SIZE-EXCLUSION CHROMATOGRAPHIC STUDY OF THE REDUCTION OF RECOMBINANT HEPATITIS-B SURFACE-ANTIGEN

The reduction of the P. pastoris-derived hepatitis B surface antigen ( HBsAg) has been investigated by size exclusion chromatography performe d in a detergent solution containing 0.3% sodium dodecyl sulfate (SDS) and 0.1 M Tris-HCl, pH 7.0. The HBsAg, reduced under different condit ions and passed through the TSK G4000 SW column (600x7.5 mm I.D.) at 0 .9 ml min(-1), was resolved into two peaks corresponding to the reduce d, monomeric, and non-reduced forms, respectively. Under these conditi ons, the antigen fraction corresponding to the HBsAg dimer can be sepa rated and completely reduced to monomers by repeated reductive treatme nt with simultaneous lipid removal. The efficiency of reduction was ma ximal after sample treatment with an equal volume of a solution contai ning 417 mM dithiothreitol, 4.2% (w/v) SDS and 16% (v/v) 2-mercaptoeth anol. In conclusion, complete reduction of recombinant HBsAg to monome r subunits is possible and depends on the efficiency of lipid removal during the reductive treatment. (C) 1998 Elsevier Science B.V. All rig hts reserved.