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 bead?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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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 (Na₂ HCO₃ ), 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

Figure 17.4.1 Photograph of a phage ELISA result. Using eight different clones that were isolated with GST‐SrcSH3 domain as the target, 25 µl of culture supernatant were added to pairs of microtiter plate wells containing GST‐SrcSH3 or GST. Retention of the phage in the wells was detected with anti‐phage antibodies (coupled to HRP) through a colorimetric assay. Six of the clones were verified to bind the SrcSH3 domain specifically.

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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.
	Grøn, H. and Hyde‐DeRuyscher, R. 2000. Peptides as tools in drug discovery. Curr. Opin. Drug Discov. Dev. 3:636‐645.
	Hyde‐DeRuyscher, R., Paige, L.A., Christensen, D.J., Hyde‐DeRuyscher, N., Lim, A., Fredericks, Z.L., Kranz, J., Gallant, P., Zhang, J., Rocklage, S.M., Fowlkes, D.M., Wendler, P.A., and Hamilton, P.T. 2000. Detection of small‐molecule enzyme inhibitors with peptides isolated from phage‐displayed combinatorial peptide libraries. Chem. Biol. 7:17‐25.
	Kay, B.K., Kurakin, A., and Hyde‐DeRuyscher, R. 1998. From peptides to drugs via phage‐display. Drug Discov. Today 3:370‐378.
	Kay, B.K., Kasanov, J., Knight, S., and Kurakin, A. 2000. Convergent evolution with combinatorial peptides. F.E.B.S. Lett. 480:55‐62.
	Linn, H., Ermekova, K.S., Rentschler, S., Sparks, A.B., Kay, B.K., and Sudol, M. 1997. Using molecular repertoires to identify high‐affinity peptide ligands of the WW domain of human and mouse YAP. Biol. Chem. 378:531‐537.
	Paige, L.A., Christensen, D.J., Grøn, H., Norris, J.D., Gottlin, E.B., Padilla, K.M., Chang, C.Y., Ballas, L.M., Hamilton, P.T., McDonnell, D.P., and Fowlkes, D.M. 1999. Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. Proc. Natl. Acad. Sci. U.S.A. 96:3999‐4004.
	Phizicky, E. and Fields, S. 1995. Protein‐protein interactions: Methods for detection and analysis. Microbiol. Rev. 59:94‐123.
	Pillutla, R.C., Hsiao, K.C., Brissette, R., Eder, P.S., Giordano, T., Fletcher, P.W., Lennick, M., Blume, A.J., and Goldstein, N.I. 2001. A surrogate‐based approach for post‐genomic partner identification. B.M.C. Biotechnol. 1:6 .
	Salcini, A.E., Confalonieri, S., Doria, M., Santolini, E., Tassi, E., Minenkova, O., Cesareni, G., Pelicci, P.G., and Di Fiore, P.P. 1997. Binding specificity and in vivo targets of the EH domain, a novel protein‐protein interaction module. Genes Dev. 11:2239‐2249.
	Scott, J.K., Huang, S.F., Gangadhar, B.P., Samoriski, G.M., Clapp, P., Gross, R.A., Taussig, R., and Smrcka, A.V. 2001. Evidence that a protein‐protein interaction ‘hot spot’ on heterotrimeric G protein betagamma subunits is used for recognition of a subclass of effectors. EMBO J. 20:767‐776.
	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.
	Songyang, Z., Fanning, A.S., Fu, C., Xu, J., Marfatia, S.M., Chishti, A.H., Crompton, A., Chan, A.C., Anderson, J.M., and Cantley, L.C. 1997. Recognition of unique carboxyl‐terminal motifs by distinct PDZ domains. Science 275:73‐77.
	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.
	Tong, A.H., Drees, B., Nardelli, G., Bader, G.D., Brannetti, B., Castagnoli, L., Evangelista, M., Ferracuti, S., Nelson, B., Paoluzi, S., Quondam, M., Zucconi, A., Hogue, C.W., Fields, S., Boone, C., and Cesareni, G. 2002. A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules. Science 295:321‐324.
	Zhao, S. and Lee, E.Y. 1997. A protein phosphatase‐1‐binding motif identified by the panning of a random peptide display library. J. Biol. Chem. 272:28368‐28372.

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

Abstract

Table of Contents

Materials

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

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

Figures

Literature Cited

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

Abstract

Table of Contents

Materials

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

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

Figures

Videos

Literature Cited