Expression and Purification of an N?terminal Fragment of the Cav1.2 Calcium Channel and Characterization of Its Interaction with Calmodulin
10.12007/j.issn.0258?4646.2017.05.004
- VernacularTitle:心肌Cav1.2钙通道NT片段的提取纯化及其与CaM的相互作用
- Author:
Jingyang SU
;
Dongxue SHAO
;
Ming LEI
;
Ze KANG
;
Jun ZHAO
;
Hantian FANG
;
Feng GUO
;
Meimi ZHAO
;
Liying HAO
;
Rui FENG
- Keywords:
NT;
extraction;
purification;
CaM;
GST pull?down assay
- From:
Journal of China Medical University
2017;46(5):397-400
- CountryChina
- Language:Chinese
-
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.
