Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures.
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.
Bickle
Marc B T
MB
Aptanomics, 181-203, Avenue Jean Jaurès, 69007 Lyon, France. marc.bickle@aptanomics.com
Dusserre
Eric
E
Moncorgé
Olivier
O
Bottin
Hélène
H
Colas
Pierre
P
D052158:Aptamers, Peptide / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D019151:Peptide Library; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D052158:Aptamers, Peptide; D019151:Peptide Library