Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization.
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.
Chan
Shzu-Wei
SW
Centre for Gene Analysis and Technology, School of BioSciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D. E., Malaysia.
Ong
Guan-Im
GI
Nathan
Sheila
S
D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D016957:Burkholderia pseudomallei / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D009500:Neutralization Tests; D010447:Peptide Hydrolases / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases