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Yeast two-hybrid assay with fluorescence reporter

형광 리포터를 활용한 효모 단백질 잡종 기법 개발

  • 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)
  • 박성균 (강원대학교 의생명과학대학 분자생명과학과) ;
  • 서수련 (강원대학교 의생명과학대학 분자생명과학과) ;
  • 황병준 (강원대학교 의생명과학대학 분자생명과학과)
  • Received : 2019.08.02
  • Accepted : 2019.08.27
  • Published : 2019.09.30

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

Acknowledgement

Supported by : 보건복지부

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