Skip to main content
SpringerLink
Log in

Baculoviral Expression of an Integral Membrane Protein for Structural Studies

  • Protocol
Macromolecular Crystallography Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 363))

Abstract

The baculovirus system has proven successful for the expression of integral membrane proteins for structural studies. A recombinant baculovirus, in which the gene of interest is placed under the control of the late-stage polyhedrin promoter, serves as the starting point for viral expansion and protein expression studies. Using large-scale insect cell culture techniques together with a filter-binding assay for protein function, the conditions of expression, purification, and solubilization can be optimized. As applied to the glutamate receptor ion channel subunit GluR2, this approach yields milligram quantities of pure, active protein, which have been used for single-particle electron microscopic analysis of the receptor structure. Detergent exchange protocols are also discussed, as a prerequisite for two-dimensional crystallization trials.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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. Montelione, G. T. and Anderson, S. (1999) Structural genomics: keystone for a Human Proteome Project. Nat. Struct. Biol. 6, 11–12.

    Article  CAS  PubMed  Google Scholar 

  2. Müller, A., MacCallum, R. M., and Sternberg, M. J. E. (2002) Structural characterization of the human proteome. Genome Res. 12, 1625–1641.

    Article  PubMed  Google Scholar 

  3. Tate, C. G. (2001) Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett. 504, 94–98.

    Article  CAS  PubMed  Google Scholar 

  4. Grisshammer, R. and Tate, C. G. (1995) Overexpression of integral membrane proteins for structural studies. Q. Rev. Biophys. 28, 315–422.

    Article  CAS  PubMed  Google Scholar 

  5. Green, T., Stauffer, K. A., and Lummis, S. C. (1995) Expression of recombinant homo-oligomeric 5-hydroxytryptamine3 receptors provides new insights into their maturation and structure. J. Biol. Chem. 270, 6056–6061.

    Article  CAS  PubMed  Google Scholar 

  6. Kuusinen, A., Arvola, M., Oker-Blom, C., and Keinänen, K. (1995) Purification of recombinant GluR-D glutamate receptor produced in Sf21 insect cells. Eur. J. Biochem. 233, 720–726.

    Article  CAS  PubMed  Google Scholar 

  7. Dale, W. E., Textor, J. A., Mercer, R. W., and Simchowitz, L. (1996) Expression and characterization of the human erythrocyte anion exchanger in a baculovirus/Sf-9 cell system. Protein Expr. Purif. 7, 1–11.

    Article  CAS  PubMed  Google Scholar 

  8. Weill, C., Autelitano, F., Guenet, C., Heitz, F., Goeldner, M., and Ilien, B. (1997) Pharmacological and structural integrity of muscarinic M2 acetylcholine receptors produced in Sf9 insect cells. Eur. J. Pharmacol. 333, 269–278.

    Article  CAS  PubMed  Google Scholar 

  9. Autry, J. M. and Jones, L. R. (1998) High-level coexpression of the canine cardiac calcium pump and phospholamban in Sf21 insect cells. Ann. N. Y. Acad. Sci. 853, 92–102.

    Article  CAS  PubMed  Google Scholar 

  10. Chinni, C., Bottomley, S. P., Duffy, E. J., Hemmings, B. A., and Stone, S. R. (1998) Expression and purification of the human thrombin receptor. Protein Expr. Purif. 13, 9–15.

    Article  CAS  PubMed  Google Scholar 

  11. Fukuzono, S., Takeshita, T., Sakamoto, T., Hisada, A., Shimizu, N., and Mikoshiba, K. (1998) Overproduction and immuno-affinity purification of myelin proteolipid protein (PLP), an inositol hexakisphosphate-binding protein, in a baculovirus expression system. Biochem. Biophys. Res. Commun. 249, 66–72.

    Article  CAS  PubMed  Google Scholar 

  12. van Huizen, R., Czajkowsky, D. M., Shi, D., Shao, Z., and Li, M. (1999) Images of oligomeric Kv beta 2, a modulatory subunit of potassium channels. FEBS Lett. 457, 107–111.

    Article  PubMed  Google Scholar 

  13. Huang, Y. W., Lu, M. L., Qi, H., and Lin, S. X. (2000) Membrane-bound human 3beta-hydroxysteroid dehydrogenase: overexpression with His-tag using a baculovirus system and single-step purification. Protein Expr. Purif. 18, 169–174.

