BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF THE INSECTICIDAL FRAGMENT OF CRYV

Two C-terminal deletion constructs were made to study the effect of su ch deletions on the biological activity of the CryV protein of Bacillu s thuringiensis subsp kurstaki. The results of feeding on neonatal lar vae of Ostrinia nubilalis (European core borer [ECB]) indicated that t he 50% lethal dose of the full-length CryV protein was 3.34 mu-g/g of diet (95% fiducial limits, 2.53 to 4.32 mu-g/g of diet). Removal of 71 amino acids (aa) from the C terminus had little effect on toxicity, w hereas deletion of 184 aa abolished the insecticidal activity of the C ryV protein completely. Truncations of the full-length CryV protein we re also generated with trypsin and the midgut protease of ECB. The pro teolytically treated products were characterized by determining their N-terminal amino acid sequences. The CryV protein was found to be clea ved by both proteases through a two-step process. Initially an interme diary form was generated which contained aa 45 of full-length CryV as its N-terminal end. The C-terminal end of this peptide was not experim entally determined. However, analysis of the deduced amino acid sequen ce of CryV indicated that the C-terminal end of the intermediary form is likely either aa 655 or 659. Further N-terminal processing of the i ntermediary form resulted in a protease-resistant core form. The core included aa 156 to aa 655 or 659. While the intermediary form retained 100% of the ECB larval toxicity, the core form exhibited only similar to 22% of the toxicity of the full-length protein.