Vector Construction,Protein Expression,Purification,and Identification of Calmodulin Mutants N2 and C2
10.12007/j.issn.0258?4646.2017.05.005
- VernacularTitle:钙调蛋白N2和C2突变体载体的构建、表达纯化和鉴定
- Author:
Shan YAN
;
Shuai LEI
;
Sichong CHEN
;
Jiahui YU
;
Xudong ZHU
;
Jiayao SUN
;
Yi DU
;
Mo LI
;
Zijian TANG
;
Liying HAO
- Keywords:
calmodulin;
N?lobe;
C?lobe;
N2;
C2;
pull?down assay
- From:
Journal of China Medical University
2017;46(5):401-405
- CountryChina
- Language:Chinese
-
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.
