Purification and preliminary characterization of agglutinogen 3 from Bordetella pertussis

One serotype antigen, agglutinogen 3, from Bordetella pertussis, (strain M2, serotype 1.3), has been purified. The purification procedure included acetone drying of cells harvested from shaking cultures. Agglutinogens were extracted in phosphate buffered saline. Crude extract was heat treated at 80 degrees C for 5 min and precipitated by ammonium sulphate between 25 and 60% saturation at 4 degrees C, providing 50% of the total activity and a five-fold purification. Further purification was attempted by gel filtration chromatography using a TSK-G3000 SW column. The ammonium sulphate precipitated fraction was also separated by anion exchange chromatography using a Mono Q HR 5/5 column. The purification work indicated that agglutinogen 3 is associated with several other substances and that this property can lead to purification difficulties. The isolation procedure was monitored by an agglutination-inhibition assay. The peak fraction from the ion exchange chromatography was purified up to 27-fold according to the specific activities (inhibition units per mg protein). The yield was only 1% due to severe loss of activity. In the gel filtration chromatography agglutinogen 3-activity eluted with a maximum activity corresponding to a molecular weight near 450,000. SDS-PAGE analysis indicated that agglutinogen 3 might have a subunit molecular weight of 20,800.