Izvorni znanstveni članak
https://doi.org/10.17113/ftb.53.03.15.3802
Maximized Autotransporter-Mediated Expression (MATE) for Surface Display and Secretion of Recombinant Proteins in Escherichia coli
Shanna Sichwart
; Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus,Westphalian Wilhelms - University, Münster, Germany
Iasson E.P. Tozakidis
; Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus,Westphalian Wilhelms - University, Münster, Germany
Mark Teese
; Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus,Westphalian Wilhelms - University, Münster, Germany
Joachim Jose
; Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus,Westphalian Wilhelms - University, Münster, Germany
Sažetak
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 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.
Ključne riječi
surface display; secretion; mCherry; OmpT; EhaA; MATE
Hrčak ID:
145449
URI
Datum izdavanja:
10.9.2015.
Posjeta: 1.862 *