Expression of an anti-Pseudomonas aeruginosa lipopolysaccharide core recombinant antibody in Escherichia coli

Abstract

Antibodies against Pseudomonas aeruginosa LPS are generally protective, however, this protection is usually serotype-specific. Thus the generation of antibodies against the more conserved core-lipid A epitopes has a potential for being more broadly cross reactive. Our laboratory has previously produced several monoclonal antibodies (mAb) against the core region and lipid A region of P. aeruginosa LPS. In this study, we cloned the immunoglobulin genes from mAb 7-4, an antibody with specificity for the inner core region of P. aeruginosa LPS. VH and VL genes of 7-4 were cloned into both of the M13-derived phagemid vectors, pComb3 and pComb8. 14 pComb3/VH/VL and 6 pComb8/V_H /V_L recombinant clones were isolated. The presence of recombinant F(ab) molecules in the periplasmic extracts of Escherichia coli was confirmed by Western immunoblots of these extracts with goat anti-mouse F(ab')2 and goat anti-mouse kappa light chain antibodies under non-reduced conditions. Recombinant 7-4 antibody also interacted with LPS prepared from a rough mutant P. aeruginosa strain AK43 in Western immunoblotting, and ELISA as well as with whole cells of AK43 in immunofluorescence. Thus, recombinant 7-4 antibodies expressed in E. coli are structurally and functionally correct.