Department of Agrobiotechnology, Agricultural and Environmental Faculty, University of Rostock, Rostock, Germany.
PLoS One. 2012;7(12):e53023. doi: 10.1371/journal.pone.0053023. Epub 2012 Dec 28.
We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T(0) transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T₂ generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.
我们评估了转基因烟草植物作为替代大肠杆菌生产重组人补体因子 5a（C5a）的方法。C5a 以前从未在植物中表达过，并且在其天然形式下在体内非常不稳定，因此有必要建立最合适的亚细胞靶向策略。我们使用强而组成型的 CaMV 35S 启动子驱动转基因表达，并比较了三种不同的亚细胞隔室。当靶向质外体（0.0002%TSP）或内质网（0.0003%TSP）时，C5a 在 T(0)转基因植物中的产量相对于总可溶性蛋白（TSP）的比例较低，但当靶向液泡时，产量高一个数量级（0.001%TSP）。通过常规育种（在 T₂代中达到 0.014%TSP）可以提高产量。当靶向质外体时，C5a 在种子和叶片中的积累水平相同，但当靶向 ER 或液泡时，C5a 的含量高 1.7 倍，尽管在表现更好的系中，这种差异不太明显。当按每克转基因植物组织的鲜重计算产量时，液泡靶向策略在种子中明显更有效，每克鲜重种子中达到 35.8 µg C5a，而每克鲜重叶片中达到 10.62 µg C5a。使用 MagnICON 载体在 N. benthamiana 中瞬时表达 C5aER 和 C5aVac 分别达到 0.2%和 0.7%的 TSP，但伴随着细胞毒性作用并诱导叶片衰老。植物提取物的 Western blot 显示与相应糖基化天然蛋白匹配的条带，生物测定表明重组 C5a 具有生物活性。
