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  • Title: Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif.
    Author: Hui CY, Guo Y, Liu L, Zheng HQ, Wu HM, Zhang LZ, Zhang W.
    Journal: Biotechnol Lett; 2019 Jul; 41(6-7):763-777. PubMed ID: 31025146.
    Abstract:
    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 Cu2+ than E. coli expressing LOA-His tag. E. coli expressing both LOT-mCherry-His tag and LOA-mCherry-His tag adhered to Cu2+ chelating sepharose beads, and adhered cells could be dissociated from the beads after excess Cu2+ 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.
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