bodies.

 A known method of producing alpha-interferon, according to which the precipitate TB is solubilized in a solution of 8 M guanidine chloride (Gdn-HCl). For renaturation, the solution was immediately diluted 4 times with 0.1 M Tris-HCl buffer pH 7.5. The suspension is centrifuged and the precipitate is discarded. 90% of the protein in the supernatant is the native monomer of alpha interferon. The process of protein purification under native conditions includes 4 chromatography and the final yield reaches 11% [1].

 A method is also known where recombinant alpha interferon is solubilized in 8 M Gdn-HCl, after which the solution is diluted with water and alpha interferon is precipitated in a sparingly soluble, oligomeric form. The precipitate was redissolved in Gdn-HCl, then diluted 2 times with water and applied to an adsorption column balanced with a solution of 3 M Gdn-HCl. The sorbed product is eluted with an aqueous solution of 0.5% Tween-20 under renaturation conditions [2].

 Both methods are based on the solubilization of inclusion bodies in a solution of strong denaturant (6-8 M Gdn-HCl) without the addition of reducing agents. Such dissolution does not break improperly closed disulfide bonds; therefore, the protein under native conditions retains a significant tendency to form oligomers and is isolated in low yield.

 There is an effective method for solubilization of inclusion bodies (prototype), which allows to obtain alpha-interferon monomer quickly and in high yield. Cells are destroyed by lysozyme, the pellet is washed with 1% Triton X-100 and dissolved in Gdn-HCl in the presence of dithiothreitol (DTT). For renaturation, the solution is diluted to a guanidine concentration of less than 3.8 M in the presence of dithiothreitol and trans-4,5-dihydroxy-1,2-dithian (DHDT). The solubilization of TB in a solution of 6 M Gdn-HCl in the presence of DTT significantly increases the yield of the native monomer. Purification of the native protein is achieved by gel permeation chromatography [3].

 However, the industrial use of this method encounters technical difficulties. Complete solubilization of TB in strong denaturant in the presence of reducing agents is achieved only in dilute solutions (protein concentration 0.5 - 1 mg / ml), in addition, reducing agents are toxic. When scaling the process, this leads to a large consumption of Gdn-HCl, large volumes already in the first stages of isolation and, accordingly, an increase in the price of the drug. In addition, the effective renaturation of alpha interferon from a solution of strong denaturant by dilution is interfered with by TB contaminants. The removal of impurities during the scaling process is especially necessary to obtain a high yield of the target protein.

 The objective of the technical solution is to develop effective and industrially suitable methods for solubilization, purification and effective renaturation of alpha-2 interferon from a producer strain forming insoluble aggregates represented by high molecular weight S-S alpha interferon oligomers.

 This problem is solved in that the destruction of the cells is carried out by ultrasound, and after solubilization of the inclusion bodies, an oxidative sulfitolysis reaction is carried out at a protein concentration of 5-10 mg / ml in solution, destroying the alpha-2-interferon oligomers. Low molecular weight impurities are separated by dialysis of the reaction mixture after sulfitolysis against 8 M urea, which allows to obtain stable yields and increases the reproducibility of the method.

 The renaturation of the sulfitated interferon derivative is carried out in the presence of 3-4 M urea using a mixture of oxidized and reduced glutathione.

 The proposed method was carried out using a producer strain obtained by transformation of E. coli SG 20050 with plasmid pIF 16 [4]. The level of biosynthesis of alpha-2-interferon in transformed cells reaches 50-60%. According to electron microscopy, inclusion cells 0.3 - 0.5 microns in size are formed in the cells. Cells are destroyed by ultrasound, centrifuged and sediment of inclusion bodies is used for further work. The precipitate of the inclusion bodies is washed with non-ionic detergents, most of the high molecular weight impurities are removed. Complete dissolution of inclusion bodies is achieved in solutions of 6-7 M Gdn-HCl.

 According to the results of electrophoresis of the obtained material in PAGE in the presence of SDS, but without preliminary treatment with 2-mercaptoethanol, the insoluble fraction is mostly oligomers crosslinked by disulfide bonds. The molecular weight of S-S oligomers is very high, they do not fractionate in 15% SDS page, remaining at the boundary between the concentrating and separating gel. On a broad-porous gel-filtration support, the material is fractionated over a wide range - up to 5 million daltons. The high degree of oligomerization of alpha-2-interferon in the composition of the inclusion bodies leads to the fact that when trying to remove the denaturing agent by dialysis against buffer solutions, alpha-2-interferon almost completely re-precipitates.

 The sulfitolysis reaction is used to restore the activity of interferon monomer after complete inactivation [5], it is also used to solubilize a number of other recombinant proteins directly from inclusion bodies [6]. To dissolve inclusion bodies containing alpha-2-interferon, the sulfitolysis reaction was not used.

 The use of oxidative sulfitolysis to break improperly closed disulfide bonds of recombinant alpha interferon in the inclusion bodies of the producer strain E. coli SG 20050 pIF 16 makes it possible to quickly and efficiently dissolve the target protein in a "concentrated" form, as a sulfitated derivative.

 Under the conditions of the sulfitolysis reaction, alpha-2-interferon in the composition of the inclusion bodies is dissolved to a monomer in a wide range of protein concentration (5 - 10 mg / ml). By SDS-electrophoresis in PAGE under non-reducing conditions, the monomer form of interferon in the product after sulfitolysis (75 - 80%) is detected mainly.

 In case of insufficient purification of the sulfitated derivative from impurities during renaturation by diluting the solution of the reaction mixture after sulfitolysis, aggregation occurs. Impurities that contaminate inclusion bodies are associated with hydrophobic groups of the target protein and inhibit renaturation. To avoid ambiguous results during renaturation and yield instability, low molecular weight impurities are removed by dialysis of the reaction mixture after sulfitolysis against a solution of weak detergent [7]. The solubility of sulfonated alpha-2-interferon monomer is maintained and increased when the strongly denaturing medium (6 - 7 M Gdn-HCl) changes to slightly denaturing medium (7 - 8 M urea). To prevent aggregation, the reaction mixture is diluted 7-10 times with a 7-8 M urea solution and dialyzed against the same solution.

 Effective renaturation is achieved by reforming disulfide bonds in the presence of a mixture of oxidized and reduced glutathione in a ratio of 1: 10. The presence of 3 to 4 M urea increases the yield of the renatured protein.

The described method for producing alpha-2-interferon from inclusion bodies makes it possible, after two-stage protein purification under native conditions, to obtain a homogeneous preparation with a specific antiviral activity of at least 1.7 • 10 ⁸ IU / mg, not containing impurity proteins (not more than 0.2 ng / μg) with a yield of 9 - 13 mg from 1 g of biomass.

 The preparative use of this method allows you to get up to 400 mg of native protein in each cycle of 30 g of biomass. The method is simple to use and combines sequential flow dialysis and chromatographic separation. The volumes of dialysis solutions sequentially increase from 200 ml in the first stages and do not exceed 3-4 l in the final stages of dialysis.

 The invention is illustrated by the following examples of specific performance.

Example 1. Removal of impurity proteins by washing the precipitate TV with nonionic detergents
30 - 40 g of the biomass of the producer strain of alpha-2-interferon E. coli SG 20050 pIF 16 is suspended in 300 - 400 ml of 50 mm Tris-HCl buffer pH 8.0, containing 2 mm EDTA. Cells are destroyed by ultrasound, centrifuged for 1 h at 12000 rpm at a temperature of 4 ^o C. For further work, a precipitate is used.

The precipitate of the inclusion bodies is washed with a solution of Triton X-100 1%, grinding the suspension in a homogenizer, and intensively stirred for 15 to 20 minutes. Centrifuged for 30 min at 12,000 rpm at a temperature of 4 ^o C.

 Similarly, the precipitate was twice suspended in a solution of 3 M urea in 50 mm Tris-HCl buffer pH 7.5. Wash solutions contain a set of solubilized high molecular weight proteins, but do not contain alpha-2-interferon. The weight of the precipitate 4 - 6 g, the content of alpha-2-interferon 78 - 80%.

Example 2. Dissolution of inclusion bodies in guanidinochloride and oxidative sulfitolysis
To the precipitate obtained in Example 1, 40-50 ml of a solution of 6-7 M Gdn-HCl in 100 mM Tris-HCl buffer pH 8.0 containing 1 mM EDTA was added and stirred vigorously until clear. For better dissolution, the suspension is triturated in a Potter homogenizer and sonicated 1 to 2 times.

 Determine the protein content by the Lowry method (the sample is diluted 200 times to exclude the influence of the detergent). The solution of inclusion bodies is diluted with 6-7 M Gdn-HCl to a protein concentration of 5-10 mg / ml and oxidative sulfitolysis is carried out. Dry samples of sodium sulfite and sodium tetrathionate (30 mg / ml and 14 mg / ml, respectively) are added to the TB solution. The reaction is carried out with stirring for 16 to 18 hours at room temperature [8]. According to the results of electrophoresis under non-reducing conditions, 75 - 80% of the protein in solution passes into the form of a sulfitated monomer in a wide range of alpha-2-interferon protein concentration in the reaction mixture (Table 1). For preparative production, a high protein concentration of 5–10 mg / ml of the reaction mixture allows one to operate with small volumes of Gdn HCl solution (100–200 ml) without reducing the yield of the modified monomer.

Example 3. Purification of the modified monomer under denaturing conditions
The reaction mixture (see Example 2) is diluted 7-10 times with a solution of 8 M urea in 50 mM Na-phosphane buffer pH 8.0 and dialyzed against the same 8 M urea buffer solution.

