Yeast two-hybrid liquid screening.
Yeast two-hybrid (YTH) method consists of a genetic trap that selects for "prey" cDNA products within a library that interact with a "bait" protein of interest. Here, we provide a protocol for YTH screening using a liquid medium screening method, which improves the sensitivity of this technique and streamlines the laborious classic screening in solid medium plates. The method uses a simple series of dilutions with established yeast strains transformed with diverse baits and complex cDNA libraries. This allows for prompt detection of positive clones revealed by liquid growth, due to activation of HIS3 reporter gene. Activation of a second reporter gene and reconstruction of the YTH interaction is highly reproducible using this system. This approach can either be performed using culture flasks or deep-well 96-well plates and the number of interactions obtained is similar, when compared to the classic method. In addition, the liquid screening method is faster and more economical for YTH screening and has the added benefit of automation if 96-well plates are used.
Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.
Elisa
E
Donnard
Erica M
EM
Queiroz
J Miguel
JM
Ortega
R Daniel
RD
Gietz
D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics; D008967:Molecular Biology / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D006836:Hydro-Lyases; C586384:HIS3 protein, S cerevisiae