title: Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage.
abstract: 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.
authors.affiliation: Institute of Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Ploss
authors.forename: Andreas
authors.initials: A
authors.lastname: Kuhn
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000821:virology*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000821:virology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D000939:Epitopes; D011993:Recombinant Fusion Proteins