Site-directed mutagenesis and expression inEscherichia coliof WMAI-1, a wheat monomeric inhibitor of insect α-amylase
✍ Scribed by Federico García-Maroto; Pilar Carbonero; Francisco GArcía-Olmedo
- Book ID
- 104617023
- Publisher
- Springer
- Year
- 1991
- Tongue
- English
- Weight
- 698 KB
- Volume
- 17
- Category
- Article
- ISSN
- 0167-4412
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✦ Synopsis
The wheat monomeric inhibitor WMAI-1 (syn. 0.28) produced in Escherichia cob using the pT7-7 expression vector has the correct N-terminal sequence and the same electrophoretic mobility and specific activity towards the a-amylase from the insect Tenebrio 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 obtainedi by insertion of G P R L P W after position 4, while insertion of EPRAPW at the same position rendered the inhibitor inactive. The substitution D / E G P R L and insertions DGP or D, at position 58, prodt~ced complete inactivation. All other mutations had only minor effects on activity.