Maximized autotransporter mediated expression (MATE) of surface display and secretion of recombinant proteins in Escherichia coli

Sichwart S, Tozakidis IE, Teese M, Jose J

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 from E.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 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.