Skip to main content
SpringerLink
Log in

Large-Scale Purification and Crystallization of Adenovirus Hexon

  • Protocol
Adenovirus Methods and Protocols

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 131))

Abstract

This chapter provides a protocol for the large-scale purification of adenovirus type 2 and 5 virions and the soluble major coat protein hexon. The purified virus particles remain intact and are suitable for vector, vaccine, or structural studies and can also be used as seed stock for further rounds of infection. The hexon may be used to produce crystals suitable for high-resolution X-ray crystallographic studies. Briefly, virus is propagated in HeLa cell suspension cultures. The infected cells are lysed, virions and hexon are separated by centrifugation, and the protein is then further purified by anion exchange chromatography. The entire purification procedure takes approx 1 wk and typically yields 1013 virus particles and 10–20 mg of highly purified hexon.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Rowe, W. P., Huebner, R. J., Gillmore, L. K., Parrott, R. H., and Ward, T. G. (1953) Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc. Soc. Exp. Biol. Med. 84, 570–573.

    CAS  PubMed  Google Scholar 

  2. Horne, R. W., Brenner, S., Waterson, A. P., and Wildy, P. (1959) The icosahedral form of an adenovirus. J. Mol. Biol. 1, 84–86.

    Article  CAS  Google Scholar 

  3. Rux, J. J. and Burnett, R. M. (2004) Adenovirus structure. Hum. Gene Ther. 15, 1167–1176.

    Article  CAS  PubMed  Google Scholar 

  4. Stewart, P. L., Burnett, R. M., Cyrklaff, M., and Fuller, S. D. (1991) Image reconstruction reveals the complex molecular organization of adenovirus. Cell 67, 145–154.

    Article  CAS  PubMed  Google Scholar 

  5. van Oostrum, J. and Burnett, R. M. (1985) Molecular composition of the adenovirus type 2 virion. J. Virol. 56, 439–448.

    PubMed  Google Scholar 

  6. White, D. O., Scharff, M. D., and Maizel, J. V., Jr. (1969) The polypeptides of adenovirus. III. Synthesis in infected cells. Virology 38, 395–406.

    Article  CAS  PubMed  Google Scholar 

  7. Pereira, H. G., Valentine, R. C., and Russell, W. C. (1968) Crystallization of an adenovirus protein (the hexon). Nature 219, 946–947.

    Article  CAS  PubMed  Google Scholar 

  8. Burnett, R. M., Grütter, M. G., and White, J. L. (1985) The structure of the adenovirus capsid. I. An envelope model of hexon at 6 Å resolution. J. Mol. Biol. 185, 105–123.

    Article  CAS  PubMed  Google Scholar 

  9. Roberts, M. M., White, J. L., Grütter, M. G., and Burnett, R. M. (1986) Three-dimensional structure of the adenovirus major coat protein hexon. Science 232, 1148–1151.

    Article  CAS  PubMed  Google Scholar 

  10. Athappilly, F. K., Murali, R., Rux, J. J., Cai, Z., and Burnett, R. M. (1994) The refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 Å resolution. J. Mol. Biol. 242, 430–455.

    Article  CAS  PubMed  Google Scholar 

  11. Rux, J. J. and Burnett, R. M. (2000) Type-specific epitope locations revealed by X-ray crystallographic study of adenovirus type 5 hexon. Mol. Therapy 1, 18–30.

    Article  CAS  Google Scholar 

  12. Rux, J. J., Kuser, P. R., and Burnett, R. M. (2003) Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution X-ray crystallographic, molecular modeling, and sequence-based methods. J. Virol. 77, 9553–9566.

    Article  CAS  PubMed  Google Scholar 

  13. Stewart, P. L., Fuller, S. D., and Burnett, R. M. (1993) Difference imaging of adenovirus: Bridging the resolution gap between X-ray crystallography and electron microscopy. EMBO J. 12, 2589–2599.

    CAS  PubMed  Google Scholar 

  14. Zubieta, C., Schoehn, G., Chroboczek, J., and Cusack, S. (2005) The structure of the human adenovirus 2 penton. Mol. Cell 17, 121–135.

    Article  CAS  PubMed  Google Scholar 

  15. Fabry, C. M. S., Rosa-Calatrava, M., Conway, J. F., et al. (2005) A quasi-atomic model of human adenovirus type 5 capsid. EMBO J. 24, 1645–1654.

