Expression and characterization of transforming protein E7 from cervical cancer-associated human papillomavirus type 31

Abstract

E7 protein is a major oncogenic factor of human papillomaviruses (HPVs) that plays a key role in virus-associated human cervical carcinogenesis. To determine the biochemical properties of the E7 protein of high-risk HPV type 31, the gene encoding the protein was cloned into a bacterial vector, pET-32a (+), to allow expression of HPV-31E7 as a thioredoxin (Trx) fusion protein in Escherichia coli BL21 (DE3). The resulting expression level of the fusion protein reached 15 ∼ 20% of the total cell protein and more than 60% of the target proteins were in soluble form upon cultivation for 6 h at 30°C in the presence of 0.5 mM IPTG. The fusion protein Trx-HPV-31E7 was effectively purified by Ni2+-chelating chromatography and analyzed by SDS-PAGE and Western blotting. After release from the fusion protein by enterokinase cleavage and purification to homogeneity, the recombinant HPV-31E7 (rHPV-31E7) was investigated for in vitro interaction with the pocket protein p107, which is known to interact with the amino-terminal portion of the protein. The immunoprecipitation studies revealed strong interactions of rHPV-31E7 protein with p107, suggesting it had binding activities and retained its conformational properties.

This is a preview of subscription content, access via your institution.

References

  1. 1.

    Margaret, E., D. McLaughlin, and K. Münger (2009) The human papillomavirus E7 oncoprotein. Virol. 384: 335–344.

    Article  Google Scholar 

  2. 2.

    Münger, K., A. Baldwin, K. M. Edwards, H. Hayakawa, C. L. Nguyen, M. Owens, M. Grace, and K. Huh (2004) Mechanisms of human papillomavirus-induced oncogenesis. J. Virol. 78: 11451–11460.

    Article  Google Scholar 

  3. 3.

    Howley, P. M. and D. R. Lowy (2001) Papillomaviruses. pp. 2231–2264. In: D. M. Knipe and P. M. Howley (eds.). Field’s Virology. vol. 2. 4th ed., Lippincott Williams and Wilkens, Philadelphia, USA.

    Google Scholar 

  4. 4.

    de Villiers, E. M. (1994) Human pathogenic papillomavirus types: An update. Curr. Top. Microbiol. Immunol. 186: 1–12.

    Google Scholar 

  5. 5.

    zur Hausen H. and E. M. de Villiers (1994) Human papillomaviruses. Annu. Rev. Microbiol. 48: 427–447.

    Article  Google Scholar 

  6. 6.

    Bosch, F. X. and S. de Sanjose (2002) Human papillomavirus in cervical cancer. Curr. Oncol. Rep. 4: 175–183.

    Article  Google Scholar 

  7. 7.

    Hawley-Nelson, P., K. H. Vousden, N. L. Hubbert, D. R. Lowy, and J. T. Schiller (1989) HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 8: 3905–3910.

    CAS  Google Scholar 

  8. 8.

    Ghittoni, R., R. Accardi, U. Hasan, T. Gheit, B. Sylla, and M. Tommasino (2010) The biological properties of E6 and E7 oncoproteins from human papillomaviruses. Virus Genes 40: 1–13.

    CAS  Article  Google Scholar 

  9. 9.

    zur Hausen, H. (2002) Papillomaviruses and cancer: From basic studies to clinical application. Nat. Rev. Cancer 2: 342–350.

    Article  Google Scholar 

  10. 10.

    Scheffner, M., B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley (1990) The E6 oncoprotein encoded by humanpapillomavirus types 16 and 18 promotes the degradation of p53. Cell 63: 1129–1136.

    CAS  Article  Google Scholar 

  11. 11.

    Dyson, N., P. M. Howley, K. Münger, and E. Harlow (1989) The human papillomavirus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Sci. 243: 934–937.

    CAS  Article  Google Scholar 

  12. 12.

    Münger, K., B. A. Werness, N. Dyson, W. C. Phelps, E. Harlow, and P. M. Howley (1989) Complex formation of human papillomavirus E7 proteinswith the retinoblastoma tumor suppressor gene product. EMBO J. 8: 4099–4105.

    Google Scholar 

  13. 13.

    Phillips, A. C. and K. H. Vousden (1997) Analysis of the interaction between human papillomavirus type 16 E7 and the TATAbinding protein, TBP. J. Gen. Virol. 78: 905–909.

