Abstract: The CRM197 protein is a variant of the diphtheria toxin(DTx, 58 kD)characterised by a single mutation(i.e. a glycine-glutamic acid substitution in position 52)that reduces its toxicity. The protein nonetheless retains the same inflammatory and immunostimulant properties as thediphtheria toxin, and it is widely used as a safe carrier in conjugatedvaccines. A new and alternative procedure for the production of full-length CRM197 in Corynebacterium diphtheriae was proposed. The gene of CRM197 was synthesized and then expressed in Corynebacterium diphtheriae. Expression vector pCKM5.1 was first electrotransformed into Escherichia coli-s-17, reisolated, and subsequently transferred into the C. diphtheriae recipient strain by conjugation. The procedure involved the use of a modified non-deferrated growth medium that allowed for fast growth of bacteria and enhanced toxin production as a result of concomitant iron depletion. The CRM197 products are secreted into the culture medium as soluble body, accounting for around 70% of the total cellular protein, then recovered by filtering or precipitation, and subsequently purified using anion-exchange chromatographic method. The purity of the final preparation reached 95% and its biological properties is maintained. Consequently, the procedure could be suitable for a large-scale industrial production.

Key words: CRM197, Corynebacterium diphtheriae conjugation, Soluble body, Depletion of iron, Anion-exchange chromatograph