Purification of E. coli-expressed HIS-tagged hepatitis B core antigen by Ni2+ -chelate affinity chromatography.
Hepatitis B virus is a major cause of human liver disease. In the case of chronic infection the virus can lead to liver cancer and cirrhosis. The virion consists of an outer envelope containing lipids of the endoplasmic reticulum and virally-encoded surface proteins. This lipoprotein shell encloses the nucleocapsid or core antigen (HBcAg), which contains the viral genome. The capsid consists of dimers of a 183-residue protein, which can be divided into an assembly (residues 1-149) and a protamin-like domain (residues 150-183), responsible for polymerization into particles and RNA packaging, respectively. Upon expression of the core gene in bacteria the products are assembled into capsids resembling those of wild type particles. A purification protocol was developed for unpolymerised (dimeric) and polymerized HBcAg by fusion of six histidine residues to a C-terminal deletion mutant of the core protein allowing the isolation of the respective antigens after denaturing Ni2+-chelate affinity chromatography and renaturing dialysis. The possible incorporation of E. coli proteins during the assembly process and the inclusion of nucleic acids can be avoided. The method might be an attractive alternative to common purification protocols of hybrid virus-like particles (VLPs) for vaccine use.
Wizemann
H
H
Max-von-Pettenkofer-Institut, Lehrstuhl Virologie, Genzentrum, Munich, Germany.
von Brunn
A
A
D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D006639:Histidine / Q000032:analysis / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009532:Nickel; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D006512:Hepatitis B Core Antigens; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel