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Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide.

Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide.
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Deb A, Johnson WA, Kline AP, Scott BJ, Meador LR, Srinivas D, Martin-Garcia JM, Dörner K, Borges CR, Misra R, Hogue BG, Fromme P, Mor TS,


Deb A, Johnson WA, Kline AP, Scott BJ, Meador LR, Srinivas D, Martin-Garcia JM, Dörner K, Borges CR, Misra R, Hogue BG, Fromme P, Mor TS, (click to view)

Deb A, Johnson WA, Kline AP, Scott BJ, Meador LR, Srinivas D, Martin-Garcia JM, Dörner K, Borges CR, Misra R, Hogue BG, Fromme P, Mor TS,

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PloS one 2017 02 2212(2) e0172529 doi 10.1371/journal.pone.0172529

Abstract

Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.

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