关键词: 白喉毒素突变体, 可溶性表达, 自动切割, 纯化

Abstract: Objective To construct a novel prokaryotic expression system, in which cross-reacting material 197 (CRM197) can be expressed as soluble form in Escherichia coli (E. coli) cytoplasm and purified by simply one-step Ni-NTA affinity purification. Methods The CRM197 coding sequence were cloned into the prokaryotic expression vector pET-32a(+) as an fusion protein with Trx tag,and 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-transfected 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 purified 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 obtain soluble CRM197 recombinant protein in E. coli has been simplified，with expression and purification efficiency improved and the production cost reduced.

Key words: diphtheria toxin mutant, soluble expression, autocleavage, purification