Heterologous Expression and Affinity Purification of Eukaryotic Membrane Proteins in View of Functional and Structural Studies: The Example of the Sarcoplasmic Reticulum Ca2+-ATPase

  • Delphine Cardi
  • Cédric Montigny
  • Bertrand Arnou
  • Marie Jidenko
  • Estelle Marchal
  • Marc le Maire
  • Christine Jaxel
Part of the Methods in Molecular Biology™ book series (MIMB, volume 601)


Heterologous SERCA1a Ca2+-ATPase (sarco-endoplasmic reticulum Ca2+-adenosine triphosphatase isoform 1a) from rabbit was expressed in yeast Saccharomyces cerevisiae as a fusion protein, with a biotin acceptor domain (BAD) linked to the SERCA C-terminus by a thrombin cleavage site. Thanks to the pYeDP60 vector, the recombinant protein was expressed under the control of a galactose-inducible promoter. Biotinylation of the protein occurred directly in yeast. Optimizing the number of galactose induction steps and increasing the amount of Gal4p transcription factor both improved expression. Lowering the temperature from 28 to 18°C during expression enhanced the recovery of detergent-extractible active protein. In the “light membrane fraction,” thought to mainly contain internal membranes, we are able to recover about 14–18 mg Ca2+-ATPase per liter of yeast culture in a bioreactor. Solubilization of this membrane fraction by n-dodecyl β-D-maltopyranoside (DDM) allowed us to recover the largest amount of active protein. The in vivo biotinylated recombinant protein was then bound to a streptavidin-Sepharose resin. Selective elution of the biotinylated SERCA1a was carried out after thrombin action on the resin-bound protein. We were able to obtain 200–500 μg/L of yeast culture of a 50% pure SERCA1a that displays an ATPase activity similar to that of the native rabbit Ca2+-ATPase. To succeed in crystallization, an additional size exclusion chromatography step was necessary. This step increases purity to 70%, removes aggregated protein and exchanges DDM for C12E8.

Key words

Biotin–streptavidin affinity Ca2+-ATPase heterologous expression membrane protein yeast bioreactor 



We would like to thank Dr. Philippe Champeil for the gift of SR membranes and for discussions. This work was supported by grants from the Commissariat à l’Energie Atomique (CEA) program Signalisation et transport membranaires and by the Agence Nationale de la Recherche (grant ANR-06-BLAN-0239-01).


  1. 1.
    Jidenko M, Nielsen RC, Sørensen TL-M, Møller JV, le Maire M, Nissen P, Jaxel C (2005) Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 102:11687–11691CrossRefPubMedGoogle Scholar
  2. 2.
    Marchand A, Winther AML, Holm PJ, Olesen C, Montigny C, Arnou B, Champeil P, Clausen JD, Vilsen B, Andersen JP, Nissen P, Jaxel C, Møller JV, le Maire M (2008) Crystal structure of D351A and P312A mutant forms of the mammalian sarcoplasmic reticulum Ca2+-ATPase reveals key events in phosphorylation and Ca2+ Release. J Biol Chem 283:14867–14882Google Scholar
  3. 3.
    Jidenko M, Lenoir G, Fuentes JM, le Maire M, Jaxel C (2006) Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain. Protein Expr Purif 48:32–42CrossRefPubMedGoogle Scholar
  4. 4.
    Cardi D. et al. In preparationGoogle Scholar
  5. 5.
    Midgett CR, Madden DR (2007) Breaking the bottleneck: eukaryotic membrane protein expression for high-resolution structural studies. J Struct Biol 160:265–274CrossRefPubMedGoogle Scholar
  6. 6.
    Ferguson AD, McKeever BM, Xu S, Wisniewski D, Miller DK, Yamin TT, Spencer RH, Chu L, Ujjainwalla F, Cunningham BR, Evans JF, Becker JW (2007) Crystal ­structure of inhibitor-bound human 5-­lipoxygenase-activating protein. Science 317:510–512Google Scholar
  7. 7.
    Jasti J, Furukawa H, Gonzales EB, Gouaux E (2007) Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH. Nature 449:316–323CrossRefPubMedGoogle Scholar
  8. 8.
    Sprang SR (2007) Structural Biology: A receptor unlocked. Nature 450:355–356Google Scholar
  9. 9.
    Pedersen BP, Buch-Pedersen MJ, Morth JP, Palmgren MG, Nissen P (2007) Crystal structure of the plasma membrane proton pump. Nature 450:1111–1114CrossRefPubMedGoogle Scholar
  10. 10.
    Drew D, Newstead S, Sonoda1 Y, Kim H, von Heijne G, Iwata S (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protocol 3:784–798Google Scholar
  11. 11.
    Centeno P, Deschamps S, Lompré A-M, Anger M, Moutin MJ, Dupont Y, Palmgren MG, Møller JV, Falson P, le Maire M (1994) Expression of the sarcoplasmic reticulum Ca2+-ATPase in yeast. FEBS Lett 354:117–122CrossRefPubMedGoogle Scholar
  12. 12.
    Lenoir G, Menguy T, Corre F, Montigny C, Pedersen PA, Thinès D, le Maire M, Falson P (2002) Over-production in yeast and rapid and efficient purification of the rabbit SERCA1a Ca2+-ATPase. Biochim Biophys Acta Biomembranes 1560:67–83CrossRefGoogle Scholar
  13. 13.
    Pompon D, Louerat B, Bronine A, Urban P (1996) Yeast expression of animal and plants P450s in optimized redox environments. Methods Enzymol 272:51–64CrossRefPubMedGoogle Scholar
  14. 14.
    Cronan JE Jr (1990) Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. J Biol Chem 265:10327–10333Google Scholar
  15. 15.
    Pouny Y, Weitzman C, Kaback HR (1998). In vitro biotinylation provides quantitative recovery of highly purified active lactose permease in a single step. Biochemistry 37:15713–15719Google Scholar
  16. 16.
    Chen DC, Yang BC, Kuo TT (1992) One-step transformation of yeast in stationary phase. Curr Genet 21:83–84CrossRefPubMedGoogle Scholar
  17. 17.
    Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425CrossRefPubMedGoogle Scholar
  18. 18.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner MD, Provenzano MD, Fugimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefPubMedGoogle Scholar
  19. 19.
    Møller JV, le Maire M (1993) Detergent binding as a measure of hydrophobic surface of integral membrane proteins. J Biol Chem 268:18659–18672PubMedGoogle Scholar
  20. 20.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  21. 21.
    Soulié S, Møller JV, Falson P, le Maire M (1996) Urea reduces the aggregation of membrane proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 236:363–364CrossRefPubMedGoogle Scholar
  22. 22.
    Fuentes JM, Lompré A-M, Møller JV, Falson P, le Maire M (2000) Clean Western blots of membrane proteins after yeast heterologous expression following a shortened version of the method of Perini et al. Anal Biochem 285:276–278CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Delphine Cardi
    • 1
  • Cédric Montigny
    • 1
  • Bertrand Arnou
    • 1
  • Marie Jidenko
    • 1
  • Estelle Marchal
    • 1
  • Marc le Maire
    • 1
  • Christine Jaxel
    • 1
  1. 1.CEA, iBiTecS – Institut de Biologie et Technologies de Saclay, University of Paris-SudGif-sur-YvetteFrance

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