Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana

Autor:	Ann E. Meyers, Suzanne M. Huddy, Brandon Weber, Edward P. Rybicki, Inga I. Hitzeroth
Rok vydání:	2018
Předmět:	0106 biological sciences 0301 basic medicine Agrobacterium Ion chromatography Nicotiana benthamiana infiltration 01 natural sciences Horseradish peroxidase Article Catalysis transient expression law.invention Inorganic Chemistry 03 medical and health sciences law Tobacco Physical and Theoretical Chemistry Codon Molecular Biology Spectroscopy horseradish peroxidase Agrobacterium tumefaciens recombinant protein chemistry.chemical_classification biology Organic Chemistry food and beverages General Medicine biology.organism_classification Recombinant Proteins Computer Science Applications 030104 developmental biology Enzyme Biochemistry chemistry biology.protein Recombinant DNA Specific activity 010606 plant biology & botany
Zdroj:	International Journal of Molecular Sciences; Volume 19; Issue 1; Pages: 115 International Journal of Molecular Sciences
ISSN:	1422-0067
DOI:	10.3390/ijms19010115
Popis:	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.
Databáze:	OpenAIRE
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