High-throughput yeast two-hybrid screening.
Charting the interactions among proteins is essential for understanding biological processes. While a number of complementary technologies for detecting protein interactions are available, the yeast two-hybrid system is one of the few that have been successfully scaled up. Two-hybrid screens have been used to construct extensive protein interaction maps for humans and several model organisms, and these maps have proven invaluable for studies on a variety of biological systems. These maps, however, have not come close to covering all proteins or interactions detectable by yeast two-hybrid. This is due in part to the difficulty of using library screening methods to sample all possible binary combinations of proteins. Ideally, every binary pair of proteins would be tested individually to ensure that every detectable interaction is identified. For organisms with large proteomes, however, this is not economically feasible and instead efficient pooling schemes must be implemented. The high-throughput two-hybrid screening methods presented here are designed to efficiently maximize coverage for selected sets of proteins or entire proteomes. We present two high-throughput screening protocols. Both methods are designed to identify interactors for any number of bait proteins expressed as DNA-binding domain (BD) fusions. The choice of which protocol to use depends largely on the nature of the available library of proteins fused to an activation domain (AD). The first protocol is appropriate for screening a library of AD clones, such as a cDNA library, a domain library, or a large pool of AD clones. By contrast, the second protocol is appropriate for screening a large array of individual sequence-verified AD clones. This protocol screens small pools of AD clones from the array in a two-phase scheme. Although the methods presented were developed using the LexA version of the yeast two-hybrid system, we include notes as appropriate to accommodate users of other versions.
Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA.
George G
GG
Roberts
Jodi R
JR
Parrish
Bernardo A
BA
Mangiola
Russell L
RL
Finley
D004247:DNA / Q000378:metabolism; D030562:Databases, Protein; D004171:Diploidy; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques / Q000295:instrumentation
D004247:DNA