    Article  CAS  PubMed  Google Scholar 

  16. Rux, J. J., Pascolini, D., and Burnett, R. M. (1999) Large scale purification and crystallization of adenovirus hexon, in Adenovirus Methods and Protocols (Methods in Molecular Medicine, vol. 21) (Wold, W. S. M., ed.), Humana Press, Totowa, NJ, pp. 259–275.

    Google Scholar 

  17. Furcinitti, P. S., van Oostrum, J., and Burnett, R. M. (1989) Adenovirus polypeptide IX revealed as capsid cement by difference images from electron microscopy and crystallography. EMBO J. 8, 3563–3570.

    CAS  PubMed  Google Scholar 

  18. Saban, S. D., Nepomuceno, R. R., Gritton, L. D., Nemerow, G. R., and Stewart, P. L. (2005) CryoEM structure at 9Å resolution of an adenovirus vector targeted to hematopoietic cells. J. Mol. Biol. 343, 526–537.

    Article  Google Scholar 

  19. Kozarsky, K. F. and Wilson, J. M. (1993) Gene therapy: Adenovirus vectors. Curr. Opin. Genet. Dev. 3, 499–503.

    Article  CAS  PubMed  Google Scholar 

  20. Seth, P. (ed.) (1999) Adenoviruses: From Basic Research to Gene Therapy Applications, R.G. Landes, Austin, TX.

    Google Scholar 

  21. Doerfler, W. and Böhm, P. (eds.) (2003) Adenoviruses: Model and Vectors in Virus Host Interactions(Current Topics in Microbiology and Immunology, vols. 272 & 273), Springer-Verlag, Berlin.

    Google Scholar 

  22. Everts, M. and Curiel, D. T. (2004) Transductional targeting of adenoviral cancer gene therapy. Curr. Gene Ther. 4, 337–346.

    CAS  PubMed  Google Scholar 

  23. Tatsis, N. and Ertl, H. C. J. (2004) Adenoviruses as vaccine vectors. Mol. Ther. 10, 616–629.

    Article  CAS  PubMed  Google Scholar 

  24. Franklin, R. M., Petterson, U., Åkervall, K., Strandberg, B., and Philipson, L. (1971) Structural proteins of adenovirus. V. Size and structure of the adenovirus type 2 hexon. J. Mol. Biol. 57, 383–395.

    Article  CAS  PubMed  Google Scholar 

  25. Cornick, G., Sigler, P. B., and Ginsberg, H. S. (1971) Characterization of crystals of type 5 adenovirus hexon. J. Mol. Biol. 57, 397–401.

    Article  CAS  PubMed  Google Scholar 

  26. Döhner, L. and Hudemann, H. (1972) Untersuchungen zur Kristallisation von Hexonen der Adenoviren der Gruppe III. Arch. Gesamte Virusforsch. 38, 279–289.

    Article  PubMed  Google Scholar 

  27. Xue, F. and Burnett, R. M. (2006) Capsid-like arrays in crystals of chimpanzee adenovirus hexon. J. Struct. Biol. 154, 217–221.

    Article  CAS  PubMed  Google Scholar 

  28. Xu, L., Benson, S. D., and Burnett, R. M. (2006) Nanoporous crystals of Chicken Embryo Lethal Orphan (CELO) adenovirus major coat protein, hexon. J. Struct. Biol. doi: 10.1016/j.jsb.2006.08.017.

    Google Scholar 

  29. Devaux, C., Adrian, M., Berthet-Colominas, C., Cusack, S., and Jacrot, B. (1990) Structure of adenovirus fibre. I. Analysis of crystals of fibre from adenovirus serotypes 2 and 5 by electron microscopy and X-ray crystallography. J. Mol. Biol. 215, 567–588.

    Article  CAS  PubMed  Google Scholar 

  30. van Raaij, M. J., Mitraki, A., Lavigne, G., and Cusack, S. (1999) A triple betaspiral in the adenovirus fibre shaft reveals a new structural motif for a fibrous protein. Nature 401, 935–938.

    Article  PubMed  Google Scholar 

  31. Xia, D., Henry, L. J., Gerard, R. D., and Deisenhofer, J. (1994) Crystal structure of the receptor-binding domain of adenovirus type 5 fiber protein at 1.7 Å resolution. Structure 2, 1259–1270.

    Article  CAS  PubMed  Google Scholar 

  32. van Raaij, M. J., Louis, N., Chroboczek, J., and Cusack, S. (1999) Structure of the human adenovirus serotype 2 fiber head domain at 1.5 A resolution. Virology 262, 333–343.

