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 integration of cDNA encoding the bait and third protein component into the yeast chromosome. A recombinant yeast strain is thus generated (screening strain) and is available for library transformation. Transformation with the library DNA is a single, efficient transformation event, allowing the cDNA library to be represented in one step. Recovery of the library plasmid from the yeast is also simplified, since it is the only episomal plasmid. Assay of trihybrid interaction and identification of positive clones is facilitated by regulating expression of the third protein component using the yeast MET3 promoter, which is repressed in the presence of exogenous methionine. Trihybrid interactions are detected only on media lacking methionine. This trihybrid system uses the standard E. coli LacZ and yeast HIS3 reporter genes and is compatible with most available Gal4 activation domain cDNA libraries. We describe the successful application of this yeast trihybrid system to the study of phosphoprotein interactions involved in T-cell signaling.
Immunology Unit, Glaxo Wellcome Medicines Research Centre, Stevenage, Hertfordshire, UK.
K J
KJ
Fuller
M A
MA
Morse
J H
JH
White
S J
SJ
Dowell
M J
MJ
Sims
D002999:Clone Cells / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D015151:Immunoblotting; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D014162:Transfection / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins