钙调蛋白镁结合位点突变体载体的构建、表达纯化及鉴定  

Vector Construction,Protein Expression,Purification and Identification of Calmodulin Mg^(2+) Binding Site Mutants

作　　者：赵美眯[1] 李卓[1] 邵冬雪[1] 梁洪玥 晏珊[1] 封瑞[1] 孙雪菲[1] 郭凤[1] 郝丽英[1] 

机构地区：[1]中国医科大学药学院药物毒理学教研室,沈阳110122

出　　处：《中国医科大学学报》2016年第5期394-397,共4页Journal of China Medical University

基　　金：国家自然科学基金(31471091;31500930);医学电生理学重点实验室开放基金(KeyME-2014-03)

摘　　要：目的构建钙调蛋白3种镁离子结合位点突变体的质粒载体,并进行表达纯化及鉴定,为深入研究钙调蛋白的生物学功能奠定基础。方法利用钙调蛋白cDNA进行点突变,制备3种镁离子结合位点突变的cDNA。将突变后的cDNA分别插入pGEX-6P-3载体,制备钙调蛋白突变体载体质粒。质粒转化大肠杆菌BL21感受态细胞,培养大肠杆菌,并诱导GST融合蛋白表达。利用Glutathione-Sepharose 4B珠子和PreScission蛋白酶分离纯化蛋白。结果酶切鉴定和DNA测序证实成功构建钙调蛋白突变体质粒;表达纯化得到的钙调蛋白突变体经电泳图鉴定纯度较高,浓度约1.0mg/mL。结论本研究成功构建了钙调蛋白镁结合位点突变体融合蛋白原核表达质粒,分离纯化可获得较高浓度较高纯度的钙调蛋白突变体。Objective To construct plasmid vectors of calmodulin （CAM） Mg2＋ binding site mutants, and to express, purify and identify the mutant proteins. Methods Three kinds of cDNAs coding for the mutated CaM were cloned into pGEX-6P-3 plasmid vectors. These recombinant plasmids were transfected into Eschedcbia coli BI221 to express GST fusion proteins of CaM mutants. The fusion proteins were purified with Glutathione-Sep- harose 4B beads and PreScission protease. Results Both enzyme digestion analysis and DNA sequence identification proved the successful con- struction of the CaM mutant plasmids. SDS-PAGE results showed the high purity of each CaM mutant protein. The concentrations of three CaM mu- tants were around 1.0 mg/mL. Conclusion Prokayotic expression vectors of CaM Mg2＋ binding site mutants were successfully developed, and the eli- gible CaM mutant proteins were obtained. This study provided an important basis for further study on CaM＇ s biological function.

关 键 词：钙调蛋白 突变体 融合蛋白 pGEX-6P-3 

分 类 号：R96[医药卫生—药理学]