Cargando…
Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies
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-bat...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369975/ https://www.ncbi.nlm.nih.gov/pubmed/32610584 http://dx.doi.org/10.3390/ijms21134625 |
_version_ | 1783560893415555072 |
---|---|
author | Humer, Diana Ebner, Julian Spadiut, Oliver |
author_facet | Humer, Diana Ebner, Julian Spadiut, Oliver |
author_sort | Humer, Diana |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7369975 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-73699752020-07-21 Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies Humer, Diana Ebner, Julian Spadiut, Oliver Int J Mol Sci Article 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. MDPI 2020-06-29 /pmc/articles/PMC7369975/ /pubmed/32610584 http://dx.doi.org/10.3390/ijms21134625 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Humer, Diana Ebner, Julian Spadiut, Oliver Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies |
title | Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies |
title_full | Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies |
title_fullStr | Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies |
title_full_unstemmed | Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies |
title_short | Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies |
title_sort | scalable high-performance production of recombinant horseradish peroxidase from e. coli inclusion bodies |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369975/ https://www.ncbi.nlm.nih.gov/pubmed/32610584 http://dx.doi.org/10.3390/ijms21134625 |
work_keys_str_mv | AT humerdiana scalablehighperformanceproductionofrecombinanthorseradishperoxidasefromecoliinclusionbodies AT ebnerjulian scalablehighperformanceproductionofrecombinanthorseradishperoxidasefromecoliinclusionbodies AT spadiutoliver scalablehighperformanceproductionofrecombinanthorseradishperoxidasefromecoliinclusionbodies |