ISOLATION OF HIGHLY PURIFIED, FUNCTIONAL CARBOXY-TERMINALLY TRUNCATEDHEPATITIS-B VIRUS MIDDLE SURFACE PROTEIN ACTIVATORS FROM EUKARYOTIC EXPRESSION SYSTEMS

Carboxy-terminally truncated hepatitis B virus (HBV) middle surface pr oteins (MHBs(t)) show a transcriptional activator function, Two differ ent subtypes of MHBs(t) activators can be distinguished: an ER-localiz ed type, represented here by MHBs(t76) (truncated at amino acid 76), a nd a cytosol-localized type, represented here by MHBs(t63). To charact erize the MHBs(t) activator on the protein level and to analyze posttr anslational modifications, we established recombinant baculoviruses en coding for fusion proteins of MHBs(t76) or MHBs(t63) and of an amino t erminal hexa-his tag. Both proteins could be obtained in high purity b y affinity chromatography using Ni-nitrilo-tri-acetate agarose. In add ition, 6H-MHBs(t76) was also isolated from transiently transfected Hep G2 cells. Both the Spodoptera frugiperda (Sig) cell-derived and the He pG2 cell-derived MHBs(t) proteins were found to be unglycosylated, A d etailed analysis of Sf9 cell-derived 6H-MHBs(t76) by electrospray-ioni zation mass spectrometry showed that a fraction of this protein is N-t erminally acetylated and phosphorylated or sulfated. Electric-field-me diated transfer of the highly purified proteins into reporter cells de monstrated that the isolated proteins are functional transcriptional a ctivators. These experiments further showed that Sf9 cell-derived and HepG2 cell-derived 6H-MHBs(t) do not differ in their functionality, Th is system allowed production and purification of functional 6H-MHBs(t) in amounts sufficient enough to allow a further detailed analysis of MHBs(t) activators on the protein level.