摘要/Abstract

摘要： 目的·利用负染电镜技术分析人源THO亚复合物结构。方法·采用共表达方式在培养的昆虫细胞里表达重组蛋白质组分，利用Ni-NTA亲和层析、Strep-Tactin亲和层析纯化和Superose凝胶过滤层析分离目的蛋白质复合物；通过负染电镜技术和单颗粒重构技术研究THO亚复合物的空间结构。结果·利用两步亲和层析纯化及一步凝胶过滤层析分离获得高纯度的、均一性良好的重组人源THOC1-THOC2-THOC3亚复合物；采用甲酸双氧铀负染后利用电镜技术初步分析了目的复合物的空间结构。结论·利用单颗粒重构技术构建了人源THOC1-THOC2-THOC3复合物的低分辨率模型。
关键词: mRNA出核转运, TREX复合物, THO复合物, 负染电镜
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


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