As the global need for nutritious food grows, more attention is spotlighted on the design of effective biopesticides. Cry1Ab is a protein toxin expressed in Bacillus thuringiensis (Bt) that targets tobacco hornworms (M. sexta). Cry1Ab binds to the Bt-R1 receptor in midgut epithelial cells and launches a signaling cascade that results in cell death. Past research demonstrates that the 12th ectodomain of Bt-R1 (EC12) is the toxin binding site (TBS). This work focuses on the interaction interface of Cry1Ab that binds to EC12, and specifically, the “Loop 3” region of Cry1Ab that has previously been shown to be important for binding and toxicity. After using co-evolutionary modeling and in vitro biochemical assays, we pinpointed six point mutations, including Cry1Ab F328E, which eliminates binding. This mutant’s lack of binding to the EC12 receptor is further supported by ELISA assays, where only the receptor should be attracted to the nickel beads, and Size Exclusion Chromatography (SEC), which is size-dependent molecular separation. Future experiments will involve testing the toxicity of this mutant in the hornworm by analyzing the hornworm’s response when Cry1Ab F328E is injected into its food. Determining this mutant’s efficiency will bring us closer to creating a new generation of smart biopesticides that will overcome costly and environmentally hazardous chemical pesticides.
