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Protein Expr Purif. 2016 Feb;118:83-91. doi: 10.1016/j.pep.2015.10.004. Epub 2015 Oct 22.

Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system.

Author information

1
Synthelis SAS, 5 avenue du Grand Sablon, 38700, La Tronche, France; TheREx Laboratory, TIMC-IMAG, UMR 5525, CNRS /UJF, University Joseph Fourier, UFR de Médecine, 38706, La Tronche, France.
2
Synthelis SAS, 5 avenue du Grand Sablon, 38700, La Tronche, France.
3
CNRS, UMR 5628 (LMGP), 3 parvis Louis Néel, 38016, Grenoble, France; University of Grenoble Alpes, Grenoble Institute of Technology, 38016, Grenoble, France.
4
Nanion Technologies GmbH, Gabrielenstraβe 9, 80636, Munich, Germany.
5
TheREx Laboratory, TIMC-IMAG, UMR 5525, CNRS /UJF, University Joseph Fourier, UFR de Médecine, 38706, La Tronche, France. Electronic address: jllenormand@chu-grenoble.fr.

Abstract

Using a cell-free expression system we produced the p7 viroporin embedded into a lipid bilayer in a single-step manner. The protein quality was assessed using different methods. We examined the channel forming activity of p7 and verified its inhibition by 5-(N,N-Hexamethylene) amiloride (HMA). Fourier transformed infrared spectroscopy (FTIR) experiments further showed that when p7 was inserted into synthetic liposomes, the protein displayed a native-like conformation similar to p7 obtained from other sources. Photoactivable amino acid analogs used for p7 protein synthesis enabled oligomerization state analysis in liposomes by cross-linking. Therefore, these findings emphasize the quality of the cell-free produced p7 proteoliposomes which can benefit the field of the hepatitis C virus (HCV) protein production and characterization and also provide tools for the development of new inhibitors to reinforce our therapeutic arsenal against HCV.

KEYWORDS:

Cell-free protein synthesis; Hepatitis C virus; Membrane protein; Proteoliposomes; Viroporin

PMID:
26477501
PMCID:
PMC5113752
DOI:
10.1016/j.pep.2015.10.004
[Indexed for MEDLINE]
Free PMC Article

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