Advertisement

Expression of Mammalian Membrane Proteins in Mammalian Cells Using Semliki Forest Virus Vectors

  • Kenneth Lundstrom
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 601)

Abstract

One of the major bottlenecks in drug screening and structural biology on membrane proteins has for a long time been the expression of recombinant protein in sufficient quality and quantity. The expression has been evaluated in all existing expression systems, from cell-free translation and bacterial systems to expression in animal cells. In contrast to soluble proteins, the expression levels have been relatively low due to the following reasons: The topology of membrane proteins requires special, posttranslational processing, folding, and insertion into membranes, which often are mammalian cell specific. Despite these strict demands, functional membrane proteins (G protein-coupled receptors, ion channels, and transporters) have been successfully expressed in bacterial, yeast, and insect cells. A general drawback observed in prokaryotic cells is that accumulation of foreign protein in membranes is toxic and results in growth arrest and therefore low yields of recombinant protein.

In this chapter, the focus is on expression of recombinant mammalian membrane proteins in mammalian host cells, particularly applying Semliki Forest virus (SFV) vectors. Replication-deficient SFV vectors are rapidly generated at high titers in BHK-21 (Baby Hamster Kidney) cells, which then are applied for a broad range of mammalian and nonmammalian cells. The SFV system has provided high expression levels of topologically different proteins, especially for membrane proteins. Robust ligand-binding assays and functional coupling to G proteins and electrophysiological recordings have made the SFV system an attractive tool in drug discovery. Furthermore, the high susceptibility of SFV vectors to primary neurons has allowed various applications in neuroscience. Establishment of large-scale production in mammalian adherent and suspension cultures has allowed production of hundreds of milligrams of membrane proteins that has allowed their submission to serious structural biology approaches. In this context, a structural genomics program for SFV-based overexpression of 100 GPCRs was established.

Key words

Large-scale production mammalian cells Semliki forest virus structural biology 

References

  1. 1.
    Strauss JH, Strauss EG (1994) The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58:491–562PubMedGoogle Scholar
  2. 2.
    Liljeström P, Garoff H (1991) A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Bio/Technology 9:1356–1360CrossRefPubMedGoogle Scholar
  3. 3.
    Lundstrom K (2003) Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins. Biochim Biophys Acta 1610:90–96CrossRefPubMedGoogle Scholar
  4. 4.
    Ehrengruber MU, Lundstrom K, Schweitzer C, Heuss C, Schlesinger S, Gähwiler BH (1999) Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc Natl Acad Sci U S A 96:7041–7046CrossRefPubMedGoogle Scholar
  5. 5.
    Lundstrom K, Michel AD, Blasey HD, Bernard AD, Hovius R, Vogel H, Surprenant A (1997) Expression of ligand-gated ion channels with the Semliki Forest virus expression system. J Receptor Signal Transduct Res 17:115–128CrossRefGoogle Scholar
  6. 6.
    Hassaine G, Wagner R, Kempf J, Cherouati N, Hassaine N, Prual C, André N, Reinhart C, Pattus F, Lundstrom K (2005) Semliki forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells. Protein Exp Purif 45:343–351CrossRefGoogle Scholar
  7. 7.
    Lundstrom K, Wagner R, Reinhart C, Desmyter A, Cherouati N, Magnin T, Zeder-Lutz G, Courtot M, Prual C, André N, Hassaine G, Michel H, Cambillau C, Pattus F (2006) Structural genomics on membrane proteins - comparison of more than 100 GPCRs in 3 expression systems. J Struct Funct Genomics 7:77–91CrossRefPubMedGoogle Scholar
  8. 8.
    Lundstrom K (2005) Biology and application of alphaviruses in gene therapy. Gene Ther 12:S92–S97CrossRefPubMedGoogle Scholar
  9. 9.
    Lundstrom K, Ehrengruber MU (2003) Semliki forest virus (SFV) vectors in neurobiology and gene therapy. Methods Mol Med 76:503–523PubMedGoogle Scholar
  10. 10.
    Lundstrom K, Mills A, Buell G, Allet E, Adami N, Liljestrom P (1994) High-level expression of the human neurokinin-1 receptor in mammalian cell lines using the Semliki Forest virus expression system. Eur J Biochem 224:917–921CrossRefPubMedGoogle Scholar
  11. 11.
    Hovius R, Tairi A-P, Blasey H, Bernard A, Lundstrom K, Vogel H (1998) Characterization of a mouse 5-HT3 receptor purified from mammalian cells. J Neurochem 70:824–834CrossRefPubMedGoogle Scholar
  12. 12.
    Scheer A, Björklöf K, Cotecchia S, Lundstrom K (1999) Expression of the α1B-adrenergic receptor and G protein subunits in mammalian cells using the Semliki Forest virus expression system. J Receptor Signal Transduct Res 19:369–378CrossRefGoogle Scholar
  13. 13.
    Polo JM, Belli BA, Driver DA, Frolov I, Sherrill S, Hariharan MJ, Townsend K, Perri S, Mento SJ, Jolly DJ, Chang SM, Schlesinger S, Dubensky TW Jr (1999) Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors. Proc Natl Acad Sci U S A 96:4598–4603CrossRefPubMedGoogle Scholar
  14. 14.
    Lundstrom K, Hermann D, Rotmann D, Schlaeger EJ (2001) Semliki Forest virus vectors in large-scale recombinant protein production: safety aspects and achievements. Cytotechnology 35:213–221CrossRefGoogle Scholar
  15. 15.
    Schlaeger E-J (1996) The protein hydrosylate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties. J Immunol Meth 194:191–199CrossRefGoogle Scholar
  16. 16.
    Schumpp B, Schlaeger E-J (1990) Optimization of cell culture conditions for high density proliferation of HL60 human promyelocytic leukemia cells. J Cell Sci 97:639–647PubMedGoogle Scholar
  17. 17.
    Berglund P, Sjöberg M, Garoff H, Atkins GJ, Sheahan BJ, Liljeström P (1993) Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Bio/Technology 11:916–920CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Kenneth Lundstrom
    • 1
  1. 1.PanTherapeuticsLutrySwitzerland

Personalised recommendations