Phage display and bacterial expression of a recombinant Fab specific for Pseudomonas aeruginosa serotype O6 lipopolysaccharide.
Immunotherapy with antibodies (Abs) against the lipopolysaccharide (LPS) of Pseudomonas aeruginosa remains an alternative to serotype-specific LPS-based vaccines due to their limited use and to antibiotics due to the intrinsic resistance to antimicrobials observed in P. aeruginosa. We have chosen a monoclonal Ab (MAb), MF23-1, that binds to the O antigen of the most clinically relevant serotype, IATS O6, for producing a recombinant antibody. Heavy (H) and light (L) chain genes were isolated from MF23-1 to form a functional Fab molecule in the periplasm of Escherichia coli and on the surface of phage by using phagemid vector pComb3. The entire kappa L chain gene was used, but the H chain gene was amplified to 2 amino acids past cysteine 128 which is involved in interchain disulfide bond formation with the L chain. The truncated H chain associated with the L chain in the periplasm of E. coli to form a functional Fab molecule that bound in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay to O6 LPS. Therefore, the remainder of the CH1 past cysteine 128 is not essential for stable formation of the Fab portion of MF23-1. This recombinant Fab (r-Fab) was shown to be specific for the LPS of the most predominant clinical isolate, serotype O6, while no cross-reactivity was detected to the LPS of the other 19 remaining serotypes. This r-Fab was also expressed on the surface of filamentous phage upon addition of helper phage to recombinant E. coli containing phagemid. Recombinant phage from clones MT13 and MT24 bound specifically to O6 LPS in ELISA. These results represent an important step toward the design of therapeutic Abs to be used against P. aeruginosa infections.
Tout
N L
NL
Department of Microbiology, University of Guelph, Ontario, Canada.
Lam
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007167:Immunotherapy; D008070:Lipopolysaccharides / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011552:Pseudomonas Infections / Q000276:immunology / Q000628:therapy; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D012703:Serotyping
D000907:Antibodies, Bacterial; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D008070:Lipopolysaccharides; D011994:Recombinant Proteins