Cell Surface Display Of Escherichia Coli Based On Fusion Of Antigen 43

Cell surface display system enables the recombinant proteins being displayed on the surface of bacterial cells as a platform for the study of proteins.The protein is convenient to access external substrates via surface display,which overcomes the difficulty caused by membrane penetration of substrates and simplifies the process of protein purification and recovery.This advantage brings many potential applications,which urgently in the high-throughput screening to whole-cell biocatalysts for chemical production.Escherichia coli(E.coli)is the most widely used microorganisms because of its easily genetic manipulation,rapid growth,and with high yield for producing heterologous proteins.In order to export the passenger to the cell surface,there were a variety of anchor proteins being developed for the surface display,which were OmpA,Blc,Ice nucleation protein(INP)and so on.Among all,autotransporters(ATs)have been reported to be one of the most attractive systems for the large and complex proteins.The anchor protein we used was antigen 43(Ag43),which belonged to the AT protein.The Ag43 from E.coli is a novel surface display system,which only few reports are related to.Therefore,it is a great potential of protein-display based on fusion of Ag43.In order to discover more functions and applications of Ag43,the backbones,fusion sites and different target proteins have demonstrated to estimate the efficiency of surface display via Ag43.In detail,we have demonstrated three different target proteins(i.e.,red fluorescent protein from Discosoma striata(RFP),formate dehydrogenase from Candida boidinii(CbFDH)and endoglucanase from Bacillus subtilis(BsCel5))on the cell surface by fusing these proteins to the Ag43 based on two different backbons(pET28a and pBAD99a).And there were two different fusion sites of Ag43.The target genes could replace the fragment of 53-137 aa or 53-699 aa,which we called 138 and 700 respectively for short.The major results are shown as follows:(1)The backbone,fusion sites and target protein showed the difference in expression level.The different target proteins had different expression levels.Because the CbFDH exhibited more complicated structure compared with the RFP and the BsCel5,the CbFDH performed obviously less expression level.We also found the proteins fused at 700 obtained higher expression levels than that fused at 138 for pET28a,while the proteins fused at 138 obtained higher expression levels than that fused at 700 for pBAD99a.(2).The RFP was surface displayed based on Ag43.The surface-displayed RFP expressed better in plasmid pET28a than that of plasmid pBAD99a.However,the intracellular RFP exhibited a darker red than the surface-displayed RFP.The fluorescent protein is easily reduced by the other materials in the external environment,as a result,loses its fluorescence.Therefore,the fluorescence of RFP would be weakened when being displayed on the cell surface.(3).The displayed BsCel5 whole-cell catalyst have an obvious superiority than intracellular' s.In the whole-cell activity assay,the displayed BsCel5 had an obvious higher activity than the intracellular BsCel5,especially the pET28a-Ag43-138-BsCel5 exhibited the highest activity.Therefore,Ag43 mediated surface display will be an alternative approach for whole-cell utilization of cellulases.(4).The CbFDH had not activity after surface displaying.The CbFDH showed no activity when it fused into Ag43 on cell surface.There is an instinct shortcoming for CbFDH-its rather low specific activity as the activity of CbFDH would be lost when displaying on the cell surface.On the other hand,the CbFDH exhibits more complicated structure compared with the other two proteins.Finally,it has been a great challenge to display cofactor-containing enzymes for a long time.(5).How to support the claim that the target proteins are displayed on the cell surface is the most important question we need to resolve.We proved the proteins were displayed on cell surface and the fusion site at 138 was more supervisory to the 700 based on whole-cell trypsin treatment.