食品科学2017，Vol.38Issue(24)：40-46,7.DOI:10.7506/spkx1002-6630-201724007
金黄色葡萄球菌M型肠毒素原核表达、纯化、鉴定及溶液构象分析
Prokaryotic Expression, Purification, Identification and Solution Conformation of Staphylococcal Enterotoxin M
摘要
Abstract
Staphylococcal enterotoxin M (SEM) is a secretory superantigen encoded by the νSa genomic islands of Staphylococcus aureus.In this study,the sem gene from S.aureus without N-terminal signal peptide was subcloned into the prokaryotic expression vector pET-28a(+)to construct the recombinant plasmid pET-28a(+)-Δ Nspsem and the recombinant expression plasmid was then transformed into E. coli Rosetta (DE3) competent cells.The positive clones, induced by IPTG,effectively expressed His-tag containing soluble ΔNspSEM fusion protein in E.coli Rosetta(DE3).The expression conditions including expression vector, time, IPTG concentration and temperature were optimized. Purified His-ΔNspSEM fusion protein was obtained by Ni2+-Sepharose affinity chromatography. Mass spectrometric analysis indicated that the amino acid sequence of the fusion protein was 96.3% similar to that of SEM. Circular dichroism revealed ΔNspSEM, whose 6 × His sequence was cleaved by thrombin, was rich in β-sheet (35%) and β-turn (21%) but low in α-helix (16%). The fluorescence emission spectrum of ΔNspSEM exhibited identical tryptophan emission peak (341 nm) with excitation at 278 and 295 nm. The time-dependent thermal stability of ΔNspSEM obtained at 100 ℃ by SDS-PAGE indicated that the recombinant protein had relatively high thermal stability. To conclude, our results showed that the ΔNspSEM recombinant protein was successfully expressed and that the purified protein exhibited a compact conformation similar to the natural one in solution, which can provide a basis for insight into the structure and function of SEM protein.关键词
金黄色葡萄球菌M型肠毒素/原核表达/纯化/质谱/圆二色谱/荧光发射谱/热稳定性Key words
staphylococcal enterotoxin M/prokaryotic expression/purification/mass spectrometry/circular dichroism/fluorescence emission spectroscopy/thermal stability分类
轻工纺织引用本文复制引用
刘骥,杨帆,田万帆,龙虎,孙思雨,周玉珊,赵燕英,唐俊妮..金黄色葡萄球菌M型肠毒素原核表达、纯化、鉴定及溶液构象分析[J].食品科学,2017,38(24):40-46,7.基金项目
国家自然科学基金面上项目(31371781) （31371781）
四川省应用基础项目(2014JY0253) （2014JY0253）
西南民族大学大学生创新创业训练计划项目(201610656053) （201610656053）
四川省教育厅项目(15ZB0482) （15ZB0482）
