PROTEIN PEPTIDE INTERACTIONS ANALYZED WITH THE YEAST 2-HYBRID SYSTEM

The yeast two-hybrid system was used to screen a library of random pep tides fused to a transcriptional activation domain in order to identif y peptides capable of binding to the retinoblastoma protein (Rb), Seve n peptides were identified, all of which contain the Leu-X-Cys-X-Glu m otif found in Rb-binding proteins, although their activity in the yeas t assay varied over a 40-fold range, Mutagenesis of the DNA encoding t wo of these peptides followed by screening in the two-hybrid system al lowed the delineation of residues apart from the invariant Leu, Cys an d Glu that affect binding to Rb, Binding affinities of a peptide and o ne of its variants to Rb, determined by surface plasmon resonance, cor related with results from the two-hybrid assay, This method offers sev eral advantageous features compared to existing technology for screeni ng peptide libraries: in vivo detection of protein-peptide interaction s, high sensitivity, the capacity for rapid genetic screening to ident ify stronger and weaker binding peptide variants, and the use of a sim ple assay (transcriptional activity) as a means to assess binding affi nity.