Assessment of non-protein impurities in potential vaccine proteins produced by Bacillus subtilis

The levels of non-protein impurities at different stages of purification of model vaccine proteins produced by Bacillus subtilis were assessed with special emphasis on peptidoglycan-wall teichoic acid and lipoteichoic acid. Intracytoplasmically produced proteins were purified by disrupting the lysozyme protoplasts using osmotic shock, depositing the inclusion bodies by low-speed centrifugation, and washing them with detergent. By this procedure most of the cell envelope-derived impurities could be removed. The final product contained less than 1% (w/w) of neutral sugars, fatty acids, phosphate, hexosamine, diaminopimelic acid and glycerol. A secreted protein was purified from the culture supernatant by successive ion-exchange and adsorption chromatography. The cell envelope-derived impurities were efficiently removed by the cation-exchanger, and the final product contained only minute amounts of non-protein components. The amounts of non-protein components such as peptidoglycan and lipoteichoic acid in proteins produced in either mode were shown to be negligible in relation to their potentially harmful biological effects.