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

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 (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
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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

Videos

Literature Cited