Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

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.

Neutralization ofBurkholderia pseudomalleiProtease by Fabs Generated through Phage Display

“…We have since found that FabC37 is able partially to inhibit proteolytic digestion of transferrin and myosin. 27) As observed in the sera ELISA and panned phage ELISA, the mice had a stronger immune response towards denatured protease (DP) than towards native protease (P), thus indirectly confirming the above speculation that the antigen was denatured following immunization. DP molecules either outnumbered P molecules, resulting in the stronger immune response, or else denaturing the protease exposed the immunodominant epitope of the antigen, thereby eliciting a stronger immune response in the animals.…”

Epitope mapping ofBurkholderia pseudomalleiserine metalloprotease: identification of serine protease epitope mimics

“…B. pseudomallei protease and IgG fractions of polyclonal antibodies specific against B. pseudomallei protease used in this study were prepared as described previously [18]. Briefly, protease was purified from brain-heart infusion (BHI, HispanLab, Cuba) broth cultures grown at 37°C for seven days, following which, cultures were subjected to ammonium sulphate precipitation (70% saturation), DEAE-Cellulose Ion Exchange chromatography (pH 8) and CM-Cellulose Ion Exchange chromatography (pH 6).…”

“…To identify the major epitope of B. pseudomallei protease, the phage display method was used whereby random 12-mer peptides were screened for affinity binding of peptides to B. pseudomallei protease specific IgG polyclonal antibodies. The antibodies were protein-A affinity-purified [18], immobilized on microtitre wells and bound phage were selected and amplified through 3-4 biopanning cycles. The eluted phage population from the first cycle of selection was amplified and used for the successive round of selection and amplification.…”

Production and Characterization of Chimeric Monoclonal Antibodies against Burkholderia pseudomallei and B. mallei Using the DHFR Expression System

“…The ultimate goal of this study is to develop superactive MAbs that can be used as therapeutics against BM and BP infections. Humanization of the target MAbs could minimize possible side effects when used as human therapeutics [21] , [22] . Therefore, the chimerization of these 3 MAbs against BP and/or BM is an initial step in the development of MAb-based therapeutics.…”