甲基CpG结合域四聚体蛋白的原核表达、纯化及鉴定  

Prokaryotic expression,purification and identification of tetrameric-protein of methyl-CpG-binding domain

作　　者：朱小华[1] 李锋[1] 陈国梁[1] 杨永生[1] 林尽染[1] 徐金华[1] 项蕾红[1] 

机构地区：[1]复旦大学附属华山医院皮肤科,上海200040

出　　处：《复旦学报（医学版）》2009年第4期450-453,共4页Fudan University Journal of Medical Sciences

基　　金：上海市科委资助课题(064119529)

摘　　要：目的在大肠埃希菌中表达甲基CpG结合域四聚体蛋白,并对其进行纯化和鉴定。方法将重组表达质粒4×MBD-pET30b+转化大肠埃希菌DH5a克隆扩增并测序鉴定后,转化大肠埃希菌BL21(DE3)后接种于LB培养基中,异丙基硫代半乳糖苷(IPTG)诱导重组蛋白的表达,表达产物经Ni-NTA亲和层析纯化,通过SDS-PAGE和Western blot鉴定蛋白表达,用此蛋白免疫染色人胚肾细胞后用荧光显微镜观察。结果克隆质粒测序结果与理论预期完全一致,SDS-PAGE显示在大肠埃希菌中表达出相对分子量为46000的目标蛋白,Western blot显示目标蛋白带有His融合标签(氨基端)和HA标签(羧基端)。荧光显微镜观察显示MBD蛋白能与细胞内的甲基化CpG基序特异性结合。结论成功表达并纯化了具有免疫原性的甲基CpG结合域四聚体蛋白,为今后进一步研究DNA甲基化奠定了基础。Objective To express,purify and identify tetrameric protein of methyl-CpG-binding domain in E. coli. Methods The recombinant plasmid 4 x MBD-pET30b ＋ were transformed into E. coli DH5a for clonal expansion and sequenced, then the tetrameric-proteins were expressed in E. coli BL21 （DE,） under the induction of IPTG. Moreover, the expression products were purified by Ni-NTA chromatography, and were determined by SDS-PAGE and Western blot. Immunostain 293T ceils with the proteins were analyzed by fluorescence microscope. Results The sequence analysis showed orientation right and was identical with the expectation. SDS-PAGE and Western blot demonstrated that the molecular weight of the tetramericprotein was 46000 with the N-terminai His-tag and the C-terminal HA-tag. The MBD proteins can bind to the intracellular CpG DNA specifically. Conclusions The tetrameric-proteins of methyl-CpG-binding domain are successfully expressed and purified in E. coll. This results establish a groundwork for the further researches on DNA methylation.

关 键 词：甲基CpG结合域 原核表达 蛋白纯化 DNA甲基化 

分 类 号：Q51[生物学—生物化学]