Plant J | 谢道昕团队发现一种高效的植物F-box蛋白表达和纯化系统

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近日，Plant Journal发表清华大学-北京大学生命科学联合中心与湖南农业大学植物激素与生长发育湖南省重点实验室在线发表题为“Efficient ASK-assisted system for expression and purification of plant F-box proteins”研究论文，建立了一种高效的植物F-box蛋白表达和纯化方法。

       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信号的响应中起重要作用。F-box蛋白是SCF（SKP1-CUL1-F-box）E3泛素连接酶复合体的关键组分之一，其能够募集特异性底物蛋白质，通过泛素化和26S蛋白酶体介导的降解反应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，因此获得具有高活性的纯化F-box蛋白并不容易。本研究发现拟南芥SKP型蛋白（ASK）能够显著改善F-box蛋白的可溶性表达并维持其生物活性。本研究建立了一种高效的ASK辅助方法来表达和纯化植物F-box蛋白。该方法满足植物F-box蛋白生物化学分析和蛋白质结晶所要求的一系列标准。

清华大学生命科学学院谢道昕和闫建斌为本文的通讯作者。

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.

http://onlinelibrary.wiley.com/doi/10.1111/tpj.13708/full

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