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http://dx.doi.org/10.7845/kjm.2019.9083

Yeast two-hybrid assay with fluorescence reporter  

Park, Seong Kyun (Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University)
Seo, Su Ryeon (Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University)
Hwang, Byung Joon (Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University)
Publication Information
Korean Journal of Microbiology / v.55, no.3, 2019 , pp. 199-205 More about this Journal
Abstract
Yeast two-hybrid (Y2H) technique has been used to study protein-protein interactions, but its application particularly to a large-scale analysis of protein interaction networks, is limited by the fact that the technique is labor-intensive, based on scoring colonies on plate. Here, we develop a new reporter for the measurement of the protein-protein interactions by flow cytometry. The yeast harboring interacting proteins can also be enriched by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). When two interacting proteins are present in the same yeast cell, a reporter protein containing 10 tandem repeats of c-myc epitope becomes localized on the surface of the cell wall, without affecting cell growth. We successful measured the surface display of c-myc epitope upon interacting p53 with SV40 T antigen by flow cytometry. Thus, the newly developed Y2H assay based on the display of c-myc repeat on yeast cell wall could be used to the simultaneous analysis of multiple protein-protein interactions without laborious counting colonies on plate.
Keywords
flow cytometry; fluorescence reporter; protein-protein interaction; yeast two-hybrid;
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