Johannes Gumpert

Use of cell wall-less bacteria (L-forms) for efficient expression and secretion of heterologous gene products

In spite of many efforts and achievements to optimize the prokaryotic expression systems, there are still general and specific problems in obtaining sufficient yields of the functionally active gene products. The main problems concern the formation of inclusion bodies, incorrect folding, toxicity for the producer cells and degradation by proteases. One way to overcome these problems is with expression systems alternative to those of Escherichia coli. For the first time, cell wall-less L-form bacteria were used to establish such an alternative expression system and test its practicability. The results showed that various recombinant proteins can be synthesized in considerable amounts as soluble, functionally active products with these cell wall-less strains.

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 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.