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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Abstract

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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Materials

Basic Protocol 1: Affinity Selection Using Protein Targets in Microtiter Dishes

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
   Adey, N.B., Mataragonon, A.H., Rider, J.E., Carter, J.M., and Kay, B.K. 1995. Characterization of phage that bind plastic from phage‐displayed random peptide libraries. Gene 156:27‐31.
   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.
   Stevens, F.J. 2001. Caveat receptor: Proteomes on display. Comb. Chem. High Throughput Screen. 4:599‐602.
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