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ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2016, Vol. 42, No. 6, pp. 631–637. © Pleiades Publishing, Ltd., 2016. Original Russian Text © V.P. Romanov, T.I. Kostromina, A.I. Miroshnikov, S.A. Feofanov, 2016, published in Bioorganicheskaya Khimiya, 2016, Vol. 42, No. 6, pp. 697–703.

A Preparative Method for Obtaining Recombinant Human Interferon α2b from Inclusion Bodies of Escherichia coli V. P. Romanovb, T. I. Kostrominaa, A. I. Miroshnikova, and S. A. Feofanovb, 1 aShemyakin–Ovchinnikov

Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia Pushchino Branch, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia

b

Received January 6, 2016; in final form, February 9, 2016

Abstract—A simple, easily reproducible, and scalable method for obtaining recombinant human interferon α2b from Escherichia coli inclusion bodies has been elaborated. It involves the following steps: preparation of producer cell biomass, isolation and washing of inclusion bodies, their dissolution with protein refolding, SP Sepharose chromatography, and DEAE Sepharose chromatography. According to the results of gel electrophoresis and reversed-phase HPLC, the purity of the protein obtained exceeds 95%. Keywords: recombinant α2b interferon, inclusion bodies, refolding, protein isolation, ion-exchange chromatography, reversed-phase HPLC DOI: 10.1134/S1068162016040154

INTRODUCTION Interferons are a collective name for a variety of glycosylated proteins produced by cells in response to viral invasion. Interferons make cells virus-resistant [1]. Interferons are classified with respect to the types of cells producing them. Interferons α are produced by peripheral blood leukocytes, and interferons β, by fibroblasts. Interferon γ is produced by stimulated T lymphocytes and NK cells [2]. Interferons are utilized in medicine as antiviral, antitumor, and immunomodulating agents [3–6]. The most commonly used interferon is α2b. It is applied to the treatment of severe viral diseases, in particular, hepatites [6, 7]. The isolation of natural interferons from donated blood is hampered by their extremely low content. In addition, there is a risk of incomplete release from viruses. Utilization of recombinant producer strains allows obtaining matter enriched in target proteins, thereby facilitating their isolation and, as long as the manufacturing practice is proper, obtaining them virus-free. Recombinant α2b interferon is a nonglycosylated protein of 165 aa. The molecule contains four cysteine residues, which, correspondingly, form two intramolecular disulfide bonds: Cys1–Cys98 and Cys29– Cys138 [8]. 1 Corresponding

[email protected]

author: phone: +7 (919)966-69-94; e-mail:

We propose a simple method of isolating recombinant α2b interferon in two ion-exchange chromatography steps. The method can be readily scaled up to a desired output. RESULTS AND DISCUSSION Recombinant α2b interferon is isolated from insoluble inclusion bodies by various methods [9–12]. Most protocols include the following steps: harvesting of producer cell biomass, cell degradation, isolation and washing of inclusion bodies, inclusion body dissolution and refolding, and interferon isolation proper. The isolation and purification include, in turn, several chromatographic steps: ion-exchange, metal chelate, etc. The methods described in [9–12] include three or more chromatography steps. Utilization of immunoaffine, metal chelate, and reverse-phase stationary phases makes the methods expensive. The multitude of purification steps with expensive sorbents is determined by the necessity of removing byproducts resulting from protein refolding aberrations. Such products include molecules with free thiol groups, molecules with nonnative disulfide bridges (scrambled forms), and oligomeric protein forms. The method of α2b interferon purification proposed herein includes two chromatography steps on common ion-exchange stationary phases. We rinsed inclusion bodies with 2 M urea and 1% Triton X-100, dissolved them in 7 M guanidinium hydrochloride, and reduced disulfide bonds with

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а b

1

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Fig. 1. Nonreducing gel electrophoresis of refolding mixture samples. Lanes: (1) reduced protein; (2) mixture immediately after dilution; (3–6), incubation for 1, 2, 3, and 4 days, respectively. The arrow indicates electrophoresis direction. Bands: a, reduced interferon; b, refolded interferon.

10 mM 2-mercaptoethanol. To refold the protein, the reduced solution in guanidium hydrochloride was diluted tenfold with cooled 50 mM Tris HCl pH 8 with 2 mM EDTA, 1 mM reduced glutathione, and 0.1 mM oxidized glutathione. The mixture was stored at 10– 12°C for three days. Later experiments were carried out without adding reduced glutathione. As shown by electrophoretic tests, this change did not alter the refolding time or protein yield. To monitor refolding, we took samples from the mixture and added 1/10 volumes of 0.5 M iodoacetamide to block free thiol residues. The samples were then analyzed by PAGE (Fig. 1).

It is apparent from Fig. 1 that the greatest degree of refolding was achieved after 2–3 days of storage at 10– 12°C. Further incubation did not increase the amount of refolded protein. Reversed-phase chromatography (RPC) of the refolded α2b interferon with sodium chloride gradient in 50 mM sodium acetate pH 5.0 (Fig. 2), which followed the chromatography on SP Sepharose, showed that the protein was heterogeneous. It contained reduced interferon and forms with nonnative disulfide bonds. We separated reduced interferon from proteins with nonnative disulfide bonds by the method described in [13]. The mixture refolded for 2–3 days was acidified with acetic acid to pH 4.5 and left at 10–12°C overnight. The precipitate was removed by centrifuging, and the supernatant was loaded onto a column with SP Sepharose. The column was then eluted as in the Experimental section. The chromatographic profile is shown in Fig. 3a. The eluted matter was analyzed by PAGE and RPC. It is seen from Figs. 3b and 4 that the first peak contained reduced interferon, interferon with nonnative disulfide bonds, and oligomeric interferon, whereas the second peak contained a single protein form. The solution of peak 2 (Fig. 3a) was run through a column with DEAE Sepharose (Fig. 5). This step was used not only for protein purification but also for concentration and transfer to a buffered solution appropriate for the production of a pharmaceutical form. According to electrophoresis in 12% polyacrylamide gel (Fig. 6) and RPC (Fig. 7), the purity of the resulting protein exceeded 95%. The results of protein purification are shown in the table. The yield of purified interferon on cell biomass basis was 2–3 mg/g. The specific activity against vesicular stomatitis virus in L68 cells was within the range from 1.5 × 108 to 2.5 × 108 IU/mg protein. The activity of our samples was consistent with the activities of samples obtained by other authors [11] and the international requirements for pharmaceutical α2b interferon [6]. EXPERIMENTAL Guanidinium hydrochloride was purchased from KMF Laborchemie Handels GmbH, Germany; Tris(hydroxymethyl)aminomethane, from Carl Roth GmbH+Co, Germany; Triton X-100, from Bio-Rad, United States; iodoacetamine, glutathione oxidized, Brillant blue G (C.I. 42655 Coomassie brilliant blue G), acrylamide, N,N'-methylenebisacrylamide, Bromophenol blue, N,N,N',N'-tetramethylethylenedi-

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15.408

A280 × 1000

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3 0

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Fig. 2. HPLC analysis of α2b interferon after chromatography on SP Sepharose in sodium chloride gradient. Curves: (1) A260; (2) A280; (3) baseline.

amine (TEMED), glycine, glycerol, 2-mercaptoethanol, and ammonium persulfate, from Sigma, United States; sodium dodecyl sulfate, from Carl Roth GmbH+Co, Germany; DEAE Sepharose FF and SP Sepharose FF, from GE HealthCare, Sweden. Inorganic salts were purchased from Panreac, Spain, and Khimmed, Russia. Water used in all experiments was purified in a water purification system Elix UV-10 (Millipore, United States). Cell biomass of the recombinant strain and inclusion bodies were collected with a CEPA-61G flowthrough centrifuge (Germany) at 17000 g. The protein was collected with a J2-21 centrifuge (Beckman, United States) with a JA-14 rotor at 12000 rpm and 6–8°C. Preparation of the Biomass of the Recombinant E. coli Strain Escherichia coli BDEES4 [14] was used as the producer strain. Biomass was grown in a 400 L bioreactor in 250 L of a nutrient medium containing 16 g/L casein hydrolysate, 10 g/L yeast extract, 5 g/L NaCl, 6 g/L dipotassium phosphate, 3 g/L monopotassium phosphate, 0.5 g/L magnesium sulfate, 5 g/L glucose, 100 mg/L ampicillin sodium salt, and 34 mg/L chloramphenicol. The pH of the medium was maintained at 7.0 with 25% aqueous ammonia. The concentration of dissolved oxygen was maintained at 50% of the maximum saturation of the medium. Growth condiRUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

