Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product.
Conditions for hyperexpression, in Escherichia coli, of the Bacillus thuringiensis var, kurstaki gene, cryIA9(c)73, encoding an insecticidal crystal protein, CryIA(c)73, were investigated by varying the promoter type, host cell, plasmid copy number, the second codon and number of terminators. The cryIA(c)73 gene was cloned into three E. coli expression vectors, pKK223-3 (Ptac promoter), pET-3a (P phi 10 promoter), and pUC19 (Ptac promoter). The level of cryIA(c)73 expression was measured by ELISA and compared to total cellular protein over growth periods of 24 and 48 h. Maximum expression levels of 284 microgram CryIA(C)73/ml (48% of cellular protein) were obtained in shake flasks with the Ptac promoter in E. coli JM103. Optimal conditions were found to be low-copy-number plasmid (pBR322 ori), 48 h of growth, in lon+ cells. A change of the gene's second codon to AAA can improve expression by two to three fold but is undetectable in the presence of a strong E. coli promoter. The cryIA(c)73 gene product, in E. coli, formed crystals with the same lattice structure as the native crystals formed in B. thuringiensis (as visualized by electron microscopy). Bioassay results (insect toxicity and specificity) of the crystal produced in E. coli were similar to that produced in B. thuringiensis.
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Department of Biochemistry, Ohio State University, Columbus 43210.
Pfister
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Dean
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D001413:Bacillus thuringiensis / Q000235:genetics* / Q000648:ultrastructure; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003001:Cloning, Molecular; D003460:Crystallization; D004731:Endotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure; D015964:Gene Expression Regulation, Bacterial; D006460:Hemolysin Proteins; D007306:Insecticides
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis