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

摘要

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.%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pET-28a(+),构建重组表达质粒pET-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-ΔNspSEM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ΔNspSEM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ΔNspSEM在278nm和295nm波长处激发时具有相同的Trp发射峰(341nm);十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明ΔNspSEM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ΔNspSEM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.