Increasing specificity in high-throughput yeast two-hybrid experiments.
Since its inception, the yeast two-hybrid (Y2H) system has proven to be an efficient system to identify novel protein-protein interactions. However, Y2H screens are sometimes criticized for generating high rates of false-positives. Minimizing false-positive interactions is especially important in proteome wide high-throughput (HT) Y2H. Here, we summarize various approaches that reduce false-positives in HT-Y2H projects. We evaluated the potential of examining putative positives after removing the prey encoding plasmid by negative selection. We found that this method reliably identifies false-positives caused by spontaneous conversion of baits into auto-activators and provides significant time-savings in HT screens. In addition, we present a method to eliminate an important source of false-positives: contaminating prey plasmids. Y2H interactors can be wrongly identified due to the presence of two or more different plasmids in the cells of a single yeast colony. Of these independent plasmids, only one encodes a genuine interactor. Contaminating plasmids are eliminated by extended culture of yeast cells under positive selection for the interaction, allowing the identification of the true interaction partner.
Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Pierre-Olivier
PO
Vidalain
Mike
M
Boxem
Hui
H
Ge
Siming
S
Li
Marc
M
Vidal
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques; D014498:Uracil / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D005656:Fungal Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D006639:Histidine; D014498:Uracil; D001616:beta-Galactosidase