钙调蛋白N2和C2突变体载体的构建、表达纯化和鉴定

摘要

目的 构建钙调蛋白(CaM)N2和C2突变体的表达性载体,表达纯化并进行活性鉴定,为进一步研究CaM与钙通道片段的相互作用奠定基础.方法 将CaM的N?lobe和C?lobe的cDNA分别与相应的N?lobe和C?lobe的cDNA相连,制备N2和C2突变体的cDNA.将N2和C2的cDNA分别插入pGEX?6p?3载体,并将重组质粒转入E.coli BL21菌株,IPTG诱导转化成功的菌株表达GST融合蛋白,超声破碎法提取,GS?4B beads纯化,SDS?PAGE电泳鉴定目的蛋白相对分子质量及纯度,pull?down实验鉴定活性.结果 酶切鉴定以及DNA测序结果证实重组质粒构建成功;SDS?PAGE电泳图显示突变体蛋白纯度和浓度均较高;pull?down实验结果证明突变体蛋白有生理活性,能与钙通道片段结合.结论 本研究成功构建了N2和C2表达质粒,并提取、纯化了有活性的钙调蛋白N2和C2突变体.%Objective To construct expression vectors of calmodulin(CaM)mutants N2 and C2,and to express,purify,and identify the mutant proteins,in order to study the interactions between CaM and calcium channels. Methods The cDNA of N?lobe and C?lobe of CaM were used to prepare the cDNA of N2 and C2. Next,the recombinant cDNAs were cloned into a pGEX?6p?3 plasmid,and the recombinant plasmids were trans?ferred into E.coli BL21 cells. The transfected BL21 cells were stimulated with IPTG. The fusion proteins were extracted by ultrasonication and puri?fied by using GS?4B beads. Finally,protein activity was identified by the pull?down assay. Results Both the restriction digestion map and the DNA sequence identification results confirmed that the recombinant plasmids were successfully constructed. SDS?PAGE results showed high purity and concentration of N2 and C2 proteins. Their activities and binding abilities with the calcium channel fragment were confirmed by the pull?down assay.Conclusion In this study,expression vectors of N2 and C2 are successfully constructed,and physiologically active N2 and C2 CaM mutant proteins are obtained.