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Identification of a Mature form and Characterization of Thermostability of a Serine-type Protease from Aquifex pyrophilus

Title
Identification of a Mature form and Characterization of Thermostability of a Serine-type Protease from Aquifex pyrophilus
Authors
Kim Y.-K.Choi I.-G.Nam W.Yu Y.G.
Ewha Authors
남원우
SCOPUS Author ID
남원우scopus
Issue Date
2000
Journal Title
Journal of Biochemistry and Molecular Biology
ISSN
1225-8687JCR Link
Citation
vol. 33, no. 6, pp. 493 - 498
Indexed
SCOPUS WOS scopus
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 90°C using a differential scanning calorimeter and circular dichroism spectrometry. The disulfide bonds were hardly reduced in the presence of reducing agents, suggesting that these disulfide bonds were located inside of the protein surface.
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자연과학대학 > 화학·나노과학전공 > Journal papers
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