Bacterial Expression of Crm197: Investigation and Optimization of Gene Expression Factors for Effective Production in E. coli

Abstract

CRM197 (Cross Reacting Material 197) is an inactive form of the C. diphtheriae exotoxin used as a carrier protein for the development and production of conjugated polysaccharide vaccines and immunotherapeutic drugs. However, the development of these research areas is not possible without an efficient and cost-effective technology to produce CRM197 of the proper quality. In this study, we developed a highly efficient method to produce recombinant CRM197 as a fusion with SUMO protein, yielding more than three grams per liter in the form of inclusion bodies. We examined the significant effect of the type of expression vector, the heterologous gene expression conditions, and cultivation on its solubility. Using a combination of reduced cultivation temperature and the promoter of the gene encoding the heat shock protein CspA, we achieved an increase in the solubility level of SUMO-CRM197 of more than 30%, with an overall biosynthesis level of more than two grams per liter. Coexpression of the target gene with the DsbC disulfide isomerase gene allowed us to obtain the target protein completely in the soluble state with a yield of more than 1.4 grams per liter. The results obtained may become the basis for the development of a promising domestic technology for the production of CRM197.