    CAS  Google Scholar 

  14. 14.

    Zwerschke, W., S. Mazurek, P. Massimi, L. Banks, E. Eigenbrodt, and P. Jansen-Durr (1999) Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein. Proc. Natl. Acad. Sci. USA 96: 1291–1296.

    CAS  Article  Google Scholar 

  15. 15.

    Schilling, B., T. De-Medina, J. Syken, M. Vidal, and K. Münger (1998) A novel human DnaJ protein, hTid-1, a homolog of the Drosophila tumor suppressor protein Tid56, can interact with the human papillomavirus type 16 E7 oncoprotein. Virol. 247: 74–85.

    CAS  Article  Google Scholar 

  16. 16.

    Brehm, A., S. J. Nielsen, E. A. Miska, D. J. McCance, J. L. Reid, A. J. Bannister, and T. Kouzarides (1999) The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth. EMBO J. 18: 2449–2458.

    CAS  Article  Google Scholar 

  17. 17.

    Liu, X., A. Clements, K. Zhao, and R. Marmorstein (2005) Structure of the human papillomavirus E7 oncoprotein and its mechanism for inactivation of the retinoblastoma tumor suppressor. J. Biol. Chem. 281: 578–586.

    Article  Google Scholar 

  18. 18.

    Ciccolini, F., G. Di Pasquale, F. Carlotti, L. Crawford, and M. Tommasino (1994) Functional studies of E7 proteins from different HPV types. Oncogene 9: 2633–2638.

    CAS  Google Scholar 

  19. 19.

    Schmitt, A., J. B. Harry, B. Rapp, F. O. Wettstein, and T. Iftner (1994) Comparison of the properties of the E6 and E7 genes of low- and high-risk cutaneous papillomaviruses reveals strongly transforming and high Rb-binding activity for the E7 protein of the low-risk human papillomavirus type 1. J. Virol. 68: 7051–7059.

    CAS  Google Scholar 

  20. 20.

    Dong, W. L., S. Caldeira, P. Sehr, M. Pawlita, and M. Tommasino (2001) Determination of the binding affinity of different human papillomavirus E7 proteins for the tumor suppressor pRb by a plate binding assay. J. Virol. Methods 98: 91–98.

    CAS  Article  Google Scholar 

  21. 21.

    Caldeira, S., W. Dong, P. Tomakidi, A. Paradiso, and M. Tommasino (2002) Human papillomavirus type 32 does not display in vitro transforming properties. Virol. 301: 157–164.

    CAS  Article  Google Scholar 

  22. 22.

    Fernando, G. J., B. Murray, J. Zhou, and I. H. Frazer (1999) Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16. Clin. Exp. Immunol. 115: 397–403.

    CAS  Google Scholar 

  23. 23.

    Rawls, J. A., R. Pusztai, and M. Green (1990) Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: Autonomous protein domains forinduction of cellular DNA synthesis and for trans-activation. J. Virol. 64: 6121–6129.

    CAS  Google Scholar 

  24. 24.

    Pahel, G., A. Aulabaugh, S. A. Short, J. A. Barnes, G. R. Painter, P. Ray, and W. C. Phelps (1993) Structural and functional characterization of the HPV16E7 protein expressed in bacteria. J. Biol. Chem. 268: 26018–26025.

    CAS  Google Scholar 

  25. 25.

    Moro, A. and P. Hernandez (1996) Bacterial expression and immunological detection of human papillomavirus type 16 E7 protein. Biochem. Biophys. Res. Commun. 226: 895–899.

    CAS  Article  Google Scholar 

  26. 26.

    Chinami, M., K. Yuge, K. Kawano, and M. Shingu (1991) Refolding and purification of human papillomavirus type 16 E7-lacZ fusion protein expressed in Escherichia coli. Protein Expr. Purif. 2: 175–178.

    CAS  Article  Google Scholar 

  27. 27.

    Patrick, D. R., K. Zhang, D. Defeo-Jones, G. R. Vuocolo, R. Z. Maigetter, M. K. Sardana, A. Oliff, and D. C. Heimbrook (1992) Characterization of functional HPV-16 E7 protein produced in Escherichia coli. J. Biol. Chem. 267: 6910–6915.

    CAS  Google Scholar 

  28. 28.

