Expression and purification of a functional human hepatitis B virus polymerase.
OBJECTIVE
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.


CONCLUSIONS
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.
Laboratory of Genetics, Department of Microbiology and Immunology, Institute of Medical Science, Chonbuk National University Medical School, Chonju, Chonbuk 561-712, South Korea.
Yang
Y
Yu
Dipendra Raj
DR
Pandeya
Meng-Lu
ML
Liu
Ming-Jie
MJ
Liu
Seong-Tshool
ST
Hong
D000255:Adenosine Triphosphate / Q000378:metabolism; D002846:Chromatography, Affinity; D003902:Detergents / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015684:Gene Products, pol / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006515:Hepatitis B virus / Q000201:enzymology* / Q000302:isolation & purification; D006801:Humans; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D019163:Reducing Agents / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
D003902:Detergents; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D011994:Recombinant Proteins; D019163:Reducing Agents; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; C010615:Nonidet P-40; D004229:Dithiothreitol