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|Title:||Conditions for homogeneous preparation of stable monomeric and oligomeric forms of activated Vip3A toxin from Bacillus thuringiensis|
Nanyang Technological University
Thailand National Center for Genetic Engineering and Biotechnology
|Keywords:||Biochemistry, Genetics and Molecular Biology|
|Citation:||European Biophysics Journal. Vol.46, No.3 (2017), 257-264|
|Abstract:||© 2016, European Biophysical Societies' Association. Bacillus thuringiensis vegetative insecticidal proteins like Vip3A have been used for crop protection and to delay resistance to existing insecticidal Cry toxins. However, little is known about Vip3A’s behavior or its mechanism of action, and a structural model is required. Herein, in an effort to facilitate future crystallization and functional studies, we have used the orthogonal biophysical techniques of light scattering and sedimentation to analyze the aggregation behavior and stability of trypsin-activated Vip3A toxin in solution. Both scattering and sedimentation data suggest that at pH 10 the toxin is monomeric and adopts an elongated shape, but after overnight incubation aggregation was observed at all pH values tested (5–12). The narrowest size distribution was observed at pH 7, but it was consistent with large oligomers of ~50 nm on average. The addition of β-d-glucopyranoside (OG) helped in achieving preparations that were stable and with a narrower particle size distribution. In this case, scattering was consistent with a 4-nm monomeric globular Vip3A form. After OG dialysis, 40-nm particles were detected, with a molecular weight consistent with homotetramers. Therefore, OG is proposed as the detergent of choice to obtain a Vip3A crystal for structural studies, either before (monomers) or after dialysis (tetramers).|
|Appears in Collections:||Scopus 2016-2017|
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