白喉毒素突变体CRM197在白喉杆菌中的表达纯化

生物技术通报 ›› 2014, Vol. 0 ›› Issue (3): 182-186.

白喉毒素突变体CRM197在白喉杆菌中的表达纯化

朱涛, 邓立功, 段磊, 常云松, 邵忠琦, 毛慧华, 宇学峰

(天津科技大学，天津 300457)

收稿日期:2013-08-23 出版日期:2014-03-29 发布日期:2014-03-31
作者简介:朱涛，男，博士，教授，研究方向:疫苗研发；E-mail:554810422@qq.com

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

Zhu Tao, Deng Ligong, Duan Lei, Chang Yunsong, Shao Zhongqi, Mao Huihua, Yu Xuefeng

(Tianjin University of Science and Technology，Tianjin 300457)

Received:2013-08-23 Published:2014-03-29 Online:2014-03-31

摘要/Abstract

摘要： 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 ? 27010 TM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。

关键词: CRM197, 白喉杆菌, 接合转移, 可溶蛋白, 铁调控, 阴离子柱

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

朱涛, 邓立功, 段磊, 常云松, 邵忠琦, 毛慧华, 宇学峰. 白喉毒素突变体CRM197在白喉杆菌中的表达纯化[J]. 生物技术通报, 2014, 0(3): 182-186.

Zhu Tao, Deng Ligong, Duan Lei, Chang Yunsong, Shao Zhongqi, Mao Huihua, Yu Xuefeng. Expression and Purification of the Diphtheria Toxin Variant CRM197 in Corynebacterium diphtheriae[J]. Biotechnology Bulletin, 2014, 0(3): 182-186.

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