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.

邵冬雪; 张晨阳; 叶苗苗; 陈帆; 郝丽英

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-transferase (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.

CELL receptors; CHIMERIC proteins; LONG QT syndrome; CALCIUM channels; MUTANT proteins

Journal of China Medical University, 2024, Vol 53, Issue 11, p967

0258-4646

Academic Journal