Binding characteristics to mosquito-larval midgut proteins of the cloned domain II-III fragment from the Bacillus thuringiensis Cry4Ba toxin.
Receptor binding plays an important role in determining host specificity of the Bacillus thuringiensis Cry delta-endotoxins. Mutations in domains II and III have suggested the participation of certain residues in receptor recognition and insect specificity. In the present study, we expressed the cloned domain II-III fragment of Cry4Ba and examined its binding characteristics to mosquito-larval midgut proteins. The 43-kDa Cry4Ba-domain II-III protein over-expressed in Escherichia coli as inclusion bodies was only soluble when carbonate buffer, pH 10.0 was supplemented with 4 M urea. After renaturation via stepwise dialysis and subsequent purification, the refolded domain II-III protein, which specifically reacts with anti Cry4Ba-domain III monoclonal antibody, predominantly exists as a beta-sheet structure determined by circular dichroism spectroscopy. In vitro binding analysis to both histological midgut tissue sections and brush border membrane proteins prepared from susceptible Aedes aegypti mosquito-larvae revealed that the isolated Cry4Ba-domain II-III protein showed binding functionality comparable to the 65-kDa full-length active toxin. Altogether, the data present the 43-kDa Cry4Ba fragment comprising domains II and III that was produced in isolation was able to retain its receptor-binding characteristics to the target larval midgut proteins.
Moonsom
Seangdeun
S
Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Chaisri
Urai
U
Kasinrerk
Watchara
W
Angsuthanasombat
Chanan
C
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D009033:Culicidae; D004064:Digestive System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007814:Larva / Q000378:metabolism; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis