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Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures.

Authors
  • Bickle, Marc B T
  • Dusserre, Eric
  • Moncorgé, Olivier
  • Bottin, Hélène
  • Colas, Pierre
Type
Published Article
Journal
Nature protocols
Publication Date
Jan 01, 2006
Volume
1
Issue
3
Pages
1066–1091
Identifiers
PMID: 17406388
Source
Medline
License
Unknown

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.

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