Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford, UK.
Chemische Biologie, Zentrum für Medizinische Biotechnologie, Fakultät für Biologie, Universität Duisburg-Essen, Universitätsstr, Essen, Germany.
Plant Biotechnol J. 2018 Oct;16(10):1797-1810. doi: 10.1111/pbi.12916. Epub 2018 May 24.
Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α-Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose-dependent. Activity-based protein profiling (ABPP) revealed that the activities of papain-like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.
农杆菌浸润的本氏烟是一种灵活且可扩展的重组蛋白 (RP) 生产平台，但由于植物蛋白酶会降解 RP，其巨大潜力受到了限制。在这里，我们在农杆菌浸润的本氏烟叶片中测试了 29 种候选蛋白酶抑制剂 (PI)，以提高三种不相关的 RP 的积累：糖基酶α-半乳糖苷酶；糖激素促红细胞生成素 (EPO)；和 IgG 抗体 VRC01。在以前描述的增强 RP 积累的 PI 中，我们发现只有半胱氨酸蛋白酶抑制剂 SlCYS8 是有效的。我们还确定了另外三种新的、不相关的 PI 可以增强 RP 的积累：N. benthamiana NbPR4、NbPot1 和人 HsTIMP，它们分别被报道抑制半胱氨酸、丝氨酸和金属蛋白酶。值得注意的是，所有三种 RP 的积累都被每种 PI 以相似的方式增强，这表明不相关 RP 的降解机制遵循共同的途径。抑制功能 HsTIMP 和 SlCYS8 是增强 RP 积累所必需的，这表明它们的靶蛋白酶可能会降解 RP。不同的 PI 可累加增强 RP 积累，但每种 PI 的效果都是剂量依赖性的。基于活性的蛋白质谱 (ABPP) 显示，新 PI 表达后，叶片中木瓜蛋白酶样 Cys 蛋白酶 (PLCPs)、丝氨酸水解酶 (SHs) 或液泡加工酶 (VPEs) 的活性不受影响，而 SlCYS8 表达特异性仅抑制 PLCP 活性。定量蛋白质组学表明，三种新的 PI 对农杆菌浸润的组织有相似的影响，它们都增加了免疫反应。NbPR4、NbPot1 和 HsTIMP 可用于研究植物蛋白酶并提高分子农业中 RP 的积累。
