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 SHI Meng,XIANG Song.Biochemical charterization of the interaction between the eRING domain in the nucleosome ubiquitin ligase Bre1 and the ubiquitin conjugating enzyme Rad6[J].Journal of Tianjin Medical University,2023,29(01):31-35.





Biochemical charterization of the interaction between the eRING domain in the nucleosome ubiquitin ligase Bre1 and the ubiquitin conjugating enzyme Rad6
(天津医科大学基础医学院生物化学与分子生物学系,天津 300070)
(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)
Bre1Ubiquitinglutathione S-transferaseprotein-protein interaction
目的:探究乳酸克鲁维酵母核小体泛素连接酶Bre1的eRING结构域与泛素转移酶Rad6的相互作用,解析Bre1的RBD结构域对该相互作用的影响。方法:在大肠杆菌中表达乳酸克鲁维酵母的GST-Rad6、Rad6、Bre1 eRING等蛋白并将它们纯化至纯度>95%,通过GST-Rad6下拉实验,结合蛋白免疫印迹实验研究GST-Rad6和Bre1的eRING结构域的相互作用及Bre1的RBD结构域对其相互作用的影响;通过SPR实验研究Rad6与Bre1的eRING结构域之间的相互作用。结果:乳酸克鲁维酵母的GST-Rad6与Bre1的eRING结构域存在相互作用,该相互作用可以被Bre1 RBD所抑制;GST蛋白与乳酸克鲁维酵母Bre1的eRING存在相互作用,不可作为该蛋白的下拉标签使用。结论:Rad6与Bre1的eRING结构域存在相互作用,该相互作用可以被RBD所抑制,GST标签不适于乳酸克鲁维酵母的eRING相互作用研究。
Objective: To explore the interaction between the eRING domain of the nucleosome ribosome ubiquitin ligase Bre1 and the ubiquitin transferase Rad6 of S. cerevisiae,elucidate the influence of RBD domain of Bre1 to this interaction. Methods:GST-tagged K. lactis Rad6,Rad6,Bre1 eRING domain and related proteins were expressed in E. coli and purified to a purity of>95%. GST Rad6 pull-down experiments and Western blotting were performed to probe the interaction between the K. lactis Bre1 eRING domain and GST-Rad6 and the effect of the Bre1 RBD domain on their interaction. SPR experiments were performed to probe the interaction between the K. lactis Bre1 eRING domain and Rad6. Results:GST-tagged K. lactis Rad6 interacted strongly with the K. lactis Bre1 eRING domain,the interaction was inhibited by the K. lactis Bre1 RBD. The GST protein alone interacts with the K. lactis Bre1 eRING domain or its N-terminal CCNR domain,so the GST protein was not suitable for the pull-down tag in this experiment. Conclusion:Rad6 interacts with Bre1-eRING domain,and the interaction can be inhibited by RBD. GST tag is not suitable for the pull-down assay in K. lactis.


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 ZHAO Jia-qi,SHI Meng,XIANG Song.Expression,purification and crystallization of Bre1-Rad6 complex[J].Journal of Tianjin Medical University,2023,29(01):83.


基金项目: 国家自然科学基金面上项目(32071205)
作者简介: 石萌(1994-),女,硕士在读,研究方向:生物学;通信作者:向嵩,E-mail:xiangsong@tmu.edu.cn。
更新日期/Last Update: 2023-02-01