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.
Highlights
The C1a isoenzyme of horseradish peroxidase (HRP) is the most abundant isoenzyme derived from horseradish (Armoracia rusticana)
By optimizing the reaction conditions and by adding disulfide bond isomerase, we successfully synthesized manganese peroxidase (MnP) with a higher specific activity than the commercial wild type enzyme, thereby suggesting that cell-free protein synthesis (CFPS) could be used as a preparative method for the efficient synthesis of disulfide bond-containing metalloenzymes, such as HRP [23]
Because HRP is a heme-containing protein and because the heme group is involved in electron transfer during catalysis, varying amounts of hemin were added into the cell-free synthesis reaction
Summary
The C1a isoenzyme of horseradish peroxidase (HRP) is the most abundant isoenzyme derived from horseradish (Armoracia rusticana). By optimizing the reaction conditions and by adding disulfide bond isomerase, we successfully synthesized MnP with a higher specific activity than the commercial wild type enzyme, thereby suggesting that CFPS could be used as a preparative method for the efficient synthesis of disulfide bond-containing metalloenzymes, such as HRP [23].
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