Vaccine 22 (2004) 1570–1575 Rapid purification of pertussis toxin (PT) and filamentous hemagglutinin (FHA) by cation-exchange chromatography Erkan Özcengiz a,∗ , Kamer Kılınç b , Özlem Büyüktanır a , Ayfer Günalp c a Bacterial Vaccines Research Laboratory, Refik Saydam Hygiene Center, 06100 Ankara, Turkey b Department of Biochemistry, Hacettepe University, 06100 Ankara, Turkey c Department of Microbiology, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey Received 8 January 2003; accepted 26 September 2003 Abstract Pertussis toxin (PT) and filamentous hemagglutinin (FHA) were purified from culture supernatant of Bordetella pertussis Saadet and Tohama strains, using CM-Sepharose CL-6B cation-exchange chromatography. By the rapid purification method described here, both proteins were separately eluted from the same column in pure forms. The PT and FHA in the extract of culture supernatant were bounded to CM-Sepharose CL-6B cation-exchange column in 50 mM phosphate buffer containing 2 M urea (Buffer A), pH 6.0. Then the PT was eluted from the column with Buffer A (pH 7.4) and after elution of the PT, the FHA was eluted with 0.5 M NaCl in 50 mM phosphate buffer. Pertussis toxin and filamentous haemagglutinin purified by this procedure were electrophoretically and immunologically identical to the reference preparations. © 2003 Elsevier Ltd. All rights reserved. Keywords: Pertussis toxin; Filamentous hemagglutinin; Purification; Vaccine 1. Introduction Pertussis toxin (PT) and filamentous haemagglutinin (FHA) which are the major components of acellular per- tussis vaccines are the extracellular proteins produced by Bordetella pertussis, the etiological agent of wooping cough. PT is a globular protein with a molecular weight of 117 000 Da. This ADP-ribosilating protein (PT) has several biological activities and is composed of five subunits des- ignated as S1, S2, S3, S4 and S5. FHA, with a molecular weight of 220 000 Da, is a nontoxic filamentous protein. It is believed to be involved in attachment to target cell surface and proves as a protective antigen [1–3]. During 1980–1990s a great deal of studies were carried for the purification and identification of PT and FHA especially aiming at the immunization studies. To date, several purifi- cation procedures have been reported by different research groups [4–14]. However, the reported methods consist of multi-step procedure and are relatively complicated purifi- cation techniques. For this reason, these antigens are quite expensive and hard to obtain. We now report a rapid, simple and inexpensive method for the purification of PT and FHA in a one-step chromatography. This method consists of an ∗ Corresponding author. ion-exchange chromatography on a CM-Sepharose CL-6B column. 2. Materials and methods 2.1. Materials B. pertussis Tohama strain was kindly provided by Dr. Sekura, Laboratory of Developmental and Molecular Im- munity, NIH, USA. B. pertussis Saadet strain was a domes- tic isolate. The PT/FHA US Standards and the reference antiserum (MR94) were kindly provided by Center for Biologics Evaluation and Research, Food and Drug Admin- istration, USA. CM-Sepharose CL-6B was from Pharmacia Fine Chemicals, Sweden. All other chemicals were of analytical grade. 2.2. Growth conditions B. pertussis Tohama or Saadet strains were grown and maintained on modified Cohen–Wheeler agar medium [15]. For toxin production, the organisms were cultivated in 200 ml of modified Morse and Bray liquid medium [16] and liquid CL medium [17] in 500 ml Fernbach flasks for 5 days at 37 ◦ C under statical conditions. 0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2003.09.040