Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha- l-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4- d + 20.5 g/l KNO₃) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.

中文翻译：

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提高芸苔毛根制造过程中重组生物制剂的产量

毛状根系统已被证明是重组蛋白生产的可行替代方案。对于顽固的蛋白质，最大限度地提高毛根培养物的生产力是必不可少的。本研究的目的是优化芜菁毛状根过程以分泌 α- l-艾杜糖醛酸酶 (IDUA)，这是一种具有医学价值的生物制品。该过程首先用表达 eGFP 的毛状根进行了优化。对于生物量优化，在改良的 Gamborg B5 培养基中实现了最高的生物量产量。对于分泌诱导，使用添加剂（1.5 g/l PVP + 1 mg/l 2,4- d  + 20.5 g/l KNO ₃) 与未诱导的对照相比，产生 3.4 倍的 eGFP 分泌。这些优化条件应用于表达 IDUA 的毛状根克隆，证实使用定义的添加剂组合时分泌的 IDUA 产量最高。IDUA 蛋白的功能，分泌的和细胞内的，通过酶活性测定得到证实。与非诱导培养基相比，使用优化的分泌培养基观察到 IDUA 活性增加了 > 150 倍。我们已经证明，我们的B. rapa rapa毛状根系统可以用来分泌顽固的蛋白质，说明毛状根在植物分子农业中的巨大潜力。