鼠伤寒沙门菌YbiB蛋白的表达纯化及结构解析  

Expression,purification and structure analysis of YbiB protein of Salmonella typhimurium

作　　者：翟莉 李冰清 贾海红 ZHAI Li;LI Bingqing;JIA Haihong(Hengshui Maternal and Child Health Hospital,Hengshui 053000,Hebei Province,China;Department of Pathogenic Biology,School of Clinical and basic Medicine,Shandong First Medical University&Shandong Academy of Medical Sciences)

机构地区：[1]衡水市妇幼保健院,河北衡水053000 [2]山东第一医科大学(山东省医学科学院)临床与基础医学院(基础医学研究所)病原生物学系

出　　处：《中国病原生物学杂志》2025年第3期271-276,共6页Journal of Pathogen Biology

基　　金：衡水市科技计划项目(No.2022014085Z)。

摘　　要：目的通过蛋白表达纯化、晶体X射线衍射技术解析鼠伤寒沙门菌YbiB蛋白的结构,为进一步探索YbiB蛋白参与的生物学过程及功能研究提供结构基础。方法分子克隆ybiB基因,插入pGL01表达载体,构建ybiB-pGL01重组质粒。诱导并纯化目标蛋白后进行晶体培养。通过上海光源SSRF光束线站收集YbiB蛋白晶体衍射数据,解析鼠伤寒沙门菌YbiB蛋白的三维结构信息。按照PDB官网的要求逐步填充YbiB蛋白的相关信息和结构参数。分析鼠伤寒沙门菌YbiB蛋白的结构特点并推测其功能。结果成功构建了融合表达质粒ybiB-pGL01并转化到BL21感受态细胞中;IPTG诱导表达YbiB蛋白,纯化后蛋白浓度为0.45mg/mL;在0.1mol/L Sodium acetate trihydrate pH 4.5,3.0mol/L Sodium chloride条件下,晶体呈长方体状;晶体衍射分辨率为2.34A;解析出鼠伤寒沙门菌YbiB的蛋白晶体结构并上传PDB数据库,PDB号为7Y3P;鼠伤寒沙门菌YbiB呈头对头的二聚体结构,且单体结构折叠成磷酸核糖基转移酶Ⅲ型家族蛋白的双叶状结构,与大肠埃希菌YbiB在结构上极其相似,有着相似的表面静电势能分布;鼠伤寒沙门菌YbiB和大肠埃希菌YbiB两个蛋白的PRPP或磷酸结合位点(天冬酰胺N89和酪氨酸Y88)、DNA结合位点(精氨酸R172和组氨酸H115)都高度保守。结论鼠伤寒沙门菌YbiB蛋白在原核表达体系中稳定表达,纯化后的蛋白也可用于晶体培养。YbiB蛋白结构成功解析并上传至PDB数据库。结构分析表明,鼠伤寒沙门菌YbiB与大肠埃希菌YbiB可能有着相似的DNA结合机制。Objective The structure of YbiB protein of Salmonella typhimurium was analyzed by protein expression and purification and crystal X-ray diffraction technique,which provides a structural basis for further exploring the biological process and function of YbiB.MethodsybiB was cloned and inserted into pGL01 expression vector to construct ybiB-pGLo1 recombinant plasmid.The target protein was induced and purified for crystal culture.The diffraction data of YbiB protein crystal were collected by Shanghai Light Source SSRF beamline station.Analysis of the three-dimensional structure of S.typhimurium YbiB.According to the requirements of the PDB official website,the relevant information and structural parameters of YbiB protein were gradually filled.The structural characteristics of S.typhimurium YbiB were analyzed and its function was speculated.Results The fusion expression plasmid ybiB-pGLO1 was successfully constructed and transformed into BL21 competent cells;The expression of YbiB protein was induced by IPTG,and the purified protein concentration was O.45 mg/mL;The crystal was cuboid under the condition of o.1 mol/L Sodium acetate trihydrate pH4.5,3.0 mol/L Sodium chloride;The crystal diffraction resolution was 2.34 A;The protein crystal structure of S.typhimurium YbiB was analyzed and uploaded to the PDB database,and the PDB number was 7Y3P;S.typhimurium YbiB presented a head-to-head dimer structure,and its monomeric structure was folded into the bilobal structure of phosphoribosyltransferase type Il family proteins.It was extremely similar to the YbiB of E.coli in structure,with a similar distribution of surface electrostatic potential energy;The PRPP or phospho-binding sites(asparagine N89 and tyrosine Y88)and DNA binding sites(arginine R172 and histidine H115)of S.typhimurium YbiBand E.coli YbiBwere highly conserved.Conclusion The S.typhimurium YbiB was stably expressed in prokaryotic expression system,and the purified protein can also be used for crystal culture.The YbiB protein structure was successfully resol

关 键 词：鼠伤寒沙门菌 YbiB蛋白 蛋白纯化 晶体培养 蛋白结构解析 

分 类 号：R51[医药卫生—内科学]