人tau融合蛋白的原核表达及纯化

目的构建人His-tau和GST-tau融合蛋白表达质粒并在大肠杆菌中诱导表达及纯化。方法以质粒pEGFP-tau为模板通过PCR扩增出人tau全长cDNA,并构建到原核表达载体pET28a和pGEX-5X-1中,挑选阳性重组子,经限制性内切酶鉴定后转化到大肠杆菌BL21中,然后用IPTG诱导表达,通过SDS-PAGE染色及Western-blot方法鉴定表达的融合蛋白。分别使用His Bind Resin和Glutathione Sepharose 4B与融合蛋白结合来纯化融合蛋白。结果成功构建原核表达质粒pET28a-tau和pGEX-5X-1-tau并在大肠杆菌BL21中诱导其大量表达。经His Bind Resin和Glutathione Sepharose 4B纯化后,可以得到较纯化的His-tau和GST-tau融合蛋白。SDS-PAGE及Western-blot分析显示,特异性的抗tau单克隆抗体(tau-5)所识别的融合蛋白分子量与理论值相近。结论构建了人tau两种原核表达的融合蛋白质粒,并高效表达和纯化了该蛋白,为进一步的tau与其它功能性蛋白的相互作用研究奠定了基础。

Expression and purification of human tau fusion proteins

Objective To construct the plasmids encoding human His-tau and GST-tau proteins in bacteria,to express and purificate them.Methods Utilizing pEGFP-tau plasmid as template,human tau cDNA was amplified by polymerase chain reaction(PCR).The expression plasmid was constructed by inserting tau cDNA into pET28a( )or pGEX-5X-1( ).The positive recombinants were identified by restriction endonuclease digestion and expressed in E.coli BL21 induced by isopropyl-beta D-thiogalactopyranoside(IPTG).The desired fusion proteins were confirmed by SDS-PAGE and Western blot.The expressed products were purified by affinity chromatography using His and GST fusion protein purification beads.Results Human tau cDNA was cloned into pET28a( )and pGEX-5X-1( )vectors and expressed in E.coli BL21 successfully.SDSPAGE analysis and Western blot showed the molecular weight of the fusion protein recognized by the specific monoclonal antibody tau-5 was that predicted.Additionally,the interest proteins were purificated prolifically.Conclusion The human tau protein is obtained by prokaryotic expression,which lays the foundation for study of its function.