Articles

Purification of the AtGrp7 RRM Domain from Arabidopsis thaliana and Its Preliminary Structure and Binding Analysis

  • CHI Xiu-juan ,
  • QIAO Xiao-ya ,
  • LIU Ying ,
  • LIU Hui-li ,
  • CHEN Lei ,
  • WANG Ji-hui ,
  • AI Xuan-jun
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  • 1. School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China;
    2. National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China;
    3. Division of Virology & Immunology, National Center for AIDS/STD Control and Prevention, Beijing 102206, China;
    4. State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan(Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences), Wuhan 430071, China

Received date: 2018-04-19

  Online published: 2018-04-24

Supported by

The Liaoning Natural Science Foundation (20170520198, 20170520043); Project Supported by the State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics (T151601).

Abstract

The glycine-rich RNA-binding protein, AtGrp7, is a component of a negative feedback loop in the circadian clock regulation of Arabidopsis thaliana. In our initial purification trial of the tobacco etch virus (TEV)-cleaved AtGrp7 RNA recognition motif (RRM) domain with the regular protocol, mixed ultraviolet signals of the target proteins and contaminants were observed. A two-step denaturing-refolding protocol was then tested, trying to solve the problem of impurities. The structure of the AtGrp71-90 RRM domain was fully recovered by quick-dilution refolding, evidenced by the fingerprint 1H-15N HSQC spectrum and CS-Rosetta model structures. Isothermal titration calorimetry (ITC) and NMR titration experiments further confirmed that the RRM domain of AtGrp71-90 had proper functions with regards to RNA/DNA binding.

Cite this article

CHI Xiu-juan , QIAO Xiao-ya , LIU Ying , LIU Hui-li , CHEN Lei , WANG Ji-hui , AI Xuan-jun . Purification of the AtGrp7 RRM Domain from Arabidopsis thaliana and Its Preliminary Structure and Binding Analysis[J]. Chinese Journal of Magnetic Resonance, 2019 , 36(1) : 1 -14 . DOI: 10.11938/cjmr20182637

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