中文摘要：

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。

英文摘要：

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 .