Functional expression, purification, characterization, and membrane reconstitution of non-structural protein 2 from hepatitis C virus.
Non-structural protein 2 (NS2) of the hepatitis C virus (HCV) is an integral membrane protein that contains a cysteine protease and that plays a central organizing role in assembly of infectious progeny virions. While the crystal structure of the protease domain has been solved, the NS2 full-length form remains biochemically and structurally uncharacterized because recombinant NS2 could not be prepared in sufficient quantities from cell-based systems. We show here that functional NS2 in the context of the NS2-NS3pro precursor protein, ensuring NS2-NS3 cleavage, can be efficiently expressed by using a wheat germ cell-free expression system. In this same system, we subsequently successfully produce and purify milligram amounts of a detergent-solubilized form of full-length NS2 exhibiting the expected secondary structure content. Furthermore, immuno-electron microscopy analyses of reconstituted proteoliposomes demonstrate NS2 association with model membranes.
Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France.
Marie-Laure
ML
Fogeron
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
David
D
Paul
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
Vlastimil
V
Jirasko
Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France.
Roland
R
Montserret
Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France.
Denis
D
Lacabanne
Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France.
Jennifer
J
Molle
Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France; RD-Biotech, Recombinant Protein Unit, Besançon, France.
Aurélie
A
Badillo
Institut Pasteur, Unit of Molecular Genetics of RNA Viruses, CNRS UMR 3569, Université Paris Diderot - Sorbonne Paris Cité, Paris, France.
Célia
C
Boukadida
Plate-Forme RIO des Microscopies, PPF ASB, Université François Rabelais and CHRU de Tours, Tours, France.
Sonia
S
Georgeault
Plate-Forme RIO des Microscopies, PPF ASB, Université François Rabelais and CHRU de Tours, Tours, France; INSERM U966, Universite François Rabelais and CHRU de Tours, Tours, France.
Philippe
P
Roingeard
Institut Pasteur, Unit of Molecular Genetics of RNA Viruses, CNRS UMR 3569, Université Paris Diderot - Sorbonne Paris Cité, Paris, France.
Annette
A
Martin
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
Ralf
R
Bartenschlager
Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France. Electronic address: f.penin@ibcp.fr.
François
F
Penin
Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, Lyon, France. Electronic address: a.bockmann@ibcp.fr.
Anja
A
Böckmann
D000595:Amino Acid Sequence; D002474:Cell-Free System / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry; D015870:Gene Expression; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology; D008081:Liposomes / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D014908:Triticum / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D003902:Detergents; D008081:Liposomes; D008563:Membrane Lipids; C097010:NS2 protein, Hepatitis C virus; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins
Cell-free protein expression; Hepatitis C virus; Lipid reconstitution; Membrane protein; Non-structural protein 2