Automation of yeast two-hybrid screening.
We have developed an automated format for screening yeast two-hybrid libraries for protein-protein interactions. The format consists of a liquid array in which pooled library subsets of yeast, expressing up to 1000 different cDNAs, are mated to a yeast strain of the opposite mating type, expressing a protein of interest. Interactors are detected by a liquid assay for beta-galacsidase following prototrophic selection. The method is demonstrated by the detection of interactions between two encoded yeast RNA polymerase subunits in simulated libraries of varied complexity. To demonstrate its utility for large scale screening of complex cDNA libraries, two nuclear receptor ligand-binding domains were screened through two cDNA libraries arrayed in pooled subsets. Screening these libraries yielded clones which had previously been identified in traditional yeast two hybrid screens, as well as several new putative interacting proteins. The formatting of the cDNA library into pooled subsets lends itself to functional subtraction of the promiscuous positive class of interactor from the library. Also, the liquid arrayed format enables electronic handling of the data derived from interaction screening, which, together with the automated handling of samples, should promote large-scale proteome analysis.
Buckholz
R G
RG
Department of Molecular Endocrinology, Glaxo Wellcome Research Institute, Research Triangle Park, North Carolina 27709, USA.
Simmons
C A
CA
Stuart
J M
JM
Weiner
M P
MP
D001331:Automation; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D015723:Gene Library; D006801:Humans; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics; D047488:Retinoid X Receptors; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics; D020798:Two-Hybrid System Techniques; D020032:Tyrphostins; D015003:Yeasts
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide