title: Yeast two-hybrid screening of cyclic peptide libraries using a combination of random and PI-deconvolution pooling strategies.
abstract: We developed a high throughput yeast two-hybrid (Y2H) assay for screening pools of combinatorial cyclic peptide preys against pools of bait proteins. The assay used the PI (pooling with imaginary tags) deconvolution pooling strategy to generate pools of baits and a random pooling strategy to generate pools of cyclic peptide preys. Haploid yeast, expressing pools of baits or preys, were arrayed and mated to each other to generate diploid arrays, where the yeast express both baits and preys. Diploid arrays were scored for activation of the Y2H reporter genes. We used this Y2H pooling strategy, referred to as 'PI-pool-on-random pool', to screen a cyclic peptide library for interactions against Bcr-Abl domains. Seven Bcr-Abl domain baits and LexA control were pooled using the PI deconvolution pooling strategy. The cyclic peptide library was randomly arrayed into pools of ~1000 members. Cyclic peptides were isolated for six of seven Bcr-Abl domain baits. The PI-pool-on-random pooling Y2H assay using high stringency Y2H reporter genes produced no false positives, while missing 20% of real interactions. The high specificity of the PI-pool-on-random pooling Y2H assay eliminates the need to validate interactions. Pooling of baits and preys allows large prey libraries to be screened against multiple baits and takes advantage of PI-deconvolution to determine protein interactions with high sensitivity and specificity. The scalability of this assay allows the peptide preys to be isolated in a high throughput manner against a large number of baits and provides an avenue for generating affinity agents against entire proteomes in the future.
authors: Department of Biochemistry,  University of Saskatchewan, Saskatoon, SK,  Canada S7N 5E5.
authors: K
authors: K
authors: Barreto
authors: A
authors: A
authors: Aparicio
authors: V M
authors: VM
authors: Bharathikumar
authors: J F
authors: JF
authors: DeCoteau
authors: C R
authors: CR
authors: Geyer
mesh_terms: D000596:Amino Acids; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000235:genetics* / Q000494:pharmacology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
chemical_list: D000596:Amino Acids; D019151:Peptide Library; D010456:Peptides, Cyclic; D016044:Fusion Proteins, bcr-abl