A SIMPLE METHOD FOR SEPARATION OF THE PROTEIN CRYSTALS FROM BACILLUS-THURINGIENSIS USING CARBOXYMETHYL CELLULOSE COLUMN CHROMATOGRAPHY

A simple and a rapid technique to separate intact crystals from spores and cell debris of Bacillus thuringiensis using a carboxymethyl cellulose (CM-cellulose) column is described. B. thuringiensis subsp. kurstaki, a toxic strain to lepidopteran insects that produces small bipyramidal crystals, and B. thuringiensis subsp. yunnanensis, a nontoxic strain producing unusually large bipyramidal and spindle-shaped crystals were used as test organisms. Spores and cell debris were washed in sodium acetate and sodium phosphate buffers and the crystals bound to the CM-cellulose were eluted with Tris-EDTA buffer, regardless of their size and shape. Light microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purity of the crystal was >98%. The percent recoveries of crystals from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. yunnanensis were approximately 14 and 25, respectively.