Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia.
Centre for Research in Biotechnology for Agriculture, University of Malaya, 50603, Kuala Lumpur, Malaysia.
Mol Biotechnol. 2020 Apr;62(4):240-251. doi: 10.1007/s12033-020-00242-2.
In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.
在过去的十年中，人们对在植物中生产重组药物蛋白的兴趣大大增加，因为植物不携带哺乳动物病毒，具有经济竞争力、易于规模化，并且能够进行重组药物蛋白所需的复杂翻译后修饰。百脉根是一种重要的多年生覆盖作物，广泛种植于油棕和橡胶种植园作为地下覆盖物。作为豆科植物，它们具有高生物量，在其栖息地茁壮成长，并能固定氮。因此，百脉根是一种具有成本效益的作物，作为大规模生产重组蛋白的平台具有理想的特性。在这项研究中，我们通过真空辅助农杆菌浸润建立了一个在百脉根中瞬时生产重组蛋白的新平台。五周龄的百脉根植物用携带编码抗弓形虫免疫球蛋白（IgG）的质粒的根癌农杆菌进行真空浸润，在不同参数下，包括三叶叶位置效应、浸润后收获天数和使用的根癌农杆菌菌株。我们的结果表明，用根癌农杆菌菌株 GV3101 对百脉根植物进行真空浸润，在浸润后 2 天产生了最高浓度的异源蛋白在其底部三叶叶中。然后使用 Western blot 和 ELISA 分析纯化的抗弓形虫 IgG。结果表明，尽管从百脉根中纯化的抗弓形虫 IgG 存在结构异质性，但它的瞬时表达水平比模型平台拟南芥高两倍。这项研究为将百脉根建立为生产重组药物蛋白的潜在平台奠定了基础。
