Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction

Abstract

Two major antigens of Bordetella pertussis, filamentous hemagglutinin (FHA) and pertussis toxin (PT), were efficiently purified from culture filtrate by exploiting their relative hydrophobicities and differences in affinity to sialic acid-containing protein. High yields of FHA (40 to 80 mg/liter) and PT (8 to 16 mg/liter) were first produced by growing the bacteria in the modified CL medium. The FHA and PT in the culture filtrate were adsorbed onto butyl-Sepharose by hydrophobic interaction at appropriately high ionic strength. Elution of the antigens was effected by decreasing their hydrophobicities with a buffer of low ionic strength. FHA was then separated from PT with an affinity column of fetuin-Sepharose. The fraction passing through the column contained purified FHA, and the fetuin-bound PT was eluted with buffered MgCl2. The FHA and PT purified by these steps were electrophoretically and serologically identical to the reference purified FHA and PT preparations. Approximately 16 to 32 mg of purified FHA and 4 to 8 mg of purified PT were obtained from 1 liter of culture filtrate. The described procedure for making FHA and PT antigens from B. pertussis for serologic and immunologic use is very simple, efficient, and reproducible.

The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model

“…PTx was purified from culture supernatants of B. pertussis strains 18323 (wild type and ptx R9K/E129G mutant) and Tohama, as previously reported ( 102 ). Rabbit anti-PTx, anti-FtsZ, anti-adenylate cyclase toxin, and anti-filamentous hemagglutinin, and rat anti-Vag8 sera were obtained by immunization of rabbits and rats with purified PTx and a purified preparation of recombinant antigens according to our previous method ( 103 ).…”

Marrow engraftment of hematopoietic stem and progenitor cells is independent of Gαi-coupled chemokine receptors

“…Purified PT was generously provided by Dr. Witold Cieplak (RIBI Immunochem, Hamilton, MT). The material utilized in these studies was purified from culture filtrates of Bordetella pertussis strain 3779 Bl 2 S 4 using Affi-Gel Blue (Bio-Rad, Hercules, CA) followed by affinity chromatography using fetuin as previously described [50]. The purified PT was maintained at a concentration of 180 g/ mL in 0.1M Tris, 0.5M NaCl, pH 8.0, and was diluted from this stock immediately prior to each experiment.…”