title: Purification and characterization of Bacillus thuringiensis var. tenebrionis insecticidal proteins produced in E. coli.
abstract: Native and single amino acid variants of the Bacillus thuringiensis var. tenebrionis insecticidal proteins were expressed in Escherichia coli, purified and examined for biological and biochemical properties. A novel, pH dependent, preferential precipitation method was implemented to purify Escherichia coli produced Bacillus thuringiensis var. tenebrionis proteins, which are active against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Cysteine residues of the native Bacillus thuringiensis var. tenebrionis protein were replaced by serine residues by site-directed mutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionis proteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteine residue (amino acid 540) caused complete inactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidal activities comparable to each other and to solubilized crystals from the original strain.
authors.affiliation: Monsanto Agricultural Company, Saint Louis, Missouri 63198.
authors.forename: S C
authors.initials: SC
authors.lastname: MacIntosh
authors.forename: S L
authors.initials: SL
authors.lastname: McPherson
authors.forename: F J
authors.initials: FJ
authors.lastname: Perlak
authors.forename: P G
authors.initials: PG
authors.lastname: Marrone
authors.forename: R L
authors.initials: RL
authors.lastname: Fuchs
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D001427:Bacterial Toxins; D001517:Coleoptera / Q000187:drug effects; D003545:Cysteine / Q000235:genetics; D004731:Endotoxins; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006460:Hemolysin Proteins; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012694:Serine / Q000235:genetics
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012694:Serine; D003545:Cysteine