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Open AccessArticle

Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies

TU Wien, Institute of Chemical, Environmental and Bioscience Engineering, Research Area Biochemical Engineering, Gumpendorfer Straße 1a, 1060 Vienna, Austria
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Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2020, 21(13), 4625; https://doi.org/10.3390/ijms21134625
Received: 21 May 2020 / Revised: 19 June 2020 / Accepted: 26 June 2020 / Published: 29 June 2020
Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications. View Full-Text
Keywords: E. coli; inclusion bodies; recombinant horseradish peroxidase; refolding; hydrophobic interaction chromatography; reversed-phase high-performance liquid chromatograpy (RP-HPLC); size exclusion HPLC (SEC-HPLC) E. coli; inclusion bodies; recombinant horseradish peroxidase; refolding; hydrophobic interaction chromatography; reversed-phase high-performance liquid chromatograpy (RP-HPLC); size exclusion HPLC (SEC-HPLC)
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Humer, D.; Ebner, J.; Spadiut, O. Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies. Int. J. Mol. Sci. 2020, 21, 4625.

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