title: Purification of recombinant hepatitis B core antigen from unclarified Escherichia coli feedstock using phage-immobilized expanded bed adsorption chromatography.
abstract: Fusion M13 phage with disulfide constrained heptapeptide, C-WSFFSNI-C, inserted into the minor coat protein (gpIII), has been selected in the current study as ligand in direct purification of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli (E. coli) feedstock. The selected fusion phage showed strong association with the surface of the core particle. In the present study, this fusion M13 phage was immobilized onto Streamline base matrix via epoxy activation and used as adsorbent to capture HBcAg from crude E. coli homogenate. The maximum binding capacity for the adsorbent was 3.76 mg/mL with equilibrium coefficient of 1.83 mg/mL. Due to the slow uptake rate of HBcAg by M13 phage-immobilized adsorbents, a modified EBAC operation with recirculation of feedstock into the expanded bed has been investigated in this study. The introduction of feedstock recirculation has led to an 18% increase in yield; however, the purity of the eluted product was reduced by 15% compared with typical EBAC operation. The level of antigenicity exhibited by the core particles purified by both EBAC operations employed in the present study was comparable to that purified using sucrose ultracentrifugation.
authors: Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors: Michelle Y T
authors: MY
authors: Ng
authors: Wen Siang
authors: WS
authors: Tan
authors: Beng Ti
authors: BT
authors: Tey
mesh_terms: D000327:Adsorption; D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry; D017104:Bacteriophage M13 / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006512:Hepatitis B Core Antigens / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008027:Light; D051379:Mice; D010316:Particle Size; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012542:Scattering, Radiation; D014508:Urea
chemical_list: D000914:Antibodies, Viral; D006512:Hepatitis B Core Antigens; D008024:Ligands; D010455:Peptides; D011994:Recombinant Proteins; D014508:Urea