Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography.
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).
Yap
Wei Boon
WB
Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Tey
Beng Ti
BT
Alitheen
Noorjahan Banu Mohamed
NB
Tan
Wen Siang
WS
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
D006512:Hepatitis B Core Antigens; D007093:Imidazoles; D011994:Recombinant Proteins; D012965:Sodium Chloride; D006639:Histidine; D013395:Sucrose; C029899:imidazole