BMB Reports
- Volume 33 Issue 6
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- Pages.493-498
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- 2000
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- 1976-670X(eISSN)
Identification of a Mature form and Characterization of Thermostability of a Serine-type Protease from Aquifex pyrophilus
- Kim, Yun-Kyeong (Structural Biology Center, Korea Institute of Science and Technology) ;
- Choi, In-Geol (Department of Chemistry and Lawrence Berkeley National Laboratory, University of California) ;
- Nam, Won-Woo (Department of Chemistry, Ewha Womans University) ;
- Yu, Yeon-Gyu (Structural Biology Center, Korea Institute of Science and Technology)
- Received : 2000.10.19
- Accepted : 2000.11.09
- Published : 2000.11.30
Abstract
Aquifex pyrophilus, a hyperthermophilic bacterium, has a serine-type protease that is located at the cell wall fraction with a mature size of 43 kDa. Molecular cloning of the protease gene revealed that it has an ORF of 619 amino acids with homologous catalytic site of serine-type proteases [Choi, I.-G., Bang, W.-K., Kim, S.-H., Yu, G. Y., J. Biol. Chem. (1999), Vol. 274, pp. 881-888]. Constructs containing different regions of the protease gene, including a alanine-substituted mutant at the active site serine, were constructed, and the factors affecting the expression level of the cloned protease gene in E. coli were examined. The presence of the C-terminus hydrophobic region of the protease hindered over-expression in E. coli. Also, the proteolytic activity of the expressed protein appeared to toxic to E. coli. An inactive form that deleted both of the N-terminal signal sequence and the C-terminal polar residues was over-expressed in a soluble form, purified to homogeneity, and its thermostability examined. The purified protein showed three disulfide bonds and three free sulfhydryl group. The thermal denaturation temperature of the protein was measured around