Purification and characterization of the entomocidal protoxin of Bacillus thuringiensis

Abstract

A procedure for purifying the insecticidal parasporal protoxin of Bacillus thuringiensis and a description of its biochemical and biophysical properties is provided. Mild alkali titration was necessary to generate a functional protoxin in a soluble form, and anion-exchange chromatography was used to remove contaminating cytoplasmic proteases that are nonspecifically bound to whole native parasporal crystals. Polyacrylamide gel electrophoresis, gel filtration chromatography, and meniscus depletion sedimentation equilibrium analysis revealed an apparent molecular weight for the protoxin of 1.34 x 10(5). The only NH2-terminal residue found was methionine. The soluble protoxin was 2.5 times more toxic to insect larvae than was the parasporal crystal. At alkaline pH the protoxin slowly converted to a low molecular weight toxin (apparent Mr = 6.8 x 10(4)). The molar specific toxicities of the protoxin and toxin were identical.