title: Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures.
abstract: Peptide aptamers are combinatorial proteins that specifically bind intracellular proteins and modulate their function. They are powerful tools to study protein function within complex regulatory networks and to guide small-molecule drug discovery. Here we describe methodological improvements that enhance the yeast two-hybrid selection and characterization of large collections of peptide aptamers. We provide a detailed protocol to perform high-efficiency transformation of peptide aptamer libraries, in-depth validation experiments of the bait proteins, high-efficiency mating to screen large numbers of peptide aptamers and streamlined confirmation of the positive clones. We also describe yeast two-hybrid mating assays, which can be used to determine the specificity of the selected aptamers, map their binding sites on target proteins and provide structural insights on their target-binding surface. Overall, 12 weeks are required to perform the protocols. The improvements on the yeast two-hybrid method can be also usefully applied to the screening of cDNA libraries to identify protein interactions.
authors.affiliation: Aptanomics, 181-203, Avenue Jean Jaurès, 69007 Lyon, France. marc.bickle@aptanomics.com
authors.forename: Marc B T
authors.initials: MB
authors.lastname: Bickle
authors.forename: Eric
authors.initials: E
authors.lastname: Dusserre
authors.forename: Olivier
authors.initials: O
authors.lastname: Moncorgé
authors.forename: Hélène
authors.initials: H
authors.lastname: Bottin
authors.forename: Pierre
authors.initials: P
authors.lastname: Colas
mesh_terms: D052158:Aptamers, Peptide / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D019151:Peptide Library; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques; D015003:Yeasts
chemical_list: D052158:Aptamers, Peptide; D019151:Peptide Library