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

Yun Kyeong Kim, In-Geol Choi, Wonwoo Nam, Yeon Gyu Yu

Research output: Contribution to journalArticle

4 Citations (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.

Original languageEnglish
Pages (from-to)493-498
Number of pages6
JournalJournal of Biochemistry and Molecular Biology
Volume33
Issue number6
Publication statusPublished - 2000 Nov 30
Externally publishedYes

Keywords

  • Aquifex pyrophilus
  • Expression
  • Hyperthermophile
  • Protease
  • Thermostability

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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