Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Assay of Epoxide Hydrolase Activity Based on PCR-linked in vitro Coupled Transcription and Translation System.

무세포 단백질합성 시스템 기반의 epoxide hydrolase 발현 및 활성 분석

  • Lee, Ok-Kyung (Department of Food Science and Technology, Kyungsung University) ;
  • Kim, Hee-Sook (Department of Food Science and Technology, Kyungsung University) ;
  • Lee, Eun-Yeol (Marine and Extreme Genome Research Center)
  • 이옥경 (경성대학교 공과대학 식품공학과) ;
  • 김희숙 (경성대학교 공과대학 식품공학과) ;
  • 이은열 (해양, 극한생물 분자유전체 연구단)
  • Published : 2005.10.01
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Abstract

Cell-free expression is a powerful tool for rapid protein analysis, enabling an efficient identification of gene without cumbersome procedure of transformation and cell culture. Epoxide hydrolase (EH) gene of Rhodotorula glutinis was simply amplified by PCR, and the resultant gene was expressed in vitro using a coupled Transcription/translation system. The cell-free expressed EH protein mixture exhibited the enantioselective hydrolysis activity toward (R)-styrene oxide, representing that cell-free protein synthesis system can be used for the rapid expression of an enantioselective enzyme for an efficient identification of the chiral activity.

Coupled transcription/translation cocktail을 이용하여 R. glutinis EH 유전자를 in vitro에서 합성하고 활성을 평가하였다. SDS-PAGE 및 immunoblotting을 통하여 45 kDa 크기의 EH 단백질이 발현되었음을 확인하였고, NBP assay 및 chiral GC 분석을 통해 발현된 단백질이 (R)-styrene oxide에 대한 입체선택성이 있음을 확인하였다. 따라서 무세포 단백질 합성 시스템을 이용하여 입체선택성을 유지시킨 EH 유전자 발현이 가능하며, 이러한 방법은 putative EH 유전자 탐색 등에 효율적으로 응용될 것이다.

Keywords

References

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