[Supplement]

¹Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University,
 Corrensstraße 48, DE-48149 Münster, Germany
²The NRW Graduate School of Chemistry, Wilhelm-Klemm-Straße 10, DE-48149 Münster, Germany
³Present address: Technical University Munich, Weihenstephaner Berg 3, DE-85354 Freising, Germany




Article history:
Received July 8, 2014
Accepted March 27, 2015



Key words: 
surface display, secretion, autotransporter, mCherry, OmpT, EhaA, MATE




Summary: 
A new optimized system for the surface display and secretion of recombinant proteins is described, termed MATE (maximized autotransporter-mediated expression). It is basedon 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 from Escherichia coli under control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherry via OmpT-mediated cleavage, using native OmpT protease activity in E. coli when 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 in E. coli.







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