串联亲和纯化原核表达载体的构建

【目的】构建串联亲和纯化原核表达载体,用于研究细菌中(生理状态或接近生理条件下的)蛋白-蛋白相互作用。【方法】设计并合成两条串联亲和标签序列,分别可以在靶蛋白N端和C端融合Protein G和链亲和素结合肽(Streptavidin binding peptide,SBP)标签;以pUC18载体为骨架,去除原有的阻遏蛋白基因,构建组成型表达载体pNTAP和pCTAP。【结果】成功构建N端和C端标签表达载体pNTAP和pCTAP,它们在大肠杆菌(Escherichia coli)BL21(DE3)、肠出血性大肠杆菌O157:H7和痢疾杆菌福氏5型M90T菌株中都可以实现表达。【结论】本实验构建的两个串联亲和纯化表达载体可以在部分革兰氏阴性细菌中表达,为研究细菌内蛋白-蛋白相互作用及致病菌毒力蛋白的作用机制奠定了基础。

Construction of prokaryotic expression vectors for tandem affinity purification

[Objective]To construct prokaryotic expression vectors suitable for tandem affinity purification to study protein-protein interactions in bacteria.[Methods]Two tandem affinity tag sequences,including the coding sequences of Protein G and streptavidin binding protein(SBP),as the N-and C-terminus of fusion proteins were designed and de novo synthesized.Constitutive expression vectors pNTAP and pCTAP were constructed using pUC18 as the backbone deleted of the lacI gene.[Results]Two expression vectors pNTAP and pCTAP were successfully constructed.pNTAP showed substantial expression of the built-in tag protein GFPuv not only in Escherichia coli BL21(DE3) but also in enterohemorrhagic Escherichia coli O157:H7 and Shigella flexneri 5a.[Conclusion]Of the two recombinant expression vectors successfully constructed,pNTAP can express the model protein for tandem affinity purification and could be used for studies of protein-protein interactions in some gram-negative pathogenic bacteria such as Escherichia coli and Shigella flexneri.