Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif

Objectives An efficient bacterial surface display system based on the anchoring motif derived from Escherichia coli ( E. coli ) outer membrane protease OmpT was developed in this study. Results Referring to the classical Lpp-OmpA (LOA) display system, the signal peptide and nine amino acids of mature Lpp were fused to the transmembrane domain comprising five β-strands of truncated OmpT to generate a novel Lpp-OmpT (LOT) display system. The C -terminal fusion strategy was used to fuse a small peptide (His tag) and red fluorescent protein (mCherry) to the C -terminus of LOT. Cell surface exposure of His tag and mCherry were compared between the LOA and LOT display systems. E. coli expressing LOT-His tag adsorbed more Cu 2+ than E. coli expressing LOA-His tag. E. coli expressing both LOT-mCherry-His tag and LOA-mCherry-His tag adhered to Cu 2+ chelating sepharose beads, and adhered cells could be dissociated from the beads after excess Cu 2+ treatment. More importantly, compared with the LOA system, a higher amount of LOT-mCherry-His tag hybrid protein was demonstrated to be localized at the outer membrane by both fluorescence spectrophotometric determination of cell fractions and cell-surface immunofluorescence assay. Conclusions These results suggest that genetically modified OmpT can be used as a potential anchoring motif to efficiently and stably display polypeptides and proteins, and that the LOT system could be used in a variety of biotechnological and industrial processes.