Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Preparation of Active Human HtrA3 in Eschrichia coli and Comparison of Proteolytic Activity between HtrA1, 2, and 3

Escherichia coli에서 효소활성을 지닌 Human HtrA3 단백질 제조와 HtrA Serine Protease 1, 2와의 효소활성 비교

  • Kim, Ji-Hwan (Research Institute of Molecular Genetics and Department of Biomedical Sciences, College of Medicine the Catholic University of Korea) ;
  • Kim, Goo-Young (Research Institute of Molecular Genetics and Department of Biomedical Sciences, College of Medicine the Catholic University of Korea) ;
  • Nam, Min-Kyung (Research Institute of Molecular Genetics and Department of Biomedical Sciences, College of Medicine the Catholic University of Korea) ;
  • Kim, Sang-Soo (Research Institute of Molecular Genetics and Department of Biomedical Sciences, College of Medicine the Catholic University of Korea) ;
  • Rhim, Hyang-Shuk (Research Institute of Molecular Genetics and Department of Biomedical Sciences, College of Medicine the Catholic University of Korea)
  • 김지환 (가톨릭대학교 생명의과학과, 분자유전학연구소) ;
  • 김구영 (가톨릭대학교 생명의과학과, 분자유전학연구소) ;
  • 남민경 (가톨릭대학교 생명의과학과, 분자유전학연구소) ;
  • 김상수 (가톨릭대학교 생명의과학과, 분자유전학연구소) ;
  • 임향숙 (가톨릭대학교 생명의과학과, 분자유전학연구소)
  • Received : 2009.09.23
  • Accepted : 2009.10.12
  • Published : 2009.12.31
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Abstract

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

본 연구에서는 Human HtrA3 (HtrA3)의 효소활성을 분자수준에서 연구하기 위해 HtrA간의 상동성과 기존에 알려진 maturation site들을 비교 분석하여 예상 mature HtrA3인 M1-HtrA3와 M2-HtrA3를 발현하는 construct를 제작하였다. pGEX system을 통해 Top10 균주에서 발현, 정제한 M1-HtrA3 단백질은 $10{\mu}g$/L를 정제할 수 있었으며 발현량 대비 1%를 회수할 수 있었다. M2-HtrA3는 M1보다 5배 가량 많은 양을 정제할 수 있었으며 발현량 대비 회수율은 3배 정도 더 높았다. $\beta$-Casein을 이용한 in vitro cleavage test를 통해 M1, M2 form 모두 protease 활성을 갖는 것을 확인하였다. 또한, $\beta$-casein cleavage를 통해 HtrA serein protease들 간의 상대적인 활성을 비교한 결과, HtrA3와 HtrA2는 HtrA1보다 약 2배 더 높은 proteolytic cleavage 활성을 보였다. 본 연구에서 정립한 protease 활성을 지닌 HtrA3의 제작과 정제 조건은 HtrA3의 substrate를 탐색을 용이하게 할 수 있을 것이며, HtrA3 연관된 질환의 발병기전과 세포 신호전달을 이해하는 연구에 활용될 수 있을 것이다.

Keywords

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