Bacterial expression of CRM197: investigation and optimization of gene expression factors for effective production in <i>E. coli</i>.

CRM197 (Cross Reacting Material 197) is an inactive form of 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 3 grams per liter in form of the inclusion bodies. We examined the significant effect of expression vector type, 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 2 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.