Purification design and practice for pertactin, the third component of acellular pertussis vaccine, from Bordetella pertussis.
Development of acellular pertussis vaccine (aPV) requires purification of several components from Bordetella pertussis. While the components pertussis toxin (PT) and filamentous hemagglutinin (FHA) have been successfully purified, the third component, pertactin, proves to be a difficult target due to its very low concentration. In order to solve its purification problem, we performed the surface potential analysis with GRASP2 program. The results demonstrated that there are two major charge patches, one negative and one positive, which are located separately on this linear protein. For this special feature, we designed a dual ion exchange chromatography strategy including an anionic exchange and a cationic exchange process for separation of pertactin from the heat extract of B. pertussis. The initial anionic exchange chromatography concentrated the product from 1.7% to 14.6%, with recovery of 80%. The second cationic exchange chromatography increased the purity to 33%, with recovery of 83%. The final purification was accomplished by hydrophobic interaction chromatography, yielding a purity of 96%. The total recovery of the three columns was 61%. Characterization of the purified antigen was performed with CD, intrinsic fluorescence, HP-SEC and western-blot, showing that the purified protein kept its natural conformation and immune-reactivity. The rationally designed process proved to be feasible, and it is suitable for large-scale preparation of the third aPV component pertactin.
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Zenglan
Z
Li
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
Yan
Y
Zhang
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Qi
Q
Wang
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Zhengjun
Z
Li
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
Yongdong
Y
Liu
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
Songping
S
Zhang
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
Guifeng
G
Zhang
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
Guanghui
G
Ma
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China. Electronic address: jluo@ipe.ac.cn.
Jian
J
Luo
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing 211800, PR China. Electronic address: zgsu@ipe.ac.cn.
Zhiguo
Z
Su
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D001886:Bordetella pertussis; D002852:Chromatography, Ion Exchange; D010567:Pertussis Vaccine / Q000737:chemistry*; D055672:Static Electricity; D010566:Virulence Factors, Bordetella / Q000737:chemistry* / Q000302:isolation & purification*
D001425:Bacterial Outer Membrane Proteins; D010567:Pertussis Vaccine; D010566:Virulence Factors, Bordetella; C068929:pertactin
Acellular pertussis vaccine; Dual ion exchange chromatography; Pertactin; Purification; Surface electrostatic potential