Vector construction and protein preparation of long QT syndrome-related C-terminal lobe of calmodulin mutant E141G
10.12007/j.issn.0258-4646.2024.11.002
- VernacularTitle:长QT综合征相关钙调蛋白突变体E141G的C末端片段载体构建和蛋白制备
- Author:
Dongxue SHAO
1
;
Chenyang ZHANG
;
Miaomiao YE
;
Fan CHEN
;
Liying HAO
Author Information
1. 中国医科大学药学院药物毒理学教研室,沈阳 110122
- Keywords:
calmodulin;
C-terminal lobe;
mutant;
fusion protein
- From:
Journal of China Medical University
2024;53(11):967-971
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a prokaryotic expression vector of of the long QT syndrome(LQTS)associated C-terminal lobe of calmodulin(CaM)mutant E141G(C-lobeE141G)and to identify the expression,purification,and activity of C-lobeE141G.Methods A cDNA fragment was inserted into a PGEX-6p-3 plasmid vector and transferred into Escherichia coli BL21 receptor cells,and glutathione-S-trans-ferase(GST)fusion protein was induced by isopropyl thio-β-D galactoside(IPTG).Glutathione-Sepharose 4B beads were used to separate and purify GST-C-lobeE141G.After removing the GST label with protease,the purity and concentration of purified C-lobeE141G were detected using SDS-PAGE and BCA,respectively.The activity of purified C-lobeE141G was detected using the GST pull-down method and patch clamp technique.Results GST-C-lobeE141G fusion protein was highly expressed,and C-lobeE141G with high purity and concentration was obtained.The purified C-lobeE141G protein not only bound to CaV1.2 calcium channels,but also rescued the channel activity from run-down in the ventricular myocytes of rat hearts.Conclusion This study successfully constructed a prokaryotic expression vector of C-lobeE141G,which provides a material basis for the study of the mechanism of LQTS mediated by C-lobe mutations in CaM.
