Expression and purification of a functional human hepatitis B virus polymerase

World J Gastroenterol. 2010 Dec 7;16(45):5752-8. doi: 10.3748/wjg.v16.i45.5752.

Abstract

Aim: To identify a method for efficient large-scale purification of functional hepatitis B virus polymerase (HBV-Pol) without addition of cellular factors.

Methods: Full-length HBV-Pol (843 amino acids) tagged with 5' end Polyhistidine was expressed at a high level in an Escherichia coli (E. coli) system. Sodium dodecyl sulfate lysis buffer was utilized to dissolve insoluble HBV-Pol, and Ni-NTA resin affinity chromatography was utilized for HBV-Pol purification. Most recombinant HBV-Pol was eluted with 100 mmol/L imidazole in the presence of NP-40, a weak detergent that keeps HBV-Pol in solution. A reducing agent was utilized throughout the purification steps to keep soluble HBV-Pol from redundant disulfide bond formation.

Results: The large-scale production of functional intact human HBV-Pol was achieved in an E. coli expression system. Purified HBV-Pol showed stable reverse transcriptase activity and DNA polymerase activity. The purified protein was of high purity and had stable reverse transcriptase activity.

Conclusion: Large-scale production of HBV-Pol in pure form should facilitate crystallization and detailed analysis of the structure and mechanism of HBV-Pol. Ability of this purification approach to obtain human HBV-Pol in an enzymatically active form should be helpful for development of drugs for treatment of chronic hepatitis B.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Chromatography, Affinity
  • Detergents / chemistry
  • Dithiothreitol / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Products, pol / biosynthesis
  • Gene Products, pol / chemistry
  • Gene Products, pol / genetics
  • Gene Products, pol / isolation & purification
  • Gene Products, pol / metabolism*
  • Hepatitis B virus / enzymology*
  • Hepatitis B virus / isolation & purification
  • Humans
  • Octoxynol
  • Polyethylene Glycols / chemistry
  • Recombinant Proteins / metabolism
  • Reducing Agents / chemistry
  • Sodium Dodecyl Sulfate / chemistry
  • Temperature
  • Time Factors

Substances

  • Detergents
  • Gene Products, pol
  • P protein, Hepatitis B virus
  • Recombinant Proteins
  • Reducing Agents
  • Sodium Dodecyl Sulfate
  • Polyethylene Glycols
  • Adenosine Triphosphate
  • Octoxynol
  • Nonidet P-40
  • Dithiothreitol