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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

29卷

期数:

2023年01期

页码:

83-87

栏目:

技术与方法

出版日期:

2023-01-20

文章信息/Info

Title:

Expression，purification and crystallization of Bre1-Rad6 complex

文章编号:

1006-8147(2023)01-0083-05

作者:

赵佳奇; 石萌; 向嵩

（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）

Author(s):

ZHAO Jia-qi; SHI Meng; XIANG Song

（Department of Biochemistry and Molecular Biology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China）

关键词:

Bre1; Rad6; 泛素化修饰; 蛋白质纯化; 蛋白质结晶

Keywords:

Bre1; Rad6; ubiquitination; protein purification; protein crystallization

分类号:

Q71

DOI:

-

文献标志码:

A

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。

Abstract:

Objective：To establish the expression， and purification protocol for the Bre1-Rad6 complex，construct the Bre1-Rad6 complex; 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 co-purification，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 1 000 crystallization 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.

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相似文献/References:

[1]石萌,向嵩.核小体泛素连接酶Bre1的eRING结构域与泛素转移酶Rad6的相互作用研究[J].天津医科大学学报,2023,29(01):31.
　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.

备注/Memo

备注/Memo:

作者简介:赵佳奇（1995-），女，硕士在读，研究方向：结构生物学；通信作者：向嵩，E-mail：xiangsong@tmu.edu.cn。

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更新日期/Last Update: 2023-02-01