中国医科大学学报

中国医科大学学报
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中国医科大学学报 ›› 2018, Vol. 47 ›› Issue (2): 97-101.doi: 10.12007/j.issn.0258-4646.2018.02.001

• 论著 •    下一篇

CaM突变体质粒的构建、表达纯化及活性鉴定

苏敬阳, 王蓉蓉, 袁媛, 李松霖, 朱正南, 黄露婷, 封瑞, 邵冬雪, 孙雪菲, 郝丽英   

  1. 中国医科大学药学院药物毒理学教研室, 沈阳 110122
  • 收稿日期:2017-06-14 出版日期:2018-02-28 发布日期:2018-01-11
  • 通讯作者: 郝丽英 E-mail:lyhao@cmu.edu.cn
  • 作者简介:苏敬阳(1992-),女,硕士研究生.
  • 基金资助:
    国家自然科学基金(31471091);大学生创新计划(201710159000213)

Construction of a Mutant CaM-expressing Plasmid,and Expression,Purification,and Activity Identification of the Recombinant Protein

SU Jingyang, WANG Rongrong, YUAN Yuan, LI Songlin, ZHU Zhengnan, HUANG Luting, FENG Rui, SHAO Dongxue, SUN Xuefei, HAO Liying   

  1. Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang 110122, China
  • Received:2017-06-14 Online:2018-02-28 Published:2018-01-11

摘要: 目的 制备钙调蛋白(CaM)突变体CaME141G的融合蛋白质粒,并进行蛋白表达、提取纯化和活性鉴定。方法 利用定点突变技术将野生型CaM的第141个氨基酸E (GAG)突变为G (GGG),再将突变体质粒转化大肠杆菌BL21感受态细胞,大量培养后诱导其GST融合蛋白表达,GS-4B beads纯化,PreScission protease酶切GST标签,采用Bradford方法测定纯化后蛋白浓度,SDS-PAGE凝胶电泳检测CaME141G蛋白的相对分子量和纯度,pull-down实验鉴定纯化后蛋白的生物活性。结果 提取纯化后的CaME141G蛋白具有较高的浓度和纯度,且能与心肌CaV1.2钙通道C末端的PreIQ蛋白片段结合,并具有CaME141G蛋白浓度依赖性,提示该蛋白具有与心肌CaV1.2钙通道结合的能力。结论 成功构建了CaME141G融合蛋白质粒,并获得了纯化后的CaME141G蛋白,为深入研究CaM突变体在心肌CaV1.2钙通道中的调节作用及其与心血管系统疾病的关系奠定了基础。

关键词: 钙调蛋白, 突变体, 质粒, 表达纯化, pull-down实验

Abstract: Objective To construct a CaME141G fusion protein-expressing plasmid,and to express,purify,and identify the activity of the recombinant protein.Methods The 141st site of the wild type CaM,E (GAG),was mutated to G (GGG),using site-specific mutagenesis technology. Escherichia coli BL-21 was transformed with the mutant plasmid. The GST-CaME141G fusion protein was mass-cultured and induced for expression. Subsequently,the GST-CaME141G fusion protein was purified using GS-4B beads. PreScission protease was applied to remove the GST,the Bradford method used to determine the concentration of purified protein,and SDS-PAGE used to detect its relative molecular weight and purity. The GST pull-down assay was used to study the protein's biological activity.Results The CaME141G protein was successfully purified at a high concentration and purity. The protein could interact with PreIQ protein fragments from the myocardial CaV1.2 calcium channel C terminal,in a CaME141G concentration-dependent manner. Therefore,CaME141G has the ability to bind with the CaV1.2 calcium channel.Conclusion This study successfully constructed a CaME141G fusion protein-expressing plasmid and purified the CaME141G protein. This lays a foundation for regulating the function of CaM mutations in the myocardial CaV1.2 calcium channel,and for the study of its relationship with diseases of the cardiovascular system.

Key words: calmodulin, mutant, plasmid, expression and purification, pull-down assay

中图分类号: 

  • R96
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