Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach
Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach
复制标题
使用合成生物学方法重建细菌中的植物泛素化级联
DOI：
10.1111/tpj.13603
复制
发表时间：
2017-08-01
期刊：
影响因子：
7.2
通讯作者：
Lu, Dongping
中科院分区：
文献类型：
--
作者：
Han, Yufang;Sun, Jianhang;Lu, Dongping
泛素化几乎调控着植物生命的方方面面。在这里，我们使用合成生物学的方法在大肠杆菌中重组了拟南芥泛素化级联。在这个系统中，植物蛋白得到表达，然后立即参与大肠杆菌细胞内的泛素化反应。此外，省略了在建立泛素化反应之前对个别泛素化成分的提纯。为了建立重组系统，我们利用Duet表达载体在大肠杆菌中与E1、E2和E3酶共表达了拟南芥泛素(Ub)和泛素化底物。通过检测环(真正有趣的新基因)型E3连接酶AIP2的自动泛素化及其底物ABI3的泛素化来评价该系统的功能。我们的结果证明了该系统的保真度和特异性。此外，我们应用这个系统通过E2接合分析评估了Ub链形成中的一个拟南芥E2子集。亲和标记的Ub允许以毫克量高效纯化Ub结合物。与以前的报道一致，在这个细菌系统中也观察到了不同的E2在Ub链组装中的不同作用。因此，该重组体系具有多种优点，可用于E3连接酶靶标的筛选或植物泛素化的详细研究。
Ubiquitination modulates nearly all aspects of plant life. Here, we reconstituted the Arabidopsis thaliana ubiquitination cascade in Escherichia coli using a synthetic biology approach. In this system, plant proteins are expressed and then immediately participate in ubiquitination reactions within E. coli cells. Additionally, the purification of individual ubiquitination components prior to setting up the ubiquitination reactions is omitted. To establish the reconstituted system, we co-expressed Arabidopsis ubiquitin (Ub) and ubiquitination substrates with E1, E2 and E3 enzymes in E. coli using the Duet expression vectors. The functionality of the system was evaluated by examining the auto-ubiquitination of a RING (really interesting new gene)-type E3 ligase AIP2 and the ubiquitination of its substrate ABI3. Our results demonstrated the fidelity and specificity of this system. In addition, we applied this system to assess a subset of Arabidopsis E2s in Ub chain formation using E2 conjugation assays. Affinity-tagged Ub allowed efficient purification of Ub conjugates in milligram quantities. Consistent with previous reports, distinct roles of various E2s in Ub chain assembly were also observed in this bacterial system. Therefore, this reconstituted system has multiple advantages, and it can be used to screen for targets of E3 ligases or to study plant ubiquitination in detail.