Construction of a Mutant CaM-expressing Plasmid,and Expression,Purification,and Activity Identification of the Recombinant Protein
10.12007/j.issn.02584646.2018.02.001
- VernacularTitle:CaM突变体质粒的构建、表达纯化及活性鉴定
- Author:
Jingyang SU
1
;
Rongrong WANG
;
Yuan YUAN
;
Songlin LI
;
Zhengnan ZHU
;
Luting HUANG
;
Rui FENG
;
Dongxue SHAO
;
Xuefei SUN
;
Liying HAO
Author Information
1. 中国医科大学药学院药物毒理学教研室
- Keywords:
calmodulin;
mutant;
plasmid;
expression and purification;
pull-down assay
- From:
Journal of China Medical University
2018;47(2):97-101
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a CaME141G fusion protein-expressing plasmid,and to express,purify,and identify the activity of the recombinant protein. Methods The 141st site of the wild type CaM,E (GAG),was mutated to G (GGG),using site-specific mutagenesis technology. Escherichia coli BL-21 was transformed with the mutant plasmid. The GST-CaME141G fusion protein was mass-cultured and induced for expression. Subsequently,the GST-CaME141G fusion protein was purified using GS-4B beads. PreScission protease was applied to remove the GST,the Bradford method used to determine the concentration of purified protein,and SDS-PAGE used to detect its relative molecular weight and purity. The GST pull-down assay was used to study the protein's biological activity. Results The CaME141G protein was successfully purified at a high concentration and purity. The protein could interact with PreIQ protein fragments from the myocardial CaV1. 2 calcium channel C terminal,in a CaME141G concentration-dependent manner. Therefore,CaME141G has the ability to bind with the CaV1. 2 calcium channel. Conclusion This study successfully constructed a CaME141G fusion protein-expressing plasmid and purified the CaME141G protein. This lays a foundation for regulating the function of CaM mutations in the myocardial CaV1. 2 calcium channel,and for the study of its relationship with diseases of the cardiovascular system.
