title: Maximized Autotransporter-Mediated Expression (MATE) for Surface Display and Secretion of Recombinant Proteins inEscherichia coli.
abstract: A new optimized system for the surface display and secretion of recombinant proteins is described, termed MATE (maximized autotransporter-mediated expression). It is based on an artificial gene consisting of the coding region for the signal peptide of CtxB, a multiple cloning site for passenger gene insertion, flanked by coding sequences for linear epitopes for monoclonal antibodies and OmpT, and factor Xa protease cleavage sites followed by a codon-optimized DNA sequence of the linker and the β-barrel of the type V autotransporter EhaA fromEscherichia coliunder control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherryviaOmpT-mediated cleavage, using native OmpT protease activity inE. coliwhen grown at 37 °C. It is the first example to show that native OmpT activity is sufficient to facilitate the secretion of a correctly folded target protein in preparative amounts obtaining 240 µg of purified mCherry from 800 mL of crude culture supernatant. Because the release of mCherry was achieved by a simple transfer of the encoding plasmid from an OmpT-negative to an OmpT-positive strain, it bears the option to use surface display for screening purposes and secretion for production of the selected variant. A single plasmid could therefore be used for continuous secretion in OmpT-positive strains or surface display in OmpT-negative strains. In conclusion, the MATE system appears to be a versatile tool for the surface display and for the secretion of target proteins inE. coli.
authors.affiliation: Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstraße 48, DE-48149 Münster, Germany.
authors.forename: Shanna
authors.initials: S
authors.lastname: Sichwart
authors.affiliation: Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstraße 48, DE-48149 Münster, Germany; The NRW Graduate School of Chemistry, Wilhelm-Klemm-Straße 10, DE-48149 Münster, Germany.
authors.forename: Iasson E P
authors.initials: IE
authors.lastname: Tozakidis
authors.affiliation: Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstraße 48, DE-48149 Münster, Germany; Present address: Technical University Munich, Weihenstephaner Berg 3, DE-85354 Freising, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Teese
authors.affiliation: Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstraße 48, DE-48149 Münster, Germany; The NRW Graduate School of Chemistry, Wilhelm-Klemm-Straße 10, DE-48149 Münster, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Jose
keywords: EhaA; MATE; OmpT; autotransporter; mCherry; secretion; surface display