Expression and purification of a functional human hepatitis B virus polymerase

Yang Yu, Dipendra Raj Pandeya, Meng Lu Liu, Ming Jie Liu, Seong Tshool Hong

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 HBVPol, 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 HBVPol in an enzymatically active form should be helpful for development of drugs for treatment of chronic hepatitis B.

Original languageEnglish (US)
Pages (from-to)5752-5758
Number of pages7
JournalWorld Journal of Gastroenterology
Volume16
Issue number45
DOIs
StatePublished - Dec 1 2010

Fingerprint

Hepatitis B virus
RNA-Directed DNA Polymerase
Escherichia coli
Reducing Agents
Chronic Hepatitis B
DNA-Directed DNA Polymerase
Crystallization
Affinity Chromatography
Sodium Dodecyl Sulfate
Disulfides
Detergents
Buffers
Amino Acids

Keywords

  • Detergent
  • Hepatitis B Virus
  • Reverse transcriptase
  • Virus polymerase

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Expression and purification of a functional human hepatitis B virus polymerase. / Yu, Yang; Pandeya, Dipendra Raj; Liu, Meng Lu; Liu, Ming Jie; Hong, Seong Tshool.

In: World Journal of Gastroenterology, Vol. 16, No. 45, 01.12.2010, p. 5752-5758.

Research output: Contribution to journalArticle

Yu, Yang ; Pandeya, Dipendra Raj ; Liu, Meng Lu ; Liu, Ming Jie ; Hong, Seong Tshool. / Expression and purification of a functional human hepatitis B virus polymerase. In: World Journal of Gastroenterology. 2010 ; Vol. 16, No. 45. pp. 5752-5758.
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