ArticlePloS one2015
Ultra-high-throughput screening of an in vitro-synthesized horseradish peroxidase displayed on microbeads using cell sorter.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 35 citations in OpenAlex.
- Peptide ligase-mediated display: A cell-free platform for tunable selection of affinity peptides.PNAS nexus · 2026Article
- Horseradish peroxidase as an electrochemical reporter protein for cell-free biosensors.Synthetic biology (Oxford, England) · 2026Article
- Cell-Free Gene Expression: Methods and Applications.Chemical reviews · 2025Review
- A sticky situation - simple method for rapid poissonian encapsulation of highly aggregation-prone microbeads in polydisperse emulsions.Frontiers in bioengineering and biotechnology · 2025Article
- Article
- A Multiplexed Cell-Free Assay to Screen for Antimicrobial Peptides in Double Emulsion Droplets.Angewandte Chemie (International ed. in English) · 2022Article
- High-throughput screening, next generation sequencing and machine learning: advanced methods in enzyme engineering.Chemical communications (Cambridge, England) · 2022Review
- Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases.Antioxidants (Basel, Switzerland) · 2022Article
- Vesicle-based cell-free synthesis of short and long unspecific peroxygenases.Frontiers in bioengineering and biotechnology · 2022Article
- Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey.Angewandte Chemie (International ed. in English) · 2021Review
- Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology.Frontiers in molecular biosciences · 2021Review
- Production of Recombinant Horseradish Peroxidase in an Engineered Cell-free Protein Synthesis System.Frontiers in bioengineering and biotechnology · 2021Article
- Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1.Protein engineering, design & selection : PEDS · 2020Article
- Accelerating the Production of Druggable Targets: Eukaryotic Cell-Free Systems Come into Focus.Methods and protocols · 2019Article
- Hierarchical design of artificial proteins and complexes toward synthetic structural biology.Biophysical reviews · 2018Review
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.