Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture.
An insecticidal protein gene from Bacillus thuringiensis var. aizawai was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteolytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resistant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CF1 cells but not dipteran (Aedes albopictus) cells. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawai toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran cell membranes, which may be the receptor for this toxin.
Haider
M Z
MZ
Department of Biochemistry, University of Cambridge, UK.
Ellar
D J
DJ
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000235:genetics*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004731:Endotoxins; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005796:Genes; D005798:Genes, Bacterial; D006460:Hemolysin Proteins; D007814:Larva; D007915:Lepidoptera
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis