|
KMID : 0613820060160071133
|
|
Journal of Life Science
2006 Volume.16 No. 7 p.1133 ~ p.1140
|
|
|
Purification of Human HtrA1 Expressed in E. coli and Characterization of Its Serine Protease Activity
|
|
Kim Kyung-Hee
Kim Sang-Soo
Kim Goo-Young
Rhim Hyang-Shuk
|
|
|
Abstract
|
|
|
|
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.
|
|
|
KEYWORD
|
|
|
HtrA1, GST fusion system, expression and purification conditions, serine protease activity
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
|