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학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
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Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (IGFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low (460 μg for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was 42℃ instead of 37℃. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200 nM of HtrA1 that cleaves half of 5 μM of β-casein during 3 hr incubation at 37℃. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.
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참고문헌 (24)
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Baldi A, A. De Luca, M. Morini, T. Battista, A. Felsani, F. Baldi , C. Catricala, A. Amantea, D. M. Noonan, A. Albini, P. G. Natali, D. Lombardi, M. G. Paggi. 2002. The HtrA1 serine protease is down-regulated during human melanoma progression and represse
Chien J, J. Staub, SI. Hu, M. R. Erickson-Johnson, F. J. Crouch, D. I. Smith, R. M. Crowl, S. H. Kaufmann, V. Shridhar. 2004. A candidate tumor suppressor HtrA1 is downregulated in ovarian cancer. Oncogene 23, 1636-1644.
Cilenti. L., M. M. Soundarapandian, G. A. Kyriazis, V. Stratico, S. Singh, S. Gupta, J. V. Bonventre, E. S. Alnernri, A. S. Zervos. 2004. Regulation of HAX-1 Antiapoptotic Protein by Omi/HtrA2 Protease during Cell Death. J. Biol. Chem. 279, 50295-50301.
Clausen T, C. Southan, M. Ehrmann. 2002. The HtrA family of proteases: implications for protein composition and cell fate. Mol. Cell 10, 443-455.
David L. Nelson and Michael M. Cox. 2000. Lehninger Principles of Biochemistry. pp. 243-269, 3rd eds., Worth Publishers. 41 Madison Avenue, New York.
Chien J, J. Staub, SI. Hu, M. R. Erickson-Johnson, F. J. Crouch, D. I. Smith, R. M. Crowl, S. H. Kaufmann, V. Shridhar. 2004. A candidate tumor suppressor HtrA1 is downregulated in ovarian cancer. Oncogene 23, 1636-1644.
Cilenti. L., M. M. Soundarapandian, G. A. Kyriazis, V. Stratico, S. Singh, S. Gupta, J. V. Bonventre, E. S. Alnernri, A. S. Zervos. 2004. Regulation of HAX-1 Antiapoptotic Protein by Omi/HtrA2 Protease during Cell Death. J. Biol. Chem. 279, 50295-50301.
Clausen T, C. Southan, M. Ehrmann. 2002. The HtrA family of proteases: implications for protein composition and cell fate. Mol. Cell 10, 443-455.
David L. Nelson and Michael M. Cox. 2000. Lehninger Principles of Biochemistry. pp. 243-269, 3rd eds., Worth Publishers. 41 Madison Avenue, New York.
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UCI(KEPA) : I410-ECN-0101-2009-470-017616918