Ultra-high-throughput screening of an in vitro-synthesized horseradish peroxidase displayed on microbeads using cell sorter

• Published in: PloS one Vol. 10; no. 5; p. e0127479
• Main Authors: Zhu, Bo, Mizoguchi, Takuro, Kojima, Takaaki, Nakano, Hideo
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20.05.2015; Public Library of Science (PLoS)
• Subjects: Amides - metabolism; Amino acids; Binding; Biochemistry; Bioremediation; Biotechnology; Calcium; Calcium ions; Cell-Free System; Cellulose; Chemical bonds; Cytometry; Deoxyribonucleic acid; DNA; E coli; Emulsions; Enzyme Assays; Enzymes; Escherichia coli; Escherichia coli - metabolism; Fine chemicals; Flow cytometry; Flow Cytometry - instrumentation; Fluorescence; Heme; Hemin; Hexadecane; High-throughput screening; High-Throughput Screening Assays - methods; Horseradish; Horseradish peroxidase; Horseradish Peroxidase - biosynthesis; Horseradish Peroxidase - chemistry; Hydrogen; Hydrogen peroxide; Kinases; Laboratories; Medical research; Microspheres; Models, Molecular; Mutants; Nanoparticles; Nucleotide sequence; Oils - pharmacology; Organic compounds; Peroxidase; Protein biosynthesis; Protein Biosynthesis - drug effects; Protein synthesis; Proteins; Recombinant Fusion Proteins - metabolism; Screening; Solubility; Surface-Active Agents - pharmacology; Water droplets; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0127479

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.