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Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

Martin Ploss1, Andreas Kuhn

  • 1Institute of Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany.

BMC Microbiology
|September 28, 2011
PubMed
Abstract

Insights

Researchers modified the M13 phage tip protein gp9 to display antigenic epitopes. This modification allows for new phage display applications, as the modified gp9 integrates into the phage and exposes the epitopes for detection.

Area of Science:

  • Molecular biology
  • Biotechnology
  • Virology

Background:

  • M13 phage filamentous bacteriophages are extruded from Escherichia coli.
  • The phage tip is composed of gp7 and gp9, with the filament made of gp8, and gp3 and gp6 terminating the structure.
  • gp3 is currently the primary protein used for phage display technology.

Purpose of the Study:

  • To investigate the modification of the amino-terminal end of M13 phage protein gp9.
  • To determine if modified gp9 is exposed and detectable on M13 phage particles for phage display.

Main Methods:

  • Insertion of sequences encoding antigenic epitopes into the amino-terminal region of gp9.
  • Utilizing the membrane insertase YidC for integration of modified gp9 into the membrane.
  • Assessing phage assembly and epitope exposure using immunogold labeling and electron microscopy, and dot blotting.

Main Results:

  • Modified gp9 proteins integrate into the membrane and expose epitopes into the periplasm.
  • Extensions up to 36 amino acids at the amino-terminal end of gp9 do not hinder membrane integration or phage assembly.
  • Antigenic tags on the modified phage tip were successfully visualized and verified using antibodies.

Conclusions:

  • The M13 phage tip protein gp9 is suitable for phage display technology.
  • Modified gp9 can be supplied in trans and complements M13 phage with amber mutations.
  • The modified phage tip is highly accessible to antibodies, enabling new display strategies.

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