Abstract
We selected 50 crystal (Cry) toxins specific to lepidopteran insects. Phylogenetic relationships of whole toxins as well as individual domains revealed that Cry1Jb and Cry1Ac are distantly placed but related to reduced cross-resistance in Helicoverpa armigera. Multiple alignments of Cry1Jb and Cry1Ac amino acid sequences showed significant differences in the composition and length of the loops of domains II and III. This was further confirmed by the superpositioning of 3D structures. cry1Jb and cry1Ac genes cloned in expression vectors were overexpressed in Escherichia coli Castellani & Chalmers, and proteins were harvested. Insect bioassays revealed that the wild-type Cry1Jb and Cry1Ac proteins showed differential specificity to H. armigera (Hubner), Spodoptera litura (Fab.) and Earias vittella F. Chimeric genes were constructed by exchanging the domains between cry1Jb and cry1Ac and overexpressed in BL21(DE3). Substitution of domain III of Cry1Jb with that of Cry1Ac enhanced its activity against H. armigera 7.8-fold. Bioassay of the parental and chimeric toxins against S. litura revealed no activity even at the discriminative dose of 100 μg/ml, but a significant difference in growth was observed. The present results, along with previous domain swapping experiments, suggest that protein engineering not only reveals the mechanism by which endotoxins work, but also generates novel toxins with enhanced toxicity/broader specificity.
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