AN ALTERNATIVE PURIFICATION PROTOCOL FOR PRODUCING HEPATITIS-B VIRUS-X ANTIGEN ON A PREPARATIVE-SCALE IN ESCHERICHIA-COLI

A truncated variant of the hepatitis B virus X gene (HBx) was cloned i nto the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was tran sformed into and expressed by the Escherichia coli strain DH5. More th an 80% of the expressed fusion protein was found in the insoluble frac tion (inclusion body) of the cell lysate. The fusion protein was selec tively extracted from the inclusion bodies with 8 M urea at pH 6.5, an d it was refolded by diluting 3-fold with deionized distilled water at 4 degrees C. The in vitro cleavage of the refolded fusion protein by factor X-a at about 2-3 mg ml(-1) in the presence of 2.66 M urea at pH 6.5 was complete. The final steps of purification involved precipitat ion of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. W ith this approach, following an inclusion body strategy and a benefici al in vitro refolding, a predominantly hydrophobic and highly disulphi de-bonded protein was produced in preparative scale for subsequent dia gnostic use. (C) 1997 Elsevier Science B.V.