Novel bacterial membrane surface display system using cell wall-less L-forms of Proteus mirabilis and Escherichia coli.
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.
Hoischen
Christian
C
Department of Molecular Biology, Institute of Molecular Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Germany. hoischen@imb-jena.de
Fritsche
Christine
C
Gumpert
Johannes
J
Westermann
Martin
M
Gura
Katleen
K
Fahnert
Beatrix
B
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
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