鼠伤寒沙门菌效应蛋白SteC核心结构域及其激酶活性研究

Probing the core domain and kinase activity of Salmonella effector protein SteC

目的鼠伤寒沙门菌效应蛋白SteC是沙门菌Ⅲ型分泌系统分泌的具有丝氨酸/苏氨酸激酶活性的蛋白,在蛋白磷酸化修饰中发挥重要作用。然而关于SteC催化磷酸化的机制尚不清楚。本研究通过分析SteC核心结构域的组成并构建原核表达体系,了解表达蛋白的性状并进行酶活性测定,为揭示SteC作为沙门菌激酶催化磷酸化的机制奠定基础。方法以生物信息学分析和液相色谱-质谱(LC-MS)联用分析技术为基础,通过基因克隆技术构建SteC蛋白原核表达体系,表达目的蛋白,使用Ni^(2+)亲和层析柱纯化SteC全长蛋白SteC-fl及C端蛋白SteC-C。采用蛋白酶裂解法,用不同浓度的胰蛋白酶对纯化的SteC-C蛋白进行酶切,选择稳定表达的区域进行LC-MS及DNAstar软件肽段序列鉴定及比对,确定SteC蛋白的核心结构域,进而构建核心结构域原核表达体系,探究蛋白的性状。使用Phos-tagSDSPAGE胶对SteC核心结构域蛋白进行酶活性验证,以确定SteC的核心区域是否具有活性。结果成功构建SteC-fl、SteC-C原核表达体系并获得目的蛋白,但SteC-fl蛋白得率低,因而选择SteC-C利用蛋白酶裂解法确定SteC稳定表达的区域。利用LM-MS方法及DNAstar软件分析SteC核心结构域(即227aa-305aa)并成功构建SteC_(227-305)原核表达体系。表达的SteC_(227-305)蛋白经Ni 2+亲和层析纯化后进行分析,其稳定性高于SteC-fl、SteC-C端,但可溶性差;使用Phostag复合胶对SteC_(227-305)蛋白进行酶活性验证,显示此结构域具有较弱的自磷酸化功能。结论成功构建鼠伤寒沙门菌效应蛋白SteC不同区域的原核表达体系(SteC-fl、SteC-C、SteC_(227-305))。利用LM-MS确定SteC核心结构域为227aa305aa,生物信息学分析显示SteC_(227-305)含有PKc_like保守结构域,稳定系数为29.23,即为稳定蛋白。SteC_(227-305)经Ni 2+亲和层析纯后其可溶性差而稳定性符合预期;Phos-tag复合胶验证其具有较弱的自磷酸化功能,为研究SteC蛋白催化磷酸盐转移的机制奠定了基础。

Objective The Salmonella typhimurium effector protein SteC is a protein secreted by the Salmonella Type III secretion system with serine/threonine kinase activity,playing an important role in protein phosphorylation modification.However,the mechanism of SteC catalyzed phosphorylation is still unclear.This study analyzed the composition of the core structural domain of SteC and constructed a prokaryotic expression system to understand the characteristics of the expressed protein and determine its enzyme activity,laying the foundation for revealing the mechanism of SteC as a Salmonella kinase catalyzing phosphorylation.Mothads Based on bioinformatics analysis and liquid chromatography-mass spectrometry(LC-MS)analysis techniques,a prokaryotic expression system for SteC protein was constructed using gene cloning technology to express the target protein.The full-length protein SteC-fl and C-terminal protein SteC-C were purified using Ni^(2+)affinity chromatography column.Using protease lysis method,purified SteC-C protein was digested with different concentrations of trypsin,and stable expression regions were selected for LC-MS and DNAstar software peptide sequence identification and comparison to determine the core domain of SteC protein.Subsequently,a prokaryotic expression system for the core domain was constructed to explore the protein's characteristics.Use Phos-tag gel to validate the enzyme activity of the SteC core domain protein to determine whether the core region of SteC is active.Results Successfully constructed the SteC-fl and SteC-C prokaryotic expression systems and obtained the target protein,but the yield of SteC-fl protein was low.Therefore,SteC-C was chosen to use protease lysis to determine the stable expression region of SteC.Using LM-MS method and DNAstar software to analyze the core structural domain of SteC(227aa-305aa)and successfully construct the prokaryotic expression system of Ste_(C_(227-305)).The expressed SteC_(227-305)protein was purified by Ni^(2+)affinity chromatography and analyzed.Its stability was higher than that of SteC-fl and Ste C-C terminus,but its solubility was poor;Enzymatic activity validation of SteC_(227-305)protein using Phos-tag composite gel showed that this domain has weak self-phosphorylation function.Conclusion Successfully constructed prokaryotic expression systems for different regions of Salmonella typhimurium effector protein SteC(SteC-fl,SteC-C,SteC_(227-305)).Using LM-MS to determine the core structural domain of SteC as 227aa-305aa,bioinformatics analysis shows that SteC_(227-305)contains PKc_Like conserved domain,with a stability coefficient of 29.23,is a stable protein.After purification by Ni^(2+)affinity chromatography,SteC_(227-305)has poor solubility and meets the expected stability;Phos tag gel has been verified to have weak self-phosphorylation function,laying a foundation for studying the mechanism of phosphate transfer catalyzed by SteC protein.