中国医科大学学报2016，Vol.45Issue(5)：394-397,4.DOI:10.12007/j.issn.0258-4646.2016.05.003
钙调蛋白镁结合位点突变体载体的构建、表达纯化及鉴定
Vector Construction,Protein Expression,Purification and Identification of Calmodulin Mg2+Binding Site Mutants
摘要
Abstract
Objective To construct plasmid vectors of calmodulin(CaM)Mg2+binding site mutants,and to express,purify and identify the mutant proteins. Methods Three kinds of cDNAs coding for the mutated CaM were cloned into pGEX⁃6P⁃3 plasmid vectors. These recombinant plasmids were transfected into Escherichia coli BL21 to express GST fusion proteins of CaM mutants. The fusion proteins were purified with Glutathione⁃Sep⁃harose 4B beads and PreScission protease. Results Both enzyme digestion analysis and DNA sequence identification proved the successful con⁃struction of the CaM mutant plasmids. SDS⁃PAGE results showed the high purity of each CaM mutant protein. The concentrations of three CaM mu⁃tants were around 1.0 mg/mL. Conclusion Prokayotic expression vectors of CaM Mg2+binding site mutants were successfully developed,and the eli⁃gible CaM mutant proteins were obtained. This study provided an important basis for further study on CaM’s biological function.关键词
钙调蛋白/突变体/融合蛋白/pGEX-6P-3Key words
calmodulin/mutant/fusion protein/pGEX-6P-3分类
医药卫生引用本文复制引用
赵美眯,李卓,邵冬雪,梁洪玥,晏珊,封瑞,孙雪菲,郭凤,郝丽英..钙调蛋白镁结合位点突变体载体的构建、表达纯化及鉴定[J].中国医科大学学报,2016,45(5):394-397,4.基金项目
国家自然科学基金（31471091，31500930）；医学电生理学重点实验室开放基金 （）
