鼠伤寒沙门菌修饰酶YdiU的表达、纯化及晶体培养  

Expression, purification, and crystallization of YdiU from Salmonella typhimurium

作　　者：马玥 杨银龙 岳盈盈[2] 宋楠楠[2] 王玮玮 贾海红 李翠玲[2] 李冰清[1,2] MA Yue;YANG Yin-long;YUE Ying-ying;SONG Nan-nan;WANG Wei-wei;JIA Hai-hong;LI Cui-ling;LI Bing-qing(School of Medicine and Life Sciences,University of Jinan,Shandong Academy of Medical Sciences,Jinan,China 250062;Institute of Basic Medicine,Shandong Academy of Medical Sciences)

机构地区：[1]济南大学山东省医学科学院医学与生命科学学院,山东济南250062 [2]山东省医学科学院基础医学研究所

出　　处：《中国病原生物学杂志》2019年第11期1241-1244,1249,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31500050,31800054)

摘　　要：目的采用大肠埃希菌原核表达体系表达并纯化YdiU蛋白,筛选蛋白质晶体,为其结构和酶活测定等研究奠定基础。方法以生物信息学预测为基础,使用PCR方法从沙门菌基因组中调取ydiU基因,构建ydiU-pGl01重组质粒,转化入大肠埃希菌BL21(DE3)中,诱导蛋白表达。并使用镍离子亲和层析柱和阴离子交换柱对YdiU蛋白进行纯化。纯化后YdiU蛋白利用16种晶体筛选试剂盒在悬滴条件下进行晶体培养。结果成功构建重组表达质粒ydiU-pGl01。IPTG诱导后,YdiU蛋白在大肠埃希菌BL21(DE3)菌株中大量表达,并多以可溶形式存在。经镍离子亲和层析和阴离子交换法纯化后得到纯度较高的蛋白溶液,浓度为3.6 mg/ml,相对分子质量约51×10^3。纯化的YdiU蛋白在0.5 mol/L Potassium thiocyanate条件下长出蛋白晶体。结论采用大肠埃希菌表达系统可表达可溶性YdiU蛋白,YdiU蛋白在阴离子交换柱上表现为峰型对称的单峰,纯度较高,可用于蛋白质晶体的筛选,为进一步解析YdiU的结构和功能研究奠定了基础。Objectives To use an E.coli prokaryotic expression system to express,purify,and screen crystals of YdiU protein and to lay a foundation for the study of its structure and assays of its enzyme activity.Methods On the basis of bioinformatic predictions,the ydiU gene was extracted from the S.typhimurium genome via a polymerase chain reaction(PCR),and a ydiU-pGl01 recombinant plasmid was constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)to induce the expression of the target YdiU protein.The YdiU protein was purified using a Ni-NTA column and an anion-exchange column.The purified YdiU protein was cultured with 16 crystal screening kits via vapor diffusion.Results The recombinant expression plasmid ydiU-pGl01 was successfully constructed.After its expression was induced with IPTG,the YdiU protein was abundantly expressed in the E.coli BL21(DE3)strain.The protein is soluble.The protein was purified via a Ni-NTA column and an anion-exchange column.The concentration of the purified YdiU protein was 6 mg/ml,and its molecular weight was about 51×10^3.The purified YdiU protein was obtained as crystals using 0.5 mol/L potassium thiocyanate.Conclusion Soluble YdiU protein can be expressed by an E.coli expression system.The YdiU protein was eluted as a symmetrical single peak from an anion-exchange column with a high level of purity,and no other proteins were noted.This approach can be used to screen protein crystals.This work has laid the foundation for further study of the function and structure of YdiU.

关 键 词：鼠伤寒沙门菌 YdiU蛋白 AMPylation修饰 表达纯化 晶体培养 

分 类 号：R378.23[医药卫生—病原生物学]