Citation
Garcia Maroto, Federico and Carbonero Zalduegui, Pilar and García Olmedo, Francisco
(1991).
Site-directed mutagenesis and expression in Escherichia coli of WMAI-1, a wheat monomeric inhibitor of insect α-amylase.
"Plant Molecular Biology", v. 17
(n. 5);
pp. 1005-1011.
ISSN 0167-4412.
https://doi.org/10.1007/BF00037140.
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 -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.