through combinatorial peptide phage display can also be valuable as
starting points for inhibitor design [6]. However, the consensus
binding motifs that are derived based on these results have been
found to be overly hydrophobic and tryptophan rich, which limits
the usability of such information for predicting interactions in a
given proteome [7]. We therefore developed a variant of phage
display termed proteomic peptide-phage display (ProP-PD) where
the displayed peptides are designed to encompass selected regions
of a given proteome [8]. The method is based on a combination of
computational library design, oligonucleotide library synthesis,
phage display, and next-generation sequencing (NGS). We have
shown that the approach can be used to successfully identify endogenous PDZ mediated interactions [9] and can be geared for the
identification of interactions that are tuned by ligand phosphorylation [10]. In this chapter we describe a comprehensive ProP-PD
protocol including (a) library construction, (b) ProP-PD selections
against PDZ domains, and (c) NGS of the peptide coding regions
of binding enriched phage pools (Fig. 1).
2 Materials
2.1 Oligonucleotide
Pool Amplification
1. Phusion high-fidelity PCR master mix with HF buffer.
2. Custom designed oligonucleotide pool from commercial
provider.
3. Agarose (molecular grade).
4. GelRed.
5. Nucleotide Removal Kit.
6. Quant-iT PicoGreen dsDNA assay kit.
7. 10Â TE buffer: 100 mM Tris–HCl, 10 mM EDTA, pH 8.0
8. Lambda phage dsDNA (100 μg/ml dsDNA).
9. White 96-well hard-shell PCR plates, thin wall.
10. CDF96 Real-Time system.
2.2 Purification
of dU-ssDNA
Phagemid
1. E. coli CJ360 cells.
2. M13K07 helper phage.
3. LB/carb agar plates: 1% tryptone (w/v), 1% NaCl (w/v), 0.5%
yeast extract (w/v), 1.5% agar (w/v), 100 μg/ml carbenicillin.
4. 2YT: 1.6% tryptone (w/v), 0.5% NaCl (w/v), 1% yeast extract
(w/v)
5. 100 mg/ml carbenicillin dissolved in deionized water. Sterile
filter.
6. 34 mg/ml chloramphenicol dissolved in 70 v/v % ethanol.
Sterile filter.
42
Susanne Lu ¨ chow et al.
starting points for inhibitor design [6]. However, the consensus
binding motifs that are derived based on these results have been
found to be overly hydrophobic and tryptophan rich, which limits
the usability of such information for predicting interactions in a
given proteome [7]. We therefore developed a variant of phage
display termed proteomic peptide-phage display (ProP-PD) where
the displayed peptides are designed to encompass selected regions
of a given proteome [8]. The method is based on a combination of
computational library design, oligonucleotide library synthesis,
phage display, and next-generation sequencing (NGS). We have
shown that the approach can be used to successfully identify endogenous PDZ mediated interactions [9] and can be geared for the
identification of interactions that are tuned by ligand phosphorylation [10]. In this chapter we describe a comprehensive ProP-PD
protocol including (a) library construction, (b) ProP-PD selections
against PDZ domains, and (c) NGS of the peptide coding regions
of binding enriched phage pools (Fig. 1).
2 Materials
2.1 Oligonucleotide
Pool Amplification
1. Phusion high-fidelity PCR master mix with HF buffer.
2. Custom designed oligonucleotide pool from commercial
provider.
3. Agarose (molecular grade).
4. GelRed.
5. Nucleotide Removal Kit.
6. Quant-iT PicoGreen dsDNA assay kit.
7. 10Â TE buffer: 100 mM Tris–HCl, 10 mM EDTA, pH 8.0
8. Lambda phage dsDNA (100 μg/ml dsDNA).
9. White 96-well hard-shell PCR plates, thin wall.
10. CDF96 Real-Time system.
2.2 Purification
of dU-ssDNA
Phagemid
1. E. coli CJ360 cells.
2. M13K07 helper phage.
3. LB/carb agar plates: 1% tryptone (w/v), 1% NaCl (w/v), 0.5%
yeast extract (w/v), 1.5% agar (w/v), 100 μg/ml carbenicillin.
4. 2YT: 1.6% tryptone (w/v), 0.5% NaCl (w/v), 1% yeast extract
(w/v)
5. 100 mg/ml carbenicillin dissolved in deionized water. Sterile
filter.
6. 34 mg/ml chloramphenicol dissolved in 70 v/v % ethanol.
Sterile filter.
42
Susanne Lu ¨ chow et al.