Centre de recherche et d'innovation sur les végétaux, Université Laval, Quebec City, QC, Canada.
Medicago Inc., Quebec City, QC, Canada.
Plant Biotechnol J. 2018 Nov;16(11):1928-1938. doi: 10.1111/pbi.12928. Epub 2018 May 2.
Cellular engineering approaches have been proposed to mitigate unintended proteolysis in plant protein biofactories, involving the design of protease activity-depleted environments by gene silencing or in situ inactivation with accessory protease inhibitors. Here, we assessed the impact of influenza virus M2 proton channel on host protease activities and recombinant protein processing in the cell secretory pathway of Nicotiana benthamiana leaves. Transient co-expression assays with M2 and GFP variant pHluorin were first conducted to illustrate the potential of proton export from the Golgi lumen to promote recombinant protein yield. A fusion protein-based system involving protease-sensitive peptide linkers to attach inactive variants of tomato cystatin SlCYS8 was then designed to relate the effects of M2 on protein levels with altered protease activities in situ. Secreted versions of the cystatin fusions transiently expressed in leaf tissue showed variable 'fusion to free cystatin' cleavage ratios, in line with the occurrence of protease forms differentially active against the peptide linkers in the secretory pathway. Variable ratios were also observed for the fusions co-expressed with M2, but the extent of fusion cleavage was changed for several fusions, positively or negatively, as a result of pH increase in the Golgi. These data indicating a remodelling of endogenous protease activities upon M2 expression confirm that the stability of recombinant proteins in the plant cell secretory pathway is pH-dependent. They suggest, in practice, the potential of M2 proton channel to modulate the stability of protease-susceptible secreted proteins in planta via a pH-related, indirect effect on host resident proteases.
细胞工程方法已被提出用于减轻植物蛋白生物工厂中意外的蛋白水解，涉及通过基因沉默或使用辅助蛋白酶抑制剂进行原位失活来设计蛋白酶活性耗尽的环境。在这里，我们评估了流感病毒 M2 质子通道对烟草原生质体叶片细胞分泌途径中宿主蛋白酶活性和重组蛋白加工的影响。首先，通过 M2 和 GFP 变体 pHluorin 的瞬时共表达实验来阐明质子从高尔基体腔中输出以促进重组蛋白产量的潜力。然后设计了一种基于融合蛋白的系统，涉及蛋白酶敏感肽接头，以连接番茄半胱氨酸蛋白酶抑制剂 SlCYS8 的无活性变体，以将 M2 对蛋白水平的影响与原位改变的蛋白酶活性联系起来。在叶片组织中瞬时表达的半胱氨酸蛋白酶融合物的分泌形式显示出可变的“融合物到游离半胱氨酸蛋白酶”切割比，与在分泌途径中针对肽接头具有不同活性的蛋白酶形式的发生一致。对于与 M2 共表达的融合物，也观察到了可变的比率，但由于高尔基体中 pH 值的增加，几种融合物的融合切割程度发生了变化，无论是正或负。这些数据表明，M2 表达后内源性蛋白酶活性的重塑证实了植物细胞分泌途径中重组蛋白稳定性是 pH 依赖性的。它们实际上表明，M2 质子通道通过对驻留蛋白酶的 pH 相关间接影响，有可能调节植物体内对蛋白酶敏感的分泌蛋白的稳定性。
