Nucleic Acids Research
Geisel School of Medicine
The yeast two-hybrid system was used to screen a library of random peptides fused to a transcriptional activation domain in order to identify peptides capable of binding to the retinoblastoma protein (Rb). Seven peptides were identified, allof which contain the Leu-X-Cys-X-Glu motif found in Rb-binding proteins, although their activity in the yeast assay variedover a 40-fold range. Mutagenesis of the DNA encoding two of these peptides followed by screening in the two-hybrid systemallowed the delineation of residues apart from the invariant Leu, Cys and Glu that affect binding to Rb. Binding affinities of a peptide and one of its variants to Rb, determined by surface plasmon resonance, correlated with results from the two-hybrid assay. This method offers several advantageous features compared to existing technology for screening peptide libraries: in vivo detection of protein-peptide interactions, high sensitivity, the capacity for rapid genetic screening to identify stronger and weaker binding peptide variants, and the use of a simple assay (transcriptional activity) as a means to assess binding affinity.
Yang M, Wu Z, Fields S. Protein-peptide interactions analyzed with the yeast two-hybrid system. Nucleic Acids Res. 1995;23(7):1152-1156. doi:10.1093/nar/23.7.1152
Dartmouth Digital Commons Citation
Yang, Meijia; Wu, Zining; and Fields, Stanley, "Protein-Peptide Interactions Analyzed with the Yeast Two-Hybrid System" (1995). Dartmouth Scholarship. 3828.