Construction Of Inner Surface Of Outer Membrane Display Vector And Application

Bacterial cell surface display is the expression of heterologous protein on the surface of living cells by recombinant DNA technology,the system is composed of carrier protein,passenger protein and bacterial host.According to the difference of the structure and properties of anchoring domain,fusion strategy have been considered in bacteria:C-terminal fusion,N-terminal fusion and sandwich fusion.Lpp-OmpA hybrid is a typical protein of C-terminal fusion.This topic analysed the structure of Lpp-OmpA hybrid,constructed a vector of displaying foreign protein at internal surface of outer membrane.By analysing the structure of cytoderm of E.coli and studying spatial structure of traditional Lpp-OmpA hybrid protein,the coding sequence of OmpA(46-90)was find which is transmembrane component of OmpA.The display fragment including signal peptide,linker and OmpA(46-90)was amplified by PCR and SOE-PCR.Transforming the base at both ends of fragment gene,so that it can produce Nde?restricition sites.The display fragment was recombinated into pET-DsbA-TD.Threonine dehydratase(TD)was displayed at internal surface of outer membrane as a passenger protein.Recombinant plasmids were purified and transformed into expression host E.coli BL21(DE3).The recombinant strains were induced by0.4 mM IPTG,SDS-PAGE analysis.After expression of the passenger protein,we assayed its activity.Accordingly,the display vector pET-Lpp having more advantages was screened.We select the L-Aspartic acid-?-decarboxylase(Asp)gene to testing the utility of pET-Lpp as a passenger protein.Asp gene were amplified from ZW vector and ligated with pET-Lpp digested by BamH?and Xho?,then transformed into E.coli DH5?.The clones with positive insert were checked by PCR analysis.Recombinant plasmids were transformed into expression host E.coli BL21(DE3).The recombinant strains were induced by 0.4 mM IPTG,SDS-PAGE analysis.The activity of Asp was assayed,which displayed at internal surface of outer membrane,we found that Asp have activity and its thermal stability is good.By contrast,Asp displayed at internal surface have a higher activity than Asp which is secreted in periplasmic space.