Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in <i>Escherichia coli</i> Yeolin Lee Jiyun Jeong Gabi Lee Jeong Hee Moon Myung Kyu Lee 10.1021/acs.analchem.6b02071.s001 https://acs.figshare.com/articles/journal_contribution/Covalent_and_Oriented_Surface_Immobilization_of_Antibody_Using_Photoactivatable_Antibody_Fc-Binding_Protein_Expressed_in_i_Escherichia_coli_i_/3827514 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 <i>E. coli</i>, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native <i>E. coli</i> system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph <i>E. coli</i> 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. 2016-08-30 00:00:00 Escherichia coli Fc-specific antibody binding proteins UV support protein expression MRS 5m Coexpression photoactivatable FcBP overexpression Photoactivatable Antibody Fc-Binding Protein Oriented Surface Immobilization report novel photoactivatable FcBPs methionyl tRNA synthetase photo-cross-linked antibody arrays sera N-terminal cysteine residues MRS 5m proteins photoactivatable FcBPs 6 xHis tag