OPTIMIZATION OF REFOLDING PROCESS FOR PRODUCTION OF RECOMBINANT CRM197 PROTEIN FROM E.COLI

Abstract

E. coli is most widely, well studied and highly characterized microorganism available for production of recombinant protein. rCRM197 (Recombinant Cross Reacting Material) is a genetically modified recombinant protein which is widely used as a carrier protein. When a recombinant protein is over expressed in heterologous expression system of E. coli, it gets misfolded and aggregated in the form of inclusion bodies. Recovery of active recombinant protein from the inclusion bodies is a cumbersome task. In the current study, rCRM197 was over expressed in E. coli based host system using optimized semi defined media. The purified inclusion bodies were isolated by optimization of Cell lysis step. Solubilization was performed using high concentration of Chaotropes at alkaline pH. A refolded and functional form of native CRM197 protein was recovered by dilution of solubilizing agent using Cystine/Cysteine redox agents in Refolding buffer. The optimized process resulted in generating high quality cost effective CRM197 with drastic reduction in the production time.