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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
引用本文: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.
作者姓名:刘骥  杨帆  田万帆  龙虎  孙思雨  周玉珊  赵燕英  唐俊妮
作者单位:(西南民族大学生命科学与技术学院,四川?成都 610041)
基金项目:国家自然科学基金面上项目(31371781);四川省应用基础项目(2014JY0253); 西南民族大学大学生创新创业训练计划项目(201610656053);四川省教育厅项目(15ZB0482)
摘    要:葡萄球菌肠毒素M是由金黄色葡萄球菌νSa基因岛编码的分泌型超抗原。本研究将截去N端信号肽的金黄色葡萄球菌M型肠毒素(staphylococcal?enterotoxin?M,SEM)蛋白编码基因亚克隆至原核表达载体p ET-28a(+),构建重组表达质粒p ET-28a(+)-ΔNspsem;随后转化感受态E.coli?Rosetta(DE3)并探讨融合蛋白最佳表达条件;利用Ni2+-Sepharose?4?Fast?Flow亲合层析纯化出His-ΔNsp SEM融合蛋白;经质谱鉴定,纯化的融合蛋白对SEM的氨基酸覆盖率达96.3%;凝血酶切除6×His标签肽后,圆二色谱分析表明ΔNsp SEM重组蛋白富含β-折叠(35%)和β-转角(21%)以及较少α-螺旋(16%)等二级结构;荧光发射谱揭示ΔNsp SEM在278?nm和295?nm波长处激发时具有相同的Trp发射峰(341?nm);十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明ΔNsp SEM重组蛋白表现出较高的热稳定性。研究结果表明重组蛋白SEM表达成功,纯化的ΔNsp SEM重组蛋白溶液构象紧密并具有接近天然状态的结构,这为深入研究SEM蛋白的结构与功能提供理论支持。

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  

Prokaryotic Expression,Purification, Identification and Solution Conformation of Staphylococcal Enterotoxin M
LIU Ji,YANG Fan,TIAN Wanfan,LONG Hu,SUN Siyu,ZHOU Yushan,ZHAO Yanying,TANG Junni.Prokaryotic Expression,Purification, Identification and Solution Conformation of Staphylococcal Enterotoxin M[J].Food Science,2017,38(24):40-46.
Authors:LIU Ji  YANG Fan  TIAN Wanfan  LONG Hu  SUN Siyu  ZHOU Yushan  ZHAO Yanying  TANG Junni
Affiliation:(College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China)
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 (+)-ΔNspsem 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.
Keywords:staphylococcal enterotoxin M  prokaryotic expression  purification  mass spectrometry  circular dichroism  fluorescence emission spectroscopy  thermal stability  
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