The delta-endotoxin proteins accumulate in Escherichia coli as a protein-DNA complex that can be dissociated by hydrophobic interaction chromatography.
The insecticidal protein CryIAc accumulated to form inclusion bodies in Escherichia coli upon overexpression of the cloned gene. The solubilized inclusion bodies contained the delta-endotoxin in association with DNA fragments of about 25 kb. The protein-DNA complex could be dissociated and the delta-endotoxin purified by hydrophobic interaction chromatography on phenyl-Sepharose. The DNA was washed out in the high-salt buffer while the delta-endotoxin was bound to the matrix and was eluted at 4 degrees C by a stepwise decreasing potassium chloride gradient. The DNA-protein complex also contained plasmids harbored by the host strain. The plasmid DNA associated with the complex became competent to transform E. coli only after it was dissociated from the delta-endotoxin. The hydrophobic interaction chromatography provides an efficient method for the purification of DNA-free activated toxin.
Chaturvedi
R
R
Molecular Biology and Genetic Engineering Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226001, India.
Bhakuni
V
V
Tuli
R
R
D001413:Bacillus thuringiensis / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006460:Hemolysin Proteins; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D004247:DNA