Mapping Protein‐Protein Interactions with Phage‐Displayed Combinatorial Peptide Libraries

Brian K. Kay1, Luisa Castagnoli2

1 Argonne National Laboratory, Argonne, Illinois, 2 University of Rome Tor Vergata, Rome
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.4
DOI:  10.1002/0471143030.cb1704s17
Online Posting Date:  February, 2003
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This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a beadā€based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

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Table of Contents

  • Basic Protocol 1: Affinity Selection Using Protein Targets in Microtiter Dishes
  • Alternate Protocol 1: Affinity Selection Using Fusion Protein Targets on Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Affinity Selection Using Protein Targets in Microtiter Dishes

  • Purified protein target(s) to be analyzed
  • 0.2 M sodium bicarbonate buffer (Na 2HCO 3), pH 8.5
  • Control targets (see )
  • Blocking buffer: 1% (w/v) BSA in PBS (see recipe for PBS)
  • Wash buffer: 0.05% (v/v) Tween 20 in PBS
  • Combinatorial phage‐display library (New England Biolabs or Felici et al., )
  • Acid elution buffer: 50 mM glycine⋅HCl, pH 2
  • Neutralization buffer: 0.2 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • Bacteria: fresh overnight culture of DH5αF′ ( appendix 3A)
  • 2× YT culture medium and top and bottom agar (see recipe)
  • Negative control protein (fusion partner)
  • Anti–bacteriophage M13 monoclonal antibody coupled to horseradish peroxidase (HRP; Amersham Pharmacia Biotech), diluted 1:5000 (v/v) in wash buffer
  • Chromogenic substrate (see recipe)
  • ELISA‐ready 96‐well microtiter plates (Costar or Immunolon, high capacity)
  • Aerosol‐resistant pipet tips
  • 5‐ml sterile tubes
  • Sterile toothpicks
  • Spectrophotometer capable of reading microtiter plates
  • Additional reagents and equipment for DNA purification and sequencing ( appendix 3A)

Alternate Protocol 1: Affinity Selection Using Fusion Protein Targets on Beads

  • 50% (w/v) slurry of glutathione‐Sepharose resin 4B (Amersham Pharmacia Biotech, Sigma) in 0.5 M NaCl/20% (v/v) ethanol (capacity 5 mg bound protein/ml resin)
  • PBS (see recipe) with 0%, 1%, and 3% (w/v) BSA
  • Glutathione‐S‐transferase (GST) fusion protein of interest (target protein)
  • TBS (optional): 50 mM Tris⋅Cl, pH 7.5 ( appendix 2A), with 150 mM NaCl
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Literature Cited

Literature Cited
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   Fack, F., Deroo, S., Kreis, S., and Muller, C.P. 2000. Heteroduplex mobility assay (HMA) pre‐screening: An improved strategy for the rapid identification of inserts selected from phage‐displayed peptide libraries. Mol. Divers. 5:7‐12.
   Felici, F., Castagnoli, L., Musacchio, A., Jappelli, R., and Cesareni, G. 1991. Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J. Mol. Biol. 222:301‐310.
   Fields, S. and Song, O. 1989. A novel genetic system to detect protein‐protein interactions. Nature (London) 340:245‐246.
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   Smothers, J.F. and Henikoff, S. 2001. Predicting in vivo protein peptide interactions with random phage display. Comb. Chem. High Throughput Screen. 4:585‐591.
   Songyang, Z., Shoelson, S.E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W.G., King, F., Roberts, T., Ratnofsky, S., and Lechleider, R.J. et al. 1993. SH2 domains recognize specific phosphopeptide sequences. Cell 72:767‐778.
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   Sparks, A., Adey, N., Cwirla, S., and Kay, B. 1996. Screening phage‐displayed random peptide libraries. In Phage Display of Peptides and Proteins: A Laboratory Manual (B.K. Kay, J. Winter, and J. McCafferty.) Academic Press, San Diego.
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