Selection by phage display of a mustard chymotrypsin inhibitor toxic to pea aphid.

L.R. Ceci, M. Volpicella, S. Conti, R. Gallerani, M.J. Beekwilder, M.A. Jongsma

    Research output: Contribution to journalArticleAcademicpeer-review

    44 Citations (Scopus)

    Abstract

    The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 ?g ml1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.
    Original languageEnglish
    Pages (from-to)557-566
    JournalThe Plant Journal
    Volume33
    DOIs
    Publication statusPublished - 2003

    Fingerprint

    Aphids
    Mustard Plant
    Acyrthosiphon pisum
    Poisons
    Peas
    Chymotrypsin
    chymotrypsin
    bacteriophages
    Bacteriophages
    Aphidoidea
    Nymph
    Libraries
    trypsin inhibitors
    Insects
    nymphs
    cattle
    Trypsin Inhibitors
    crop (digestive system)
    Random Allocation
    Aphis gossypii

    Keywords

    • serine proteinase-inhibitor
    • amino-acid-sequence
    • protease inhibitors
    • cysteine proteinase
    • acyrthosiphon-pisum
    • transgenic plants
    • reactive-site
    • alba l.
    • adaptation
    • resistance

    Cite this

    Ceci, L.R. ; Volpicella, M. ; Conti, S. ; Gallerani, R. ; Beekwilder, M.J. ; Jongsma, M.A. / Selection by phage display of a mustard chymotrypsin inhibitor toxic to pea aphid. In: The Plant Journal. 2003 ; Vol. 33. pp. 557-566.
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    title = "Selection by phage display of a mustard chymotrypsin inhibitor toxic to pea aphid.",
    abstract = "The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 ?g ml1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.",
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    year = "2003",
    doi = "10.1046/j.1365-313X.2003.01645.x",
    language = "English",
    volume = "33",
    pages = "557--566",
    journal = "The Plant Journal",
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    Selection by phage display of a mustard chymotrypsin inhibitor toxic to pea aphid. / Ceci, L.R.; Volpicella, M.; Conti, S.; Gallerani, R.; Beekwilder, M.J.; Jongsma, M.A.

    In: The Plant Journal, Vol. 33, 2003, p. 557-566.

    Research output: Contribution to journalArticleAcademicpeer-review

    TY - JOUR

    T1 - Selection by phage display of a mustard chymotrypsin inhibitor toxic to pea aphid.

    AU - Ceci, L.R.

    AU - Volpicella, M.

    AU - Conti, S.

    AU - Gallerani, R.

    AU - Beekwilder, M.J.

    AU - Jongsma, M.A.

    PY - 2003

    Y1 - 2003

    N2 - The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 ?g ml1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.

    AB - The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 ?g ml1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.

    KW - serine proteinase-inhibitor

    KW - amino-acid-sequence

    KW - protease inhibitors

    KW - cysteine proteinase

    KW - acyrthosiphon-pisum

    KW - transgenic plants

    KW - reactive-site

    KW - alba l.

    KW - adaptation

    KW - resistance

    U2 - 10.1046/j.1365-313X.2003.01645.x

    DO - 10.1046/j.1365-313X.2003.01645.x

    M3 - Article

    VL - 33

    SP - 557

    EP - 566

    JO - The Plant Journal

    JF - The Plant Journal

    SN - 0960-7412

    ER -