생명과학회지 (Journal of Life Science)

  • 제19권8호
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  • Pages.1039-1046
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  • 2009
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
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대장균 시스템을 이용한 Arabidopsis 막 단백질 cytochrome P450 p-coumarate-3hydroxylase (C3H) 활성형의 과발현 및 분리정제

High Yield Bacterial Expression and Purification of Active Cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis Membrane Protein

  • 양희정 (부산대학교 자연과학대학 생명과학과) ;
  • 김완연 (부산대학교 자연과학대학 생명과학과) ;
  • 윤영주 (부산대학교 자연과학대학 생명과학과) ;
  • 윤지원 (부산대학교 자연과학대학 생명과학과) ;
  • 권태우 (부산대학교 자연과학대학 생명과학과) ;
  • 윤혜숙 (세종대학교 생명과학대학 생명공학부 생명공학과) ;
  • 윤부현 (부산대학교 자연과학대학 생명과학과)
  • Yang, Hee-Jung (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Kim, Wan-Yeon (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Yun, Young-Ju (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Yoon, Ji-Won (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Kwon, Tae-Woo (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Youn, Hye-Sook (Department of Bioscience & Biotechnology/institute of Bioscience, Sejong University) ;
  • Youn, Bu-Hyun (College of Natural Sciences, Department of Biological Sciences, Pusan National University)
  • 발행 : 2009.08.30
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초록

다양한 천연물의 합성대사에 관여하는 식물 cytochrome P450 (P450s)은 그 기능적 다양성에도 불구하고, 이들 효소의 광범위한 기질 특이성을 설명해 줄 수 있는 구조분석에 대해서는 충분한 연구가 이루어지지 못하고 있는 실정이다. 식물 p-coumarate 3-hydroxylase (C3H)에 의해 매개되는 효소 반응은 lignin 과 다양한 phenylpropanoid 부산물들의 생합성에 매우 중요한 것으로 여겨지지만, 막 단백질인 C3H의 발현 및 정제가 효과적으로 이루어지지 못하여, 활성을 측정하기 위한 분석방법이 체계화 되지 못하고 있다. C3H의 작용기작과 기질특이성에 대해 폭넓은 이해를 위한 구조분석의 선행단계는 활성을 갖는 C3H를 밀리그램 단위로 분리, 정제하는 실험적 방법을 확립하는 것이라 할 수 있다. 이를 위해, 본 연구에서는 다양한 돌연변이 방법을 도입하여 식물 막단백질 C3H를 대장균 시스템에서 효과적으로 발현 및 정제할 수 있는 시스템을 사용하였다. 변형된 cytochrome P450 C3H ($C3H_{mod}$)을 세포막으로부터 고농도의 염완충용액을 이용하여 계면활성제 없이 추출하였으며, 2단계 chromatography를 통해 활성을 유지한 상태로 분리할 수 있었다. 이러한 실험적 기법은 NMR 및 X-ray crystallography와 같은 구조분석을 통한 C3H의 효과적인 분석에 적용될 수 있을 것이며, 또한 다른 식물 cytochrome P450 단백질의 효과적인 분석에도 적용 될 수 있을 것이다.

The cytochrome P450s (P450s) metabolizing natural products are among the most versatile biological catalysts known in plants, but knowledge of the structural basis for their broad substrate specificity has been limited. The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants however, all attempts to express and purify the protein corresponding C3H gene have failed. As a result, no conditions suitable for the unambiguous assay of the enzyme are known. The detailed understanding of the mechanism and substrate-specificity of C3Hdemands a method for the production of active protein on the milligram scale. We have developed a bacterial expression and purification system for the plant C3H, which allows for the quick expression and purification of active wild-type C3H via introduction of combinational mutagenesis. The modified cytochrome P450 C3H ($C3H_{mod}$) could be purified in the absence of detergent using immobilized metal affinity chromatography and size exclusion chromatography following extraction from isolated membranes in a high salt buffer and catalytically activated. This method makes the use of isotopic labeling of C3H for NMRstudies and X-ray crystallography practical, and is also applicable to other plant cytochrome P450 proteins.

키워드

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