tions: stirring at 400 rpm, airflow 250 L/min, temperature 37°C. After the culture liquid had reached the optical density 3.0, isopropyl thiogalactopyranoside (0.25 mM) was added to induce the synthesis of α2b interferon. Three hours after the induction, the mixture was cooled to 13°C, and cells were harvested in a flow-through centrifuge. Inclusion bodies. The biomass pellet (2.1 kg) was suspended in 20 L of a solution containing 50 mM Tris HCl pH 8.0, 0.05 M EDTA, and 2 M urea. Cells were degraded with a 1MC4 flow-through homogenizer (Manton–Gaulin, Switzerland) at 600 bar. Inclusion bodies were collected by centrifuging in a flow-through centrifuge. The pellet was stored at –30°C. Inclusion body rinsing and dissolution. The frozen inclusion body pellet was suspended in 500 mL of 50 mM Tris HCl with 1% Triton X-100. The suspension was agitated at room temperature for 30 min and centrifuged. The resulting pellet was dissolved in 200 mL of 50 mM Tris HCl pH 8.0 with 7 M guanidinium hydrochloride. 2-Mercaptoethanol was added to 10 mM, and the mixture was left at room temperature overnight. Optimization of refolding conditions. The solution of inclusion bodies reduced with 2-mercaptoethanol was diluted tenfold with 50 mM Tris HCl pH 8.0 with 0.1 mM oxidized glutathione. The mixture was incubated at 10–12°C for three days. Samples were taken from the mixture on a daily basis. Iodoacetamide (0.5 M) Vol. 42

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A280 0.6 0.5 5 mМ KCl

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Fig. 3. Preparative chromatography on SP Sepharose in stepwise potassium chloride gradient (see Experimental). Chromatography control: (a) optical density profile; (b) nonreducing gel electrophoresis of eluted fractions.

was added to the concentration 0.05 M, and the mixture was analyzed by nonreducing PAGE (Fig. 1). Chromatography on SP Sepharose with linear sodium chloride gradient elution. Twenty milliliters of the mixture obtained at the protein refolding step were mixed with 20 mL of purified water and acidified with

acetic acid to pH 4.5. The resulting suspension was left at 10–12°C overnight. The precipitate was separated by centrifuging. The supernatant (40 mL) was loaded onto a 10-mL column with SP Sepharose equilibrated with 50 mM sodium acetate pH 4.5. The column was washed with 20 mL of the equilibration buffer, and the

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Fig. 4. RPC profiles of protein peaks obtained at the step of chromatography on SP Sepharose with stepwise potassium chloride gradient. Superimposed curves for peaks 1 and 2 (Fig. 3): Numerals correspond to peak numbers.

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Fig. 5. Profile of α2b interferon chromatography on DEAE Sepharose. Curves: (1) NaCl gradient; (2) A280.

protein was eluted with 10 volumes of linear NaCl gradient. Fractions of 5 mL volume were collected. Preparative chromatography on SP Sepharose with stepwise sodium chloride gradient elution. The mixture obtained at the refolding step was acidified with acetic acid to pH 4.5. The suspension was left at 10–12°C overnight. The precipitate was separated by centrifuging. The RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

supernatant (about 2 L) was loaded onto a column with SP Sepharose equilibrated with 50 mM sodium acetate pH 4.5–4.7 at the rate 200–300 mL/h. The column was consecutively eluted with the following solutions: 20 mM sodium acetate pH 4.5–4.7 (2 L); 5 mM sodium phosphate pH 6.1 (2 L); and 5 mM sodium phosphate buffers pH 6.1 containing 5 mM KCl (440 mL), 20 mM KCl Vol. 42

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Fig. 7. RPC profile of the prepared interferon α2b after chromatography on DEAE Sepharose. Curves: (1) A280; (2) acetonitrile gradient.

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Fig. 6. Gel electrophoresis of the prepared α2b interferon. Sample volumes: 5 μL on lanes 1 and 4; 20 μL on lanes 2 and 5. Lane 3: protein molecular weight ladder, from top to bottom, kDa: 97.5, 68, 45, 31, 21.5, 14.5. Samples 1–3 were denatured under reducing conditions; samples 4, 5, under nonreducing conditions.

tion buffer. Interferon was eluted with linear NaCl gradient (0–0.2 M, 1000 mL) in the equilibration buffer. Fractions of 16 mL in volume were collected. Fractions with interferon were combined and filtered through a 0.22 μm nitrocellulose filter (Minisart N, 0.2 μm, Sartorius, Germany). Analytical Methods

