Site-directed mutagenesis and expression in Escherichia coli of WMAI-1, a wheat monomeric inhibitor of insect alpha-amylase.
The wheat monomeric inhibitor WMAI-1 (syn. 0.28) produced in Escherichia coli using the pT7-7 expression vector has the correct N-terminal sequence and the same electrophoretic mobility and specific activity towards the alpha-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 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.
García-Maroto
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F
Catedra de Bioquímica y Biología Molecular, E.T.S. Ingenieros Agrónomos-UPM, Madrid.
Carbonero
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P
García-Olmedo
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F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D007700:Kinetics; D007814:Larva; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D013712:Tenebrio / Q000201:enzymology*; D014908:Triticum / Q000235:genetics*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011994:Recombinant Proteins; C071391:WMAI-1; D000516:alpha-Amylases