title: Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography.
abstract: Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8 mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500 mM imidazole and 1.5 M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP).
authors.affiliation: Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Wei Boon
authors.initials: WB
authors.lastname: Yap
authors.forename: Beng Ti
authors.initials: BT
authors.lastname: Tey
authors.forename: Noorjahan Banu Mohamed
authors.initials: NB
authors.lastname: Alitheen
authors.forename: Wen Siang
authors.initials: WS
authors.lastname: Tan
mesh_terms: D000327:Adsorption; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D010316:Particle Size; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D013395:Sucrose; D014783:Viscosity
chemical_list: D006512:Hepatitis B Core Antigens; D007093:Imidazoles; D011994:Recombinant Proteins; D012965:Sodium Chloride; D006639:Histidine; D013395:Sucrose; C029899:imidazole