title: Separation of pure and immunoreactive virus-like particles using gel filtration chromatography following immobilized metal ion affinity chromatography.
abstract: A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease virus (IBDV) with six additional histidine residues at its C-terminus. The ultimate goal was the development of an efficient subunit vaccine against IBDV infection. The particles within the infected High-Five (Hi-5) cell lysates were partially purified by employing immobilized metal ion (Ni(2+)) affinity chromatography (IMAC). The initial step could recover approximately 85% of immunoreactive rVP2H proteins but failed to separate the rVP2H particles from the free rVP2H proteins or its degraded products. To separate the particulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultracentrifugation. Both were able to produce extremely pure rVP2H particles with a buoyant density close to 1.27 g/cm(3). However, the former method can process a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was routinely obtained from a 500 mL Hi-5 cell culture broth. The separation of the particulate form from the free form of rVP2H proteins exposes their respective immunogenicity to induce the virus-neutralizing antibodies and the ability to protect chickens from IBDV infection. Additionally, the abundant quantities of pure rVP2H particles coupled with their uniform dimensions facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus.
authors: Graduate Institute of Agricultural Biotechnology and The Institute of Biochemistry, National Chung Hsing University, Taichung, Taiwan.
authors: Y S
authors: YS
authors: Cheng
authors: M S
authors: MS
authors: Lee
authors: S Y
authors: SY
authors: Lai
authors: S R
authors: SR
authors: Doong
authors: M Y
authors: MY
authors: Wang
mesh_terms: D016367:Baculoviridae / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D011994:Recombinant Proteins / Q000302:isolation & purification; D014461:Ultracentrifugation; D014771:Virion / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins