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 into the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was transformed into and expressed by the Escherichia coli strain DH5. More than 80% of the expressed fusion protein was found in the insoluble fraction (inclusion body) of the cell lysate. The fusion protein was selectively extracted from the inclusion bodies with 8 M urea at pH 6.5, and 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 Xa 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 precipitation of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. With this approach, following an inclusion body strategy and a beneficial in vitro refolding, a predominantly hydrophobic and highly disulphide-bonded protein was produced in preparative scale for subsequent diagnostic use.
Marczinovits
I
I
Institute of Microbiology, Albert Szent-Györgyi Medical University, Szeged, Hungary.
Somogyi
C
C
Patthy
A
A
Németh
P
P
Molnár
J
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006511:Hepatitis B Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D054334:Viral Regulatory and Accessory Proteins
D000911:Antibodies, Monoclonal; D006146:Guanidines; D006511:Hepatitis B Antigens; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D015951:Factor Xa; D019791:Guanidine