Zhu Wenjun, Hong Tao, Wang Weikai, Wei Yuchen, Mao Xueling, Qiu Xiaoting
Key Laboratory of Applied Marine Biotechnology of Ministry of Education, School of Marine Sciences, Ningbo University, Ningbo, 315211, China.
Key Laboratory of Applied Marine Biotechnology of Ministry of Education, School of Marine Sciences, Ningbo University, Ningbo, 315211, China.
Protein Expr Purif. 2019 Feb;154:112-117. doi: 10.1016/j.pep.2018.09.008. Epub 2018 Sep 18.
Carboxyl-terminal repeat domain (CTD) of the largest subunit Rpb1 of RNA polymerace II is essential for transcription regulation. Heptapeptide repeat of CTD of Rpb1 is phosphorylated by carboxyl-terminal repeat domain kinase (CTDK-I), composed of CTK1, CTK2 and CTK3, in order to regulate transcription and transcription associated processes. The yeast specific protein CTK3 binds to cyclin CTK2 to form a heterodimer serving as a regulational factor to control CTK1 activity by binding to CTK1. Structural information of CTK2-CTK3 complex is yet to be elucidated. Here, we report the co-expression of CTK2-CTK3 complex from Saccharomyces cerevisiae with N-terminal His-tag in CTK3 in Escherichia coli (E. coli), purification of the complex by four chromatographic steps and crystallization of the complex as well as the diffraction data collection and processing. This study provides some essential information and a guide for structural and functional study of CTK2-CTK3 complex and CTDK-I in the future.
RNA聚合酶II最大亚基Rpb1的羧基末端重复结构域（CTD）对于转录调控至关重要。Rpb1的CTD七肽重复序列由CTK1、CTK2和CTK3组成的羧基末端重复结构域激酶（CTDK-I）磷酸化，以调控转录及与转录相关的过程。酵母特异性蛋白CTK3与细胞周期蛋白CTK2结合形成异二聚体，作为调控因子通过与CTK1结合来控制CTK1的活性。CTK2 - CTK3复合物的结构信息尚未阐明。在此，我们报道了在大肠杆菌（E. coli）中共同表达来自酿酒酵母的带有CTK3中N端组氨酸标签的CTK2 - CTK3复合物，通过四个色谱步骤纯化该复合物，以及该复合物的结晶、衍射数据收集和处理。本研究为未来CTK2 - CTK3复合物和CTDK-I的结构与功能研究提供了一些重要信息和指导。
