title: Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator.
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.
authors.affiliation: a Donnelly Center for Cellular and Biomolecular Research , University of Toronto , Toronto , Ontario , Canada.
authors.forename: Amandeep K
authors.initials: AK
authors.lastname: Gakhal
authors.affiliation: b Department of Biochemistry and Biophysics , Cystic Fibrosis Treatment and Research Center, University of North Carolina , Chapel Hill , NC , USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Jensen
authors.affiliation: c Program in Molecular Structure & Function , The Hospital for Sick Children , Toronto , ON , Canada.|d Department of Biochemistry , University of Toronto, Toronto , ON , Canada.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Bozoky
authors.affiliation: e Department of Physiology and Biochemistry , McGill University , Montreal , QC , Canada.
authors.forename: Ariel
authors.initials: A
authors.lastname: Roldan
authors.affiliation: e Department of Physiology and Biochemistry , McGill University , Montreal , QC , Canada.
authors.forename: Gergely L
authors.initials: GL
authors.lastname: Lukacs
authors.affiliation: c Program in Molecular Structure & Function , The Hospital for Sick Children , Toronto , ON , Canada.|d Department of Biochemistry , University of Toronto, Toronto , ON , Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Forman-Kay
authors.affiliation: b Department of Biochemistry and Biophysics , Cystic Fibrosis Treatment and Research Center, University of North Carolina , Chapel Hill , NC , USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Riordan
authors.affiliation: a Donnelly Center for Cellular and Biomolecular Research , University of Toronto , Toronto , Ontario , Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D000915:Antibody Affinity; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D019151:Peptide Library; D010766:Phosphorylation; D000072417:Protein Domains; D015202:Protein Engineering; D017510:Protein Folding; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: Cystic fibrosis conductance regulator; membrane protein; phage display; protein engineering; synthetic antibody