title: Ultra-high-throughput screening of an in vitro-synthesized horseradish peroxidase displayed on microbeads using cell sorter.
abstract: The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhu
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Mizoguchi
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kojima
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakano
mesh_terms: D000577:Amides / Q000378:metabolism; D002474:Cell-Free System; D004655:Emulsions; D057075:Enzyme Assays; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry / Q000295:instrumentation*; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D006735:Horseradish Peroxidase / Q000096:biosynthesis* / Q000737:chemistry; D008863:Microspheres; D008958:Models, Molecular; D009821:Oils / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013501:Surface-Active Agents / Q000494:pharmacology
chemical_list: D000577:Amides; D004655:Emulsions; D009821:Oils; D011993:Recombinant Fusion Proteins; D013501:Surface-Active Agents; D006735:Horseradish Peroxidase