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형광 리포터를 활용한 효모 단백질 잡종 기법 개발

Yeast two-hybrid assay with fluorescence reporter

  • 박성균 (강원대학교 의생명과학대학 분자생명과학과) ;
  • 서수련 (강원대학교 의생명과학대학 분자생명과학과) ;
  • 황병준 (강원대학교 의생명과학대학 분자생명과학과)
  • 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)
  • 투고 : 2019.08.02
  • 심사 : 2019.08.27
  • 발행 : 2019.09.30

초록

Yeast two-hybrid는 특정 단백질에 대한 상호작용 파트너 단백질의 선별을 위한 방법으로 개발되었다. 하지만 대규모 단백질 상호작용체 분석을 수행하기에 요구되는 노동과 대량의 한천배지 사용에 따른 문제에 의해 널리 사용되지 못하고 있다. 따라서 본 연구에서는 새로운 리포터 시스템을 yeast two-hybrid 방법에 도입하여 fluorescence-activated cell sorting (FACS) 또는 magnetic-activated cell sorting (MACS)를 이용하여 상호작용 파트너 단백질을 포함하는 효모 클론을 손쉽게 선별할 수 있도록 하였다. 새로운 리포터 시스템은 c-myc 항원 결정기가 총 10번 반복되는 형태로 효모 표면에 발현되도록 하였으며, p53과 SV40 T항원을 이용한 실험을 통하여 리포터 단백질의 정상적인 발현을 flow cytometry 분석을 통하여 확인하였다. 따라서, 새로운 리포터 시스템을 도입한 yeast two-hybrid 방법은 대규모 상호작용체 분석을 위해 필요한 노력을 현저히 줄일 수 있을 것으로 기대한다.

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.

키워드

참고문헌

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