Efficient recovery of recombinant CRM197 expressed as inclusion bodies in E.coli

Abstract

CRM197, which retains the same inflammatory and immune-stimulant properties as diphtheria toxin but with reduced toxicity, has been used as a safe carrier in conjugated vaccines. Expression of recombinant CRM197 in E. coli is limited due to formation of inclusion bodies. Soluble expression attempts in Bacillus subtilis, P. fluorescens, Pichia pastoris, and E. coli were partially unsuccessful or did not generate yields sufficient for industrial scale production. Multiple approaches have been attempted to produce CRM197 in E. coli, which has attractive features such as high yield, simplicity, fast growth, etc., including expression of oxidative host, concurrent expression of chaperones, or periplasmic export. Recently, alternative methods for recovery of insoluble proteins expressed in E. coli were reported. Compared to traditional denaturation/refolding, these methods used the non-denaturing solubilization agent, N-lauroylsarkosine to obtain higher recovery yields of native proteins. Based on this work, here, we focused on solubilization of CRM197 from E. coli inclusion bodies. First, CRM197 was expressed as inclusion bodies by high-level expression of recombinant CRM197 in E. coli (126.8 mg/g dcw). Then bioactive CRM197 was isolated from these inclusion bodies with high yield (108.1 mg/g dcw) through solubilization with N-lauroylsarkosine including Triton X-100 and CHAPS, and purified by Ni-affinity chromatography and size-exclusion chromatography. In this study, we present a cost-effective alternative for the production of bioactive CRM197 and compare our recovery yield with yields in other production processes.

Investigating the Deoxyribonuclease Activity of CRM197 with Site-Directed Mutagenesis

“…Park and co-workers showed that the presence of this purification tag did not disrupt CRM197 folding or activity. 11 The constructed plasmid was transformed into BL21 (DE3) cells (Lucigen) and plated onto LB media supplemented with ampicillin. A single colony was picked and cultured overnight at 37 °C/200 rpm in 2 mL of LB media supplemented with ampicillin.…”

Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts

“…Both classical and non-classical IBs contain relatively pure and intact recombinant proteins, and several approaches have been reported to recover biologically active proteins from these aggregated forms. Usually, during an in vitro refolding procedure, IBs are denatured by dissolving with a high or mild concentration of chaotropic denaturants such as urea, guanidine hydrochloride (GdnHCl) [ 16 ], or alternately with non-ionic or ionic detergents, such as Triton X-100, N-lauroylsarcosine, dimethylsulfoxide (DMSO) and a low percentage (~5%) of n-propanol [ 17 ]. In addition, dithiothreitol (DTT) or 2-mercaptoethanol was added to reduce undesirable inter- and/or intra-molecular disulfide bonds [ 18 ].…”

Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process

“…High concentration of chaotropic agent results in complete denaturation of insoluble IBs which are further subjected to a single refolding step to recover soluble protein [16]. Alternatively, soluble protein can also be recovered directly from non-classical IBs by using non-denaturing solubilizing agents such as N -lauroyl sarcosine, dimethylsulfoxide (DMSO) and low percentage (~ 5%) of n -propanol [7, 17, 18]. Several studies have mentioned earlier that the recovery yield of soluble protein from bacterial IBs depends on its nature and the strength of denaturing agents which are applied during the solubilization process [7, 19].…”