DataSheet_1_Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures.pdf

The COVID-19 pandemic, caused by the worldwide spread of SARS-CoV-2, has prompted the scientific community to rapidly develop efficient and specific diagnostics and therapeutics. A number of avenues have been explored, including the manufacture of COVID-related proteins to be used as reagents for diagnostics or treatment. The production of RBD and Spike proteins was previously achieved in eukaryotic cells, mainly mammalian cell cultures, while the production in microbial systems has been unsuccessful until now. Here we report the effective production of SARS-CoV-2 proteins in two plant model systems. We established transgenic tobacco BY-2 and Medicago truncatula A17 cell suspension cultures stably producing the full-length Spike and RBD recombinant proteins. For both proteins, various glycoforms were obtained, with higher yields in Medicago cultures than BY-2. This work highlights that RBD and Spike can be secreted into the culture medium, which will impact subsequent purification and downstream processing costs. Analysis of the culture media indicated the presence of the high molecular weight Spike protein of SARS-CoV-2. Although the production yields still need improvement to compete with mammalian systems, this is the first report showing that plant cell suspension cultures are able to produce the high molecular weight Spike protein. This finding strengthens the potential of plant cell cultures as production platforms for large complex proteins.

由严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）全球大流行引发的新型冠状病毒肺炎（COVID-19）疫情，促使全球科研界快速研发高效且具有特异性的诊断方法与治疗手段。目前科研团队已探索了多种技术路径，包括制备用于诊断或治疗的新冠相关蛋白试剂。此前，受体结合域（Receptor Binding Domain, RBD）与刺突蛋白（Spike）已在真核细胞（主要为哺乳动物细胞培养体系）中成功表达，但截至目前，微生物表达体系仍无法实现这两类蛋白的有效生产。本研究首次报道了在两种植物模型系统中高效表达SARS-CoV-2蛋白的成果：我们构建了可稳定表达全长刺突蛋白与受体结合域重组蛋白的转基因烟草BY-2悬浮细胞系，以及蒺藜苜蓿（Medicago truncatula）A17悬浮细胞系。两类蛋白均获得了多种糖型，且在蒺藜苜蓿培养体系中的蛋白产量高于烟草BY-2体系。本研究证实，RBD与刺突蛋白可被分泌至细胞培养基中，这将对后续纯化工艺及下游处理成本产生直接影响。对细胞培养基的分析结果显示，其中存在SARS-CoV-2的高分子量刺突蛋白。尽管当前蛋白产量仍需提升，方能与哺乳动物表达体系形成竞争，但本研究首次证实植物细胞悬浮培养体系可生产高分子量刺突蛋白。这一发现进一步强化了植物细胞培养作为大型复杂蛋白生产平台的应用潜力。