Bre1与Rad6形成的复合物的表达、纯化和结晶  

Expression,purification and crystallization of Bre1-Rad6 complex

作　　者：赵佳奇 石萌 向嵩 ZHAO Jia-qi;SHI Meng;XIANG Song(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]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

出　　处：《天津医科大学学报》2023年第1期83-87,共5页Journal of Tianjin Medical University

摘　　要：目的:建立Bre1及Rad6的表达和纯化方法,构建Bre1-Rad6的复合物,寻找复合物的结晶条件,为使用X射线晶体学的方法解析复合物的结构奠定基础。方法:使用大肠杆菌表达系统表达蛋白质;使用共纯化的方法组装复合物;使用气象扩散的方法结晶复合物。结果:建立了来源于Lodderomyces elongisporus的Bre1(LeBre1)氮端与Rad6相互作用的结构域(RBD)和Rad6(LeRad6)在大肠杆菌中大量表达的方法;通过使用共纯化的方法,成功组装了LeBre1 RBD-Rad6复合物,并进一步通过两步凝胶过滤层析纯化的方法精细纯化,获得了纯度超过95%的蛋白复合物;使用气象扩散的方法结晶复合物,在18℃通过对1 000多个结晶条件的筛选,确定了复合物的两种结晶条件,进一步对该条件优化,获得了质量相对较好的晶体。结论:成功的构建了LeBre1 RBD-Rad6复合物的表达、纯化和结晶的方法,为使用X射线晶体学方法解析复合物的结构奠定了基础。O bjective:To establish the expression,and purification protocol for the Bre1-Rad6 complex,construct the Bre1-Rad6complex;search for its crystallization condition to provide a starting point for structure determination of the complex with X-ray crystallography. Methods:The Escherichia coli host was used for the recombinant expression of protein,the complex was assembled by copurification,the vapor diffusion method was used for complex crystallization. Results:The Lodderomyces elongisporus Bre1(LeBre1)N-terminal Rad6 binding domain(RBD)and Rad6(LeRad6)can be expressed separately in large amounts in Escherichia. coli cells.The LeBre1 RBD-Rad6 complex was assembled with affinity co-purification. The complex was further purified to more than 95% purity with two-step gel filtration chromatography. The compound was crystallized by the method of meteorological diffusion. More than 1 000crystallization conditions were screened at 18℃,two crystallization conditions for the complex were identified. After optimizing the crystallization condition,relatively good quality crystals were obtained. Conclusion:The expression,purification and crystallization protocol for the LeBre1 RBD-Rad6 complex is established. Such protocol provides a starting point for future structural determination of the complex with X ray crystallography.

关 键 词：Bre1 Rad6 泛素化修饰 蛋白质纯化 蛋白质结晶 

分 类 号：Q71[生物学—分子生物学]