title: Conditions for homogeneous preparation of stable monomeric and oligomeric forms of activated Vip3A toxin from Bacillus thuringiensis.
abstract: 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).
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, 73170, Nakhon Pathom, Thailand.
authors.forename: Thittaya
authors.initials: T
authors.lastname: Kunthic
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.
authors.forename: Wahyu
authors.initials: W
authors.lastname: Surya
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Pahonyothin Road, Khlong Luang, 12120, Pathumthani, Thailand.
authors.forename: Boonhiang
authors.initials: B
authors.lastname: Promdonkoy
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore. jtorres@ntu.edu.sg.
authors.forename: Jaume
authors.initials: J
authors.lastname: Torres
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, 73170, Nakhon Pathom, Thailand. panadda.boo@mahidol.ac.th.
authors.forename: Panadda
authors.initials: P
authors.lastname: Boonserm
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D008970:Molecular Weight; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D018411:Spodoptera; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D007306:Insecticides; C447139:Vip3A protein, Bacillus thuringiensis; D014357:Trypsin
keywords: Analytical ultracentrifugation sedimentation velocity; Bacillus thuringiensis; Bioassay; Light scattering; Vegetative insecticidal proteins; Vip3A