Vector Construction,Protein Expression,Purification and Identification of Calmodulin Mg2+Binding Site Mutants

VernacularTitle:钙调蛋白镁结合位点突变体载体的构建、表达纯化及鉴定
Author: Meimi ZHAO ; Zhuo LI ; Dongxue SHAO ; Hongyue LIANG ; Shan YAN ; Rui FENG ; Xuefei SUN ; Feng GUO ; Liying HAO
Publication Type:Journal Article
Keywords: calmodulin; mutant; fusion protein; pGEX-6P-3
From: Journal of China Medical University 2016;45(5):394-397
CountryChina
Language:Chinese
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.