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

作者：Fleetwood Filippa; Andersson Ken G; Stahl Stefan; Lofblom John^*

来源：Microbial Cell Factories, 2014, 13(1): 179.

DOI：10.1186/s12934-014-0179-z

• Affibody molecule
• Bacterial display
• Directed evolution
• Combinatorial protein engineering
• AIDA-I
• Autotransporter
• FACS
• Secreted protein production
• E. coli
• Phage display

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. %26lt;br%26gt;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. %26lt;br%26gt;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.

• 出版日期2014-12-30