Heterologous Expression and Affinity Purification of Eukaryotic Membrane Proteins in View of Functional and Structural Studies: The Example of the Sarcoplasmic Reticulum Ca2+-ATPase
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 wordsBiotin–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).
- 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
- 4.Cardi D. et al. In preparationGoogle Scholar
- 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
- 8.Sprang SR (2007) Structural Biology: A receptor unlocked. Nature 450:355–356Google Scholar
- 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
- 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.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