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.
Himanen
J P
JP
Department of Molecular Bacteriology, National Public Health Institute, Helsinki, Finland.
Sarvas
M
M
Helander
I M
IM
D001412:Bacillus subtilis / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification*; D002241:Carbohydrates / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods; D002845:Chromatography / Q000379:methods; D003902:Detergents; D004340:Drug Contamination; D005227:Fatty Acids / Q000032:analysis; D006801:Humans; D008070:Lipopolysaccharides / Q000032:analysis*; D037342:Pertussis Toxin; D010710:Phosphates / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification*; D013682:Teichoic Acids / Q000032:analysis*; D014614:Vaccines, Synthetic / Q000032:analysis* / Q000302:isolation & purification; D010566:Virulence Factors, Bordetella / Q000302:isolation & purification*
D001425:Bacterial Outer Membrane Proteins; D002241:Carbohydrates; D003902:Detergents; D005227:Fatty Acids; D008070:Lipopolysaccharides; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D013682:Teichoic Acids; D014614:Vaccines, Synthetic; D010566:Virulence Factors, Bordetella; C083388:pertussis toxin, S1 subunit; C083389:pertussis toxin, S4 subunit; C009900:lipoteichoic acid; D037342:Pertussis Toxin