人胱硫醚β合酶I278T突变体的表达、纯化及鉴定  

Expression,purification and identification of I278T-mutant human cystathionine β synthase

作　　者：牛卫宁[1] 许乐[1] 羊梦林[1] 曹珊珊[1] 

机构地区：[1]西北工业大学生命学院,陕西西安710072

出　　处：《国际检验医学杂志》2014年第9期1089-1091,共3页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(20802057);西北工业大学基础研究基金资助项目(JC201161);西北工业大学研究生创业种子基金资助项目(Z2013186)资助

摘　　要：目的探讨在大肠埃希菌中重组表达和纯化人胱硫醚β合酶CBS（I278T）突变体。方法采用重叠延伸聚合酶链反应（PCR）定点突变技术构建突变质粒pGEX4T-1-CBS（I278T）,在含有3%乙醇的培养基中诱导表达,亲和层析纯化得到突变CBS（I278T）蛋白,测定纯化蛋白的活性、紫外可见吸收光谱、蛋白粒径及Zeta电位。结果成功构建了质粒pGEX4T-1-CBS（I278T）。CBS（I278T）蛋白产率、比活性及酶活性回收率分别为2.3mg/L、21.4U/mg、22.6%。终浓度为1mmol/L的S-腺苷甲硫氨酸（AdoMet）对突变CBS（I278T）蛋白没有激活作用。紫外可见吸收光谱分析显示纯化的突变CBS（I278T）存在429nm和550nm的血红素结合蛋白特征吸收峰。蛋白平均粒径为7.5～10.1nm,主要以四聚体形式存在,Zeta电位为-16.3mV。结论成功建立了在大肠埃希菌中表达、纯化及鉴定突变CBS（I278T）的方法。Objective To investigate the expression and purification I278T-mutant human cystathionineβsynthase（CBS） in E . coli .Methods Site-directed mutagenesis by overlap extension using the polymerase chain reaction （PCR） was employed to construct mutant plasmids pGEX4T-1-CBS（I278T） ,which was induced and expressed in a medium containing 3% ethanol ,purified by affinity chromatography to obtain mutated CBS （I278T） protein .The activity ,UV-visible absorption spectroscopy ,protein particle size and Zeta potential of the purified protein were measured .Results Plasmid pGEX4T-1-CBS（I278T） was successfully constructed .The yield ,the specific activity and activity recovery of purified mutant CBS （I278T ） protein were 2 .3 mg/L ,21 .4 U/mg and 22 .6% .S-adenosylmethionine（AdoMet） with final concentration of 1 mmol/L showed no activation toward mutant CBS （I278T） protein .Ac-cording to UV-visible absorption spectroscopy analysis ,purified mutant CBS（I278T） had characteristic absorption peaks at 429 nm and 550 nm for heme-binding proteins .Protein average particle size was 7 .5 -10 .1 nm ,mainly in the form of tetramers ,and Zeta potential was - 16 .3 mV .Conclusion The methods of expression ,purification and identification of I278T-mutant human cystathionineβsynthase in E .coli were successfully established .

关 键 词：胱硫醚Β合酶 突变 粒径 ZETA电位 

分 类 号：R346[医药卫生—基础医学]