Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography

Wei Boon Yap, Beng Ti Tey, Noorjahan Banu Mohamed Alitheen, Wen Siang Tan

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26 Citations (Scopus)

Abstract

Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500mM imidazole and 1.5. M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP).

Original languageEnglish
Pages (from-to)3473-3480
Number of pages8
JournalJournal of Chromatography A
Volume1217
Issue number21
DOIs
Publication statusPublished - May 2010
Externally publishedYes

Keywords

  • His-tagged HBcAg
  • IMA-EBAC
  • Streamline Chelating
  • Unclarified bacterial homogenate
  • Virus-like particles

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