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.
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.
authors.affiliation: Division of Protein technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden. filippaf@kth.se.
authors.forename: Filippa
authors.initials: F
authors.lastname: Fleetwood
authors.affiliation: Division of Protein technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden. ken2@kth.se.
authors.forename: Ken G
authors.initials: KG
authors.lastname: Andersson
authors.affiliation: Division of Protein technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden. stefans@biotech.kth.se.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.affiliation: Division of Protein technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden. lofblom@kth.se.
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
mesh_terms: D018830:Adhesins, Escherichia coli / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D058947:Bacterial Secretion Systems / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli; D001425:Bacterial Outer Membrane Proteins; D058947:Bacterial Secretion Systems; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C499588:ompT protein, E coli; D010447:Peptide Hydrolases