根据先进的治疗药物产品(ATMP)和细胞农业选择,医疗保健和营养正面临范式转变。这两种选择都严重依赖于某种动物细胞培养,例如自体干细胞。这些文化需要各种生长因子,如白细胞介素-6和8(IL-6/8),在一个纯粹的,安全和可持续的形式,可以以可扩展的方式提供。植物似乎非常适合这项任务,因为小蛋白的纯化可以很容易地通过膜分离来实现。人/动物病原体不会在植物中复制,可以使用门内农业或农业实践扩大生产。这里,我们通过首先优化IL-6/8在烟草中翻译的密码子使用来说明这种能力。,以及在高通量植物细胞包(PCP)测定中测试非翻译区和靶向不同亚细胞区室的产物对表达的影响。在叶绿体中,IL-6累积达6.9±3.8(SD,n=2)和14.4±7.4mgkg-1(SD,在IL-8的情况下观察到n=5)。当将IL-8表达转移到整个植物中时,积累量为12.3±1.5mgkg-1(SD,n=3)。提取和澄清后,使用两阶段方法纯化IL-8,所述两阶段方法包括具有100kDa和10kDa截留膜的超滤/渗滤步骤,随后是IMAC精制步骤。纯度,收率和回收率为97.8%,6.6mgkg-1和38%,分别。我们评估了拟议的纯化方法去除内毒素的能力,以确保植物制造的生长因子与细胞培养物的相容性。
Healthcare and nutrition are facing a paradigm shift in light of advanced therapy medicinal products (ATMPs) and cellular agriculture options respectively. Both options heavily rely on some sort of animal cell culture, e.g. autologous stem cells. These cultures require various growth factors, such as interleukin-6 and 8 (IL-6/8), in a pure, safe and sustainable form that can be provided in a scalable manner. Plants seem well suited for this task because purification of small proteins can be readily achieved by membrane separation, human/animal pathogens do not replicate in plants and production can be scaled up using in-door farming or agricultural practices. Here, we illustrate this capacity by first optimizing the codon usage of IL-6/8 for translation in Nicotiana spp., as well as testing the effect of untranslated regions and product targeting to different sub-cellular compartments on expression in a high-throughput plant cell pack (PCP) assay. In the chloroplast, IL-6 accumulated up to 6.9±3.8 (SD, n=2) and 14.4±7.4 mg kg-1 (SD, n=5) were observed in case of IL-8. When transferring IL-8 expression into whole plants, accumulation was 12.3±1.5 mg kg-1 (SD, n=3). After extraction and clarification, IL-8 was purified using a two-stage process consisting of an ultrafiltration/diafiltration step with 100 kDa and 10 kDa cut off membranes followed by an IMAC polishing step. The purity, yield and recovery were 97.8%, 6.6 mg kg-1 and 38%, respectively. We evaluated the ability of the proposed purification process to remove endotoxins to ensure the compatibility of plant-made growth factors with cell culture.