New Strategy for Solubilization and Refolding of Recombinant Human Interferon α2b Inclusion Bodies from E. coli Gene Overexpression System

Abstract

Objective: New Strategy for solubilization and refolding of recombinant human Interferon α2b inclusion bodies was established to obtain a high solubilization and refolding rate of recombinant human Interferon α2b inclusion bodies from E. coli Gene Overexpression System.Method: The IFN-α2b inclusion bodies were solubilized with solubilizing buffer of 2mol/L urea. Refolding was performed via two steps, diluting by pulse adding the solubilized IFN α2b sample and dialysing it slowly using ultrafiltration 5K step by step. Finally refolded IFN α2b was purified through Cu 2+ chelate affinity chromatography. Results: The urea concentration of 2mol/L in the solubilizing buffer gave the solubilization rate of 89.73%. Diluting the solubilized IFN α2b sample by pulse adding gave the refolding rate of 78.53%. The multi steps dialysis through ultrafiltration membrane 5K gave the antiviral activity recovery of 98.73%. The purification through one step Cu 2+ chelate affinity chromatography raised the specific activity of IFN sample to 1.4×10 8 U/mg protein and 12% SDS-PAGE showed single band of purified IFN-α2b at expected MW height, whose purity was 99.8%. Conclusion: Large-scale production of recombinant human Interferon α2b (IFN-α2b) in E. coli with a thermoinducible overexpression system was established by applying an effective solubilizing and refolding processes of interferon α2b inclusion bodies (IBs). Solubilization and refolding rate of IFN-α2b IBs were 89.71% and 79.87% respectively. Refolded IFN-α2b was purified through one-step immobilized metal affinity chromatography to give a pure bioactive IFN-α2b with the specific activity of 1.4×10 8 IU/mg protein and with the recovery rate over 52.89%.

Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies & evaluations on its in vitro & in vivo bioactivity

“…Since the existence of the native-like secondary structure was revealed, many researchers have developed mild solubilisation methods ( Chura-Chambi et al., 2019 ; Datta et al., 2013 ; Patra et al., 2000 ; Singh et al., 2008 , 2012 ). It is assumed that by retaining the native-like secondary structure, it will be easier to fold the protein into its native form, leading to increased protein recovery from the IBs ( Yon et al., 2018 ). In this study, we utilised a single freeze-thawing process as described by Qi et al.…”

“…Solubilisation of IBs and refolding of the solubilised protein into a native state are the most important steps in recovering bioactive protein from IBs ( Singh et al., 2015 ). Mostly, IBs are solubilised using a high concentration of urea or guanidine hydrochloride ( Clark, 2001 ) to unfold the protein structure and expose the hydrophobic patch causing the formation of aggregates, thus resulting in a low recovery of bioactive protein ( Upadhyay et al., 2014 ; Yon et al., 2018 ). Mostly, IBs are solubilized using high concentration of urea or guanidine hydrochloride ( Clark, 2001 ).…”

“…They will unfold the protein structure and expose the hydrophobic patch. The usage of high urea concentration is often resulting in low recovery of bioactive protein due to the formation of aggregates during refolding step ( Upadhyay et al., 2014 ; Yon et al., 2018 ). Solubilisation of IBs using milder conditions can retain the native-like secondary structure, thus making it easier to restore the structure of solubilised protein into the native state, reducing protein aggregation during refolding and increasing bioactive protein recovery ( Sahdev et al., 2008 ; Upadhyay et al., 2016 ).…”