DEVELOPMENT OF A YEAST TRIHYBRID SCREEN USING STABLE YEAST STRAINS AND REGULATED PROTEIN EXPRESSION

We describe a yeast trihybrid system that facilitates rapid screening of cDNA libraries. Novel yeast vectors were developed that direct inte gration of cDNA encoding the bait and third protein component into the yeast chromosome. A recombinant yeast strain is thus generated (scree ning strain) and is available for library transformation. Transformati on with the library DNA is a single, efficient transformation event, a llowing the cDNA library to be represented in one step. Recovery of th e library plasmid from the yeast is also simplified, since it is the o nly episomal plasmid. Assay of trihybrid interaction and identificatio n of positive clones is faciliated by regulating expression of the thi rd protein component using the yeast MET3 promoter which is repressed in the presence of exogenous methionine. Trihybrid interactions are de tected only on media lacking methionine. This trihybrid system uses th e standard E. coli LacZ and yeast HIS3 reporter genes and is compatibl e with most available Ga14 activation domain cDNA libraries. We descri be the successful application of this yeast trihybrid system to the st udy of phosphoprotein interactions involved in T-cell signaling.