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Preparation of Active Human HtrA3 in Eschrichia coli and Comparison of Proteolytic Activity between HtrA1, 2, and 3  

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)
Publication Information
Korean Journal of Microbiology / v.45, no.4, 2009 , pp. 291-299 More about this Journal
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.
Keywords
GST-fusion system; HtrA3; serine protease activity;
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