截短人胱硫醚β-合成酶的可溶性表达、纯化及活性鉴定  

Expression,Purification and Characterization of Truncated Human Cystathionine β-synthase

作　　者：王利群[1] 顾雅平[1] 曹利民[1,2] 汪庆[1] 于海燕 奚学志[1] 孙培培[1] 沈关心[3] 

机构地区：[1]常州大学制药与生命科学学院,常州213164 [2]常州二十一世纪生物技术研究所有限公司,常州213164 [3]华中科技大学同济医学院基础医学院免疫学系,武汉430030

出　　处：《华中科技大学学报（医学版）》2012年第6期697-703,共7页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：科技部国际合作资助项目(No.2011DFR30700)

摘　　要：目的构建截短人胱硫醚β-合成酶(T-hCBS)基因原核表达载体,得到可溶性的T-hCBS蛋白,为同型半胱氨酸检测试剂盒的开发打下基础。方法通过优化T-hCBS基因,克隆并构建重组表达质粒pET15b/T-hCBS,转化大肠埃希菌BL21。使用Ni 2+亲和层析柱对重组蛋白进行纯化,再通过HiTrap脱盐柱脱盐,采用比色法检测目的蛋白活性。结果重组蛋白在大肠埃希菌中可以高效表达,产量为44mg/L,比活约1 100U/mg。15%SDS-PAGE显示其相对分子质量为45kD,与预计大小一致。表达菌体超声破碎后上清及纯化的蛋白溶液均呈现红色。结论成功克隆和表达了T-hCBS蛋白,并得到高产高酶活性的可溶性蛋白,对同型半胱氨酸检测试剂盒的开发有一定的潜在应用价值。Objective To construct the truncated human cystathionine β-synthase（T-hCBS） express vector,and obtain the soluble recombinant protein T-hCBS.Methods The T-hCBS gene was cloned into pET15b vector to construct the recombinant plasmid pET15b/T-hCBS which was then transformed into E.coli BL21 cells for expression.The expressed protein was purified by metal（Ni2＋） chelating affinity chromatography and desalted by HiTrap desalting column.Colorimetry was used to determine the activity of the recombinant protein.Results The recombinant T-hCBS protein was over-expressed in E.coli,with a yield of 44 mg/L and the activity of about 1 100 U/mg.The relative molecular mass was 45 kD by 15%SDS-PAGE,which was consistent with the expected size.The expressed bacterium,the lysate supernatant and the purified protein samples had visible red color,showing obvious activity of the heme protein.Conclusion The T-hCBS was successfully cloned and expressed with high enzyme activity,which could be useful for developing homocysteine kits.

关 键 词：胱硫醚Β-合成酶 重组 可溶性蛋白 表达 

分 类 号：R345.5[医药卫生—基础医学]