›› 2020, Vol. 40 ›› Issue (1): 1-.doi: 10.3969/j.issn.1674-8115.2020.01.001

• Original article (Basic research) •     Next Articles

Electron microscopic study of the human THOC1-THOC2-THOC3 subcomplex

LIU Yong-mei, TAN Ming, LEI Ming   

  1. Shanghai Institute of Precision Medicine, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
  • Online:2020-01-28 Published:2020-03-05
  • Supported by:
    National Natural Science Foundation of China (31525007).

Abstract: Objective · To study the structure of the human THOC1-THOC2-THOC3 subcomplexnegative-staining electron microscopy. Methods · The recombinant protein components were co-expressed in cultured insect cells, and the protein complex was isolated via sequential Ni-NTA and Strep-Tactin affinity purification followedSuperose gel filtration chromatography. The purified protein sample was then subjected to negative-staining electron microscopy and single particle image analysis. Results · The recombinant human THOC1-THOC2-THOC3 complex with high purity and good homogeneity was obtainedusing a tandem affinity purification scheme with Ni-NTA and streptavidin-based chromatography. Preliminary study on the structure of human THOC1-THOC2-THOC3 subcomplex was achievedelectron microscopy using negative staining with uranium formate. Conclusion · A low resolution model of human THOC1-THOC2-THOC3 subcomplex was achievedsingle particle reconstruction.

Key words: mRNA nuclear transport, TREX complex, THO complex, negative-staining electron microscopy