Purification and characterization of Bacillus thuringiensis vegetative insecticidal toxin protein(s)

Bacillus thuringiensis subsp. aegypti C18 is an Egyptian isolate, obtained from dead pink bollworm larvae. Insecticidal active proteins against different insect were purified from BtaC18 strain during vegetative states. Both the bacterial pellet and cell-free supernatant obtained during vegetative growth had insecticidal activity against black cutworm (BCW). Bioassays revealed that the pellet after 48 h of growth is more potent and toxic against BCW. The toxin in the pellet was active at very high temperatures but lost toxicity after boiling or autoclaving. Proteins extracted from the BtaC18 pellet were further purified by ammonium sulfate precipitation, and the 40% fraction was then subjected to fast protein liquid chromatography (FPLC). Seven major protein peaks were detected after FPLC (Pi- a, b, c, d, e, f and g). Pic protein fraction was active against BCW with an estimated LC50 = 26 ng cm(-2) , Pid protein killed 50% of European corn borer (ECB) at 46 ng cm(-2) , and Pif showed insecticidal activity against western corn root worm (WCRW) with estimated LC50 was 94 ng cm(-2) . Based on the significant and high toxicity of Pic against BCW and Pif against WCRW, the 88- and 44-kDa proteins were further characterized by N-terminal amino acid sequencing.

Significance and impact of the study: Insecticidal activity of Bacillus thuringiensis subsp. aegypti was determined, and its vegetative insecticidal protein was subjected to FPLC for protein purification. This work contributes to improve understanding the different toxins secreted during vegetative growth of Bt. Moreover, the N-terminal amino acid sequences of 88-kDa protein was only 92% identical to that of vip3A, and for 44 kDa was 92% identical with Cry35a, suggesting that we might have identified a new genes. Finally, we have proven these proteins to be novel insecticidal agents that may complement the use of known insecticidal proteins derived from Bacillus.