Gengenbach B B, Müschen C R, Buyel J F
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany.
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany; Institute for Molecular Biotechnology, Worringerweg 1, RWTH Aachen University, 52074, Aachen, Germany.
Protein Expr Purif. 2018 Nov;151:46-55. doi: 10.1016/j.pep.2018.06.003. Epub 2018 Jun 14.
Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones. We therefore used a design of experiments approach to test the suitability of three plant-based systems for the expression of satisfactory quantities of recombinant PHACTR1, namely transient expression in tobacco (Nicotiana tabacum) BY-2 plant cell packs (PCPs), whole N. benthamiana leaves and BY-2 cell lysate (BYL). The highest yield was achieved using the BYL: up to 120 mg product kg biomass equivalent within 48 h of translation. This was 1.3-fold higher than transient expression in N. benthamiana together with the silencing inhibitor p19, and 6-fold higher than the PCP system. The presence of Triton X-100 in the extraction buffer increased the recovery of PHACTR1 by 2-200-fold depending on the conditions. PHACTR1 was incompatible with biomass blanching and was stable for less than 16 h in raw plant extracts. Purification using a DDK-tag proved inefficient whereas 15% purity was achieved by immobilized metal affinity chromatography.
心血管疾病是发病和死亡的常见原因，尤其是在工业化国家。人类磷酸酶和肌动蛋白调节剂1（PHACTR1）可能与这类疾病有关，但其精确的调节功能仍不清楚，因为它有大量潜在的相互作用伙伴。同样的现象使得这种蛋白质难以在哺乳动物细胞中表达，而且它还是一种内在无序的蛋白质，由于缺乏伴侣蛋白，在细菌中表达时可能会聚集。因此，我们采用实验设计方法来测试三种基于植物的系统表达足够量重组PHACTR1的适用性，即烟草（Nicotiana tabacum）BY-2植物细胞包（PCP）中的瞬时表达、本氏烟草全叶和BY-2细胞裂解物（BYL）。使用BYL获得了最高产量：在翻译48小时内，每千克生物量当量可产生高达120毫克的产物。这比在本氏烟草中与沉默抑制剂p19一起瞬时表达高出1.3倍，比PCP系统高出6倍。提取缓冲液中Triton X-100的存在使PHACTR1的回收率提高了2至200倍，具体取决于条件。PHACTR1与生物质热烫不相容，在未加工的植物提取物中稳定性不到16小时。使用DDK标签进行纯化效率不高，而通过固定化金属亲和色谱法可达到15%的纯度。
