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-β 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-β inclusion bodies prior to solubilization. The inclusion bodies solubilization process was performed by denaturants and reducing agents; guanidine-hydrochloride, urea, β-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-β. 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-β 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.

Antibiotic-free expression system for the production of human interferon-beta protein

“…Recombinant hIFN-β is a therapeutic protein and there is still a need for the development of cost effective and safe system for its production. Various attempts to develop expression and production protocols for achieving higher yield of rhIFN-β are still underway (Allen et al 2015;Ashnagar et al 2014;Moradian et al 2013;Morowvat et al 2015). The choice of expression system is a key in the development of new protein biopharmaceuticals and each expression system has its own advantages and disadvantages (Baneyx et al 1999;Huang et al 2012).…”

Interferon-Based Biopharmaceuticals: Overview on the Production, Purification, and Formulation

“…After cell lysis, inclusion bodies are isolated from soluble proteins by centrifugation [ 60 ] and are then subjected to different washing steps, e.g., with detergents (Tween 20 [ 89 ] or triton X-100 [ 60 ]), low concentrations of urea [ 60 , 90 ], or sodium deoxycholate [ 91 ]. Subsequently, solubilization occurs in a high concentration of denaturing agents such as urea (6–8 M) [ 92 ] or guanidine hydrochloride (6 M) [ 60 , 90 ] and can be enhanced at alkaline pH [ 5 , 49 ].…”

“…A wide range of techniques have been successfully applied to this end, including sonication [56,57,59], bead milling [60], and high-pressure homogenization [46]-this last technique also being suitable at an industrial scale [61]. In cases where IFN is transported to the periplasm, selective disruption of the outer membrane is crucial to avoid complete lysis, thus ensuring that the target protein is recovered After cell lysis, inclusion bodies are isolated from soluble proteins by centrifugation [60] and are then subjected to different washing steps, e.g., with detergents (Tween 20 [89] or triton X-100 [60]), low concentrations of urea [60,90], or sodium deoxycholate [91]. Subsequently, solubilization occurs in a high concentration of denaturing agents such as urea (6-8 M) [92] or guanidine hydrochloride (6 M) [60,90] and can be enhanced at alkaline pH [5,49].…”