Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana
AbstractHorseradish 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
- Supplementary File 1:
PDF-Document (PDF, 253 KB)
Share & Cite This Article
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.
Huddy SM, Hitzeroth II, Meyers AE, Weber B, Rybicki EP. Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana. International Journal of Molecular Sciences. 2018; 19(1):115.Chicago/Turabian Style
Huddy, Suzanne M.; Hitzeroth, Inga I.; Meyers, Ann E.; Weber, Brandon; Rybicki, Edward P. 2018. "Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana." Int. J. Mol. Sci. 19, no. 1: 115.
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.