The Yeast Two-Hybrid Assay

 

John Mordacq & Roberta Ellington

Tested Studies in Laboratory Teaching, 2008, Volume 29

Abstract

This laboratory exercise investigates the yeast two-hybrid assay as it is used to identify protein-protein interactions. The two proteins being tested are called the bait and the prey. The cDNA that codes for the bait protein has been subcloned into a plasmid vector containing the coding sequences for the DNA-binding domain for the yeast GAL4 transcription factor. The cDNA that codes for the prey protein has been subcloned into a separate vector containing the coding sequences for the activation domain of GAL4. If these chimeric proteins interact, they result in the reconstitution of the GAL4 transcription factor as the DNA-binding domain and activation domain are held together by the protein-protein interaction between the bait and the prey proteins. Interactions are observed in a strain of yeast that is auxotrophic for histidine and adenine biosynthesis. This yeast strain contains histidine and adenine reporter genes found downstream from the yeast UAS sequence (UAS is the binding site for GAL4). This selection allows for the screening of protein interactions on media lacking histidine and/or adenine.

Keywords:  yeast, two-hybrid

University of Kentucky (2007)