title: Construction and characterization of two anti-sweetener single chain antibodies using radioligand binding, fluorescence and circular dichroism spectroscopy.
abstract: Two single-chain antibodies (scFv) that bind the superpotent sweetener ligand, NC-174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, kappa) and NC10.14 (IgG, lambda). 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 Ni(2+)-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 beta 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.
authors.affiliation: Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843, USA.
authors.forename: D W
authors.initials: DW
authors.lastname: Pledger
authors.forename: T C
authors.initials: TC
authors.lastname: Brodnicki
authors.forename: B L
authors.initials: BL
authors.lastname: Graham
authors.forename: S
authors.initials: S
authors.lastname: Tetin
authors.forename: D M
authors.initials: DM
authors.lastname: Kranz
authors.forename: D S
authors.initials: DS
authors.lastname: Linthicum
mesh_terms: D000085:Acetates / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006146:Guanidines / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D013050:Spectrometry, Fluorescence; D013549:Sweetening Agents
chemical_list: D000085:Acetates; D000911:Antibodies, Monoclonal; D006146:Guanidines; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D013549:Sweetening Agents; C055966:immunoglobulin Fv; C083643:NC 174