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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

  • Chan, Shzu-Wei (Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore) ;
  • Ong, Guan-Im (Centre for Gene Analysis and Technology, School of BioSciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia) ;
  • Nathan, Sheila (Centre for Gene Analysis and Technology, School of BioSciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia)
  • Published : 2004.09.30
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Abstract

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

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