생명과학회지 (Journal of Life Science)

  • 제15권6호
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  • Pages.916-922
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  • 2005
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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대장균과 포유류 세포 내에서 parkin의 발현 양상에 관한 연구

The Expression Patterns of Human Parkin in E. codi and Mammalian Cells

  • 남민경 (가톨릭대학교 생명의과학과, 분자유전학연구소) ;
  • 박혜민 (고려대학교 생명과학대학 생명과학부) ;
  • 최주연 (가톨릭대학교 분자유전학연구소) ;
  • 박효진 (가톨릭대학교 분자유전학연구소, 고려대학교 생명과학대학 생명과학부) ;
  • 정광철 (연세대학교 이과대학 생물학과) ;
  • 강성만 (고려대학교 생명과학대학 생명과학부) ;
  • 임향숙 (가톨릭대학교 생명의과학과, 분자유전학연구소)
  • Nam Min-Kyung (Department of Biomedical Sciences, Research Institute of Molecular Genetics, the Catholic University of Korea) ;
  • Park Hye-Min (School of Life Science and Biotechnology, Korea University) ;
  • Choi Ju-Youn (Research Insitute of Molecular Genetics, the Catholic University of Korea) ;
  • Park Hyo-Jin (Research Institute of Molecular Genetics, The Catholic University of Korea, School of Life Science and Biotechnology, Korea University) ;
  • Chung Kwang Chul (Department of Biology, College of Sciences, Yonsei University) ;
  • Kang Seong man (School of Life Science and Biotechnology, Korea University) ;
  • Rhim Hyangshuk (Department of Biomedical Sciences, Research Institute of Molecular Genetics, the Catholic University of Korea)
  • 발행 : 2005.12.01
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초록

E3 ligase로 알려진 Parkin은 protein quality control에서 중요한 역할을 할 뿐만 아니라, 이런 quality control system의 이상으로 나타나는 퇴행성 뇌질환에도 밀접한 연관성이었다. 이와 같이 생체의 필수적인 업무를 담당하는 Parkin의 기능을 생화학적 측면에서 연구하기 위해서는 고 순도의 단백질을 다량 정제할 수 있는 시스템이 필요하나, 아직까지 Parkin의 발현 양상과 정제법에 관한 연구가 미흡한 상태이다. 본 연구에서는 pCEX system을 이용하여 Parkin을 대장균에서 overexpression시켜 단일 스텝으로 정제할 수 있는 방법을 정립하였다. 저온의 배양조건에서 0.01 mM의 IPTC로 발현을 유도한 결과 $90\%$ 이상의 순도를 가지는 완전한 크기의 Parkin을 정제할 수 있었다. 또한, 여러 tag을 갖는 Parkin plasmid를 제작하였을 뿐만 아니라, 이들을 HEK293 세포에 transfection하여 Parkin의 발현 양상을 비교 분석하였다. 그 결과 Parkin의 N-말단에 pretense에 민감한 절단 부위가 존재한다는 사실을 확인하였다. 본 연구에서 정립한 Parkin 정제법과 포유류 세포에서 Parkin의 발현 양상에 대한 결과는 Parkin의 기질을 탐색하고,그들이 Parkin의 효소 활성 및 기능에 미치는 영향을 조사하기 위한 다양한 연구에 활용할 수 있을 것이다.

Parkin, known as an E3 ubiquitin ligase, has essential role in protein quality control, and its severe dysfunction leads to neurodegenerative disorders. Human Parkin was excessively degraded when expressed in Escherichia coli under the conventional induction condition ($37^{\circ}C$ culture condition with 0.5 mM IPTG). To optimize the induction and culture conditions for recombinant human Parkin and develop a rapid method for the Parkin purification, we expressed Parkin by using PCEX system at the different culture temperatures and IPTC concentrations. The intact Parkin protein was purified to approximately $90\%$ purity with suitable amounts of protein under the optimal culture condition ($25^{\circ}C$E with 0.01 mM IPTG). Additionally, we constructed various parkin plasmids with different tagging systems and investigated their expression patterns in HEK293 cells. We found that the proteolytically sensitive site is localized within a ubiquitin-like domain of Parkin. This study developes a method for generating useful reagents to investigate biochemical properties of Parkin.

키워드

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피인용 문헌

  1. Purification of Human HtrA1 Expressed in E. coli and Characterization of Its Serine Protease Activity vol.16, pp.7, 2006, https://doi.org/10.5352/JLS.2006.16.7.1133