title: High efficiency prokaryotic expression and purification of a portion of the hepatitis C core protein and analysis of the immune response to recombinant protein in BALB/c mice.
abstract: Hepatitis C virus (HCV) produces chronic persistent liver infection in 1-2% of the U.S. population and is the leading cause of end stage liver disease in patients presenting for liver transplantation at our center. Efforts to cure persistent HCV infection are frequently unsuccessful, so the development of a HCV vaccine is a high priority. HCV envelope proteins are hypervariable so production of a recombinant surface antigen vaccine such as is available for hepatitis B is not likely to confer widespread, high level protective immunity. As the most highly conserved structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 and 163 amino acids of the core region. Deletion of the 23 amino acids (aa) from aa141-163 led to a marked increase in the efficiency of protein production from < 1 to 3-4 mg/liter for both systems studied. Protein purification was accomplished using affinity chromatography (MBP) or inclusion body isolation (GST) as determined by SDS-PAGE gels and immunotransblot with HCV core protein-specific monoclonal antibody. Finally, the immune response to recombinant protein was assessed in BALB/c mice using a MBP HCV core fusion protein and an ELISA developed using GST HCV core protein as a target. In all mice of this strain, serum anti-HCV core antibody titer increased to 10(-4), two logs above background, following immunization in conjunction with Freund's complete adjuvant. These results represent an encouraging first step toward production of a core protein vaccine. Recombinant core protein is a useful tool to study the immune response to core protein and may be useful to further study the epidemiology and biology of the HCV virus.
authors.affiliation: Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109-0680, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Hitomi
authors.forename: W M
authors.initials: WM
authors.lastname: McDonnell
authors.forename: J R
authors.initials: JR
authors.lastname: Baker
authors.forename: F K
authors.initials: FK
authors.lastname: Askari
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D004195:Disease Models, Animal; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006526:Hepatitis C / Q000097:blood / Q000276:immunology* / Q000821:virology; D018937:Hepatitis C Antibodies / Q000097:blood / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011387:Prokaryotic Cells; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D004279:DNA, Viral; D018937:Hepatitis C Antibodies; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus; D005982:Glutathione Transferase