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Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture
Author(s) -
Haider M. Z.,
Ellar D. J.
Publication year - 1987
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1987.tb00527.x
Subject(s) - biology , bacillus thuringiensis , spodoptera , sf9 , trypsin , biochemistry , escherichia coli , proteases , microbiology and biotechnology , inclusion bodies , toxin , proteinase k , aedes albopictus , aedes aegypti , recombinant dna , gene , bacteria , dna , genetics , botany , larva , enzyme
Summary An insecticidal protein gene from Bacillus thuringiensis var. aizawal 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 proteloytic 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‐resist‐ant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CFI ceils but not dipteran (Aedes albopictus) calls. 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. aizawal 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 ceil membranes, which may be the receptor for this toxin.