All experiments were performed in theE

All experiments were performed in theE. to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant UNC 0224 wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface ofE. colifor diverse applications. The display of peptides on the surface of bacteria has become very attractive for a variety of applications such as the development of recombinant bacterial vaccines (32,33,34) and the screening of polypeptide libraries for protein-protein interactions (5,27,36). InEscherichia coli, the outer membrane proteins OmpA, LamB, and PhoE and also the flagellar and fimbrial proteins flagellin, FimH, and PapA (for a review, see reference18) have been used to display peptides or proteins on the cell surface. However, insertion of peptides longer than 60 amino acids was shown to perturb the conformation of LamB and PhoE Mmp28 (1,11), resulting in interference with proper cell UNC 0224 surface localization. Similarly, the subunits of cellular appendages were also reported not to be suitable for the display of larger polypeptides (for a review, see reference18). Although the lambda receptor is restricted for the size of insertion, it had been shown that a diverse variety of peptides could be displayed on the surface when fused to LamB (11). Subsequently, the adhesin AIDA-I UNC 0224 (39) and the TraT protein (10) fromEscherichia coli, as well as the ice-nucleation protein ofPseudomonas mobilis(26), were used to display heterologous polypeptides on the surface ofE. coli. Whereas only peptide sequences of up to 100 amino acids were examined for display using the TraT protein, the AIDA-I and the ice-nucleation protein were shown to be capable of displaying a full-length protein. For the latter two proteins, only a few individual examples were examined UNC 0224 for surface display. In addition, the AIDA-I and the ice-nucleation fusion proteins were generated by C-terminal addition, while peptides were always inserted within loops of the bacteriophage receptors. E. colipossesses numerous outer membrane proteins which are involved in different activities to acquire nutrients from the outside milieu. Hydrophilic substrates with molecular masses below 700 Da diffuse through channels formed by the porins OmpC and OmpF, sucrose enters the cell via the ScrY protein (46), and nucleosides through the Tsx pore (4). In contrast, receptor-mediated transport requires the binding of substrates to a receptor, and translocation across the outer membrane is energy and TonB dependent. FhuA facilitates the uptake of ferrichrome (13), FepA transports ferric enterobactin (37,45), and BtuB mediates uptake of vitamin B12(21). The elucidation of the three-dimensional structures of UNC 0224 outer membrane proteins has shown that they in general consist of numerous antiparallel -barrels connected by turns exposed to the periplasm and loops facing the exterior (29). While the -barrel structure anchors the protein within the outer membrane, the flexible extracellular loops are well suited to accommodate and display foreign peptide inserts on the cell surface. Importantly, the function of outer membrane proteins as phage and colicin receptors demonstrates that the loops are accessible to extracellular ligands of considerably different sizes. In addition, it indicates that even large structures might be efficiently and stably linked to the bacterial surface via outer membrane proteins. The ferrichrome and phage T5 receptor FhuA exposes 11 loops to the extracellular milieu and 10 turn regions to the periplasm (14,35). Most of these structures have been predicted by mutagenic and subsequent functional analyses of mutant FhuA proteins (28). These studies have also shown that small peptide insertions in loops 4, 5, and 10 of the ferrichrome receptor did not interfere with the sensitivity for phage T5 and colicin M, which is an indication of the proper conformation and assembly of the fusion protein in the outer membrane. Although the three-dimensional structure has not been solved for the vitamin B12receptor fromE..