Cloning, Functional Expression in Pichia pastoris, and Purification of Potato Cystatin and Multicystatin

S. Annadana, B. Schipper, J. Beekwilder, N. Outchkourov, M. Udayakumar, M.A. Jongsma

    Research output: Contribution to journalArticleAcademicpeer-review

    12 Citations (Scopus)

    Abstract

    In the tubers and leaves of potato, Solanum tuberosum, cysteine protease inhibitors are thought to play roles in the defence against herbivores and in regulating physiological processes like senescence and cell death. The cDNAs for two such inhibitors, potato multicystatin (PMC) with 8 cystatin domains and potato cystatin (PC) with a single domain, were cloned and expressed in the yeast Pichia pastoris. PC yielded on average 100 mg of purified active protein from 1 l of culture supernatant. Purification to homogeneity was done in one step by cation exchange. The apparent equilibrium dissociation constant (Ki) for papain was 0.1 nM. Cloning of the PMC cDNA was successful despite apparent toxicity for Escherichia coli and a high frequency of recombination events in RecA- strains of E. coli. In yeast, the expression of the cloned full length PMC gene was poor compared to that of the single domain.
    Original languageEnglish
    Pages (from-to)118-123
    JournalJournal of Bioscience and Bioengineering
    Volume95
    Issue number2
    DOIs
    Publication statusPublished - 2003

    Fingerprint

    Cystatins
    Pichia
    Cloning
    Solanum tuberosum
    Yeast
    Escherichia coli
    Purification
    Organism Cloning
    Papain
    Cell death
    Complementary DNA
    Cysteine Proteinase Inhibitors
    Toxicity
    Ion exchange
    Genes
    Positive ions
    Proteins
    Cations
    Yeasts
    Physiological Phenomena

    Keywords

    • cysteine proteinase-inhibitor
    • molecular-cloning
    • equistatin
    • crystals
    • domains
    • plants
    • genes

    Cite this

    Annadana, S. ; Schipper, B. ; Beekwilder, J. ; Outchkourov, N. ; Udayakumar, M. ; Jongsma, M.A. / Cloning, Functional Expression in Pichia pastoris, and Purification of Potato Cystatin and Multicystatin. In: Journal of Bioscience and Bioengineering. 2003 ; Vol. 95, No. 2. pp. 118-123.
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    abstract = "In the tubers and leaves of potato, Solanum tuberosum, cysteine protease inhibitors are thought to play roles in the defence against herbivores and in regulating physiological processes like senescence and cell death. The cDNAs for two such inhibitors, potato multicystatin (PMC) with 8 cystatin domains and potato cystatin (PC) with a single domain, were cloned and expressed in the yeast Pichia pastoris. PC yielded on average 100 mg of purified active protein from 1 l of culture supernatant. Purification to homogeneity was done in one step by cation exchange. The apparent equilibrium dissociation constant (Ki) for papain was 0.1 nM. Cloning of the PMC cDNA was successful despite apparent toxicity for Escherichia coli and a high frequency of recombination events in RecA- strains of E. coli. In yeast, the expression of the cloned full length PMC gene was poor compared to that of the single domain.",
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    author = "S. Annadana and B. Schipper and J. Beekwilder and N. Outchkourov and M. Udayakumar and M.A. Jongsma",
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    Cloning, Functional Expression in Pichia pastoris, and Purification of Potato Cystatin and Multicystatin. / Annadana, S.; Schipper, B.; Beekwilder, J.; Outchkourov, N.; Udayakumar, M.; Jongsma, M.A.

    In: Journal of Bioscience and Bioengineering, Vol. 95, No. 2, 2003, p. 118-123.

    Research output: Contribution to journalArticleAcademicpeer-review

    TY - JOUR

    T1 - Cloning, Functional Expression in Pichia pastoris, and Purification of Potato Cystatin and Multicystatin

    AU - Annadana, S.

    AU - Schipper, B.

    AU - Beekwilder, J.

    AU - Outchkourov, N.

    AU - Udayakumar, M.

    AU - Jongsma, M.A.

    PY - 2003

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    AB - In the tubers and leaves of potato, Solanum tuberosum, cysteine protease inhibitors are thought to play roles in the defence against herbivores and in regulating physiological processes like senescence and cell death. The cDNAs for two such inhibitors, potato multicystatin (PMC) with 8 cystatin domains and potato cystatin (PC) with a single domain, were cloned and expressed in the yeast Pichia pastoris. PC yielded on average 100 mg of purified active protein from 1 l of culture supernatant. Purification to homogeneity was done in one step by cation exchange. The apparent equilibrium dissociation constant (Ki) for papain was 0.1 nM. Cloning of the PMC cDNA was successful despite apparent toxicity for Escherichia coli and a high frequency of recombination events in RecA- strains of E. coli. In yeast, the expression of the cloned full length PMC gene was poor compared to that of the single domain.

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    KW - molecular-cloning

    KW - equistatin

    KW - crystals

    KW - domains

    KW - plants

    KW - genes

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