白喉毒素突变体CRM197在白喉杆菌中的表达纯化

Expression and Purification of the Diphtheria Toxin Variant CRM197 in Corynebacterium diphtheriae

CRM197是一种白喉毒素突变体,第52位的甘氨酸突变为谷氨酸,作为载体蛋白广泛用于疫苗开发。将阐述一种新的生产CRM197方法。将合成的CRM197基因片段克隆到表达载体pCKM4.1中,表达质粒pCKM5.1电转化至大肠杆菌E.coli S17-1中,通过结合转移转化至白喉杆菌(ATCC?27010TM的白喉杆菌)中,载体上的导肽序列可以使得CRM197作为可溶性蛋白分泌到胞外表达,CRM197蛋白可占到菌体总蛋白的70%。增强对铁的调控,进一步优化培养基及发酵条件以提高产量。经过Q膜、硫酸铵沉淀、阴离子交换纯化步骤获得纯度达到95%的CRM197样品,提高了蛋白得率,节约了纯化时间和成本。

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.