Abstract

The yeast two-hybrid system (Y2H) is a powerful tool to identify protein-protein interactions. Here we describe array-based two-hybrid methods that use defined libraries of open reading frames (ORFs) as opposed to random genomic or cDNA libraries. The array-based Y2H system is well suited for interactome studies of existing ORFeomes or subsets thereof, preferentially in a recombination-based cloning system. Array-based Y2H screens efficiently reduce false positives by using built-in controls, retesting, and evaluation of background activation. Hands-on time and the amount of used resources grow exponentially with the number of tested proteins; this is a disadvantage for large genome sizes. For large genomes, random library screen may be more efficient in terms of time and resources, but not as comprehensive as array screens, and they require an efficient sequencing facility. However, large array screens require some extent of automation although they can be carried out manually on smaller scales. Future-generation Y2H plasmid constructs including tightly regulated expression systems and features that facilitate biochemical characterization will provide more efficient and powerful tools to identify interacting proteins.