Construction and characterization of two anti-sweetener single chain antibodies using radioligand binding, fluorescence and circular dichroism spectroscopy

• Published in: Journal of molecular recognition 1999-07, Vol.12 (4), p.258-266
• Main Authors: Pledger, David W., Brodnicki, Thomas C., Graham, Brent L., Tetin, Sergey, Kranz, David M., Linthicum, D. Scott
• Format: Article
• Language: English
• Subjects: Acetates - immunology; Amino Acid Sequence; Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; antibody; Antibody Affinity; Antigen-Antibody Reactions; Circular Dichroism; Cloning, Molecular; Guanidines - immunology; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - immunology; Immunoglobulin Fragments - chemistry; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - immunology; Mice; Molecular Sequence Data; Molecular Structure; monoclonal; Protein Structure, Secondary; Radioligand Assay; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - immunology; single-chain; Spectrometry, Fluorescence; spectroscopy; sweetener; Sweetening Agents
• ISSN: 0952-3499; 1099-1352
• DOI: 10.1002/(SICI)1099-1352(199907/08)12:4<258::AID-JMR464>3.0.CO;2-A

Two single‐chain antibodies (scFv) that bind the superpotent sweetener ligand, NC‐174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, κ) and NC10.14 (IgG, λ). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25‐amino‐acid polypeptide linker, and expressing them in E. coli using the pET‐11a system. The recombinant proteins were purified using Ni2+–NTA–agarose by virtue of a hexahistidine sequence introduced to the C‐terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far‐UV CD spectra of both scFv possessed predominantly β character, as did those of the Fab, and the near‐UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent. Copyright © 1999 John Wiley & Sons, Ltd.