title: Quantification of Bacillus thuringiensis Vip3Aa16 Entomopathogenic Toxin Using Its Hemolytic Activity.
abstract: Vegetative insecticidal proteins produced by some Bacillus thuringiensis strains are specifically toxic to different agricultural pests such as the polyphagous Spodoptera and several other Lepidopteran insects, but one of the major problems found in the use of these biopesticides was the lack of an easy and credible method of quantification of such secreted toxins. Heterologous expression of B. thuringiensis Vip3Aa16 toxin was performed in Escherichia coli then the protein was purified by chromatography. Using blood agar as well as blood agar overlay (zymogram assay), we reported, for the first time, the capacity of Vip3Aa16 to induce hemolysis. The hemolytic activity of this protein was shown to be relatively stable after treatment at 40 °C and at a range of pH between 6.5 and 9. Moreover, a linear relationship was shown between hemolysis levels and Vip3Aa16 concentrations. The model established in the present study could quantify Vip3A toxin as a function of hemolytic activity and the assay proposed showed to be a simple and low-cost method to readily assess Vip3A toxins in liquid cultures and facilitate the use of this kind of bioinsecticides in pest management programs.
authors.affiliation: Laboratory of Biopesticides, Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3018, Sfax, Tunisia.
authors.forename: Hanen
authors.initials: H
authors.lastname: Boukedi
authors.affiliation: Laboratory of Biopesticides, Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3018, Sfax, Tunisia.
authors.forename: Saoussen
authors.initials: S
authors.lastname: Ben Khedher
authors.affiliation: Higher Institute of Biotechnology of Sfax, University of Sfax, P.O. Box 261, 3038, Sfax, Tunisia.
authors.forename: Dhouha
authors.initials: D
authors.lastname: Ghribi
authors.affiliation: Laboratory of Biopesticides, Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3018, Sfax, Tunisia.
authors.forename: Mariam
authors.initials: M
authors.lastname: Dammak
authors.affiliation: Laboratory of Biopesticides, Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3018, Sfax, Tunisia.
authors.forename: Slim
authors.initials: S
authors.lastname: Tounsi
authors.affiliation: Department of Biology, Faculty of Sciences and Arts-Khulais, University of Jeddah, Jeddah, Saudi Arabia. mesratilobna@yahoo.com.
authors.forename: Lobna
authors.initials: L
authors.lastname: Abdelkefi-Mesrati
mesh_terms: D000898:Antibiosis / Q000235:genetics*; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism*; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D007306:Insecticides; D055550:Protein Stability; D011994:Recombinant Proteins; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007306:Insecticides; D011994:Recombinant Proteins; C447139:Vip3A protein, Bacillus thuringiensis
keywords: Bacillus thuringiensis; Hemolytic activity; Quantification; Vegetative insecticidal protein; Zymogram