생명과학회지 (Journal of Life Science)

  • 제14권1호
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  • Pages.26-31
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  • 2004
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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Yeast two-hybrid system을 이용한 Ref-1 (redox factor-1) 결합 단백질의 분리 및 동정

Detection of Ref-1 (Redox factor-1) Interacting Protein Using the Yeast Two-hybrid System

  • 이수복 (부산대학교 자연과학대학 분자생물학과) ;
  • 김규원 (서울대학교 약학대학 생화학교실) ;
  • 배문경 (부산대학교 자연과학대학 분자생물학과) ;
  • 배명호 (부산대학교 자연과학대학 분자생물학과) ;
  • 정주원 (부산대학교 자연과학대학 분자생물학과) ;
  • 안미영 (부산대학교 자연과학대학 분자생물학과) ;
  • 김영진 (부산대학교 자연과학대학 분자생물학과)
  • 발행 : 2004.02.01
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초록

본 연구는 redox regulator로 알려 진 Ref-1 (Redox factor-1)과 결합하는 새로운 단백질을protein-protein interaction의 원리를 이용한 방법인 yeast two-hybrid assay로 검색, 동정하고, 검색된 단백질의 in vitro, in vivo 기능을 규명하는 데 그 목적을 두고, mouse 11-day Embryo cNA library를 prey로, full length REF-1을 bait로 하여 yeast strain 인 HF7C에 cotransformatiom시킨 후 histidine, leucine, tryptophan이 결핍된 SD plate에서 키워 자란 yeast transformants를 $\beta$-galactosidaseassay하여 screening하여 분리한 세 개의 clone중 한 clone이 DNA sequencing으로 확인한 결과 mouse thioredoxin임을 확인하였다.

Redox factor-1 (Ref-1), known as a redox regulator, controls the DNA binding of AP-1 and is activated in HT29 colon cancer cells by hypoxia in vitro. REF-1 also increases tile DNA binding affinity of Hypoxia-inducible Factor-lalpha$ (HIF-lalpha$), HIF-like Factor (HLF) and early growth response-1 (Egr-1) which induce expression of the genes involved in angiogenesis, so that we speculate that REF-1 may play a role in hypoxia-induced angiogenesis. In this research we tried to detect novel proteins interacting with REF-1 using Yeast two-hybrid system using full-length REF-1 cDNA as bait. As result of such screening we detected 3 positive clones. DNA sequencing and GeneBank search revealed that one of the clones contained the same sequences as M.musculus cDNA for tioredoxin.

키워드

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