Lv Mingyu, Zhu Yingzi, Wang Jiawen, Zhang Haihong, Wang Xiaodan, Zuo Tao, Liu Donglai, Zhang Jingyao, Wu Jiaxin, Kong Wei, Yu Xianghui
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People's Republic of China.
Protein Expr Purif. 2013 Oct;91(2):112-8. doi: 10.1016/j.pep.2013.07.015. Epub 2013 Aug 3.
Tetherin/BST-2/CD317 inhibits HIV-1 release from infected cells, while HIV-1 Vpu efficiently antagonizes tetherin based on intermolecular interactions between the transmembrane domains of each protein. In this study, we successfully partially purified His-tagged tetherin with a glycophosphatidylinositol deletion (delGPI) and His-tagged full-length Vpu from transiently transfected 293T cells using affinity chromatography. The in vitro interaction between these purified proteins was observed by a pull-down assay and ELISA. Detection of the Vpu/tetherin interaction by ELISA is a novel approach that would be advantageous for inhibitor screening in vitro. Successful co-purification of the tetherin/Vpu complex also provides a basis for further structural studies.
tetherin/BST-2/CD317可抑制HIV-1从受感染细胞中释放，而HIV-1 Vpu则基于每种蛋白质跨膜结构域之间的分子间相互作用有效地拮抗tetherin。在本研究中，我们使用亲和层析法从瞬时转染的293T细胞中成功部分纯化了带有糖基磷脂酰肌醇缺失（delGPI）的His标签tetherin和His标签全长Vpu。通过下拉试验和ELISA观察这些纯化蛋白之间的体外相互作用。通过ELISA检测Vpu/tetherin相互作用是一种新方法，有利于体外抑制剂筛选。成功共纯化tetherin/Vpu复合物也为进一步的结构研究提供了基础。
