Directed mutagenesis of selected regions of a Bacillus thuringiensis entomocidal protein.
Comparison of the sequences of Bacillus thuringiensis entomocidal toxins of widely differing specificity reveals six conserved domains. The role of one of the most highly conserved domains (D1) located near the N-terminus has been investigated by site directed mutagenesis at two positions. Although preliminary results indicate that the capacity of the mutants to bind to putative receptors on the plasma membrane of susceptible cells was unaffected, toxicity in vivo was reduced by 70-80%. The role of the highly hydrophobic segment exposed at the N-terminus of the toxin after proteolytic activation was investigated by substituting two aspartate residues for phenylalanine and valine located adjacent to each other in the centre of this segment. The toxicity of the resulting mutant protein was only 40% of the unmutated toxin but again preliminary results suggest that binding to putative receptors was unaffected. These results suggest that regions close to the N-terminus of this and similar toxins may play an important role in the membrane insertion event which is believed to follow receptor recognition.
Ahmad
W
W
Department of Biochemistry, University of Cambridge, U.K.
Ellar
D J
DJ
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics* / Q000494:pharmacology; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007313:Insecta; D007306:Insecticides; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011498:Protein Precursors / Q000235:genetics* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
C029103:Bacillus thuringiensis protoxin; D001427:Bacterial Toxins; D007306:Insecticides; D011498:Protein Precursors; D011994:Recombinant Proteins