title: Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.
abstract: A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kojima
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Mizoguchi
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Ota
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Jumpei
authors.initials: J
authors.lastname: Hata
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Homma
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhu
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Hitomi
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Electronic address: hnakano@agr.nagoya-u.ac.jp.
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakano
mesh_terms: D055162:Biocatalysis; D002474:Cell-Free System; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D008863:Microspheres; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D004800:Enzymes, Immobilized; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA; D010544:Peroxidases; C051129:manganese peroxidase; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins
keywords: Cell-free protein synthesis; Flow cytometry; Manganese peroxidase; OR consensus DNA; Transglutaminase 2; scCro