title: Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system.
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.
authors.affiliation: 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.
authors.forename: Thomas
authors.initials: T
authors.lastname: Soranzo
authors.affiliation: Synthelis SAS, 5 avenue du Grand Sablon, 38700, La Tronche, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Cortès
authors.affiliation: CNRS, UMR 5628 (LMGP), 3 parvis Louis Néel, 38016, Grenoble, France; University of Grenoble Alpes, Grenoble Institute of Technology, 38016, Grenoble, France.
authors.forename: Flora
authors.initials: F
authors.lastname: Gilde
authors.affiliation: Nanion Technologies GmbH, Gabrielenstraβe 9, 80636, Munich, Germany.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Kreir
authors.affiliation: CNRS, UMR 5628 (LMGP), 3 parvis Louis Néel, 38016, Grenoble, France; University of Grenoble Alpes, Grenoble Institute of Technology, 38016, Grenoble, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Picart
authors.affiliation: TheREx Laboratory, TIMC-IMAG, UMR 5525, CNRS /UJF, University Joseph Fourier, UFR de Médecine, 38706, La Tronche, France. Electronic address: jllenormand@chu-grenoble.fr.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Lenormand
mesh_terms: D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D017510:Protein Folding; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D008081:Liposomes; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins
keywords: Cell-free protein synthesis; Hepatitis C virus; Membrane protein; Proteoliposomes; Viroporin