Purification and hydrophobic properties of the δ-endotoxin in the parasporal crystal produced by Bacillus thuringiensis var. entomocidus

Parasporal crystals of Bacillus thuringiensis var. entomocidus were separated from spores and other cell debris by the water-chloroform biphase procedure. The solubilization and fractionation were carried out under mild conditions at 4°C. Crystals were solubilized in 0.01 M dithiothreitol and 0.2 M glycine NaOH buffer at pH 10.0. The solution was treated overnight with 0.01 M Tris-HCl buffer, pH 5.5, containing 0.1% Triton N-101 and 0.1% sodium cholate, and then placed on a Sepharose 6B column, equilibrated, and later developed with the same buffer. Under these conditions, four fractions were obtained, one of which had a molecular weight ranging from 60,000 to 70,000, and demonstrated a high insecticidal activity on second instar larvae of Spodoptera litioralis. The LC₅₀ value of this fraction was a half of that of the solubilized crystals. The other three fractions had a lower activity. The active fraction was further fractionated on an octyl-Sepharose 4B resin. Elution of this column with the same buffer separated the proteins into two fractions. The first eluted fraction was highly active, while the second demonstrated a very low activity. The active fraction was further purified by loading on a short column of octyl-Sepharose 4B and eluted with a linear gradient of the same detergents. Under these conditions, the highly active fraction gave a sharp and symmetrical peak that revealed five close bands at the pH range of 6.1-6.5 on isoelectric focusing gel electrophoresis.