人 DEAD-box RNA 解旋酶 DDX21 在不同解旋状态下的蛋白纯化、结晶和结构测定。
Protein purification, crystallization, and structure determination of human DEAD-box RNA helicase DDX21 in different unwinding states.
机构信息
State Key Laboratory of Genetic Engineering, School of Life Sciences and Huashan Hospital, Engineering Research Center of Gene Technology of MOE, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai 200438, China.
Department of Chemistry, The Hong Kong University of Science and Technology, Hong Kong, China.
出版信息
STAR Protoc. 2022 Aug 19;3(3):101642. doi: 10.1016/j.xpro.2022.101642. eCollection 2022 Sep 16.
RNA helicase DDX21 plays vital roles in ribosomal RNA processing and the regulation of host innate immunity during virus infection. Here, we describe the optimized protocols for nucleic acid-free protein purification and crystallization of DDX21 in its different unwinding states. Rational design of the flexible region within the helicase core, and biophysical approach to characterize interactions between DDX21 and RNA, leads to successful crystallization of DDX21. This protocol can be applied to the crystallography of other DExD/H-box RNA helicases. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).
RNA 解旋酶 DDX21 在核糖体 RNA 加工和病毒感染期间宿主固有免疫的调节中发挥重要作用。在这里，我们描述了无核酸的蛋白纯化和 DDX21 在不同解旋状态下结晶的优化方案。在解旋酶核心内的柔性区域进行合理设计，并采用生物物理方法来表征 DDX21 与 RNA 之间的相互作用，这使得 DDX21 的结晶得以成功。该方案可应用于其他 DExD/H 盒 RNA 解旋酶的晶体学研究。有关该方案使用和实施的完整详细信息，请参考 Chen 等人。（2020 年）。
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