Abstract
Human HBV polymerase has been expressed in reticulocyte lysate system. The expressed protein shows the DNA-dependent DNA polymerase activity. In vitro transcription and translation produces a major protein product with an apparent molecular weight of approximately 100 kD. The HBV DNA polymerase has been characterized biochemically in the condition that the contaminating cellular DNA polymerases were fairly suppressed by aphidicolin and NEM. The polymerization reaction is optimal at pH 7.5 and 37°C and the polymerase requires either MnCl2 or MgCl2, with a preference for MnCl2. The protein represented an optimal activity in the presence of either 75 mM NaCl or 100 mM KCl, with a higher activity at 75 mM NaCl than 100 mM KCl. Study of the polymerizing activity of the deleted versions of the polymerase protein suggests that the terminal protein is essential for full polymerase function and the spacer region may decrease the stability of the P protein.
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Summers J. and Mason W.S., Cell 29, 403–415, 1982.
Miller R.H., Marion P.L., and Robinson S.W., Virology 139, 64–72, 1984.
Ganem D. and Varmus H.E., Annu Rev Biochem 56, 651–693, 1987.
Khudyakov Y.E. and Makhov A.M., FEBS Lett 243, 115–118, 1989.
Wang G.H. and Seeger C., Cell 71, 663–670, 1992.
Zoulim F. and Seeger C., J Virol 68, 6–13, 1994.
Lanford R.E., Notvall L., and Beames B., J Virol 69, 4431–443, 1995.
Toh H., Hayashida H., and Miyata T., Nature 305, 827–829, 1983.
Radziwill G., Zentgraf H., Schaller H., and Bosch V., Virology 163, 123–132, 1988.
Li J.-S., Cova L., Buckland R., Lambert V., Deleage G., and Trepo C., J Virol 63, 4965–4968, 1989.
Chang L.J., Hirsch R.C., Ganem D., and Varmus H.E., J Virol 64, 5553–5558, 1990.
Kaplan P.M., Greenman R.L., Gerin J.L., Purcell R.H., and Robinson W.S., J Virol 12, 995–1005, 1973.
Howe A.Y.M., Elliott J.F., and Tyrrell D.L.J., Biochem Biophys Res Commun 189, 1170–1176, 1992.
Tavis J.E. and Ganem D., Proc Natl Acad Sci USA 90, 4107–4111, 1993.
Wang G.H. and Seeger C., J Virol 67, 6507–6512, 1993.
Tavis J.E., Perri S., and Ganem D., J Virol 68, 3536–3543, 1994.
Lanford R.E., Notvall L., Lee H., and Beams B., J Virol 71, 2996–3004, 1997.
Jeong J.-H., Kwak D.-S., Rho H.-M., and Jung G., Biochem Biophys Res Commun 223, 264–271, 1996.
Wei X. and Peterson D.L., J Biol Chem 271, 32617–32622, 1996.
Lee Y.-I., Hong Y.-B., Kim Y., Rho H.-M., and Jung G., Biochem Biophys Res Commun 233, 401–407, 1997.
Rho H.-M., Kim K., Hyun S.-W., and Kim Y.-S., Nucleic Acids Res 17, 2124, 1989.
Lee H.-J., Kwon Y.-T., Rho H.-M., and Jung G., Biotechnol Lett 15, 821–826, 1993.
Sanger F., Nickelen S., and Coulson A.R., Proc Natl Acad Sci USA 74, 5463–5467, 1977.
Ikegami S., Taguchi T., Ohashi M., Oguro M., Nagano H., and Mano Y., Nature 275, 485–459, 1978.
Goto Y., Yamashita T., Arens M., Takahashi T., and Hashimoto T., Japan J Med Sci Biol 37, 9–18, 1984.
Helgstrand E., Eriksson B., Johansson N.G., Lannero B., Larsson A., Misiorny A., Noren J.O., Sjoberg B., Stenberg K., Stening G., Stridh S., Oberg B., Alenius S., and Philipson L., Science 291, 819–821, 1978.
Staschke K. and Colacino J., J Virol 68, 8265–8269, 1994.
Hu J. and Seeger C., Methods in Enzymol 275, 195–208, 1996a.
Tavis J.E., Massey B., and Gong Y., J Virol 72, 5789–5796, 1998.
Hu J. and Seeger C., Proc Natl Acad Sci USA 93, 1060–1064, 1996b.
Radziwill G., Tucker W., and Schaller H., J Virol 64, 613–620, 1990.
Faruqi A.F., Roychoudhury S., Greenberg R., Israel J., and Shih C., Virology 64, 764–768, 1991.
Tanese N. and Goff S.P., Proc Natl Acad Sci USA 85, 1777–1781, 1988.
Johnson M.S., McClure M.A., Feng D.-F., Gray J., and Doolittle R.F., Proc Natl Acad Sci USA 83, 7648–7652, 1986.
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Kim, Y., Jung, G. Active Human Hepatitis B Viral Polymerase Expressed in Rabbit Reticulocyte Lysate System. Virus Genes 19, 123–130 (1999). https://doi.org/10.1023/A:1008175107309
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DOI: https://doi.org/10.1023/A:1008175107309