title: Rapid purification of Bordetella pertussis toxin by alternating affinity and hydrophobic chromatography.
abstract: Pertussis toxin was purified to homogeneity from a 2-day culture supernatant of Bordetella pertussis by stepwise elution from three columns of, consecutively, Blue Sepharose, phenyl Sepharose, and hydroxyapatite. The toxin was eluted from Blue Sepharose and hydroxyapatite by high ionic strength and from phenyl Sepharose with low ionic strength and with 17% glycerol. Toxin fractions from one chromatographic column were immediately charged on the next column, saving laborious and time-consuming concentration or dialysis steps. Based on peptide composition (after sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and on HPLC profile (under nondenaturing conditions), the toxin was already practically pure after two steps, the third hydroxyapatite column serving only to separate the whole native toxin from any free S1 subunit. Recovery was estimated from the capacity of the preactivated toxin (and any preexisting free S1 subunit) to catalyze the ADP-ribosylation of the guanine nucleotide binding protein Ni in rat pancreatic plasma membranes: of the total capacity initially present in the culture medium, 23% could be recovered as pure native toxin with the present procedure. Besides, the nondenaturing HPLC method used to check the purity of the native toxin appeared to be superior to classical acidic polyacrylamide gel electrophoresis.
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008218:Lymphocytosis / Q000209:etiology; D051379:Mice; D010179:Pancreas / Q000378:metabolism; D037342:Pertussis Toxin; D051381:Rats; D010566:Virulence Factors, Bordetella / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin