Expression and purification of a functional human hepatitis B virus polymerase
- Author:
- No author available
- Journal Title:
- WORLD JOURNAL OF GASTROENTEROLOGY
- Issue:
- 45
- DOI:
- 10.3748/wjg.v16.i45.5752
- Key Word:
- Hepatitis B Virus;Virus polymerase;Reverse transcriptase;Detergent
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 HBVPoi, 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 HBVPoi in an enzymatically active form should be helpful for development of drugs for treatment of chronic hepatitis B.
- [1]Kim SS,Cho YH,Rho HM,etc.Expression of stable hepatitis B viral polymerase associated with GRP94 in E. coli.[J].2000,145(7).
- [2]M, Weber,V, Bronsema,H, Bartos,etc.Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription.[J].1994,68.
- [3]Park SG,Jung G.Human hepatitis B virus polymerase interacts with the molecular chaperonin Hsp60.[J].2001,75(15).
- [4]Han YS,Choi J,Park YI,etc.Expression of the active human and duck hepatitis B virus polymerases in heterologous system of Pichia methanolica.[J].2002,55(2).
- [5]Ji Hoon Jeong,Dong Seok Kwak,Hyune Mo Rho,etc.The Catalytic Properties of Human Hepatitis B Virus Polymerase[J].1996,223(2).
- [6]Rivkin A.Entecavir: A new nucleoside analogue for the treatment of chronic hepatitis B.[J].2007,43(4).
- [7]Beames B.,Lanford RE.,Notvall L..Nucleotide Priming and Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Expressed in Insect Cells[J].1995,69(7).
- [8]Beck J,Nassal M.Efficient Hsp90-independent in vitro activation by Hsc70 and Hsp40 of duck hepatitis B virus reverse transcriptase, an assumed Hsp90 client protein.[J].2003,278(38).
- [9]R, Ling,D, Mutimer,M, Ahmed,etc.Selection of mutations in the hepatitis B virus polymerase during therapy of transplant recipients with lamivudine.[J].1996,24.
- [10]Jung G,Kim Y.Active human hepatitis B viral polymerase expressed in rabbit reticulocyte lysate system.[J].1999,19(2).
- [11]Siddiqui A,Qadri I.Expression of hepatitis B virus polymerase in Ty1-his3AI retroelement of Saccharomyces cerevisiae.[J].1999,274(44).
- [12]Guang-Hua Wang,Christoph Seeger.The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis[J].1992,71(4).
- [13]JulianA.Chesshyre,AlanR.Hipkiss.Low temperatures stabilize interferon α-2 against proteolysis inMethylophilus methylotrophusandEscherichia coli[J].1989,31(2).
- [14]Hu J,Seeger C,Toft DO.Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids.[J].1997,16(1).
- [15]Wei X,Peterson DL.Expression, purification, and characterization of an active RNase H domain of the hepatitis B viral polymerase.[J].1996,271(51).