    Article  CAS  PubMed  Google Scholar 

  14. Rao, U. S., Mehdi, A., and Steimle, R. E. (2000) Expression of amiloride-sensitive sodium channel: a strategy for the coexpression of multimeric membrane protein in Sf9 insect cells. Anal. Biochem. 286, 206–213.

    Article  CAS  PubMed  Google Scholar 

  15. Tierney, M. L. and Unwin, N. (2000) Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor. J. Mol. Biol. 303, 185–196.

    Article  CAS  PubMed  Google Scholar 

  16. Antaramian, A., Butanda-Ochoa, A., Vazquez-Martinez, O., Diaz-Munoz, M., and Vaca, L. (2001) Functional expression of recombinant type 1 ryanodine receptor in insect cells. Cell Calcium 30, 9–17.

    Article  CAS  PubMed  Google Scholar 

  17. Werten, P. J., Hasler, L., Koenderink, J. B., et al. (2001) Large-scale purification of functional recombinant human aquaporin-2. FEBS Lett. 504, 200–205.

    Article  CAS  PubMed  Google Scholar 

  18. Safferling, M., Tichelaar, W., Kümmerle, G., et al. (2001) First images of a glutamate receptor ion channel: oligomeric state and molecular dimensions of GluRB homomers. Biochemistry 40, 13,948–13,953.

    Article  CAS  PubMed  Google Scholar 

  19. Massotte, D. (2003) G protein-coupled receptor overexpression with the baculovirus-insect cell system: a tool for structural and functional studies. Biochim. Biophys. Acta 1610, 77–89.

    Article  CAS  PubMed  Google Scholar 

  20. Warne, T., Chirnside, J., and Schertler, G. F. (2003) Expression and purification of truncated, non-glycosylated turkey beta-adrenergic receptors for crystallization. Biochim. Biophys. Acta 1610, 133–140.

    Article  CAS  PubMed  Google Scholar 

  21. Joyce, K. A., Atkinson, A. E., Bermudez, I., Beadle, D. J., and King, L. A. (1993) Synthesis of functional GABAA receptors in stable insect cell lines. FEBS Lett. 335, 61–64.

    Article  CAS  PubMed  Google Scholar 

  22. Farrell, P. J., Lu, M., Prevost, J., Brown, C., Behie, L., and Iatrou, K. (1998) High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector. Biotechnol. Bioeng. 60, 656–663.

    Article  CAS  PubMed  Google Scholar 

  23. Higgins, M. K., Demir, M., and Tate, C. G. (2003) Calnexin co-expression and the use of weaker promoters increase the expression of correctly assembled Shaker potassium channel in insect cells. Biochim. Biophys. Acta 1610, 124–132.

    Article  CAS  PubMed  Google Scholar 

  24. Jarvis, D. L. (2003) Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology 310, 1–7.

    Article  CAS  PubMed  Google Scholar 

  25. Frank, J., Radermacher, M., Penczek, P., et al. (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struc. Biol. 116, 190–199.

    Article  CAS  Google Scholar 

  26. van Heel, M., Harauz, G., and Orlova, E. V. (1996) A new generation of the IMAGIC image processing system. J. Struc. Biol. 116, 17–24.

    Article  Google Scholar 

  27. Ludtke, S. J., Baldwin, P. R., and Chiu, W. (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struc. Biol. 128, 82–97.

    Article  CAS  Google Scholar 

  28. Schiöth, H. B., Kuusinen, A., Muceniece, R., Szardenings, M., Keinänen, K., and Wikberg, J. E. (1996) Expression of functional melanocortin 1 receptors in insect cells. Biochem. Biophys. Res. Commun. 221, 807–814.

    Article  PubMed  Google Scholar 

  29. Keinänen, K., Wisden, W., Sommer, B., et al. (1990) A family of AMPA-selective glutamate receptors. Science 249, 556–560.

    Article  PubMed  Google Scholar 

  30. Sommer, B., Keinänen, K., Verdoorn, T. A., et al. (1990) Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science 249, 1580–1585.

    Article  CAS  PubMed  Google Scholar 

  31. Madden, D. R., Abele, R., Andersson, A., and Keinänen, K. (2000) Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain. Eur. J. Biochem. 267, 4281–4289.

    Article  CAS  PubMed  Google Scholar 

  32. Ausubel, F. M., Brent, R., Kingston, R. E., et al. (eds.) (1999) Short Protocols in Molecular Biology, John Wiley, New York, NY.

    Google Scholar 

  33. O’Reilly, D. R., Miller, L. K., and Luckow, V. A. (1994) Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press, New York, NY.