    Kasher, M. S., M. Wakulchik, J. A. Cook, and M. C. Smith (1993) One-step purification of recombinant human papillomavirus type 16 E7 oncoprotein and its binding to the retinoblastoma gene product. Biotechniques 14: 630–641.

    CAS  Google Scholar 

  29. 29.

    Laemmli, U. K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    CAS  Article  Google Scholar 

  30. 30.

    Towbin, H., T. Staehelin, and J. Gordon (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA. 76: 4350–4354.

    CAS  Article  Google Scholar 

  31. 31.

    Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248–254.

    CAS  Article  Google Scholar 

  32. 32.

    Fiedler, M., B. Campo-Fernández, A. Laich, B. Moser, P. Stöckl, P. Jansen-Dürr, and W. Zwerschke (2006) Purification and characterization of the E7 oncoproteins of the high-risk human papillomavirus types 16 and 18. J. Virol. Methods 134: 30–35.

    CAS  Article  Google Scholar 

  33. 33.

    Jiang, X. Y., H. P. Chen, W. L. Yang, Y. Liu, W. Liu, J. W. Wei, H. B. Tu, X. J. Xie, L. Wang, and A. L. Xu (2003) Functional expression and characterization of an acidic actinoporin from sea anemone Sagartia rosea. Biochem. Biophys. Res. Commun. 312: 562–570.

    CAS  Article  Google Scholar 

  34. 34.

    Liu, W. H., L. Wang, Y. L. Wang, L. S. Peng, W. Y. Wu, W. L. Peng, X. Y. Jiang, H. B. Tu, H. P. Chen, P. O. Yang, and A. L. Xu (2003) Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp. Toxicon. 41: 793–801.

    CAS  Article  Google Scholar 

  35. 35.

    Peng, L. S., X. F. Zhong, J. X. Ou, S. L. Zheng, J. Liao, L. Wang, and A. L. Xu. (2004) High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris. J. Biotechnol. 108: 185–192.

    CAS  Article  Google Scholar 

  36. 36.

    Cho, C. W., S. H. Park, and D. H. Nam (2001) Production and purification of single chain human insulin precursors with various fusion peptides. Biotechnol. Bioproc. Eng. 6: 144–149.

    CAS  Article  Google Scholar 

  37. 37.

    Sang, B. C. and M. S. Barbosa (1992) Single amino acid substitutions in “low-risk” human papillomavirus (HPV) type 6 E7 protein enhance features characteristic of the “high-risk” HPV E7 oncoproteins. Proc. Natl. Acad. Sci. USA. 89: 8063–8067.

    CAS  Article  Google Scholar 

  38. 38.

    Münger, K., C. L. Yee, W. C. Phelps, J. A. Pietenpol, H. L. Moses, and P. M. Howley (1991) Biochemical and biological differences between E7 oncoproteins of the high- and low risk human papillomavirus types aredetermined by amino-terminal sequences. J. Virol. 65: 3943–3948.

    Google Scholar 

  39. 39.

    Heck, D. V., C. L. Yee, P. M. Howley, and K. Münger (1992) Efficiency of binding the retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the human papillomaviruses. Proc. Natl. Acad. Sci. USA. 89: 4442–4446.

    CAS  Article  Google Scholar 

  40. 40.

    Davies, R., R. Hicks, T. Crook, J. Morris, and K. Vousden (1993) Human papillomavirus type-16 E7 associates with a histone H1 kinase and with p107 through sequences necessary for transformation. J. Virol. 67: 2521–2528.

    CAS  Google Scholar 

  41. 41.

    Dyson, N., P. Guida, K. Münger, and E. Harlow (1992) Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins. J. Virol. 66: 6893–6902.

    CAS  Google Scholar 

  42. 42.

    McIntyre, M. C., M. N. Ruesch, and L. A. Laimins (1996) Human papillomavirus E7 oncoproteins bind a single form of cyclin E in a complex with cdk2 and p107. Virol. 215: 73–82.

    CAS  Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to In Seok Bang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Choi, S.B., Kang, Y.S., Bang, S.K. et al. Expression and characterization of transforming protein E7 from cervical cancer-associated human papillomavirus type 31. Biotechnol Bioproc E 18, 1109–1115 (2013). https://doi.org/10.1007/s12257-013-0467-1

Download citation

Keywords

  • E7 oncoprotein
  • human papillomaviruses
  • fusion protein
  • prokaryotic expression