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Applied and Environmental Microbiology, February 2002, p. 525-531, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.525-531.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Novel Bacterial Membrane Surface Display System Using Cell Wall-Less L-Forms of Proteus mirabilis and Escherichia coli

Christian Hoischen,1* Christine Fritsche,1 Johannes Gumpert,1 Martin Westermann,2 Katleen Gura,1 and Beatrix Fahnert3

Department of Molecular Biology, Institute of Molecular Biotechnology,1 Department of Applied Microbiology, Hans-Knöll-Institute for Natural Products Research, D-07745 Jena,3 Department of Ultrastructure Research, Friedrich-Schiller-University Jena, D-07740 Jena, Germany2

Received 12 June 2001/ Accepted 5 November 2001

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 µg 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.

* Corresponding author. Mailing address: Department of Molecular Biology, Institute of Molecular Biotechnology, Beutenbergstr. 11, D-07745 Jena, Germany. Phone: 49-3641-656305. Fax: 49-3641-656310. E-mail: hoischen{at}imb-jena.de.

Applied and Environmental Microbiology, February 2002, p. 525-531, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.525-531.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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