Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies

Iman Esmaili , Hamid Mir Mohammad Sadeghi, Vajihe Akbari

Abstract


Reteplase is a non-glycosylated and recombinant form of tissue type plasminogen activator, which is produced in Escherichia coli. However, its overexpression usually leads to formation of inactive aggregates or inclusion bodies. In the present study, we report on the development of optimized processes for isolation, solubilization, and refolding of reteplase inclusion bodies to recover active protein. After protein overexpression in E. coli BL21 (DE3) inclusion bodies were isolated by cell disruption and repeated wash of pellet with buffer containing Triton X-100. To solubilize the inclusion bodies, different types, concentrations, pHs, and additives of denaturing agents were used. Rapid micro dilution method was applied for refolding of solubilized reteplase. Different chemical additives including sugars, alcohols, polymers, detergents, amino acids, kosmotropic, and chaotropic salts, reducing agents, and buffering agents were used in the refolding buffer. To evaluate the biological activity of refolded reteplase, an indirect chromogenic assay was performed. The best solubilizing agent for dissolving reteplase inclusion bodies was 6 M urea at pH 12. The optimized buffer for refolding of solubilized reteplase was found to be 1.15 M glucose, 9.16 mM imidazole, and 0.16 M sorbitol which resulted in high yield of biologically active protein. Our results indicate type, concentration, and pH of solvent and type, concentration, and combination of chemical additives can significantly influence the yield of inclusion bodies solubilization and refolding.


Keywords


Chemical chaperone; Inclusion bodies; Refolding additives; Reteplase.

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Rosamond W, Flegal K, Furie K, Go A, Greenlund K, Haase N, et al. Heart disease and stroke statistics--2008 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2008;117(4):e25-e146.

Gaziano TA, Bitton A, Anand S, Abrahams-Gessel S, Murphy A. Growing epidemic of coronary heart disease in low- and middle-income countries. Curr Probl Cardiol. 2010;35(2):72-115.

Rutherford JD, Braunwald E. Thrombolytic therapy in acute myocardial infarction. Chest. 1990;97(4 Suppl):136s-145s.

Tebbe U, Michels R, Adgey J, Boland J, Caspi A, Charbonnier B, et al. Randomized, double-blind study comparing saruplase with streptokinase therapy in acute myocardial infarction: The COMPASS Equivalence Trial. Comparison Trial of Saruplase and Streptokinase (COMASS) Investigators. J Am Coll Cardiol. 1998;31(3): 487-493.

Mattes R. The production of improved tissue-type plasminogen activator in Escherichia coli. Semin Thromb Hemost. 2001;27(4):325-336.

Khodabakhsh F, Dehghani Z, Zia MF, Rabbani M, Mir Mohammad Sadeghi H. Cloning and expression of functional reteplase in Escherichia coli top10. Avicenna J Med Biotechnol. 2013;5(3):168-75

Yamaguchi H, Miyazaki M. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules. 2014;4(1):235-251.

Sørensen HP, Sperling-Petersen HU, Mortensen KK. Dialysis strategies for protein refolding: preparative streptavidin production. Protein Expr Purif. 2003;31(1):149-154.

Yoshii H, Furuta T, Yonehara T, Ito D, Linko YY, Linko P. Refolding of denatured/reduced lysozyme at high concentration with diafiltration. Biosci Biotechnol Biochem. 2000;64(6):1159-1165.

Gu Z, Weidenhaupt M, Ivanova N, Pavlov M, Xu B, Su ZG, et al. Chromatographic methods for the isolation of, and refolding of proteins from, Escherichia coli inclusion bodies. Protein Expr Purif. 2002;25(1):174-179.

Alibolandi M, Mirzahoseini H. Chemical assistance in refolding of bacterial inclusion bodies. Biochem Res Int. 2011;2011:631607.

Shiraki K, Kudou M, Fujiwara S, Imanaka T, Takagi M. Biophysical effect of amino acids on the prevention of protein aggregation. ‎J Biochem. 2002;132(4):591-595.

