Production of Ligand-Specific Mutants Using a Yeast Two-Hybrid Mating Assay

Acquiring functional knowledge from protein-protein interaction studies often necessitates the production of binding-impaired mutants and the study of their effects in biological systems. In many cases, multiple ligands compete for binding to the same protein domain and it becomes useful to produce specific mutations that prevent binding to one ligand but not to the others. We have combined PCR mutagenesis and a two hybrid mating assay to produce a screening strategy that has already proven useful in isolating ligand-specific mutants of the Grb10 and Raf-1 signaling proteins.