鼠伤寒沙门菌效应蛋白SteC的原核表达、纯化与晶体培养

Prokaryotic expression,purification and crystallization of Salmonella effector protein SteC

目的鼠伤寒沙门菌效应蛋白SteC是沙门菌Ⅲ型分泌系统中唯一的一种激酶,然而其与以往发现的激酶同源性均较低,其发挥激酶功能的机制尚不清楚。本研究通过基因克隆、原核表达截短鼠伤寒沙门菌效应蛋白SteC-C端催化结构域,获得纯化蛋白并进行晶体培养,为揭示SteC作为激酶催化磷酸化的机制奠定基础。方法以鼠伤寒沙门菌效应蛋白SteC生物信息学分析为基础,在SteC-C端催化结构域进行片段截取,并将此结构域中仅有的第276位半胱氨酸突变为丝氨酸,利用基因克隆技术分别构建SteC C1-pGl01和SteC C1_(C276S)-pGl01两种重组质粒,在大肠埃希菌原核体系中进行表达。依次使用镍离子亲和层析柱、凝胶层析柱进行蛋白纯化。使用12种晶体试剂盒筛选适合SteC活性(Holo)与非活性(Apo)状态晶体生长的条件,从而获得SteC不同类型的蛋白质晶体,并使用additive试剂盒寻找更利于蛋白晶体生长的条件,从而获得单晶性良好的晶体。结果鼠伤寒沙门菌效应蛋白SteC C1催化结构域相对分子质量大小为19.7×10^(3),在原核表达体系中蛋白可溶性好、浓度高,但是蛋白性质不稳定存在二聚化现象,其突变体证明SteC Cys-276是SteC C1导致二聚化的原因。两种蛋白均在2.0 mol/LAmmonium sulfate,0.01 mol/L Magnesium sulfate heptahydrate,0.05 mol/LSodium cacodylate trihydrate(pH 6.5)条件结晶,且SteC C1_(C276S)晶体结晶时间更快,添加0.1mol/L碘化钠试剂后更有助于蛋白晶体的生长。结论成功构建SteC C1及SteC C1_(C276S)原核表达系统并获得蛋白进行结晶培养。硫酸铵盐有助于SteC C1结构域及其突变体晶体的形成且突变体更容易结晶。SteC C1结构域蛋白晶体的培养为SteC蛋白质空间结构的解析及进一步揭示其催化磷酸化的分子机制奠定了基础。

Objective The Salmonella typhimurium effector protein SteC is the only kinase in the Salmonella type Il secretion system.However,its homology with previously discovered kinases is low,and its mechanism of kinase function is still unclear.In this study,through gene cloning and prokaryotic expression of the truncated SteC-C terminal catalytic domain of the Salmonella typhi m urium effector protein,the purified protein was obtained and crystallized,laying the foundation for revealing the mechanism of SteC as a kinase that catalyzes phosphorylation.Methods Based on the bioinformatics analysis of the effector protein SteC of Salmonella ty phim urium,fragments were intercepted in the SteC-C terminal catalytic domain,and the only 276^(th)cysteine in this domain was mutated to serine.Two recombinant plasmids,SteC C1-pG101 and SteC Cl_(c276s)-pG101 were constructed by cloning technology,and expressed in the prokaryotic system of Escherichia coli.Protein purification was performed using nickel ion affinity chromatography column and gel chromatography column sequentially.Using twelve kinds of crystal kits to screen the conditions suitable for the crystal growth of SteC active(Holo)and inactive(Apo)states,so as to obtain different types of SteC protein crystals.And then using additive kit to find conditions that are more conducive to protein crystal growth,so as to obtain crystal with good single crystallinity.Results The relative molecular mass of the Salmonella typhi m urium effector protein SteC C1 was 19.7×10^(3).In the prokaryotic expression system,the protein had good solubility and high concentration,but the protein was unstable and had dimerization phenomenon.The mutants proved that SteC Cys-276 is responsible for dimerization of SteC C1.Both proteins were crystallized under the conditions of 2.0 mol/L Ammonium sulfate,0.01 mol/L Magnesium sulfate heptahydrate,0.05 mol/L Sodium cacodylate trihydrate(pH 6.5),and the crystallization time of SteC C1_(c276s)crystal was faster,adding 0.1 mol/L iodide Sodium reagent is more conducive to the growth of protein crystals.Conclusion The prokaryotic expression system of SteC C1 and SteC C1_(c276s)was successfully constructed and the protein was obtained for crystallization.Ammonium sulfate helps the crystal formation of SteC C1 domain and its mutants,and the mutants are easier to crystallize.The culture of SteC C1 domain protein crystals laid a foundation for analyzing the spatial structure of SteC protein and further revealing the molecular mechanism of its catalytic phosphorylation.