title: Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli.
abstract: Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photo-cross-linked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photo-cross-linked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides, and nanobeads, which are required for development of sensitive immunosensors.
authors: Hazards Monitoring Bionano Research Center, ‡Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, and §Department of Nanobiotechnology, University of Science and Technology , 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors: Yeolin
authors: Y
authors: Lee
authors: Hazards Monitoring Bionano Research Center, ‡Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, and §Department of Nanobiotechnology, University of Science and Technology , 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors: Jiyun
authors: J
authors: Jeong
authors: Hazards Monitoring Bionano Research Center, ‡Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, and §Department of Nanobiotechnology, University of Science and Technology , 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors: Gabi
authors: G
authors: Lee
authors: Hazards Monitoring Bionano Research Center, ‡Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, and §Department of Nanobiotechnology, University of Science and Technology , 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors: Jeong Hee
authors: JH
authors: Moon
authors: Hazards Monitoring Bionano Research Center, ‡Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, and §Department of Nanobiotechnology, University of Science and Technology , 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
authors: Myung Kyu
authors: MK
authors: Lee
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000941:Antigens / Q000097:blood / Q000276:immunology; D001386:Azides / Q000737:chemistry / Q000528:radiation effects; D002352:Carrier Proteins / Q000737:chemistry / Q000528:radiation effects*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000528:radiation effects; D004926:Escherichia coli / Q000276:immunology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000528:radiation effects*; D006801:Humans; D007118:Immunoassay; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000276:immunology; D008715:Methionine / Q000031:analogs & derivatives / Q000737:chemistry / Q000528:radiation effects; D008718:Methionine-tRNA Ligase / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D001386:Azides; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D007141:Immunoglobulin Fc Fragments; D008715:Methionine; D008718:Methionine-tRNA Ligase