Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm

Manabu Sugimoto, Nobuyoshi Nakajima

    Research output: Contribution to journalArticlepeer-review

    43 Citations (Scopus)

    Abstract

    An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by post-translational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.

    Original languageEnglish
    Pages (from-to)1575-1580
    Number of pages6
    JournalBioscience, Biotechnology and Biochemistry
    Volume65
    Issue number7
    DOIs
    Publication statusPublished - Jul 1 2001

    Keywords

    • Earthworm
    • Fibrinolytic enzyme
    • Gene expression
    • Nucleotide sequence
    • Serine protease

    ASJC Scopus subject areas

    • Biotechnology
    • Analytical Chemistry
    • Biochemistry
    • Applied Microbiology and Biotechnology
    • Molecular Biology
    • Organic Chemistry

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