title: Characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris.
abstract: The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexahistidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni(2+)-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellman's reagent allowed the measurement respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with respectively, a diameter of 34 and 28-nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect anti-HBc antibodies in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.
authors.affiliation: BioMérieux-Pierre Fabre, Chemin de l'Orme, 69280, Marcy l'Etoile, France.
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Watelet
authors.forename: Martine
authors.initials: M
authors.lastname: Quibriac
authors.forename: Dominique
authors.initials: D
authors.lastname: Rolland
authors.forename: Gaspard
authors.initials: G
authors.lastname: Gervasi
authors.forename: Marie
authors.initials: M
authors.lastname: Gauthier
authors.forename: Michel
authors.initials: M
authors.lastname: Jolivet
authors.forename: Odile
authors.initials: O
authors.lastname: Letourneur
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006509:Hepatitis B / Q000175:diagnosis*; D006510:Hepatitis B Antibodies / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics / Q000276:immunology; D006801:Humans; D007118:Immunoassay; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D006510:Hepatitis B Antibodies; D006512:Hepatitis B Core Antigens; D011994:Recombinant Proteins