Cloning, Expression, And Purification Of The Diphtheria Toxin Vari-Ant CRM197 In Corynebacterium Diphtheria

Diphtheria toxoid (DT) and tetanus toxoid (TT) are wildly used as protein carriers in preparation of polysaccharide-protein conjugate vaccines in China. DT and TT are derived from tetanus toxin and diphtheria toxin respectively through formaldehyde treatment, which can be a time consuming process when a large amount of the toxic material will be handled. Residual toxicity in the DT or TT preparations is also a major concern. CRM197, a nontoxic mutant of diphtheria toxin, has been successfully used as a carrier in several conjugate vaccine products manufactured abroad. A large amount of data accumulated so far demonstrated that CRM197 is safe and highly immugenic in human.In this study, we generated a recombinant Corynebacterium diphtheriae strain, in which the expression of CRM197 was increased, through genetic engineering of the parent strain C. diphtheriae ATCC 27010. A fermentation process for the recombinant CRM197 production strain as well as a purification process for the CRM197 protein were developed.To create the recombinant C. diphtheriae strain, the CRM197 gene from the strain ATCC 27010 was cloned and inserted into the Escherihia coli-C. diphtheriae shuttle vector, pCKM4.1, resulted in pCKM5.1. The plasmid pCKM5.1 was transformed into the E.coli strain S17-1 and subsequently transferred into C. diphtheriae ATCC 27010 through conjugation. Analysis of the cell lysate of the recombinant strain using SDS-PAGE and coomassie blue staining showed an intense protein band with the molecular weight of 58 kDa. This protein reacted strongly with anti-CRM197 antibody when analyzed using Western blotting.A new and effective production process for CRM197 from the recombinant C. diphtheriae strain was also developed in this study. The procedure described here involved the use of a modified growth medium that would allow for fast growth of bacterial cells and enhanced toxin production as a result of concomitant iron depletion. The CRM197 products were secreted into the culture medium, accounting for approximately 70% of the total protein in the supernatant. The CRM197 protein was then recovered by filtering or precipitation, and subsequently purified using anion-exchange chromatography. The purity of CRM197 prepared using the process reached 95%. The manufacturing process developed in this study may be used in a large scale industrial production of CRM197.