Qualitative and Quantitative Assessment of Interactions

Abstract

Interactions between proteins in the yeast two-hybrid system are scored by testing for expression of the reporter genes. In the protocols presented, the reporter genes are LacZ and HIS3, but other types are also available. The expression of the reporter genes is under the control of the upstream activating sequence for GAL4. The interacting pair is expressed as fusion proteins with the DNA-binding domain (DBD) or activation domain (AD) of GAL4. In a “hunt” for novel or unknown proteins from a cDNA library that specifically interact with a protein of interest, cDNA library proteins are expressed as fusion proteins with GAL4 AD (referred to as target or prey), and the protein of interest is expressed as a fusion protein with the GAL4 DBD (referred to as bait). Interaction between bait and target fusion proteins results in activation of the LacZ and HIS3 reporter genes resulting in (1) growth of the strain on medium lacking histidine and (2) transcriptional activation of the LacZ reporter leading to production of β-galactosidase. The use of two different reporter genes under the control of different promoters eliminates many false positive AD/library fusion proteins, particularly those that do not bind to the bait fusion proteins but instead interact directly with promoter sequences flanking the GAL4-binding sites or with proteins bound to the flanking sequences. Putative true positive clones should be tested further to determine whether they can activate the reporter genes only in the presence of the GAL4 DBD bait protein. Yang et al 1.