title: High-yield production of a chimeric glycoprotein based on permuted E1 and E2 HCV envelope ectodomains.
abstract: In this report it is described for the first time the expression and purification of large quantities of a soluble and correctly folded chimeric recombinant protein, E2661E1340, containing the permuted Hepatitis C virus (HCV) glycoprotein ectodomains E1 (amino acids 192-340) and E2 (amino acids 384-661). Using the baculovirus/insect cell expression system, 8mg of secreted protein were purified from 1L of culture media, a yield 4 times higher than the described for its counterpart E1341E2661. This permuted chimeric protein is glycosylated and possesses a high tendency to self-associate. The fluorescence emission spectrum indicates that Trp residues occupy a relatively low hydrophobic environment. The secondary structure was determined by deconvolution of the far-UV circular dichroism spectrum yielding 13% α-helix structure, 49% extended structure and 38% non-ordered structure. E2661E1340 binds to antibodies present in human sera from HCV-positive patients, a binding that is blocked at different levels by a rabbit anti-E2661 antibody. All these structural and antigenic features of E2661E1340 are very similar to those described for E1340E2661, Thus, this high-yield isolated chimeric protein may be a valuable tool to study the first steps of the HCV infection.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Tello
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain.
authors.forename: Mar
authors.initials: M
authors.lastname: Rodríguez-Rodríguez
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Yélamos
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain.
authors.forename: Julián
authors.initials: J
authors.lastname: Gómez-Gutiérrez
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: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid 28040, Spain. Electronic address: pacog@bbm1.ucm.es.
authors.forename: Francisco
authors.initials: F
authors.lastname: Gavilanes
mesh_terms: D000818:Animals; D016367:Baculoviridae; D002942:Circular Dichroism; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D016174:Hepacivirus / Q000235:genetics; D018937:Hepatitis C Antibodies / Q000276:immunology; D006801:Humans; D007313:Insecta; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000235:genetics*
chemical_list: C087035:E1 protein, Hepatitis C virus; D018937:Hepatitis C Antibodies; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus
keywords: Baculovirus; E1; E2; Envelope protein; Glycosylation; Hepatitis C virus