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  • Title: An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains.
    Author: Fleetwood F, Andersson KG, Ståhl S, Löfblom J.
    Journal: Microb Cell Fact; 2014 Dec 30; 13():179. PubMed ID: 25547008.
    Abstract:
    BACKGROUND: Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. RESULTS: The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection using FACS from a 1:100,000 background yielded around 20,000-fold enrichment in a single round and high viability of the isolated bacteria after sorting. CONCLUSIONS: The new expression vectors are promising for combinatorial engineering of Affibody molecules and the strategy for small-scale production of soluble recombinant proteins has the potential to increase throughput of the entire discovery process.
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