    Google Scholar 

  34. Keinänen, K., Kohr, G., Seeburg, P. H., Laukkanen, M. L., and Oker-Blom, C. (1994) High-level expression of functional glutamate receptor channels in insect cells. Biotechnology (N.Y.) 12, 802–806.

    Article  Google Scholar 

  35. Safferling, M. (2000) Expression und Charakterisierung von Neurotransmitterrezeptoren am Beispiel des nikotinischen α7-Acetylcholinrezeptors und des ionotropen Glutamatrezeptors GluRB, Ph.D., University of Kaiserslautern, Kaiserslautern, Germany, May 2000.

    Google Scholar 

  36. Tichelaar, W., Safferling, M., Keinänen, K., Stark, H., and Madden, D. R. (2004) The three-dimensional structure of a nonotropic glutamate receptor reveals a dimer-of-dimers assembly. J. Mol. Biol. 344, 435–442.

    Article  CAS  PubMed  Google Scholar 

  37. Scarborough, G. A. (1994) Large single crystals of the Neurospora crassa plasma membrane H+-ATPase: an approach to the crystallization of integral membrane proteins. Acta Crystallogr. D50, 643–649.

    CAS  Google Scholar 

  38. Auer, M., Scarborough, G. A., and Kühlbrandt, W. (1999) Surface crystallisation of the plasma membrane H+-ATPase on a carbon support film for electron crystallography. J. Mol. Biol. 287, 961–968.

    Article  CAS  PubMed  Google Scholar 

  39. Kühlbrandt, W. (1992) Two-dimensional crystallization of membrane proteins. Q. Rev. Biophys. 25, 1–49.

    Article  PubMed  Google Scholar 

  40. Hasler, L., Heymann, J. B., and Engel, A. (1998) 2D crystallization of membrane proteins: rationales and examples. J. Struct. Biol. 121, 162–171.

    Article  CAS  PubMed  Google Scholar 

  41. Mosser, G. (2001) Two-dimensional crystallogenesis of transmembrane proteins. Micron 32, 517–540.

    Article  CAS  PubMed  Google Scholar 

  42. Rigaud, J.-L., Chami, M., Lambert, O., Levy, D., and Ranck, J.-L. (2000) Use of detergents in two-dimensional crystallization of membrane proteins. Biochim. Biophys. Acta 1508, 112–128.

    Article  CAS  PubMed  Google Scholar 

  43. Ringler, P., Heymann, B., and Engel, A. (2000) Two-dimensional crystallization of membrane proteins. In: Membrane Transport: A Practical Approach, (Baldwin, S. A., ed.), Oxford University Press, Oxford, UK.

    Google Scholar 

  44. Stahlberg, H., Fotiadis, D., Scheuring, S., et al. (2001) Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins. FEBS Lett. 504, 166–172.

    Article  CAS  PubMed  Google Scholar 

  45. Lévy, D., Mosser, G., Lambert, O., Moeck, G. S., Bald, D., and Rigaud, J.-L. (1999) Two-dimensional crystallization on lipid layer: a successful approach for membrane proteins. J. Struct. Biol. 127, 44–52.

    Article  PubMed  Google Scholar 

  46. Lebeau, L., Lach, F., Vénien-Bryan, C., et al. (2001) Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer. J. Mol. Biol. 308, 639–647.

    Article  CAS  PubMed  Google Scholar 

  47. Invitrogen Corporation. (2002) Bac-to-Bac® Baculovirus Expression System Manual. Version C, p. 5.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, Dartmouth Medical School, Hanover, NH

    Dean R. Madden

  2. Ion Channel Structure Group, Max Planck Institute for Medical Research, Heidelberg, Germany

    Dean R. Madden & Markus Safferling

  3. Bayer CropScience AG, Monheim, Germany

    Markus Safferling

Authors
  1. Dean R. Madden
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Markus Safferling
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT

    Sylvie Doublié

Rights and permissions

Reprints and permissions

Copyright information

© 2007 Humana Press Inc.

About this protocol

Cite this protocol

Madden, D.R., Safferling, M. (2007). Baculoviral Expression of an Integral Membrane Protein for Structural Studies. In: Walker, J.M., Doublié, S. (eds) Macromolecular Crystallography Protocols. Methods in Molecular Biology, vol 363. Humana Press. https://doi.org/10.1007/978-1-59745-209-0_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-209-0_3

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-292-6

  • Online ISBN: 978-1-59745-209-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation