Protein purification, crystallization, and structure determination of human DEAD-box RNA helicase DDX21 in different unwinding states

Zijun Chen; Jinqing Huang; Jixi Li

doi:10.1016/j.xpro.2022.101642

Protein purification, crystallization, and structure determination of human DEAD-box RNA helicase DDX21 in different unwinding states

STAR Protoc. 2022 Aug 19;3(3):101642. doi: 10.1016/j.xpro.2022.101642. eCollection 2022 Sep 16.

Authors

Zijun Chen¹, Jinqing Huang², Jixi Li³

Affiliations

¹ 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.
² Department of Chemistry, The Hong Kong University of Science and Technology, Hong Kong, China.
³ 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. Electronic address: lijixi@fudan.edu.cn.

Abstract

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).

Keywords: Molecular Biology; Protein Biochemistry; Protein expression and purification; Structural Biology; X-ray Crystallography.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Crystallization
DEAD-box RNA Helicases* / chemistry
Humans
RNA Processing, Post-Transcriptional
RNA* / metabolism
RNA, Ribosomal / metabolism

Substances

RNA, Ribosomal
RNA
DDX21 protein, human
DEAD-box RNA Helicases