中国医科大学学报2018，Vol.47Issue(2)：97-101,5.DOI:10.12007/j.issn.02584646.2018.02.001
CaM突变体质粒的构建、表达纯化及活性鉴定
Construction of a Mutant CaM-expressing Plasmid,and Expression,Purification,and Activity Identification of the Recombinant Protein
摘要
Abstract
Objective To construct a CaME141G fusion protein-expressing plasmid,and to express,purify,and identify the activity of the recombinant protein. Methods The 141st site of the wild type CaM,E (GAG),was mutated to G (GGG),using site-specific mutagenesis technology. Escherichia coli BL-21 was transformed with the mutant plasmid. The GST-CaME141G fusion protein was mass-cultured and induced for expression. Subsequently,the GST-CaME141G fusion protein was purified using GS-4B beads. PreScission protease was applied to remove the GST,the Bradford method used to determine the concentration of purified protein,and SDS-PAGE used to detect its relative molecular weight and purity. The GST pull-down assay was used to study the protein's biological activity. Results The CaME141G protein was successfully purified at a high concentration and purity. The protein could interact with PreIQ protein fragments from the myocardial CaV1. 2 calcium channel C terminal,in a CaME141G concentration-dependent manner. Therefore,CaME141G has the ability to bind with the CaV1. 2 calcium channel. Conclusion This study successfully constructed a CaME141G fusion protein-expressing plasmid and purified the CaME141G protein. This lays a foundation for regulating the function of CaM mutations in the myocardial CaV1. 2 calcium channel,and for the study of its relationship with diseases of the cardiovascular system.关键词
钙调蛋白/突变体/质粒/表达纯化/pull-down实验Key words
calmodulin/mutant/plasmid/expression and purification/pull-down assay分类
医药卫生引用本文复制引用
苏敬阳,郝丽英,王蓉蓉,袁媛,李松霖,朱正南,黄露婷,封瑞,邵冬雪,孙雪菲..CaM突变体质粒的构建、表达纯化及活性鉴定[J].中国医科大学学报,2018,47(2):97-101,5.基金项目
国家自然科学基金(31471091) （31471091）
大学生创新计划(201710159000213) （201710159000213）
