The GST fusion expression of L-type calcium channel α_(1C) gene segments in E.coli

Objective To construct a prokaryotic recombinant vector for mouse L-type calcium channelα_(1C)C-terminus and to detect its expression in BL21.Methods The coding sequence of mouseα_(1C)C-terminus(1808-2139)was amplified with specific primers and cloned into the glutathione-S-transferase(GST)fusion expression vector pGEX-KG.Then the recombinant plasmid was transformed into E.coli BL21 and the expression of GST fusion protein was induced by adding isopropylthiogalactoside(IPTG).Results Restriction digestion of the constructed recombinant plasmid revealed the existence of gene segments about 1000 or 5000 bp in length,which were in accordance with what had been expected and the result of sequencing showed that the coding sequence C-terminus of mouseα_(1C)subunit was correct.A protein band with a molecular weight of about 70 ku was induced by IPTG in the recombinant plasmid.Western blot assay revealed that the GST fusion protein could be specifically recognized byα_(1C)C-terminus antibody.Conclusion The coding sequence of mouseα_(1C)C-terminus has been successfully cloned into the GST fusion expression vector pGEX-KG and efficiently expressed in E.coli BL21,which laythe foundation for further purifying and studyingα_(1C) interactive protein.