Cav1.2通道CT1片断突变体的质粒构建和蛋白表达

Plasmid Construction and Protein Expression of CT1 Fragment Mutants of Cav1.2 Channel

目的构建心肌Cav1.2通道CT1片段及其突变体与谷胱甘肽转移酶(GST)重组的融合蛋白原核表达载体,并进行蛋白表达和纯化。方法以豚鼠Cav1.2通道CT1质粒(p GEX-6p-3/CT1)为模板,采用定点突变技术构建CT1 T1603A和CT1 T1603D两种突变体质粒。转化大肠杆菌BL21感受态细胞,大量培养后用IPTG诱导GST融合蛋白表达,分离纯化后采用SDS-PAGE检测CT1及其突变体蛋白的相对分子量和纯度。结果 CT1片段及其突变体融合蛋白得到了正确、大量表达,提取纯化后的CT1片段及其突变体融合蛋白具有较高的纯度。结论成功构建了Cav1.2通道CT1片断及其突变体融合蛋白原核表达载体,获得了CT1突变体融合蛋白,为深入研究CT1片断在Cav1.2通道调节中的作用和机制奠定基础。

Objective To construct prokaryotic expression vectors of the CT1 fragment of Cav1.2 channel and its mutants for CT1-GST fusion protein expression and purification. Methods Taking plasmid of p GEX-6p-3/CT1 as template,two mutational plasmids（CT1-T1603 A and CT1-T1603D）with site directed mutagenesis were constructed. The plasmids were then transformed to E.coli BL21 competent cells,and the transformants were induced with IPTG for the expression of GST fusion proteins of CT1 fragment and its mutants. SDS-PAGE was performed to determine the relative molecular weight and purity. Results Mutated bases corresponding to the target amino acid site were confirmed by c DNA sequence. High purity of GST-CT1 fusion protein and its mutants were successfully obtained. Conclusion Prokaryotic expression vectors of CT1 fragment and its mutants were constructed,and the fusion proteins were successfully expressed were obtained. These results provided a basis for further studies of the function of CT1 fragment in the regulation for Cav1.2 channel and its mechanism.