Extremely thermostable serine-type protease from Aquifex pyrophilus. Molecular cloning, expression, and characterization

In-Geol Choi, Won Gi Bang, Sung Hou Kim, Yeon Gyu Yu

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A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Set-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105 °C, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent.

Original languageEnglish
Pages (from-to)881-888
Number of pages8
JournalJournal of Biological Chemistry
Issue number2
Publication statusPublished - 1999 Jan 8


ASJC Scopus subject areas

  • Biochemistry

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