title: Novel bacterial membrane surface display system using cell wall-less L-forms of Proteus mirabilis and Escherichia coli.
abstract: We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 microg ml(-1), with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.
authors.affiliation: Department of Molecular Biology, Institute of Molecular Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Germany. hoischen@imb-jena.de
authors.forename: Christian
authors.initials: C
authors.lastname: Hoischen
authors.forename: Christine
authors.initials: C
authors.lastname: Fritsche
authors.forename: Johannes
authors.initials: J
authors.lastname: Gumpert
authors.forename: Martin
authors.initials: M
authors.lastname: Westermann
authors.forename: Katleen
authors.initials: K
authors.lastname: Gura
authors.forename: Beatrix
authors.initials: B
authors.lastname: Fahnert
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D007740:L Forms / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods; D011513:Proteus mirabilis / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; C118373:CcmA protein, bacteria; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus