人TSPAN12蛋白大胞外片段的可溶表达和纯化  

Soluble Expression and Purification of the Large Extracellular Loop of the Human TSPAN12 Protein from Escherichia Coli

作　　者：串俊兰 谢天[3,4] 王刚刚[3,4] 杨正林 Junlan Chuan;Tian Xie;Ganggang Wang;Zhenglin Yang(Sichuan Provincial Key Laboratory for Human Disease Gene Study,School of Medicine,University of Electronic Science and Technology of China,Chengdu 610072,China;Personalized Drug Therapy Key Laboratory of Sichuan Province,School of Medicine,University of Electronic Science and Technology of China,Chengdu 610072,China;Key Laboratory of Environmental and Applied Microbiology of Chinese Academy of Sciences,Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China;Key Laboratory of Environmental Microbiology of Sichuan Province,Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China)

机构地区：[1]电子科技大学医学院人类疾病基因研究四川省重点实验室,成都610072 [2]电子科技大学医学院个体化药物治疗四川省重点实验室,成都610041 [3]中国科学院成都生物研究所环境微生物四川省重点实验室,成都610041 [4]中国科学院成都生物研究所环境生物四川省重点实验室,成都610041

出　　处：《中华眼视光学与视觉科学杂志》2019年第12期881-887,共7页Chinese Journal Of Optometry Ophthalmology And Visual Science

基　　金：国家自然科学基金(81430008,81790643);电子科技大学附属医院四川省人民医院青年人才基金(2016QN01)。

摘　　要：目的:构建人源四次跨膜超家族蛋白12大胞外片段(TSPAN12-LEL)的原核表达质粒,诱导表达和纯化后获得可溶的重组蛋白。方法:实验研究。首先将目的基因克隆至pMal-c2x载体上,再经PCR扩增、酶切、连接,将带麦芽糖结合蛋白(MBP)标签的融合蛋白编码序列克隆至pETDuet-1载体的第一个多克隆位点,同时将大肠杆菌的DsbC基因克隆至pETDuet-1载体的第2个多克隆位点,与重组蛋白共表达,促进二硫键的正确形成。将重组质粒转化至OrigamiB(DE3)菌株中,用异丙基-β-D硫代半乳糖苷诱导表达,经交联直链淀粉树脂亲和层析和阴离子交换层析纯化重组蛋白。结果:成功构建重组表达质粒pETDuet-1-MBP-TSPAN12 LEL-DsbC,诱导表达后在蛋白上清中得到大量带MBP标签的融合蛋白(MBP-TSPAN12 LEL),经SDS-PAGE检测该融合蛋白分子量约为60 kD,与理论大小相符,经亲和层析和阴离子交换层析后能获得大量、高纯度可溶的重组蛋白。结论:经过优化,与DsbC蛋白共表达时,带MBP标签的TSPAN12蛋白大胞外片段可以实现在大肠杆菌中可溶表达。Objective:To construct a prokaryotic expression plasmid for the large extracellular loop(LEL)of human TSPAN12 protein and obtain highly purified soluble recombinant proteins.Methods:In this experimental study,the coding sequence of Tspan12 LEL was first cloned into pMal-c2x to link with the coding sequence of the maltose binding protein(MBP).Then the DNA of MBP-tagged Tspan12 LEL(MBP-TSPAN12 LEL)was cloned to multiple cloning site 1 of vector pETDuet-1 after PCR amplification,restriction of enzyme digestion and T4 ligase reaction.DNA of DsbC was cloned into multiple cloning site 2 of vector pETDuet-1 and co-expressed with MBP-TSPAN12 LEL in order to facilitate disulfide bond formation.After transforming the recombinant plasmid into OrigamiB(DE3),MBP-TSPAN12 LEL was expressed by isopropyl-β-d-thiogalactoside induction and purified by amylose resin affinity chromatography and anion exchange chromatography.Results:Sequencing results suggested that recombinant plasmid was successfully constructed.SDS-PAGE showed that the molecular weight of the soluble MBP-TSPAN12 LEL was about 60 kD.Abundant,soluble and highly purified fusion protein was acquired after affinity and anion exchange chromatography.Conclusions:The experimental results prove that co-expression DsbC with MBP-TSPAN12 LEL is a practicable way to produce soluble large extracellular loop of human Tspan12 protein in Escherichia coli.

关 键 词：人源四次跨膜超家族蛋白12 麦芽糖结合蛋白 原核表达 可溶蛋白 纯化 

分 类 号：R73[医药卫生—肿瘤]