Next Article in Journal
Sphingolipids in Ventilator Induced Lung Injury: Role of Sphingosine-1-Phosphate Lyase
Next Article in Special Issue
Fusion Proteins of NKG2D/NKG2DL in Cancer Immunotherapy
Previous Article in Journal
The Role of Microglia in Diabetic Retinopathy: Inflammation, Microvasculature Defects and Neurodegeneration
Previous Article in Special Issue
Simple Purification of Nicotiana benthamiana-Produced Recombinant Colicins: High-Yield Recovery of Purified Proteins with Minimum Alkaloid Content Supports the Suitability of the Host for Manufacturing Food Additives
Article Menu
Issue 1 (January) cover image

Export Article

Open AccessArticle
Int. J. Mol. Sci. 2018, 19(1), 115; https://doi.org/10.3390/ijms19010115

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana

1
Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa
2
Aaron Klug Centre for Imaging Analysis, University of Cape Town, Rondebosch 7701, South Africa
3
Institute for Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
*
Author to whom correspondence should be addressed.
Received: 30 November 2017 / Revised: 24 December 2017 / Accepted: 30 December 2017 / Published: 1 January 2018
(This article belongs to the Special Issue Recombinant Proteins)
Full-Text   |   PDF [1437 KB, uploaded 1 January 2018]   |  

Abstract

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme. View Full-Text
Keywords: horseradish peroxidase; transient expression; Nicotiana benthamiana; Agrobacterium tumefaciens; infiltration; recombinant protein horseradish peroxidase; transient expression; Nicotiana benthamiana; Agrobacterium tumefaciens; infiltration; recombinant protein
Figures

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

Supplementary material

SciFeed

Share & Cite This Article

MDPI and ACS Style

Huddy, S.M.; Hitzeroth, I.I.; Meyers, A.E.; Weber, B.; Rybicki, E.P. Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana. Int. J. Mol. Sci. 2018, 19, 115.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top