RFP-AWP1原核表达载体的构建及表达特性研究  

Expression of Human AWP1-DsRed Fusion Protein in Prokaryotic Cells and Its Characteristic Investigation

作　　者：曹永宽[1] 莫永炎[2] 薛刚[1] 王培红[1] 姜勇[2] 田伏洲[1] 

机构地区：[1]成都军区总医院全军普通外科中心,四川成都610083 [2]南方医科大学休克微循环重点实验室,广州510515

出　　处：《华西医学》2008年第4期701-703,共3页West China Medical Journal

摘　　要：目的:建立红RFP-AWP1融合基因的原核表达载体,研究其表达蛋白特性。方法:将克隆的人AWP1(associated with protein kinase Crelated kinase 1,AWP1)和水母的红色荧光蛋白(redfluorescence protein,RFP)的cD-NA插入到原核表达载体pET-17b的多克隆位点(mutiple clone sites,MCS)中,经AWP1 cDNA的PCR鉴定和E.coli DE3中的表达鉴定后,E.coli DE3表达AWP1-RFP融合蛋白,荧光显微镜观察其表达特性及与GST-beads的pull-down实验。结果:成功构建了载体pET-AWP1-DsRed1,在大肠杆菌中获得了高效表达的AWP1-RFP融合蛋白,其活菌液在荧光显微镜下呈火山熔岩状的亮红色,干固的细菌呈斑片状红色荧光;AWP1-RFP融合蛋白可与GST-beads结合,并在激发波长为508nm的条件下呈红色荧光,而在波长为488 nm激发光条件下可见橙黄色荧光。结论:在体外验证了AWP1-RFP融合蛋白具有可视性表达的特点,同时发现其能与GST-beads结合,为pull-down AWP1及其相关蛋白提供了一条新途径,为研究AWP1-RFP融合蛋白在真核细胞中的表达、定位及蛋白-蛋白相互作用等具有指导意义。Objective：To express AWP1（associated with protein kinase C related kinase 1,AWP1）-DsRed fusion protein in E.coli DE3 and observe its characteristics.Methods：Cloned AWP1 and red fluorescence protein（RFP）cDNA fused gene was subcloned into the multiply clone sites（MCS） of pET-17b vector.After identified by the assay of PCR,the resultant plasmids were transformed into E.coli DE3.The expression of AWP1-RFP fusion protein and the binding test with GST-beads（pull-down）were observed through the RFP by fluorescent microscopy.Results：Vectors pET-AWP1-DsRed were constructed successfully;And,AWP1-RFP was expressed high-efficiently;Under the fluorescent microcopy,the expressing bacteria solution was red fluorescence just like the lava while a volcano was springing;dried expressing bacteria was red spot fluorescence.AWP1-RFP in the splitting bacteria solution was capability of binding with GST-beads because red fluorescence were seen on the beads;and under the condition of exciting green fluorescence,yellow fluorescence were seen on the beads.Conclusion：Expression of AWP1-RFP fusion protein was visualized in Vitro;And the protein AWP1-RFP showed the capability of binding with GST-beads and changing of its emission,which may provide a new way to pull down AWP1 and other proteins associated with AWP1.These results would play a guiding role on using the RFP to investigate the biological functions,such as expression,localization and protein-protein interaction,of AWP1.

关 键 词：人 AWP1 红色荧光蛋白 GST-beads 大肠杆菌 

分 类 号：R346[医药卫生—基础医学]