长QT综合征相关钙调蛋白突变体E141G的C末端片段载体构建和蛋白制备  

Vector construction and protein preparation of long QT syndrome-related C-terminal lobe of calmodulin mutant E141G

作　　者：邵冬雪 张晨阳 叶苗苗 陈帆 郝丽英 SHAO Dongxue;ZHANG Chenyang;YE Miaomiao;CHEN Fan;HAO Liying(Department of Pharmaceutical Toxicology,Pharmacy College of China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学药学院药物毒理学教研室,沈阳110122

出　　处：《中国医科大学学报》2024年第11期967-971,共5页Journal of China Medical University

基　　金：辽宁省科学技术计划(2022JH2/20200069);西南医科大学心血管医学研究所医学电生理教育部重点实验室开放基金(KeyME-2018-02)。

摘　　要：目的构建长QT综合征(LQTS)相关钙调蛋白(CaM)突变体E141G的C末端片段(C-lobe_(E141G))原核表达载体并进行蛋白表达、纯化及活性鉴定。方法将C-lobe_(E141G) cDNA片段插入PGEX-6p-3质粒载体后转化大肠杆菌BL21感受态细胞,异丙基硫代-β-D半乳糖苷(IPTG)诱导谷胱甘肽-S-转移酶(GST)融合蛋白表达。利用Glutathione-Sepharose 4B beads对融合蛋白进行分离纯化,经蛋白酶切除GST标签后,分别采用SDS-PAGE及BCA方法检测纯化后的目的蛋白纯度及浓度,GST pull-down方法和膜片钳技术检测纯化后蛋白活性。结果GST-C-lobe_(E141G)融合蛋白高表达,纯化后获得高纯度、高浓度C-lobe_(E141G)蛋白。纯化后C-lobe_(E141G)蛋白具有能与CaV1.2型钙通道结合并维持大鼠心室肌细胞通道开放的活性。结论成功构建可以表达生物活性蛋白的C-lobe_(E141G)原核表达载体,可为CaM的C-lobe位点突变介导的LQTS机制研究提供材料基础。Objective To construct a prokaryotic expression vector of of the long QT syndrome(LQTS)associated C-terminal lobe of calmodulin(CaM)mutant E141G(C-lobe_(E141G))and to identify the expression,purification,and activity of C-lobe_(E141G).Methods A cDNA fragment was inserted into a PGEX-6p-3 plasmid vector and transferred into Escherichia coli BL21 receptor cells,and glutathione-S-transferase(GST)fusion protein was induced by isopropyl thio-β-D galactoside(IPTG).Glutathione-Sepharose 4B beads were used to separate and purify GST-C-lobe_(E141G).After removing the GST label with protease,the purity and concentration of purified C-lobe_(E141G) were detected using SDS-PAGE and BCA,respectively.The activity of purified C-lobe_(E141G) was detected using the GST pull-down method and patch clamp technique.Results GST-C-lobe_(E141G) fusion protein was highly expressed,and C-lobe_(E141G) with high purity and concentration was obtained.The purified C-lobe_(E141G) protein not only bound to CaV1.2 calcium channels,but also rescued the channel activity from run-down in the ventricular myocytes of rat hearts.Conclusion This study successfully constructed a prokaryotic expression vector of C-lobe_(E141G),which provides a material basis for the study of the mechanism of LQTS mediated by C-lobe mutations in CaM.

关 键 词：钙调蛋白 C末端片段 突变体 融合蛋白 

分 类 号：R96[医药卫生—药理学]