title: Expression, refolding and bio-structural analysis of a tetravalent recombinant dengue envelope domain III protein for serological diagnosis.
abstract: Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of β-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections.
authors.affiliation: Immunoassays Research & Development, Raw Materials, bioMérieux, Chemin de l'Orme, 69280 Marcy l'Etoile, France.
authors.forename: Maxime
authors.initials: M
authors.lastname: Combe
authors.affiliation: Immunoassays Research & Development, Raw Materials, bioMérieux, Chemin de l'Orme, 69280 Marcy l'Etoile, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Lacoux
authors.affiliation: Immunoassays Research & Development, Raw Materials, bioMérieux, Chemin de l'Orme, 69280 Marcy l'Etoile, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Martinez
authors.affiliation: Immunoassays Research & Development, Raw Materials, bioMérieux, Chemin de l'Orme, 69280 Marcy l'Etoile, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Méjan
authors.affiliation: Immunoassays Research & Development, Raw Materials, bioMérieux, Chemin de l'Orme, 69280 Marcy l'Etoile, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Luciani
authors.affiliation: Immunoassays Research & Development, Raw Materials, bioMérieux, Chemin de l'Orme, 69280 Marcy l'Etoile, France. Electronic address: soizic.daniel@biomerieux.com.
authors.forename: Soizic
authors.initials: S
authors.lastname: Daniel
mesh_terms: D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D003715:Dengue / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D003716:Dengue Virus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014764:Viral Proteins
keywords: Bacterial expression; Dengue; Diagnosis; Protein refolding