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

1. FACS of library displayed on OmpT-neg strain FACS 2. Small-scale production in OmpT-pos strain Affibody molecules Fluorophore-labeled target OmpT protease 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 Fleetwood et al. Fleetwood et al. Microbial Cell Factories (2014) 13:179 DOI 10.1186/s12934-014-0179-z Fleetwood et al. Microbial Cell Factories (2014) 13:179 DOI 10.1186/s12934-014-0179-z RESEARCH Open Access 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 Filippa Fleetwood†, Ken G Andersson†, Stefan Ståhl and John Löfblom* 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. Keywords: Affibody molecule, Bacterial display, Directed evolution, Combinatorial protein engineering, AIDA-I, Autotransporter, FACS, Secreted protein production, E. coli, Phage display * Correspondence: † Equal contributors Division of Protein technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden © 2014 Fleetwood et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fleetwood et al. Microbial Cell Factories (2014) 13:179 Background Protein engineering using combinatorial libraries is a well-established approach for selection of specific affinity proteins (e.g. recombinant antibody fragments). Selections from such libraries are commonly performed using various display technologies (e.g. phage display), creating a physical link between phenotype and genotype [1]. Cell surface display technologies (e.g. yeast and bacterial display) have several properties that are suitable for library applications, such as the convenience of using selfamplifying living cells [2]. The large particle size combined with a multivalent display format is particularly important since it allows for single cells to be sorted using fluorescence-activated cell sorting (FACS), enabling real-time visualization of the selection process, and quantitative isolation of high-affinity binders [3]. This is not possible using for example phage or ribosome display. We have previously developed a Gram-positive bacterial display technology based on expression of recombinant proteins on the surface of Staphylococcus carnosus [4,5]. The selection system has been successfully employed for generation of high-affinity single domain antibodies [6] and other alternative affinity proteins, including Affibody molecules [4,7-9]. Affibody molecules are based on a small three-helical domain (58 aa) with advantageous properties such as high thermal stability, rapid and reversible folding, high solubility, no cysteines, flexible pharmacokinetics and high tolerance for multiple mutations [10-12]. In a number of previous studies, a wide array of specific Affibody molecules with high affinity for various antigens has been selected using phage display, Gram-positive bacterial display and ribosome display [4,7,13]. A common challenge with all microbial-displayed combinatorial libraries is to reach a sufficiently high number of transformants during library construction. Gram-negative bacteria, such as Escherichia coli, are typically demonstrating higher transformation frequencies than for example yeast or Grampositive bacteria and have the same potential as phage display for creation of really large libraries [14-16]. E. coli also has a rapid growth rate [17] and is the most well-characterized host for expression of recombinant proteins and is hence attractive for library applications. In this study, we therefore sought to design and evaluate an E. coli display method for Affibody libraries to complement the existing toolbox of technologies for engineering of new Affibody molecules. Several formats for display of recombinant proteins and peptides on E. coli have been described previously [18-26]. However, although E. coli has several valuable properties, one of the major challenges is to enable the recombinant protein of interest to success (...truncated)