title: A yEGFP-based reporter system for high-throughput yeast two-hybrid assay by flow cytometry.
abstract: The yeast two-hybrid (Y2H) assay is a widely used method to study protein-protein interactions, a major objective in postgenome research. Despite the tremendous utility of the Y2H assay, several issues including accuracy, speed, automation, and cost-effectiveness limit its application in high-throughput analysis. We have created an improved Y2H assay reporter system by integrating the codon-optimized yeast enhanced green fluorescent protein (yEGFP) gene into the ADE2 locus of the AH109 yeast strain and evaluated reporter expression using the strong and weak triggers via flow cytometry. We also performed flow cytometry-based Y2H assays in liquid cultures as well as in a cDNA library screening. We have shown that yEGFP, but not EGFP, is a sensitive Y2H reporter for flow cytometric detection. We also show that this yEGFP-based Y2H could be easily adapted to high-throughput format without conventional agar plating. Moreover, our data demonstrate that this system can be used for cDNA library screening. We have developed sensitive and efficient flow cytometry-based Y2H assay system that is well suited for large-scale protein-protein interaction identification and characterization. When compared with the conventional plate and filter membrane-based nutrient and colorimetric analysis, our flow cytometric assay offers convenient, quantitative, and faster reporter analysis compatible with existing liquid handling robots.
authors.affiliation: National Flow Cytometry Resource, Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Jianhong
authors.initials: J
authors.lastname: Zhou
authors.forename: Weon
authors.initials: W
authors.lastname: Bae
authors.forename: Claire K
authors.initials: CK
authors.lastname: Sanders
authors.forename: John P
authors.initials: JP
authors.lastname: Nolan
authors.forename: Hong
authors.initials: H
authors.lastname: Cai
mesh_terms: D000362:Agar / Q000737:chemistry; D001419:Bacteria / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D015723:Gene Library; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D000362:Agar