ANALYSIS OF NON-ACTIVE ENGINEERED BACILLUS-THURINGIENSIS CRYSTAL PROTEINS

Crystal proteins of Bacillus thuringiensis are known for their insecti cidal specificity. This specificity is, to a large extent, determined by the interaction of the proteins with high-affinity binding sites on the epithelial membrane of the midgut of sensitive insects. In partic ular, domain II of the three domains of the toxic moiety has been impl icated in specificity. To determine which sequences of the protein are involved in binding, loops of domain II which terminate in the molecu lar apex of CryIA(b) were replaced by the corresponding regions of Cry IE, a protein with different binding characteristics and insect specif icity. In contrast to expression of the wild-type genes, expression of the mutant alleles in Escherichia coli resulted in the formation of b iologically inactive, insoluble aggregates. Although these aggregates could be solubilized in vitro using urea, in contrast to the wild-type CryIA(b), the mutant proteins did not correctly refold as is shown by their increased protease sensitivity and lack of biological activity. The results indicate that engineering CryI proteins, based on the Cry IIIA structure, is likely to prove difficult, particularly since the c onformation of CryIIIA and CryI proteins might differ in domain II.