    Article  PubMed  Google Scholar 

  33. Bewley, M. C., Springer, K., Zhang, Y. B., Freimuth, P., and Flanagan, J. M. (1999) Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR. Science 286, 1579–1583.

    Article  CAS  PubMed  Google Scholar 

  34. Durmort, C., Stehlin, C., Schoehn, G., et al. (2001) Structure of the fiber head of Ad3, a non-CAR-binding serotype of adenovirus. Virology 285, 302–312.

    Article  CAS  PubMed  Google Scholar 

  35. Schoehn, G., Fender, P., Chroboczek, J., and Hewat, E. A. (1996) Adenovirus 3 penton dodecahedron exhibits structural changes of the base on fibre binding. EMBOJ. 15, 6841–6846.

    Google Scholar 

  36. Cepko, C. L. and Sharp, P. A. (1982) Assembly of adenovirus major capsid protein is mediated by a nonvirion protein. Cell 31, 407–415.

    Article  CAS  PubMed  Google Scholar 

  37. Molinier-Frenkel, V., Lengagne, R., Gaden, F., et al. (2002) Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response. J. Virol. 76, 127–135.

    Article  CAS  PubMed  Google Scholar 

  38. Pettersson, U., Philipson, L., and Höglund, S. (1967) Structural proteins of adenoviruses. I. Purification and characterization of the adenovirus type 2 hexon antigen. Virology 33, 575–590.

    Article  CAS  PubMed  Google Scholar 

  39. Dowdle, W. R., Lambriex, M., and Hierholzer, J. C. (1971) Production and evaluation of a purified adenovirus group-specific (hexon) antigen for use in the diagnostic complement fixation test. Appl. Microbiol. 21, 718–722.

    CAS  PubMed  Google Scholar 

  40. Boulanger, P. A. and Puvion, F. (1973) Large-scale preparation of soluble adenovirus hexon, penton and fiber antigens in highly purified form. Eur. J. Biochem. 39, 37–42.

    Article  CAS  PubMed  Google Scholar 

  41. Grütter, M. and Franklin, R. M. (1974) Studies on the molecular weight of the adenovirus type 2 hexon and its subunit. J. Mol. Biol. 89, 163–178.

    Article  PubMed  Google Scholar 

  42. Jörnvall, H., Pettersson, U., and Philipson, L. (1974) Structural studies of adenovirus type-2 hexon protein. Eur. J. Biochem. 48, 179–192.

    Article  PubMed  Google Scholar 

  43. Siegel, S. A., Hutchins, J. E., and Witt, D. J. (1987) Purification of adenovirus hexon by high performance liquid chromatography. J. Virol. Meth. 17, 211–217.

    Article  CAS  Google Scholar 

  44. Challberg, S. S. and Ketner, G. (1981) Deletion mutants of adenovirus 2: Isolation and initial characterization of virus carrying mutations near the right end of the viral genome. Virology 114, 196–209.

    Article  CAS  PubMed  Google Scholar 

  45. McPherson, A. (1982) Preparation and Analysis of Protein Crystals, Wiley, New York.

    Google Scholar 

  46. Owens, J., Walthall, B., and Murphy, T. (1993) The effects of freshly prepared cell culture medium vs stored cell culture medium on high-density cell culture. Am. Biotech. Lab. 11, 64–66.

    CAS  Google Scholar 

  47. Blanche, F., Cameron, B., Somarriba, S., Maton, L., Barbot, A., and Guillemin, T. (2001) Stabilization of recombinant adenovirus: Site-directed mutagenesis of key asparagine residues in the hexon protein. Anal. Biochem. 297, 1–9.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. The Wistar Institute, Philadelphia, PA

    John J. Rux

  2. The Wistar Institute, Philadelphia, PA

    Roger M. Burnett

Authors
  1. John J. Rux
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roger M. Burnett
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Saint Louis University School of Medicine, St. Louis, Missouri

    William S. M. Wold  & Ann E. Tollefson  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2007 Humana Press Inc, Totowa, NJ

About this protocol

Cite this protocol

Rux, J.J., Burnett, R.M. (2007). Large-Scale Purification and Crystallization of Adenovirus Hexon. In: Wold, W.S.M., Tollefson, A.E. (eds) Adenovirus Methods and Protocols. Methods in Molecular Medicine™, vol 131. Humana Press. https://doi.org/10.1007/978-1-59745-277-9_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-277-9_17

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-901-7

  • Online ISBN: 978-1-59745-277-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation