Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis
AbstractVip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices. View Full-Text
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Bel, Y.; Banyuls, N.; Chakroun, M.; Escriche, B.; Ferré, J. Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis. Toxins 2017, 9, 131.
Bel Y, Banyuls N, Chakroun M, Escriche B, Ferré J. Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis. Toxins. 2017; 9(4):131.Chicago/Turabian Style
Bel, Yolanda; Banyuls, Núria; Chakroun, Maissa; Escriche, Baltasar; Ferré, Juan. 2017. "Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis." Toxins 9, no. 4: 131.
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