中国医科大学学报2017，Vol.46Issue(5)：397-400,4.DOI:10.12007/j.issn.0258⁃4646.2017.05.004
心肌Cav1.2钙通道NT片段的提取纯化及其与CaM的相互作用
Expression and Purification of an N?terminal Fragment of the Cav1.2 Calcium Channel and Characterization of Its Interaction with Calmodulin
摘要
Abstract
Objective To investigate a method for the purification of the N?terminal peptide fragment(NT)of the myocardial calcium channel Cav1.2,and characterize its interaction with calmodulin(CaM). Methods EscherichiacoliBL?21 cells were transformed with plasmid pGEX?6p?3/NT harboring the NT?GST fusion gene. The cells harboring pGEX?6p?3/NT were cultured and protein expression was induced with isopropyl?β?D?thiogalactoside(IPTG). Then,the GST?NT fusion protein was purified by using glutathione Sepharose 4B(GS?4B)beads. GST was cleaved off with the PreScission protease,and SDS?PAGE was performed to detect the purity and relative molecular weight of the purified peptide. Further, GST pull?down assay was performed to characterize the interaction of the NT peptide with CaM. Results SDS?PAGE analysis showed that the NT peptide was successfully purified,with high purity. Results of the GST pull?down assay showed that the NT peptide could interact with CaM. Conclusion This study establishes a method for the purification of the NT peptide and lays the foundation for further research on the interaction partners and biological functions of NT.关键词
NT/提取/纯化/CaM/GSTpull⁃down实验Key words
NT/extraction/purification/CaM/GST pull⁃down assay分类
医药卫生引用本文复制引用
苏敬阳,封瑞,邵冬雪,雷明,康泽,赵君,方涵田,郭凤,赵美眯,郝丽英..心肌Cav1.2钙通道NT片段的提取纯化及其与CaM的相互作用[J].中国医科大学学报,2017,46(5):397-400,4.基金项目
国家自然科学基金(31400981,31471091) （31400981,31471091）
大学生创新计划项目(2015069,201510159068,201510159047,201610159000064) （2015069,201510159068,201510159047,201610159000064）
