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Updated: May 9, 2026
Identification of Protein Interacting Partners Using Tandem Affinity Purification
Published on: February 25, 2012
Purification of eukaryotic tetherin/Vpu proteins and detection of their interaction by ELISA
Mingyu Lv1, Yingzi Zhu, Jiawen Wang
1National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People's Republic of China.
Abstract:
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.
