Construction of and Protein Preparation from a Recombinant Plasmid Containing the Distal Fragment of the Distal C-terminus of the Cav1.2 Channel
10.12007/j.issn.0258-4646.2017.10.001
- VernacularTitle:Cav1.2钙通道C末端远端片段dDCT重组质粒的构建及蛋白制备
- Author:
Huiyuan HU
1
;
Shuai LEI
;
Deri SUN
;
Xuanxuan SUN
;
Shan YAN
;
Shihao LIU
;
Jian WANG
;
Ying WANG
;
Yue LI
;
Liying HAO
Author Information
1. 中国医科大学药学院药物毒理学教研室
- Keywords:
Cav1.2 Channel;
distal fragment of the distal C-terminus;
pull-down assay
- From:
Journal of China Medical University
2017;46(10):865-868
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant plasmid vector containing the distal fragment of the distal C-terminus (dDCT) of the Cav 1.2 channel,and express,extract,and purify dDCT protein and characterize its biological activity.Methods dDCT cDNA was ligated into the pGEX-6p-1 vector to create a recombinant plasmid that was subsequently transformed into Escherichia coli BL21 competent cells.Expression of GST-dDCT fusion protein from this plasmid was induced with isopropy-β-D-thiogalactoside,and the resulting protein was purified using glutathione-sepharose 4B beads.The biological activity of dDCT was analyzed by GST pull-down assay.Results The recombinant plasmid was verified by restriction enzyme digestion and sequencing.The concentration and purity of the dDCT protein,which was extracted by ultrasonication,were high enough to detect dDCT activity.The binding of dDCT to CT1 was determined to be concentration-dependent.Conclusion The recombinant dDCT plasmid was successfully constructed,providing the fundamental basis for future studies on mechanisms of Cav 1.2 channel autoregulation.
