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Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.
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Njengele Z, Kleynhans R, Sayed Y, Mosebi S,


Njengele Z, Kleynhans R, Sayed Y, Mosebi S, (click to view)

Njengele Z, Kleynhans R, Sayed Y, Mosebi S,

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Protein expression and purification 2016 08 31128() 109-14 doi 10.1016/j.pep.2016.08.018

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.

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