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

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.

Description

Title: Site-directed mutagenesis and expression in Escherichia coli of WMAI-1, a wheat monomeric inhibitor of insect α-amylase
Author/s:
  • Garcia Maroto, Federico
  • Carbonero Zalduegui, Pilar
  • García Olmedo, Francisco
Item Type: Article
Título de Revista/Publicación: Plant Molecular Biology
Date: November 1991
ISSN: 0167-4412
Volume: 17
Subjects:
Freetext Keywords: -amylase inhibitor - expression inE. coli - glycosylation versus activity - insect -amylase - mutagenesis - wheat
Faculty: E.T.S.I. Agrónomos (UPM) [antigua denominación]
Department: Biotecnologia [hasta 2014]
Creative Commons Licenses: Recognition - No derivative works - Non commercial

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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.

More information

Item ID: 6040
DC Identifier: http://oa.upm.es/6040/
OAI Identifier: oai:oa.upm.es:6040
DOI: 10.1007/BF00037140
Official URL: http://www.springerlink.com/content/t424k2x2r82828ll/
Deposited by: Memoria Investigacion
Deposited on: 16 Feb 2011 15:13
Last Modified: 20 Apr 2016 14:41
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