Optimizing Primary Recovery and Refolding of Human Interferon-b from Escherichia coli Inclusion Bodies

Abstract

Background: The refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. Objectives: The purpose of this study was optimization of recombinant human interferon-b purification in order to achieve higher efficiency, yield, and a product with a better and more suitable biological activity. Materials and Methods: Triton X-100 and sodium deoxycholate were used to wash the recombinant human interferon-b inclusion bodies prior to solubilization. The inclusion bodies solubilization process was performed by denaturants and reducing agents; guanidine-hydrochloride, urea, b-mercaptoethanol and dithiothritol. Results: The best recovery was obtained in the presence of 0.5% TritonX-100 (v/v). Low concentrations of urea only gave a marginal improvement on the refolding of recombinant human interferon-b. Successful refolding was achieved by gradient elution (decreasing the guanidine-hydrochloride concentration) in the presence of L-arginine. Partial purification was also achieved continuously, and recombinant human interferon-b was recovered with 93.5% purity. The interferon prepared in this project was biologically active and inhibited the replication of vesicular stomatitis virus in Hela cells, when compared to the standard interferon. Conclusions: In this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg. Conclusion: In this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH = 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg.