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 g 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 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 endotoxin-poor organisms and can provide a novel platform for numerous applications.The overexpression of recombinant proteins, which remain bound to the outer surface of the bacterial cells as accessible and functional active molecules, offers new applications in biotechnology and medicine. Among these are the development of diagnostics and vaccines, adhesin-receptor interaction studies, the generation of peptide libraries, the immobilization of enzymes, and the expression of heavy metal-binding peptides and antibody fragments (6,10,25,33). The surface display systems follow various strategies for anchoring. In gramnegative bacteria, outer membrane proteins (21), pili and flagella (26), modified lipoproteins (6, 7, 11), ice nucleation proteins (17, 23), and autotransporters (19,27,35) have been used as anchors. In gram-positive bacteria, surface anchors have been derived from lipoproteins, cell wall proteins, or S-layer proteins (24,32,33). However, depending on the displayed protein and the desired application, each system has its own advantages and disadvantages, such as the size limitation of the displayed protein, mislocalization or formation of inclusion bodies, association with lipopolysaccharides (LPS), or destabilization of the outer membrane (10,17,22). There is still a need for further developments to increase the repertoire of applications of surface display systems (21).Stable protoplast type L-form bacteria have up to now not been considered as a system for surface display, although they exhibit several interesting features for such applications. They have lost irreversibly the ability to form cell wall structures and periplasmic compartments, and their cells are surrounded only by a cytoplasmic membrane (CM). Cell biological properties of the strains and the lipid composition of their CM are well characterized (13,16). Furthermore, the L-form strains of Proteus mirabilis LVIWEI and Escherichia coli LWFϩWEI have been used for the efficient overexpr...

Boosting Secretion of Extracellular Protein by Escherichia coli via Cell Wall Perturbation

“…Peptidoglycan is the main constituent of the cell wall and contributes to cell structure robustness and stability (10). Bacterial L-forms, representing the most drastic example of disturbing the cell surface structure, have been used to improve the secretion of murein staphylokinase and penicillin G acylase (11,12). Bacterial L-forms are formed by completely deleting the cell wall through natural or artificial induction (e.g., by penicillin) (13).…”

Secretion of recombinant proteins from E. coli

“…The advantages of these cell types are that the cells lack extracellular proteases and protein secretion limitation through the outer membrane is not an issue [196]. However, the protein yields from this strategy were low, generating only a few mg/L of fusion protein [198]. With regard to protein production, E. coli L-form cells were able to express and secrete proteins including penicillin G acylase [196] and a mini-antibody, however, the yield was comparably low (less than 0.5 mg/L functional mini-antibody) [197].…”

“…E. coli L-form cells were also tested for their use as membrane surface display system, since the lack of the cell wall (components) avoids human inflammatory processes, which is especially advantageous for diagnostic and medical applications. However, the protein yields from this strategy were low, generating only a few mg/L of fusion protein [198]. Recently, research focus has moved away from E. coli L-form cells, presumably due to the low yields and difficult handling.…”

Recent Progress and Future Prospects in Protein Display Technologies as Tools for Proteomics

“…Because the self-assembly of these appendages requires intact both N-and C-termini, polypeptides can only be inserted within a permissive loop of the flagellin or pili proteins. Fusion of proteins with cytoplasmic membrane proteins, such as LacY and SecY, is an alternative approach (Hoischen et al, 2002). As in the case of flagella or pili components, both the N-and C-termini of most outermembrane proteins are required for their outer membrane targeting, and thus it is generally impossible to display polypeptides in an end-to-end fashion.…”