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