title: Purification of eukaryotic tetherin/Vpu proteins and detection of their interaction by ELISA.
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.
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People's Republic of China.
authors.forename: Mingyu
authors.initials: M
authors.lastname: Lv
authors.forename: Yingzi
authors.initials: Y
authors.lastname: Zhu
authors.forename: Jiawen
authors.initials: J
authors.lastname: Wang
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhang
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Wang
authors.forename: Tao
authors.initials: T
authors.lastname: Zuo
authors.forename: Donglai
authors.initials: D
authors.lastname: Liu
authors.forename: Jingyao
authors.initials: J
authors.lastname: Zhang
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Wu
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
authors.forename: Xianghui
authors.initials: X
authors.lastname: Yu
mesh_terms: D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D057809:HEK293 Cells; D054298:Human Immunodeficiency Virus Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000074010:Bone Marrow Stromal Antigen 2; D000086082:Viroporin Proteins
chemical_list: D015703:Antigens, CD; D058851:GPI-Linked Proteins; D054298:Human Immunodeficiency Virus Proteins; D011994:Recombinant Proteins; D054334:Viral Regulatory and Accessory Proteins; C094572:BST2 protein, human; D000074010:Bone Marrow Stromal Antigen 2; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins
keywords: ELISA; HIV-1; Purification; Tetherin; Vpu