Expression and characterization of an enantioselective antigen-binding fragment directed against α-amino acids

• Design and expression of a stereoselective Fab.
• Amplification of variable heavy and light chain domains from mRNA of parent monoclonal antibody.
• Construction of constant domains based on database searches.
• Overlap extension PCR to produce Fab.
• Fab binding properties comparable to those displayed by the whole parent monoclonal antibody.

This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-l-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to l-amino acids but not to recognize the corresponding d-enantiomers.