Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
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Proteolysis of the Reverse Transcriptase of Hepatitis B Virus by Lon Protease in E. coli

  • Han, Joo-Seok (Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University) ;
  • Park, Jae-Yong (Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University) ;
  • Hwang, Deog-Su (Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University #AU=Joo Seok Han )
  • Published : 2001.09.01
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Abstract

Hepatitis B virus (HBV) polymerase, which possesses the activities of terminal binding, DNA polymerase, reverse transcriptase and RNaseH, has been shown to accomplish viral DNA replication through a pregenomic intermediate. Because the HBV polymerase has not been purified, the expression of HBV polymerase was examined in an E. coli expression system that is under the regulation of arabinose operon. The expressed individual domain containing terminal binding protein, polymerase, or RNaseH turned out to be insoluble. The activities of those domains were not able to be recovered by denaturation and renaturation using urea or guanidine-HCI. The expressed reverse transcriptase containing the polymerase and RNaseH domains became extensively degraded, whereas the proteolysis was reduced in a Ion- mutant. These results indicate that Lon protease proteolyzes the HBV reverse transcriptase expressed in E. coli.

Keywords

References

  1. Blum HE, Galun E, Liang TJ, von Weizsacker F, and Wands JR (1991) Naturally occurring missence mutation in the polymerase gene terminating hepatitis B virus replication. J Virol 65: 1836-1842
  2. Blum HE, Liang TJ, Galun E, and Wands JR (1991) Persistence of hepatitis B viral DNA after serological recovery from hepatitis B virus infection. Hepatology 14: 56-63 https://doi.org/10.1002/hep.1840140110
  3. Burnette WN (1981) Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195-203 https://doi.org/10.1016/0003-2697(81)90281-5
  4. Cho G, Park S-G, and Jung G (2000) Localization of HSP90 binging sites in the human hepatitis B virus polymerse. Biochem Biophys Res Commun 269: 191-196 https://doi.org/10.1006/bbrc.2000.2240
  5. Enfors S-O (1992) Control of in vivo proteolysis in the production of recombinant proteins. Trends Biotech 10: 310-315 https://doi.org/10.1016/0167-7799(92)90256-U
  6. Ganem D and Varmus HE(1987) The molecular biology of the hepatitis B viruses. Annu Rev Biochem 56: 651-693 https://doi.org/10.1146/annurev.bi.56.070187.003251
  7. Georgiou G (1995) Expression of proteins in bacteria. In: Cleland J1 and Craik CS (ed), Principles and Practice of Protein Engineering. Butteworths, New York, pp 101-127
  8. Goff SA and Goldberg AL (1985) Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell 41: 587-595 https://doi.org/10.1016/S0092-8674(85)80031-3
  9. Goldberg AL, Moerschell RP, Chung CH, and Maurizi MR (1994) ATP-dependent protease La (Lon) from Escherichia coli. Meth Enzymol 244: 350-375 https://doi.org/10.1016/0076-6879(94)44027-1
  10. Gottesman S (1989) Genetics of proteolysis in Escherichia coli. Annu Rev Genet 23: 163-198 https://doi.org/10.1146/annurev.ge.23.120189.001115
  11. Guzman L-M, Belin D, Carson MJ, and Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J Bacteriol 177: 4121-4130
  12. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557-580 https://doi.org/10.1016/S0022-2836(83)80284-8
  13. Khudyakov YE and Mahkov AM (1989) Prediction of terminal protein and ribonuclease H domains in the gene P product of hepadnaviruses. FEBS Lett 243: 115-118 https://doi.org/10.1016/0014-5793(89)80110-3
  14. Kim SS, Shin HJ, Cho YH, and Rho HM (2000) Expression of stable hepatitis B viral polymerase associated with GRP94 in E. Coli. Arch Virol 145: 1305-1320 https://doi.org/10.1007/s007050070092
  15. Lee YI, Hong YB, Kim Y, Rho HM, and Jung G (1997) RNase H activity of human hepatitis B virus polymerase expressed in Esherichia coli. Biophys Res Commun 233: 401-407 https://doi.org/10.1006/bbrc.1997.6467
  16. Maurizi MR (1992) Proteases and protein degradation in Escherichia coli. Experientia 48: 178-201 https://doi.org/10.1007/BF01923511
  17. Meissner PS, Sisk WP, and Berman ML (1987) Bacteriophage cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci USA 84: 4171-4175 https://doi.org/10.1073/pnas.84.12.4171
  18. Lien J-M, Aldrich CE, and Mason WS (1986) Evidence that a capped oligoribonucleotide is the primer for duck hepatitis B virus plus-strand DNA synthesis. J Virol 57: 229-236
  19. OFarrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007-4021
  20. Phillips TA, Van Bogelen RA, and Neidhardt FC (1984) Ion gene product of Escherichia coli is a heat-shock protein. J Bacteriol 159: 283-287
  21. Radziwill G, Tucker W, and Schaller H (1990) Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. J Virol 64: 613-620
  22. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: a Laboratory Manaul. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
  23. Seol JH, Back SH, Kang MS, Ha DB, and Chung JH (1995) Distincive roles of the two ATP-binding sites in ClpA, the ATPase component of protease Ti in Escherichia coli. J Biol Chem 270: 8087-8092 https://doi.org/10.1074/jbc.270.14.8087
  24. Summers J and Mason WS (1982) Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29: 403-415 https://doi.org/10.1016/0092-8674(82)90157-X
  25. Tomoyasu T, Gamer J, Bukau B, Kanemori M, Mori H, Rutman JA, Oppenheim BA, Yura T, Yamanaka K, Niki H, Hirage S, and Ogura T (1995) Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma-32. EMBO J 14: 2551-2560
  26. Wawrzynow D, Wojtkowiak D, Marzsalek J, Benecki B, Jonsen M, Graves B, Georgopoulos C, and Zylicz M (1995) The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone. EMBO J 14: 1867-1877
  27. Wei X and Peterson DL (1996) Expression, purification, and characterization of an active RNaseH domain of the hepatitis B viral polymerase. J Biol Chem 271: 32617-32622 https://doi.org/10.1074/jbc.271.51.32617