Centre de recherche en horticulture, Département de phytologie, Université Laval, Québec City, Québec, Canada.
PLoS One. 2013 Jul 23;8(7):e70203. doi: 10.1371/journal.pone.0070203. Print 2013.
The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ~3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.
在利用农杆菌浸润法开发出高产的瞬时蛋白表达系统后，临床上有用的蛋白在植物中的表达得到了增强。现在需要更多地了解宿主植物的发育和新陈代谢如何影响重组蛋白的数量和质量。内源性蛋白水解是决定植物中重组蛋白稳定性和产量的关键因素。在这里，我们研究了广泛使用的表达宿主烟草 Nicotiana benthamiana 叶片中半胱氨酸（C1A）和天冬氨酸（A1）蛋白酶谱与靶向细胞分泌途径的鼠 IgG，C5-1 的生产之间的关系。农杆菌浸润显著改变了 C1A 和 A1 蛋白酶沿叶片年龄梯度的分布，叶片年龄与全细胞和质外体蛋白提取物中蛋白水解水平之间存在相关性。番茄半胱氨酸酶抑制剂 SlCYS8 与 C5-1 共表达后，在通常的 6 天孵育期后，抗体产量增加了近 40%，达到每株植物约 3 毫克。在最老的叶片中，SlCYS8 没有表现出积极的效果，这与 C1A 蛋白酶活性的增加和抑制剂的表达率非常低相一致。相比之下，在较年轻的叶片中进行 8-10 天的孵育后，C5-1 的产量额外增加了 40%，此时 SlCYS8 的高表达得以维持。这些发现证实，共表达重组蛋白酶抑制剂是提高植物中重组蛋白产量的一种很有前途的策略，但通过解决叶片年龄和其他类别的蛋白酶的影响，进一步改进这种方法还有机会。
