Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins
Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins
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DOI：
10.1002/biot.201500271
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发表时间：
2016-04-01
影响因子：
4.7
通讯作者：
Buyel, Johannes Felix
中科院分区：
文献类型：
--
作者：
Arfi, Zulfaquar Ahmad;Hellwig, Stephan;Buyel, Johannes Felix
在植物中生产生物制药蛋白需要高效的下游加工步骤，以去除杂质，如宿主细胞蛋白（HCP）和细菌在瞬时表达过程中产生的外源内毒素。因此，我们努力开发从植物提取物中去除内毒素的有效程序，并随后使用提取物产生检测广泛HCP的抗体。首先，我们耗尽了过量的蛋白质核酮糖-1，5-二磷酸羧化酶/加氧酶（RuBisCO），PEG沉淀达到了最佳效果，防止了针对该蛋白质的显性免疫反应。我们发现，与单独使用的单个血清相比，用耗尽前或耗尽后提取物免疫的兔血清的混合物检测到更多的HCP。我们还开发了一个强大的内毒素去除程序，使用多粘菌素B从野生型植物的提取物或纤维流过滤和EndoTrap蓝的组合渗透与根癌农杆菌的烟草植物。我们产生的抗体将是有用的质量和性能评估，在未来的过程中的发展和我们目前的方法可以很容易地转移到其他表达系统，使他们在植物分子农业领域的有用。
The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre-depleted or post-depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber-flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.