CRH/INAF, Pavillon des Services (INAF), Université Laval, Québec, QC, Canada.
Plant Biotechnol J. 2010 Feb;8(2):142-54. doi: 10.1111/j.1467-7652.2009.00470.x. Epub 2009 Dec 28.
We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in-built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359-368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI-expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%-40% depending on the light regime. Out of approximately 700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were up-regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase-PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two- to threefold in SlCDI-expressing lines. These observations, along with the unrelated biological functions assigned to MS-identified proteins up-regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human alpha(1)-antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by approximately 2.5-fold in SlCDI-expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.
我们之前曾报道过番茄组织蛋白酶 D 抑制剂 (SlCDI) 作为一种内建稳定因子的潜力，可用于保护转基因植物叶片粗提物中的重组蛋白（植物生物技术杂志 4, 359-368）。在此，我们记录了 SlCDI 用于原位保护马铃薯叶片中蛋白质的潜力。对照和 SlCDI 表达马铃薯品系的总蛋白分析表明，slcdi 转基因表达对叶片蛋白含量有积极影响，平均相对增加 35%-40%，具体取决于光照条件。在 2-D 凝胶上检测到的大约 700 种蛋白质中，只有 20 种在蛋白质特异性基础上表现出明显改变的水平，而大多数蛋白质在叶片鲜重基础上被上调，尽管上调幅度不同。尽管 SlCDI 表达系中 Rubisco 激活酶的转录本水平相似，但定量逆转录酶-PCR 分析表明，测试和对照系叶片中的蛋白水平增加了两到三倍。这些观察结果，以及分配给在叶片中上调的与 MS 鉴定的蛋白质的不相关的生物学功能，以及蛋白酶分析显示 SlCDI 表达系的蛋白提取物中的蛋白酶体活性略有增加，表明 SlCDI 在植物体内具有普遍的、不依赖蛋白酶体的蛋白质稳定作用。人α(1)-抗糜蛋白酶的瞬时表达分析也表明 SlCDI 对异源蛋白具有稳定作用，导致 SlCDI 表达系中人类蛋白的净水平增加约 2.5 倍。这些数据总体上说明了 SlCDI 作为一种体内蛋白质稳定因子在转基因植物系统中的潜力，可用于提高蛋白质水平和重组蛋白积累。
