Phage display and bacterial expression of a recombinant Fab specific for Pseudomonas aeruginosa serotype O6 lipopolysaccharide

Abstract

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 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 C H 1 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.

Antibody Binding to the O-Specific Antigen of Pseudomonas aeruginosa O6 Inhibits Cell Growth

“…The variable region coding sequences of MAbs MF23-1, 7-4, and N1F10 were PCR amplified from total hybridoma RNA using degenerate primers, cloned, and sequenced using Sanger's method. The sequences of MAbs S20 and MF23-1 were compared with those previously published (10,49). Antibody variable-region sequences were analyzed using IMGT V-QUEST (50).…”

Synthesis of Ligand-Specific Phage-Display ScFv against the Herbicide Picloram by Direct Cloning from Hyperimmunized Mouse

“…Immunoglobulin genes have been successfully cloned from hybridoma cell lines that produce Abs specific to antigens ranging from human self-antigens () and pathogens ( , ) to small chemical molecules ( , ). Obtaining and maintaining a stable hybridoma cell line, however, is labor intensive and in some cases problematic ().…”

Bacterial Expression and Characterization of a Picloram-Specific Recombinant Fab for Residue Analysis

“…Total RNA (5 µg in 20 µL) was mixed with 11 µL of bulk firststrand reaction mix, 1 µL (0.2 µg/mL) of oligo-dT primer, and 1 µL of 200 mM dithiothreitol. The mixture was incubated at 37 °C for 1 h. Five microliters of the product was used as a template for the amplification of heavy (H) and light (L) genes by Polymerase Chain Reaction (PCR) using the conditions and universal primers described previously (Tout and Lam, 1997). By incorporating the SpeI and XhoI sites on the H chain * Author to whom correspondence should be addressed [telephone (519) 824-4120, ext.…”