Characterization of the insecticidal protein from Bacillus thuringiensis: The importance of DNA-protein interactions.
|Title:||Characterization of the insecticidal protein from Bacillus thuringiensis: The importance of DNA-protein interactions.|
|Authors:||Bietlot, Henri P.|
|Abstract:||Many strains of Bacillus thuringiensis produce a crystalline inclusion during sporulation which is toxic to insect larvae. The major component of crystals toxic to lepidopteran larvae is a 130-kDa protein, the protoxin. Following ingestion by susceptible insect larvae, protoxin is proteolysed by larval gut proteinases to yield a 58-70 kDa toxic fragment, toxin. A procedure was developed to prepare purified toxin for chemical characterization. Toxin generated by bovine trypsin was shown to be composed of the amino acid residues that span position 29-623 of the protoxin. The results obtained from competitive labelling experiments on the protoxin show that the functional groups of the lysine and tyrosine residues do not exhibit regular titration behaviour over the pH range of 7 to 10. These results indicate that the majority of these groups are not free in solution but are involved in inter and intra molecular interactions. During purification by ion exchange chromatography of the bovine generated toxin, it was discovered that the toxin could be separated into two components. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1). Only one major protoxin component was observed, and it was found to be associated with DNA. The DNA from the T2 toxin varied in size from 100 to 300 base pairs, whereas the crystal and the solubilized protoxin contain 20-kilobase DNA as the major DNA component. DNase treatment converted the T2 toxin to the DNA-free T1 toxin. In contrast, the DNA in the crystal and the solubilized protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl. The protoxin and DNA appeared to elute as a complex with a molecular mass of greater than $2\times 10\sp6$ Da on gel-filtration chromatography. No toxin was generated from the protoxin with trypsin after extensive digestion of the protoxin with DNase or dissociation of the DNA by succinylation of the lysine residues. It is proposed that DNA binds to the carboxyl terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|