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

Abstract

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 

Notes

Acknowledgments

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).

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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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