Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa.
Aaron Klug Centre for Imaging Analysis, University of Cape Town, Rondebosch 7701, South Africa.
Int J Mol Sci. 2018 Jan 1;19(1):115. doi: 10.3390/ijms19010115.
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 (); 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 codon-adapted full-length isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the gene were synthesized, and their expression in was evaluated using an -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 in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.
辣根过氧化物酶（HRP）是一种在分子生物学和诊断产品行业中使用的商业上重要的试剂酶。它通常从辣根（）的根部中纯化得到；然而，这种作物仅在季节性供应，产量不稳定且通常较低，并且该产品是同工酶的混合物。在瞬时转化的烟草中进行高水平表达可能是解决这些问题的一种方法。在这项研究中，合成了一种经过密码子适应的全长同工酶基因，以及 C 末端截短的和 N 和 C 末端都截短的 基因，并使用 -介导的瞬时表达系统评估了它们在烟草中的表达。还评估了与沉默抑制子（NSs）构建体共浸润对 HRP C 表达水平的影响。使用全长或 C 末端截短的 HRP C 构建体始终可以获得最高水平的 HRP C。通过离子交换色谱法对 HRP C 进行纯化，总体收率为 54%，Reinheitszahl 值>3，比活度为 458 U/mg。仅在五天内即可在烟草中大量生产 HRP C，为生产这种具有商业重要性的酶提供了一种替代、可行且可扩展的系统。
