A protein engineering of Bacillus thuringiensis δ-endotoxin by conjugating with 4"-O-succinoyl abamectin

Conjugation of Bacillus thuringiensis δ-endotoxin (Bt toxin) with other toxins for insect pest control has been proposed as a new efficient strategy with increasing insecticidal toxicity and target range and delay the onset of insect resistance. A modified method was investigated by conjugating Bt toxin with 4"-O-succinoyl abamectin to form a new biocide which was named as BtA. 'Zero-length' cross-linker EDC in combination with NHS activated 4"-O-succinoyl abamectin and extended half-life period of active intermediate for binding to Bt toxin. The dissociation constant for 4"-O-succinoyl abamectin binding to Bt toxin was 6.44 μM by fluorescence quenching analysis. BtA showed a higher insecticidal toxicity against Plutella xylostella, while the relative-toxicity multiple of BtA to Bt toxin was calculated as 5.6. The interaction between Bt toxins with their receptors played a key role in toxicity of Bt toxins. The binding analysis showed the dissociation rate for the binding of BtA to its receptors (7.495 × 10(-3) S(-1)) was twice slower than that of Bt toxin (1.695 × 10(-2) S(-1)). The relative dissociation constant of BtA to Bt toxin was only 29% for the binding to the receptors. These results demonstrated that BtA bound to the receptor in BBMV with significantly higher affinity compared with Bt toxin.