中国医科大学学报2017，Vol.46Issue(5)：401-405,5.DOI:10.12007/j.issn.0258⁃4646.2017.05.005
钙调蛋白N2和C2突变体载体的构建、表达纯化和鉴定
Vector Construction,Protein Expression,Purification,and Identification of Calmodulin Mutants N2 and C2
摘要
Abstract
Objective To construct expression vectors of calmodulin(CaM)mutants N2 and C2,and to express,purify,and identify the mutant proteins,in order to study the interactions between CaM and calcium channels. Methods The cDNA of N?lobe and C?lobe of CaM were used to prepare the cDNA of N2 and C2. Next,the recombinant cDNAs were cloned into a pGEX?6p?3 plasmid,and the recombinant plasmids were trans?ferred into E.coli BL21 cells. The transfected BL21 cells were stimulated with IPTG. The fusion proteins were extracted by ultrasonication and puri?fied by using GS?4B beads. Finally,protein activity was identified by the pull?down assay. Results Both the restriction digestion map and the DNA sequence identification results confirmed that the recombinant plasmids were successfully constructed. SDS?PAGE results showed high purity and concentration of N2 and C2 proteins. Their activities and binding abilities with the calcium channel fragment were confirmed by the pull?down assay.Conclusion In this study,expression vectors of N2 and C2 are successfully constructed,and physiologically active N2 and C2 CaM mutant proteins are obtained.关键词
钙调蛋白/N⁃lobe/C⁃lobe/N2/C2/pull⁃down实验Key words
calmodulin/N⁃lobe/C⁃lobe/N2/C2/pull⁃down assay分类
医药卫生引用本文复制引用
晏珊,郝丽英,雷帅,陈思充,于佳慧,祝旭东,孙嘉瑶,杜逸,李墨,唐子鉴..钙调蛋白N2和C2突变体载体的构建、表达纯化和鉴定[J].中国医科大学学报,2017,46(5):401-405,5.基金项目
国家自然科学基金(31471091) （31471091）
大学生创新计划项目(201610159052,201510159047,2015048,2015023) （201610159052,201510159047,2015048,2015023）
