title: Photoactivable antibody binding protein: site-selective and covalent coupling of antibody.
abstract: Here we report new photoactivable antibody binding proteins, which site-selectively capture antibodies and form covalent conjugates with captured antibodies upon irradiation. The proteins allow the site-selective tagging and/or immobilization of antibodies with a highly preferred orientation and omit the need for prior antibody modifications. The minimal Fc-binding domain of protein G, a widely used antibody binding protein, was genetically and chemically engineered to contain a site-specific photo cross-linker, benzophenone. In addition, the domain was further mutated to have an enhanced Fc-targeting ability. This small engineered protein was successfully cross-linked only to the Fc region of the antibody without any nonspecific reactivity. SPR analysis indicated that antibodies can be site-selectively biotinylated through the present photoactivable protein. Furthermore, the system enabled light-induced covalent immobilization of antibodies directly on various solid surfaces, such as those of glass slides, gold chips, and small particles. Antibody coupling via photoactivable antibody binding proteins overcomes several limitations of conventional approaches, such as random chemical reactions or reversible protein binding, and offers a versatile tool for the field of immunosensors.
authors.affiliation: BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Daejeon 305-600, Korea.
authors.forename: Yongwon
authors.initials: Y
authors.lastname: Jung
authors.forename: Jeong Min
authors.initials: JM
authors.lastname: Lee
authors.forename: Jung-won
authors.initials: JW
authors.lastname: Kim
authors.forename: Jeongwon
authors.initials: J
authors.lastname: Yoon
authors.forename: Hyunmin
authors.initials: H
authors.lastname: Cho
authors.forename: Bong Hyun
authors.initials: BH
authors.lastname: Chung
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry* / Q000528:radiation effects; D001426:Bacterial Proteins / Q000737:chemistry* / Q000528:radiation effects; D001577:Benzophenones / Q000138:chemical synthesis / Q000737:chemistry; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000528:radiation effects; D008969:Molecular Sequence Data; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D001577:Benzophenones; C034284:IgG Fc-binding protein, Streptococcus; D007141:Immunoglobulin Fc Fragments; D001710:Biotin; C047723:benzophenone