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Background: Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results: The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions: Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies. Background The bacteriophage M13 is assembled during a secretion process in the cytoplasmic membrane of Escherichia coli. Membrane inserted phage proteins contact the single stranded phage DNA in an helical array and pass through the outer membrane by a porin-like structure composed of gp4 [1]. In the inner membrane a protein complex probably consisting of gp1, gp11 and thioredoxin catalyses the assembly process [2]. First, membrane inserted gp7 and gp9 proteins form a tip structure [3] that is extended by a multiple array of gp8 proteins, the major coat protein. Gp8 is synthesised as a precursor protein, termed procoat, that is inserted into the inner membrane by the YidC protein [4,5] and is then processed by leader peptidase [6]. After processing, the transmembrane coat proteins assemble into oligomers and bind to the viral DNA forming the nascent phage filament [7,8]. This filament traverses the outer membrane through the gp4 complex [1]. Finally, the membrane inserted gp3 and gp6 proteins are assembled onto the extruding phage at the proximal end of the virion terminating phage assembly. The gp3 protein has been extensively used for the phage display technology. Since gp3 is engaged in the adsorption of the phage onto the host cell certain restrictions on the infectivity of the modified phage have to be encountered [9]. This might be different for gp9 modifications since this protein is localized at the distal end of the filamentous phage particle. Previously, it has been shown that gp9 is accessible in the phage particle [3]. Therefore, gp9 might be a good target for phage display technology [10]. In addition, an attractive idea is to have both ends supplied with functional peptide moieties applicable as molecular measures or bifunctional binders. * Correspondence: [email protected] Institute of Microbiology and Molecular Biology, Garbenstrasse 30, University of Hohenheim, 70599 Stuttgart, Germany Ploss and Kuhn BMC Microbiology 2011, 11:211 http://www.biomedcentral.com/1471-2180/11/211 © 2011 Ploss and Kuhn; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gp9 is a 32 amino acid long protein that is synthesised without a signal sequence. It is thought that the membrane-inserted protein displays its N-terminus into the periplasm. However, the first amino-terminal 17 residues are hydrophobic and it is questionable whether the protein spans the entire bilayer. One possibility to explore this is to fuse hydrophilic peptides onto the Nterminus. When these modified gp9 proteins are inserted into the membrane their amino-terminal region can be analysed whether they are exposed in the periplasm. Therefore, we have fused short antigenic peptides to the N-terminus of gp9 between the residues 2 and 3. They extend the protein by 17 to 36 amino acid residues. The proteins are inserted into the membrane and efficiently assemble onto phage progeny particles since they can substitute for the wild-type protein. Also, the antigenic epitopes are detectable with gold-labelled antibodies by electron microscopy.