SITE-DIRECTED MUTAGENESIS OF CYSTEINE RESIDUES OF HEPATITIS-B SURFACE-ANTIGEN ANALYSIS OF 2 SINGLE MUTANTS AND THE DOUBLE MUTANT

The structure of hepatitis B surface antigen (HBsAg) is-mainly maintai ned by an intricate disulfide network, responsible for most of its str uctural and antigenic properties. Characterization of three cysteine-r eplacement mutants of HBsAg has been performed by both structural and immunological methods. Replacement of Cys121 or Cys124 with serine res ults in mutant proteins that show diminished binding titres to both mo noclonal antibodies and to a polyclonal serum, indicating that a struc tural change has taken place. Circular dichroism analysis shows that t he substitution of either of these two residues also diminishes the he lical content of the protein. However, the double mutant, in which bot h cysteine residues have been simultaneously changed, reverts the prop erties of the single mutations, and shows similar behaviour to the wil d-type protein. Both the single and double cysteine mutants are effici ently glycosylated and secreted from Chinese hamster ovary cells and, in all cases, the mutant proteins assemble into spherical particles of similar buoyant density to both the wild-type and serum-derived HBsAg .