Ke CY, Yin DY, Sun WJ, Zhang QZ. Refolding of denatured/reduced lysozyme by aromatic thiols in the absence of small molecule disulfide. Res Chem Intermed. 2015;41(8):5859-5868.

Sharma GS, Singh LR. Polyols have unique ability to refold protein as compared to other osmolyte types. Biochemistry (Mosc). 2017;82(4):465-473.

Zardeneta G, Horowitz PM. Protein refolding at high concentrations using detergent/phospholipid mixtures. Anal Biochem. 1994;218(2):392-398.

Wedemeyer WJ, Welker E, Narayan M, Scheraga HA. Disulfide bonds and protein folding. Biochemistry. 2000;39(15):4207-4216.

Sehl LC, Nguyen HV, Berleau LT, Arcila P, Bennett WF, Keyt BA. Locating the unpaired cysteine of tissue-type plasminogen activator. Protein Eng. 1996;9(3):283-290.

Khodabakhsh F, Dehghani Z, Zia MF, Rabbani M, Mir Mohammad Sadeghi H. Cloning and expression of functional reteplase in Escherichia coli top10. Avicenna J Med Biotechnol. 2013;5(3):168-175.

Mir Mohammad Sadeghi H, Rabbani M, Rismani E, Moazen F, Khodabakhsh F, Dormiani K, et al. Optimization of the expression of reteplase in Escherichia coli. Res Pharm Sci. 2011;6(2):87-92.

Rodriguez-Carmona E, Villaverde A, Garcia-Fruitos E. How to break recombinant bacteria: does it matter? Bioeng Bugs. 2011;2(4):222-225.

Rodríguez-Carmona E, Cano-Garrido O, Seras-Franzoso J, Villaverde A, García-Fruitós E. Isolation of cell-free bacterial inclusion bodies. Microb Cell Fact. 2010;9:71. Doi: 10.1186/1475-2859-9-71.

Middelberg AP. Preparative protein refolding. Trends Biotechnol. 2002;20(10):437-443.

Maachupalli-Reddy J, Kelley BD, De Bernardez Clark E. Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme. Biotechnol Prog. 1997;13(2):144-150.

Kelly ST, Zydney AL. Effects of intermolecular thiol-disulfide interchange reactions on bsa fouling during microfiltration. Biotechnol Bioeng. 1994;44(8):972-982.

Zhang Z, Zhang Y, Yang K. Mechanism of enhancement of prochymosin renaturation by solubilization of inclusion bodies at alkaline pH. Sci China C Life Sci. 1997;40(2):169-175.

Singh SM, Upadhyay AK, Panda AK. Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli. J Chem Technol Biotechnol. 2008;83(8):1126-1134.

Gabrielczyk J, Kluitmann J, Dammeyer T, Jördening HJ. Effects of ionic strength on inclusion body refolding at high concentration. Protein Expr Purif. 2017;130:100-106.

Shi R, Pan Q, Guan Y, Hua Z, Huang Y, Zhao M, et al. Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein. Arch Biochem Biophys. 2007;459(1):122-128.

Buck M. Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Q Rev Biophys. 1998;31(3):297-355.

Upadhyay V, Singh A, Jha D, Singh A, Panda AK. Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent. Microb Cell Fact. 2016;15(1):100-113.

Sasahara K, Nitta K. Effect of ethanol on folding of hen egg-white lysozyme under acidic condition. Proteins. 2006;63(1):127-135.

Abe M, Abe Y, Ohkuri T, Mishima T, Monji A, Kanba S, et al. Mechanism for retardation of amyloid fibril formation by sugars in Vλ6 protein. Protein Sci. 2013;22(4):467-474.

Xie G, Timasheff SN. Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured than for the native protein. Protein Sci. 1997;6(1):211-221.

Wang W, Wang YJ, Wang DQ. Dual effects of Tween 80 on protein stability. Int J Pharm. 2008;347(1-2):31-38.


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