title: Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein.
abstract: Hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, are involved in the first steps of virus infection. The E2 ectodomain can be produced as an isolated form (E2661). However, there is some concern about its proper conformation and the role that E1 can play as a chaperone for the folding of E2. In order to verify this fact we have expressed a chimeric protein (E1tmbE2) based on the full-length E1 sequence followed by the E2 ectodomain using the baculovirus-insect cells system. The E2 ectodomain is folded in the presence of the E1, proteolytically processed by cellular proteases and secreted to cell culture media (E2661p), while the E1 protein is retained into the cell due to its transmembrane sequence. The purification of E2661p from culture media was facilitated by a His tag introduced in its amino terminus. Both E2661 and E2661p glycoproteins shared very similar structural features, monitored by spectroscopic and antigenic studies. Moreover, their functional properties, tested by means of CD81 binding, were almost indistinguishable, indicating that the E2 ectodomain constitutes an independent folding unit.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Ortega-Atienza
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Lombana
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain.
authors.forename: Julián
authors.initials: J
authors.lastname: Gómez-Gutiérrez
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Yélamos
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, United States.
authors.forename: Darrell L
authors.initials: DL
authors.lastname: Peterson
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain. Electronic address: pacog@bbm1.ucm.es.
authors.forename: Francisco
authors.initials: F
authors.lastname: Gavilanes
mesh_terms: D016367:Baculoviridae; D002460:Cell Line; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D000072417:Protein Domains; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: C087035:E1 protein, Hepatitis C virus; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus
keywords: E2 ectodomain; E2 glycoprotein; Hepatitis C virus envelope proteins; Protein structure