title: An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein.
abstract: Using phage-display technology, a novel binding protein (Z-affibody) showing selective binding to the RSV (Long strain) G protein was selected from a combinatorial library of a small alpha-helical protein domain (Z), derived from staphylococcal protein A (SPA). Biopanning of the Z-library against a recombinant fusion protein comprising amino acids 130-230 of the G protein from RSV-subgroup A, resulted in the selection of a Z-affibody (Z(RSV1)) which showed G protein specific binding. Using biosensor technology, the affinity (K(D)) between Z(RSV1) and the recombinant protein was determined to be in the micromolar range (10(-6) M). Interestingly, the Z(RSV1) affibody was demonstrated to also recognize the partially (54%) homologous G protein of RSV subgroup B with similar affinity. Using different recombinant RSV G protein derived fragments, the binding was found to be dependent on the presence of the cysteinyl residues proposed to be involved in the formation of an intramolecular disulfide-constrained loop structure, indicating a conformation-dependent binding. Results from epitope mapping studies, employing a panel of monoclonal antibodies directed to different RSV G protein subfragments, suggest that the Z(RSV1) affibody binding site is located within the region of amino acids 164-186 of the G protein. This region contains a 13 amino acid residue sequence which is totally conserved between subgroups A and B of RSV and extends into the cystein loop region (amino acids 173-186). The potential use of the RSV G protein-specific Z(RSV1) affibody in diagnostic and therapeutic applications is discussed.
authors.affiliation: Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Hansson
authors.forename: J
authors.initials: J
authors.lastname: Ringdahl
authors.forename: A
authors.initials: A
authors.lastname: Robert
authors.forename: U
authors.initials: U
authors.lastname: Power
authors.forename: L
authors.initials: L
authors.lastname: Goetsch
authors.forename: T N
authors.initials: TN
authors.lastname: Nguyen
authors.forename: M
authors.initials: M
authors.lastname: Uhlén
authors.forename: S
authors.initials: S
authors.lastname: Ståhl
authors.forename: P A
authors.initials: PA
authors.lastname: Nygren
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000737:chemistry* / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D015374:Biosensing Techniques; D018604:Epitope Mapping; D015707:HN Protein; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000276:immunology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000276:immunology; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry / Q000276:immunology*; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D015707:HN Protein; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G