Insecticidal Activity And Trypsin Sensitivity Of Bacillus Thuringiensis Vip3 Mutant Proteins

Vip3s are novel toxin isolated from Bacillus thuringiensis (Bt). They are expressed and secreted out off cells during the vegetative growth stage. Their amino acid sequence shares no similarity with any known Btδ-endotoxins and the mode of action of Vip3s is believed to be different from 8-endotoxins. Most of them are highly active against broad species of Lepidopteran insects. Therefore, Vip3s might represent a new source of insecticidal toxins discovered after the 8-endotoxins from Bacillus thuringi ensis. However, very few studies were reported so far on biochemistry and activity improvement of Vip3 by creating mutants.In this study, Vip3 mutants with deletion, addition or mutations at the very end of the C-terminus were generated from vip3AcAa, an chimeric Vip3 gene created by Fang et al (2007). The insecticidal activities of the E. coli expressed proteins of these mutants were assayed, and their sensitivity to trypsin digestion was also determined. It was found that the deletion of the last three amino acid residues (SLK) or addition of 8 extra amino acid residues, Leu-Glu-His-His-His-His-His-His, at the C-terminus rendered the Vip3 mutants unstable to trypsin and total loss of insecticidal activity against beet armyworm and cotton bollworm. Furthermore, by PCR with a degenerated primer, we obtained 15 different mutants with mutations at the last two residues. Only one mutant, Vip3m11, with the mutation from IK to LR increased its activity substantially against beet armyworm and cotton bollworm. All other mutants were inactive. The active mutant was found to be resistant to high concent- ration of trypsin, while the inactive ones were highly unstable to trypsin. This study suggests that the C-terminus of Vip3 is critical to its insecticidal activity and might be the region for protein engineering for activity improvement.At the same time, we obtained the DNA fragment which encodes the 62-kDa polypeptide by tuncating a 5' end DNA fragment encoding the N-terminal 198 amino acid residues of the vip3AcAa gene. The E. coli expressed 62-kDa polypeptide was not active agaisnt neither cotton bollworm nor beet armyworm. Furthermore, it was found to be highly sensitive to trypsin digestion, which is consistant with the notion that inactive Vip3s are senstive to typsin. This study suggests that the N-terminal part of 198 amino acid residues may be required to form the 62-kDa active core polypeptide. Although the N-terminal deletion Vip3 mutant shares the same amino acid sequnce to the core polypeptide generated by trypsin digestion, they were acturally different.