INVESTIGAION OF PRODUCTION OF A NON-TOXIC DIPHTHERIA TOXIN VARIANT CRM197 IN ESCHERICHIA COLI CELLS

Abstract

The aim of research: to synthesize the gene, coding the non-toxic variant of diphtheria toxin CRM197, to explore its expression in Escherichia coli cells, to construct bacterial strain - producer of recombinant CRM197 and to develop the method for purification of this CRM197 protein from the biomass of bacteria. Materials and methods of research. the gene of CRM197 was synthesized by chemical and enzymatic methods. Expression vector pColdII-CRM197 construction and assembly were performed by standard genetic engineering methods. CRM197 production by Escherichia coli cells was explored by electrophoresis in polyacrylamide gel and immunoblotting. The recombinant CRM197 was purified with the use of metal-chelate chromatography. Results. The application of the pColdII expression vector made possible to produce recombinant protein after decrease of the cell cultivation temperature from 37° C to 16° C. The method for purification of CRM197 recombinant protein was developed and the protein preparation with the purity 97% was obtained, having molecular mass 60 kDa; this protein was coupled with polyclonal antibodies against diphtheria toxin in the immunoblot. Conclusion. Successful production of CRM197 recombinant protein was reached with the use of pColdII vector at the decreased cell cultivation temperature. The purified CRM197 demonstrated nuclease activity pointing its proper folding. Production of the purified recombinant CRM197 protein allows its use for development of new conjugate vaccines.

Keywords

CRM197 / Escherichia coli / diphtheria toxin / CRM197 / cspA promoter / Escherichia coli / metallochelating chromatography

RIGHTS & PERMISSIONS

Dukhovlinov I.V., Bogomolova E.G., Dobrovolskaya O.V., Ishuk C.A., Fedorova E.A., Klimov N.A., Simbirtsev A.S.