基础医学与临床2018，Vol.38Issue(6)：815-820,6.
原核表达及一步法制备可溶性白喉毒素突变体CRM197
Prokaryotic expression and one-step purification of soluble diphtheria toxin variant CRM197
摘要
Abstract
Objective To construct a novel prokaryotic expression system, in which cross-reacting material 197 (CRM197) can be expressed in a soluble form in Escherichia coli(E. coli) cytoplasm and purified simply by one-step Ni-NTA affinity purification. Methods The CRM197 coding sequence was cloned into the prokaryotic expres-sion vector pET-32a(+) as an fusion protein with Trx tag,the HRV3C(human rhinovirus 3C) protease recognition sequence and 6 histidine sequence were added to the N-terminal of CRM197.HRV3C protease gene was cloned into another prokaryotic expression plasmid pGArasd. Both plasmids were co-transformed into E. coli Origami B (DE3) and induced mildly at 15℃. CRM197 recombinant protein was purified by Ni-NTA affinity matrix. Results The free soluble His-tagged CRM197 protein was released by cleavage of the accompanying expressed HRV3C protease after the CRM197 fusion protein was expressed. After one-step affinity purification recombinant CRM197 protein with a purity of almost 95% was obtained. Outcoming of the final preparation incubated with DNA indicated the pu-rified CRM197 recombinant protein has deoxyribonuclease activity. Conclusions By constructing a novel double-plasmid auto-cleavage prokaryotic expression system in this study, the production process of obtaining soluble CRM197 recombinant protein in E. coli has been simplified,with expression and purification efficiency improved and the production cost reduced.关键词
白喉毒素突变体/可溶性表达/自动切割/纯化Key words
diphtheria toxin mutant/soluble expression/autocleavage/purification分类
生物科学引用本文复制引用
高宇辉,邓唯唯..原核表达及一步法制备可溶性白喉毒素突变体CRM197[J].基础医学与临床,2018,38(6):815-820,6.基金项目
中国医学科学院医学与健康科技创新工程(2017-I2M-3-022) （2017-I2M-3-022）
