Barbante Alessandra, Irons Sarah, Hawes Chris, Frigerio Lorenzo, Vitale Alessandro, Pedrazzini Emanuela
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Via Bassini 15, 20133 Milan, Italy.
Plant Biotechnol J. 2008 Aug;6(6):560-75. doi: 10.1111/j.1467-7652.2008.00342.x. Epub 2008 Apr 28.
The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.
重组疫苗在转基因植物中的积累水平具有蛋白质特异性，并且受到目标亚细胞区室的强烈影响。人类免疫缺陷病毒蛋白Nef（负因子）是抗病毒疫苗开发的一个有前景的靶点，它是一种胞质蛋白，在转基因烟草中积累水平较低，并且当导入分泌途径时甚至更不稳定，这可能是由于非胞质环境中的折叠缺陷所致。为了提高Nef的积累，开发了一种新策略，将该分子锚定在内质网（ER）膜的胞质面上。为此，将Nef序列与哺乳动物ER细胞色素b5的C末端结构域融合，细胞色素b5是一种长寿的尾锚定（TA）蛋白。在许多独立的转基因烟草植株中，这一方法持续使Nef的积累增加了三倍多。mRNA水平的实时聚合酶链反应和蛋白质脉冲追踪分析表明，增加并非由更高的转录水平引起，而是由增强的蛋白质稳定性所致。亚细胞分级分离和免疫细胞化学表明，Nef-TA积累在内质网膜上。哺乳动物或植物ER细胞色素b５的过表达导致形成堆叠的膜结构，这与之前在哺乳动物细胞中进行的类似实验中观察到的情况相同；然而，Nef-TA并未改变烟草细胞中的膜组织。最后，利用工程化的凝血酶切割位点，Nef可以在体外通过其尾锚被去除。这些结果为利用尾锚提高植物细胞质中外源蛋白的稳定性而不干扰细胞功能开辟了道路。