(700 mL), and 40 mM KCl (300 mL). Fractions of 20 mL in volume were collected (Fig. 3a). The eluted fractions were analyzed by nonreducing PAGE. Fractions with properly refolded interferon (Nos. 58–68, corresponding to the second optical density peak) were combined (Fig 3b). Chromatography on DEAE Sepharose. The combined fractions (about 200 mL) were diluted twofold with sodium phosphate pH 7.4. The solution was loaded onto a 100-mL column with DEAE Sepharose equilibrated with 10 mM sodium phosphate pH 7.4. The column was washed with 300 mL of the equilibra-

Gel electrophoresis under reducing and nonreducing conditions was carried out in 12% polyacrylamide gel as in [15]. Reversed-phase chromatography was performed in the systems Alliance Dissolution HPLC (Waters) or ACTAPurifier (GEHealthCare) in a Diasorb C18 column, 4.6 × 250 mm, 10 μm (Biokhimmak ST, Russia). Solution A: 0.1% trifluoroacetic acid; solution B: acetonitrile in 0.1% trifluoroacetic acid. Gradient: 40–60% solution B. Mobile phase volume: 10 column volumes.

Isolation of α2b interferon Step Inclusion body dissolution Mixture after refolding Chromatography on SP Sepharose Chromatography on DEAE Sepharose

Volume, mL

Protein concentration, mg/mL

Total protein, mg

Yield, %

200 4200 885 258

5.65 0.25 0.43 1.03

1129 1050 381 265.7

100 93 33.7 23.2

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The activity of the prepared interferon against vesicular stomatitis virus was tested in L-68 cells as described in [16]. ACKNOWLEDGMENTS This work was supported by the Federal Targeted Program “Research and Development Works in the Top-Priority Areas of the Science and Technology Sector of Russia in 2007–2012.” REFERENCES 1. Isaac, A. and Lindemann, J., Proc. R. Soc. B, 1957, vol. 147, pp. 258–267. 2. Roitt, I.M., Essential Immunology, London: Departments of Immunology and Rheumatology Research University College and Middlesex School of Medicine University College, 1989. 3. Nagabhushan, T.L. and Trotta, P.P., Ull-Mann’s Encyclopedia of Industrial Chemistry, 5th ed., New York: VCH Verlag, 1989, A14, pp. 365–380. 4. Sen, G.C. and Lengyel, P., J. Biol. Chem., 1992, vol. 267, pp. 5017–5020. 5. Ortaldo, J.R., Mason, A., Rehberg, E., Moschera, J., Kelder, B., Pestka, S., and Herberman, R.B., J. Biol. Chem., 1983, vol. 258, pp. 15011–15015.

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6. Walsh, C., Biopharmaceuticals. Biochemistry and Biotechnology, 2nd ed., West Sussex PO19 8SQ, England: Wiley, 2003. 7. Nedogoda, V.V., Novye Lekarstva Novosti Farmakoterapii, 2000, no. 6, pp. 3–16. 8. Morehead, H., Johnston, P.D., and Wetzel, R., Biochemistry, 1984, vol. 23, pp. 2500 – 2507. 9. Hochuli, E. Process for producing alpha-interferon, Eur. Patent no. 0679718 A2, 1996. 10. D’Andrea, M.J., Purification of monomeric interferon, US Patent no. 4765903, 1995. 11. Beldarrain, A., Cruz, Y., Navarro, M., and Gil, M., Biotechnol. Appl. Biochem., 2001, vol. 33, pp. 173–182. 12. Thratcher, D. and Panayotatos, N., in Methods Enzymol., Colowick, S.P. and Kaplan, N.O., Eds., London: Ac. Press, 1986, vol. 119, pp. 166–177. 13. Scapol, S. and Viscomi, G.C., Process for the purification of pharmacologically active proteins through cationic exchange chromatography, US Patent no. 6866782, 2005. 14. Bairamashvili, D.I., Vorob’ev, I.I., Gabibov, A.G., Demin, A.V., Miroshnikov, A.I., Mart’yanov, V.A., Ponomarenko, N.A., and Shuster, A.M., RF Patent No. 2258081, Byull. Izobret., no. 22, 2005. 15. Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685. 16. Kolokol’tsov, A.A., Frolov, V.G., Gur’ev, V.P., and Kovrigina, M.A., RF Patent no. 2121364, Byull. Izobret., 1998, no. 31.

Translated by V. Gulevich

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