Brief Article
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World J Gastroenterol. Dec 7, 2010; 16(45): 5752-5758
Published online Dec 7, 2010. doi: 10.3748/wjg.v16.i45.5752
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
Yang Yu, Dipendra Raj Pandeya, Meng-Lu Liu, Ming-Jie Liu, Seong-Tshool Hong, Laboratory of Genetics, Department of Microbiology and Immunology, Institute of Medical Science, Chonbuk National University Medical School, Chonju, Chonbuk 561-712, South Korea
Author contributions: Yu Y and Pandeya DR contributed equally to this work; Yu Y and Pandeya DR performed the experiment and analyzed the data; Liu ML and Liu MJ critically revised the paper; Hong ST designed the research; Pandeya DR, Yu Y and Hong ST wrote the paper.
Supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 2010, Grants No. 08-1-28
Correspondence to: Seong-Tshool Hong, Associate Professor, Laboratory of Genetics, Department of Microbiology and Immunology, Institute of Medical Science, Chonbuk National University Medical School, Chonju, Chonbuk 561-712, South Korea. seonghong@chonbuk.ac.kr
Telephone: +82-63-2703105 Fax: +82-63-2703105
Received: June 11, 2010
Revised: August 6, 2010
Accepted: August 13, 2010
Published online: December 7, 2010
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.

Keywords: Hepatitis B Virus, Virus polymerase, Reverse transcriptase, Detergent