Soluble expression of horseradish peroxidase in Escherichia coli and its facile activation
Section snippets
Materials
All the chemicals and plant-derived HRP (Type II) were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise stated. Kits for cloning and DNA preparations were from Enzynomics (Daejeon, Korea) and CosmoGenetech (Seoul, Korea), respectively. Primers for the polymerase chain reactions were synthesized from CosmoGenetech.
Vector construction
The nucleotide sequences of primers and genes used in this study are presented as Supplementary information accompanying this paper. For the construction of
Soluble expression of HRP
Since refolding of HRP followed by the activation with heme and Ca2+ had been demonstrated (21), it seemed plausible that similar activation would be possible once the soluble expression of HRP was achieved. Directing synthesized HRP to the periplasm of E. coli for proper disulfide formation and thus for soluble expression could be one way to achieve the goal. It was shown that partially active HRP could be expressed in the periplasm of E. coli 21, 22. An independent study with a similar
Discussion
Here we successfully demonstrated a new strategy to produce active HRP in E. coli. Using the solubility-enhancing fusion partner, the target protein was overexpressed in a soluble form. Subsequent activation with Ca2+, hemin, and GSSG afforded the molar specific activity comparable or better than the plant-derived, commercial HRP. We routinely recovered more than 7.2 mg of purified PGK-HRP from 100 mL of culture, which could be improved by employing a bioreactor with optimized feeding
Acknowledgments
This work was supported by National Research Foundation of Korea (NRF-2017R1D1A1B03033679).
References (35)
- et al.
Horseradish peroxidase: a valuable tool in biotechnology
Biotechnol. Annu. Rev.
(2003) - et al.
Direct electron transfer in the system gold electrode-recombinant horseradish peroxidases
J. Electroanal. Chem.
(2001) - et al.
Ethanol biosensors based on alcohol oxidase
Biosens. Bioelectron.
(2005) - et al.
Oxidative activation of indole-3-acetic acids to cytotoxic species- a potential new role for plant auxins in cancer therapy
Biochem. Pharmacol.
(2001) - et al.
Oxidation of indole-3-acetic acid by horseradish peroxidase induces apoptosis in G361 human melanoma cells
Cell. Signal.
(2004) - et al.
Detoxification of phenolic solutions with horseradish peroxidase and hydrogen peroxide
Water Res.
(2002) Asymmetric enzymatic oxidoreductions in organic solvents
Curr. Opin. Biotechnol.
(2003)Horseradish peroxidase: a modern view of a classic enzyme
Phytochemistry
(2004)- et al.
Baculovirus expression and characterization of catalytically active horseradish peroxidase
Arch. Biochem. Biophys.
(1992) - et al.
High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture
Process Biochem.
(2005)
Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme
J. Biol. Chem.
A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli
J. Biotechnol.
Protein production by auto-induction in high density shaking cultures
Protein Expr. Purif.
Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli
Protein Expr. Purif.
Production strategies for active heme-containing peroxidases from E. coli inclusion bodies-a review
Biotechnol. Rep. (Amst.)
Hydrogen peroxide sensing with horseradish peroxidase-modified polymer single conical nanochannels
Anal. Chem.
Biosensor based on platinum chips for glucose determination
Anal. Chim. Acta
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