title: A secretory system for bacterial production of high-profile protein targets.
abstract: Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.
authors.affiliation: Facility for Protein Science and Technology, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. alexander.kotzsch@cpr.ku.dk
authors.forename: Alexander
authors.initials: A
authors.lastname: Kotzsch
authors.forename: Erik
authors.initials: E
authors.lastname: Vernet
authors.forename: Martin
authors.initials: M
authors.lastname: Hammarström
authors.forename: Jens
authors.initials: J
authors.lastname: Berthelsen
authors.forename: Johan
authors.initials: J
authors.lastname: Weigelt
authors.forename: Susanne
authors.initials: S
authors.lastname: Gräslund
authors.forename: Michael
authors.initials: M
authors.lastname: Sundström
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D058947:Bacterial Secretion Systems / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D018272:Porins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D058947:Bacterial Secretion Systems; D029968:Escherichia coli Proteins; C109237:OmpF protein; D033902:Periplasmic Binding Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; C074783:osmY protein, E coli; C078167:OMPA outer membrane proteins