Construction of prokaryotic expression vectors for tandem affinity purification

Abstract:

Abstract:［Objective］To construct prokaryotic expression vectors suitable for tandem affinity purification to study proteinprotein 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．