Plant Molecular Biology

, Volume 17, Issue 5, pp 1005–1011 | Cite as

Site-directed mutagenesis and expression inEscherichia coli of WMAI-1, a wheat monomeric inhibitor of insect α-amylase

  • Federico García-Maroto
  • Pilar Carbonero
  • Francisco GArcía-Olmedo
Article

Abstract

The wheat monomeric inhibitor WMAI-1 (syn. 0.28) produced inEscherichia coli using the pT7-7 expression ventor has the correct N-terminal sequence and the same electrophoretic mobility and specific activity towards the α-amylase from the insectTenebrio molitor as the native WMAI-1 isolated from wheat. This confirms that the native inhibitor is not glycosylated and contradicts claims that a putative glycosyl moiety was essential for inhibition. Thirteen mutants have been obtained at six different sites. Substitution of the highly conserved N-terminal S by the sequence ARIRAR increased the pre-incubation time required for maximum activity. A similar result was obtained by insertion of GPRLPW after position 4, while insertion of EPRAPW at the same position rendered the inhibitor inactive. The substitution D/EGPRL and insertions DGP or D, at position 58, produced complete inactivation. All other mutations had only minor effects on activity.

Key words

α-amylase inhibitor expression inE. coli glycosylation versus activity insect α-amylase mutagenesis wheat 

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Copyright information

© Kluwer Academic Publishers 1991

Authors and Affiliations

  • Federico García-Maroto
    • 1
  • Pilar Carbonero
    • 1
  • Francisco GArcía-Olmedo
    • 1
  1. 1.Catedra de Bioquímica y Biología MolecularE.T.S. Ingenieros Agrónomos-UPMMadridSpain

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