An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli

Ilona Marczinovits, Csilla Somogyi, András Patthy, Péter Németh, János Molnár

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A truncated variant of the hepatitis B virus X gone (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°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 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.

Original languageEnglish
Pages (from-to)81-88
Number of pages8
JournalJournal of Biotechnology
Issue number2
Publication statusPublished - Aug 11 1997



  • Escherichia coli
  • Hepatitis B virus
  • High yield
  • Recombinant
  • Refolding
  • X protein

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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