Erratum to "characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris".
The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexa-histidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni2+-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 antibodies to HBcAg (anti-HBc) 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.
Watelet
Bénédicte
B
BioMérieux-Pierre Fabre, Chemin de l'Orme, 69280, Marcy l'Etoile, France.
Quibriac
Martine
M
Rolland
Dominique
D
Gervasi
Gaspard
G
Gauthier
Marie
M
Jolivet
Michel
M
Letourneur
Odile
O
D000595:Amino Acid Sequence; D004279:DNA, Viral; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D006510:Hepatitis B Antibodies / Q000097:blood / Q000276:immunology*; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000235:genetics / Q000276:immunology / Q000648:ultrastructure; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019251:Nucleocapsid; D019590:Nucleocapsid Proteins; D010843:Pichia; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D004279:DNA, Viral; D006510:Hepatitis B Antibodies; D006512:Hepatitis B Core Antigens; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; C099600:nucleocapsid protein, Hepatitis virus