Prokaryotic expression of chloride channel ClC-2 fusion protein and its phosphorylation in vitro
- VernacularTitle:氯通道ClC-2融合蛋白的原核表达及体外磷酸化
- Author:
Yajuan ZHENG
;
Hua XIN
;
Weizhong WANG
- Publication Type:Journal Article
- Keywords:
Chloride channel ClC-2;
Fusion proteins;
Mitogen activated protein kinase(MAPK);
Protein phosphorylation in vitro
- From:
Chinese Journal of Immunology
2000;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate if chloride channel ClC-2 could be phosphorylated by mitogen activated protein kinase(MAPK) for further study of its regulation mechanism in proliferation and differentiation of cells.Methods:The coding sequence containing the cytosolic C-terminus of ClC-2 was amplified from pSPORT1/ClC-2 plasmid,including rabbit ClC-2 cDNA, by polymerase chain reaction(PCR),the fragment was cloned into pGEX-4T-1 plasmid for the construction of GST-tagged fusion protein expressing vector, pGEX-4T-1/ClC-2CT.After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E.coli BL21. The expression of GST-tagged fusion protein was induced with IPTG and purified with Gluthathion Sepharose 4B affinity chromatography. Then the phosphorylation of ClC-2 by MAPK was examined by using phosphorylation assays in vitro.Results:The construction of pGEX-4T-1/ClC-2CT recombinant vector was proved by enzyme digestion and sequencing. The purified fusion protein GST/ClC-2CT could be phosphorylated by MAPK, however the GST could not.Conclusion:Chloride channel ClC-2 can be phosphorylated by MAPK.
