Yeast Two-Hybrid Screening
Part of the
Methods in Molecular Biology™
book series (MIMB, volume 362)
Yeast two-hybrid screening represents a sensitive in vivo method for the identification and analysis of protein-protein interactions. The principle is based on the ability of a separate DNA-binding domain (DNA-BD) and activation domain (AD) to reconstitute a functional transactivator when brought into proximity. In the MATCHMAKER yeast two-hybrid system, a bait protein is expressed as a fusion to the GAL4 DNA-BD, whereas the prey protein is expressed as a fusion to the GAL4 AD. When a bait and a prey protein interact, the DNA-BD and AD form a functional transactivator, resulting in activation of reporter gene expression in yeast reporter strains. The method described in this chapter can be used to identify novel protein interactions, analyze protein-protein interactions between two known proteins, as well as dissect interacting protein domains.
Key WordsYeast two-hybrid MATCHMAKER system GAL4 DNA-binding domain GAL4 activation domain reporter gene yeast transformation auxotrophy bait protein prey protein cDNA library protein expression yeast mating diploids LacZ
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