Jiang Min-Chao, Hu Chung-Chi, Hsu Wei-Li, Hsu Tsui-Ling, Lin Na-Sheng, Hsu Yau-Heiu
Ph.D. Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung, Taiwan.
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
Front Plant Sci. 2020 Nov 12;11:594758. doi: 10.3389/fpls.2020.594758. eCollection 2020.
Plant viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in . In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in . The results revealed that the fusion of a native extensin secretory signal (SS) to the N-terminal of mIFNγ (designated SS mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of leaf tissues. The addition of 10 units of 'Ser-Pro' motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SS mIFNγ (designated SS mIFNγ(SP)) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SS mIFNγ, respectively. The purified soluble SS mIFNγ(SP) protein was glycosylated with abundant complex-type N-glycan attached to residues N and N, and exhibited biological activity against and replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic , which produced secreted SS mIFNγ(SP) protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags.
植物病毒可作为表达载体，用于在植物中高效生产药用蛋白。然而，目标蛋白的下游加工和翻译后修饰仍然是主要挑战。我们之前开发了一种源自水稻条纹花叶病毒（BaMV）的表达系统，命名为pKB19，并证明了其在水稻中生产人成熟干扰素γ（mIFNγ）的适用性。在本研究中，我们旨在通过引入各种植物来源的信号肽来提高可溶性和分泌型mIFNγ的产量。此外，我们分析了改良的pKB19表达系统在水稻中表达的mIFNγ的糖基化模式和生物学活性。结果表明，将天然水稻伸展蛋白分泌信号（SS）与mIFNγ的N端融合（命名为SS mIFNγ），导致水稻叶片组织的细胞内（IC）或质外体洗液（AWF）组分中蛋白质的积累水平最高。在SS mIFNγ的C端添加10个单位的羟脯氨酸-O-糖基化肽（HypGPs）的“Ser-Pro”基序（命名为SS mIFNγ(SP)），其溶解度分别比mIFNγ和SS mIFNγ提高了近2.7倍和1.5倍。纯化的可溶性SS mIFNγ(SP)蛋白被大量复杂型N-聚糖糖基化，连接在N和N残基上，并在人细胞培养系统中表现出针对病毒和病毒复制的生物学活性。此外，从转基因水稻建立了悬浮细胞培养物，其产生的分泌型SS mIFNγ(SP)蛋白适用于下游加工。这些结果表明，基于BaMV的载体系统通过引入适当的融合标签，作为生产治疗性蛋白的有用替代方法具有适用性。
