Development of an optimized interaction-mating protocol for large-scale yeast two-hybrid analyses

Abstract

Background: Protein-protein interactions have decisive roles in almost all aspects of the structural and functional organization of cells. But in spite of the increasing amount of complete genome sequence data, the ability to predict protein function from sequences alone is limited. Therefore comprehensive analysis of protein-protein interactions, as derived from the yeast two-hybrid mating system, will yield valuable information for functional biology on a proteomic scale. Results: We have developed an optimized interaction mating protocol for the yeast two-hybrid system, which gives increased mating efficiencies. This significantly reduces the effort and cost of cDNA library screening and allows multiple parallel approaches. Improved preincubation conditions before mating, and optimal cell densities and cell ratios enable almost quantitative mating of the yeast cells carrying the cDNA library. We have proved the applicability of this technology using 20 bait proteins to screen an Arabidopsis thaliana cDNA library, in spite of bait-dependent variations in mating efficiency. Conclusions: The improved yeast two-hybrid interaction-mating protocol presented here allows the multiple parallel screening of cDNA libraries. It can be carried out without specialized equipment and has the potential to be standardized and automated.