Facile preparation and characterization of the toxin from Bacillus thuringiensis var. kurstaki

We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to easter...

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Bibliographic Details
Published in:Biochemical journal 1989-05, Vol.260 (1), p.87-91
Main Authors: Bietlot, H, Carey, P.R, Choma, C, Kaplan, H, Lessard, T, Pozsgay, M
Format: Article
Language:English
Subjects:
Bacillus thuringiensis
Bacillus thuringiensis - analysis
Bacterial Proteins - isolation & purification
Bacterial Toxins - isolation & purification
Choristoneura fumiferana
Endotoxins - isolation & purification
Hemolysin Proteins
isoelectric focusing
papain
Pest Control, Biological
Protein Conformation
Protein Precursors - isolation & purification
proteolysis
strain differences
toxins
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Summary:We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj2600087