novel bacterial membrane surface display system using cell wallless lforms 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 wallless lform 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 lform strains overexpressed fusion proteins in amounts of 1 to 100 microg ml1 with higher expression for those with homologous anchor motifs various experimental approaches eg 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 immunogoldlabeled 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 endotoxinpoor organisms and can provide a novel platform for numerous applications