Can Ag43 Mediate Surface Display Efficiently in Escherichia coli?

Authors: 
Ng, I. S., National Cheng Kung University
Guo, Y., Xiamen University
Kao, P. H., National Cheng Kung University

Cell surface display system enables the recombinant proteins being displayed on the surface of bacterial cells. The protein is convenient to access external substrates via surface display, and overcomes the difficulty caused by membrane penetration of substrates. This feature has many potential applications, which urgent in the high-throughput screening to whole-cell biocatalysts for biofuels production. Various microorganisms are used for surface displaying recombinant proteins, including yeast, lactic acid bacteria and Escherichia coli. Among all, E. coli is the most widely used microorganisms because of its history in genetic manipulation, intrinsic advantages of rapid growth, and 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 in E. coli, which are OmpT, PgsA, Blc, AIDA-I, ice nucleation protein (INP) and autotransporter proteins (ATs). ATs are a family of bacterial proteins composed of an N-terminal passenger domain and a C-terminal b-barrel-forming domain that is integrated into the outer membrane and thereby facilitates the surface display of the passenger domain. The ATs system we used to display recombinant protein is E. coli AT Antigen 43 (Ag43) via genetic replacement of the native passenger domain. Ag43-mediated display has been used in some studies of single-chain antibody (scFv) and recombinant chymotrypsin (rChyB) recently. However, these researches rarely show how to optimize the expression and performance of recombinant protein mediated by Ag43. The key factors may involve with promoters, target protein types and even the position at which passenger fused. In this study, we have successfully constructed a series of recombinant plasmids to display different proteins (i.e., red fluorescent protein from Discosoma striata (RFP), formate dehydrogenase from Candida boidinii (CbFDH) and endo-1,4-beta glucanase from Bacillus subtilis (BaCel5)). By adopted various backbones with three promoters, and fused at 138 aa and 700 aa of Ag43, we have different expression level for the construction selected. Firstly, no activity of CbFDH fused into Ag43 might cause by oxidoreductase would inactive due to the oxygen effect when being displayed on the cell surface. The recombinant RFP expressed in pET28a is better than pBAD99a, which can be observed by the color of cell turn red. All recombinant BaCel5 mediated by Ag43 produce a transparent hydrolytic zone in the congo red test. In our present result, surface display mediated by Ag43 is not a universal system but can be a potential tool in mutagenesis screening in the hydrolytic enzymes.