Département de phytologie, Université Laval, Pavillon des Services (INAF), Québec, QC, Canada.
Plant Biotechnol J. 2012 Jan;10(1):83-94. doi: 10.1111/j.1467-7652.2011.00643.x. Epub 2011 Sep 5.
Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ∼45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway.
重组蛋白在植物细胞分泌途径中面临着主要的限制，包括蛋白水解处理会破坏其结构完整性。在这里，我们展示了蛋白酶抑制剂作为重组蛋白向叶片质外体迁移的原位稳定剂的潜力。评估了拟南芥、水稻和烟草原生质体的基因组数据，以确定细胞分泌途径中蛋白酶家族的相对发生率。然后使用模式平台烟草进行瞬时表达分析，以测试蛋白酶抑制剂稳定靶向质外体的蛋白的效率。目前的基因组数据表明，在分泌途径中存在来自几个家族的蛋白酶，包括 A1 和 A22 天冬氨酸蛋白酶；C1A 和 C13 半胱氨酸蛋白酶；以及 S1、S8 和 S10 丝氨酸蛋白酶。体外蛋白酶分析证实了 N. benthamiana 叶片中存在各种蛋白酶，特别是 A1 和 S1 型活性优先沉积在质外体中。因此，A1/S1 蛋白酶抑制剂，番茄组织蛋白酶 D 抑制剂（SlCDI）的瞬时表达和分泌，在细胞区室中改变了 A1 和 S1 蛋白酶的活性，同时使叶片质外体蛋白含量增加了约 45%，并提高了在质外体中共分泌的一种鼠源性诊断抗体 C5-1 的积累。C1A 和 C13 半胱氨酸蛋白酶抑制剂 SlCYS9 对质外体蛋白酶和蛋白含量没有影响，但在植物体内稳定了 C5-1，可能在分泌途径的上游。这些数据总体上证实了蛋白酶抑制剂在原位保护重组蛋白沿植物细胞分泌途径方面的潜力。
