MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Provincial Key Laboratory for Crop Germplasm Innovation and Utilization, Hunan Agricultural University, Changsha, 410128, China.
Plant J. 2017 Nov;92(4):736-743. doi: 10.1111/tpj.13708. Epub 2017 Oct 6.
Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins.
泛素介导的蛋白质降解在植物生长发育以及对环境和内源性信号的响应中起着至关重要的作用。F-box 蛋白是 SCF（SKP1-CUL1-F-box 蛋白）E3 泛素连接酶复合物的关键组成部分之一，它可以招募特定的底物蛋白进行随后的泛素化和 26S 蛋白酶体介导的降解，以调节发育过程和信号网络。然而，由于其不稳定的蛋白质结构，获得具有高活性的纯化 F-box 蛋白并不容易。在这里，我们发现拟南芥 SKP 样蛋白（ASKs）可以显著提高 F-box 蛋白的可溶性表达并保持其生物活性。我们建立了一种有效的 ASK 辅助表达和纯化植物 F-box 蛋白的方法。该方法满足了植物 F-box 蛋白生化分析或蛋白质结晶所需的广泛标准。
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