title: Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.
abstract: Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli.
authors: Centre for Metal-based Drug Discovery, Advanced Materials Division, Mintek, Randburg, 2125, South Africa; Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2050, South Africa.
authors: Zikhona
authors: Z
authors: Njengele
authors: Centre for Metal-based Drug Discovery, Advanced Materials Division, Mintek, Randburg, 2125, South Africa.
authors: Ronel
authors: R
authors: Kleynhans
authors: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2050, South Africa.
authors: Yasien
authors: Y
authors: Sayed
authors: Centre for Metal-based Drug Discovery, Advanced Materials Division, Mintek, Randburg, 2125, South Africa. Electronic address: salerwem@mintek.co.za.
authors: Salerwe
authors: S
authors: Mosebi
mesh_terms: D015703:Antigens, CD / Q000737:chemistry*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry*; D004926:Escherichia coli; D058851:GPI-Linked Proteins / Q000737:chemistry; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000737:chemistry*; D054298:Human Immunodeficiency Virus Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D054334:Viral Regulatory and Accessory Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000074010:Bone Marrow Stromal Antigen 2; D000086082:Viroporin Proteins
chemical_list: D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D058851:GPI-Linked Proteins; D000949:Histocompatibility Antigens Class II; D054298:Human Immunodeficiency Virus Proteins; D011994:Recombinant Proteins; D054334:Viral Regulatory and Accessory Proteins; C094572:BST2 protein, human; D000074010:Bone Marrow Stromal Antigen 2; C045084:invariant chain; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins
keywords: HIV-1; Over-expression; Purification; Refolding; Solubilization; Vpu