38th Annual Meeting of the Society for Invertebrate Pathology

Previously, critical surface-exposed loop residues (P₃₈₉ in β₆-β₇ loop, E₄₁₇ in β₈-β₉ loop, Y₄₅₅ and N₄₅₆ in β₁₀-β₁₁ ) in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin have been demonstrated to be involved in larvicidal activity. In this study, further mutagenic analysis was carried out to investigate a correlative effect among these critical loop residues on Cry4Ba toxicity. Double mutants, P389A/E417A (β₆-β₇/β₈-β₉ loops), E417A/Y455A and E417A/N456A (β₈-β₉/β₁₀-β₁₁ loops) were constructed via PCR-based mutagenesis and highly expressed in Escherichia coli as 130-kDa protoxins in the form of inclusion bodies with yields comparable to the wild type toxin. When E. coli cells expressing each double mutant toxin was determined their toxicity against Aedes aegypti mosquito larvae, an almost complete lose in larvicidal activity was observed from all these double mutant toxins. In addition the double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. This results suggested that all these critical loop residues (P₃₈₉, E₄₁₇, Y₄₅₅ and N₄₅₆) are correlatively in larvicidal activity of the Cry4Ba toxin.