Example 4. Renaturation of the modified monomer
As shown by the experimental results (see Table 2), the presence of 3–4 M urea increases the yield of the native monomer after renaturation, and the volume increases by a factor of 2.5–2.

The dialyzed solution obtained in Example 3 is diluted 2 times with 50 mM Na-phosphate buffer pH 8.0 containing reduced and oxidized glutathione (concentrations of 1 and 0.1 mM, respectively). The solution was incubated for 24 - 48 hours at 4 - 10 ^o C and dialyzed against 50 mm Na-phosphate buffer pH 8.0 and then against 100 mm Na-acetate buffer pH 4.6. The precipitate was collected by centrifugation and discarded.

Example 5. Purification of alpha-2-interferon under native conditions to homogeneity
In the process of renaturation and consecutive dialyses, the correct conformation of the alpha-interferon molecule is formed.

 To purify the protein to homogeneity, an alpha-interferon solution is applied to a CM-52 cellulose column and eluted with a linear gradient of salt (0 - 0.3 M) NaCl in 100 mM Na-acetate buffer pH 4.6.

 Separation of the monomeric form from di-, trimers of alpha-interferon is carried out by gel filtration on Sephadex G-50 in 100 mM Na-acetate buffer, pH 4.6, containing 0.12 M NaCl.

A two-stage purification under native conditions is sufficient to obtain a preparation with the following quality indicators:
- the purity of the drug is not less than 95% in reducing conditions and not less than 90% in non-reducing conditions (according to the results of SDS-electrophoresis in SDS page);
- specific antiviral activity of alpha-2-interferon is not less than 1.7 • 10 ⁸ IU / mg;
- the drug is non-toxic in cell culture and in animals;
- the content of impurity proteins is not more than 0.2 ng / μg of protein;
- the content of impurities of nucleic acids is not more than 40 pg / mg protein.

 The yield of the final product varies from 9 to 13 mg from 1 g of biomass.

Industrial applicability
An effective and scalable method of solubilization, purification, and effective renaturation of alpha-2-interferon from a producer strain that forms insoluble aggregates represented by high molecular weight alpha-interferon oligomers gives an opportunity to obtain a homogeneous preparation with specific antiviral activity after two-step protein purification under native conditions not less than 1.7 • 10 ³ IU / mg, not containing impurity proteins (not more than 0.2 ng / μg) with a yield of 9-13 mg from 1 g of biomass.

 Using this method allows you to get up to 400 mg of native protein in each cycle of 30 g of biomass. The method is simple to use and combines sequential flow dialysis and chromatographic separation. The volumes of dialysis solutions consistently increase from 200 ml in the first stages and do not exceed 3-4 l in the final stages of dialysis.

Sources of information
1. Thatcher DR, Panayotatos N. Purification of Recombinant Human IFN-α2. Methods in Enzymology, 1986, v. 119, p. 166-177.

 2. Application of Germany N 3511011 "Method for the isolation and purification of alpha interferon", publ. 10/02/86.

 3. Wilson M.J., Freedman R. B., Fitton J. E. Recovery, Refolding and Purification α2-Interferon. Biochemical Society Transactions 1988, 76, 1, p. 58-60.

 4. RF patent N 2054041 "Recombinant plasmid DNA pIF16 encoding mature human leukocyte interferon-alpha-2-strain E. coli-producer of mature human leukocyte interferon-alpha-2". BI N 4, publ. 02/10/96.

 5. Fanger J. A., Pestka S. Procedure for Reduction and Reoxidation of Human Leukocyte Interferon. Methods in Enzymology, 1986, v. 119, p. 248-255.

 6. Marston F.A. The purification of eukaryotic polypeptides synthesized in Escherichia coli - Biochem. J. 1986, 240 (1), 1-12.

 7. US patent N 4512922 "Cleaning and maintaining the activity of precipitated heterologous proteins", publ. 04/23/85.

 8. Panayotis G.K., et al. Studies of the Synthesis of Insulin from Natural and Synthetic A and B Chains. I. Splitting of Insulin and Isolation of the S-Sulfonated Derivatives of the A and B Chains. Biochemistry 1967, v. 6, N 9, p. 635.

Claims (1)

 A method of producing recombinant interferon-alpha-2 from insoluble inclusion bodies, including cell destruction, removal of impurity proteins by washing the precipitate with a nonionic detergent, solubilization of inclusion bodies with 6-7 M Gdn-HCl, renaturation of the obtained product and chromatographic purification to obtain the target product, characterized in that the destruction of cells is carried out by ultrasound, and after the solubilization of the inclusion bodies, an oxidative sulfitolysis reaction is carried out at a protein concentration of 5-10 mg / ml in a solution of 6-7 M Gdn-HCl, the sulfite obtained anced derivative dialyzed against 7 - 8 M urea and annealed mixture of oxidized and reduced glutathione at a ratio of 1: 10 in the presence of 3 - 4 M urea.

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