类泛素化修饰蛋白SUMO3/GST融合蛋白表达载体的构建及表达  

Construction of small ubiquitin-like modifiers SUMO3/pGEX-4T-1 plasmid and expression of GST/SUMO3 fusion protein in E.coli

作　　者：杨南扬 缪璇 伍贤军 刘金美 辻一郎 李晓萌[1] 

机构地区：[1]东北师范大学生命科学学院遗传与细胞研究所,吉林长春130024 [2]日本东北大学医学部公共卫生学院

出　　处：《吉林大学学报（医学版）》2011年第6期1010-1014,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30871301;30700827);科技部国际合作项目资助课题(2010DFA31430);教育部新世纪优秀人才支持计划资助课题(NCET-10-0316);高校基本科研业务费专项资金资助课题(09SSXT035;10SSXT147;10JCXK004);吉林省科技厅科研基金资助课题(20080731;200905116)

摘　　要：目的:构建pGEX-4T-1/SUMO3重组表达质粒,并在大肠杆菌中表达和纯化出类泛素化修饰蛋白SUMO3/GST融合蛋白。方法:采用PCR技术利用pcDNA3.1-SUMO3质粒为模板,扩增出SUMO3全长编码序列(312bp),并将其重组于谷胱甘肽硫转移酶(GST)融合蛋白表达质粒pGEX-4T-1中,酶切、测序鉴定获得重组质粒。将重组质粒转化E.coli BL21,异丙基β-D-硫代半乳糖苷(IPTG)诱导产生GST-SUMO3的融合蛋白,并纯化获得相对分子质量为38 000的融合蛋白。结果:通过BamHⅠ和XhoⅠ双酶切,确定了重组质粒pGEX-4T-1/SUMO3中包含有312bp的小片段,并测序鉴定该插入片段序列与SUMO3序列一致;在终浓度为0.1mmol·L-1的IPTG诱导时,GST-SUMO3的融合蛋白也被成功地表达和纯化。结论:成功地制备和纯化了类泛素化修饰蛋白SUMO3/GST融合蛋白,为SUMO3多克隆抗体的制备提供了抗原基础,同时也为SUMO3及SUMO第二类家族功能的深入研究提供了保证。Objective To construct pGEX-4T-1/SUMO3 recombinant plasmid in order to express the full length of small ubiquitin-like modifier 3(SUMO3) in E.coli and purify the GST-SUMO3 fusion protein.Methods The DNA sequence of SUMO3(full length: 312 bp) was amplified by PCR using the template plasmid pcDNA3.1/SUMO3 and was then cloned into the expression vector pGEX-4T-1.After identified by restriction enzyme digestion and sequencing,the recombinant clone was transformed into the competent expression cells of E.coli BL21.GST-SUMO3 fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain a fusion protein with molecular weight of 38 000.Results It was identified that the recombinant expression vector of pGEX-4T-1/SUMO3 contained a 312 bp insert fragment by BamHⅠ and XhoⅠ double digestion and the insert fragment showed exactly consistant sequence with SUMO3.The fusion protein of SUMO3 combined with GST was successfully expressed and purified with 0.1 mmol·L-1 IPTG induction.Conclusion The SUMO3 protein combined with GST-tag is gained successfully,which provides the basis for the preparation of SUMO3 antibody and the further functional research of SUMO3.

关 键 词：类泛素化蛋白3 GST融合蛋白 蛋白纯化 

分 类 号：Q78[生物学—分子生物学]