Abstract
Membrane proteins (MPs) are challenging to study from a biochemical standpoint owing to the difficulties associated with the isolation of these proteins from the membranes they are embedded in. Even for the expression of closely-related homologues, protocols often require to be adjusted. Prominently, the solubilization step and the stabilization of recombinant proteins during the purification process are key issues, and remain a serious bottleneck. Here, we present a method for the expression and the purification of the human ATP8B1/CDC50A lipid flippase complex. Selection of the right Saccharomyces cerevisiae strain proved to be a critical step for the successful purification of this complex. Likewise, the use of cholesteryl hemisuccinate, a cholesterol analogue, contributed to significantly increase the yield of purification. We hope that the simple method described here can help researchers to succeed in the expression of other mammalian difficult-to-express lipid flippases and, by extension, help in the production of other membrane proteins whose isolation has so far proven difficult.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Abbreviations
- BAD:
-
Biotin acceptor domain
- BCA:
-
Bicinchoninic acid
- BSA:
-
Bovin serum albumin
- CHS:
-
Cholesteryl hemisuccinate
- DDM:
-
n-dodecyl-β-D-maltopyranoside
- MPs:
-
Membrane proteins
- SDS-PAGE:
-
Sodium dodecylsulfate polyacrylamide gel electrophoresis
References
Drew D, Newstead S, Sonoda Y et al (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3:784–798
Montigny C, Azouaoui H, Jacquot A et al (2014) Overexpression of membrane proteins in Saccharomyces cerevisiae for structural and functional studies: a focus on the rabbit Ca2+-ATPase Serca1a and on the yeast lipid “Flippase” complex Drs2p/Cdc50p. In: Mus-Veteau I (ed) Membrane proteins production for structural analysis. Springer, New York, pp 133–171
Dilworth MV, Piel MS, Bettaney KE et al (2018) Microbial expression systems for membrane proteins. Methods 147:3–39
Lee HJ, Lee HS, Youn T et al (2022) Impact of novel detergents on membrane protein studies. Chem 8:980–1013
Scharff-Poulsen P, Pedersen PA (2013) Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography. PLoS One 8:e76851
Molbaek K, Scharff-Poulsen P, Helix-Nielsen C et al (2015) High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae. Microb Cell Factories 14:15
Lenoir G, Dieudonné T, Lamy A et al (2018) Screening of detergents for stabilization of functional membrane proteins. Curr Protoc Protein Sci 93:e59
Agasid MT, Robinson CV (2021) Probing membrane protein-lipid interactions. Curr Opin Struct Biol 69:78–85
Bryde S, Hennrich H, Verhulst PM et al (2010) CDC50 proteins are critical components of the human class-1 P4-ATPase transport machinery. J Biol Chem 285:40562–40572
Montigny C, Lyons J, Champeil P et al (2016) On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport. Biochim Biophys Acta (BBA) – Mol Cell Biol Lipids 1861:767–783
Lenoir G, Menguy T, Corre F et al (2002) Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase. Biochim Biophys Acta 1560:67–83
Cardi D, Montigny C, Arnou B et al (2010) Heterologous expression and affinity purification of eukaryotic membrane proteins in view of functional and structural studies: the example of the sarcoplasmic reticulum Ca(2+)-ATPase. Methods Mol Biol 601:247–267
Azouaoui H, Montigny C, Jacquot A et al (2016) Coordinated overexpression in yeast of a P4-ATPase and its associated Cdc50 subunit: the case of the Drs2p/Cdc50p lipid flippase complex. Methods Mol Biol 1377:37–55
Tropea JE, Cherry S, Waugh DS (2019) Expression and purification of soluble His(6)-tagged TEV protease. Methods Mol Biol 498:297–307
Dieudonné T, Herrera SA, Laursen MJ et al (2022) Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders. elife 11:e75272
Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Azouaoui H, Montigny C, Ash MR et al (2014) A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate. PLoS One 9:e112176
Sehgal P, Olesen C, Møller JV (2016) ATPase activity measurements by an enzyme-coupled spectrophotometric assay. In: Bublitz M (ed) P-type ATPases. Springer, New York, pp 105–109
Acknowledgements
We would like to thank warmly Joseph Lyons for stimulating discussions (Aarhus University, Denmark). We are grateful to Joost Holthuis (University of Osnabrück, Germany) for kindly providing the ATP8B1 and CDC50A cDNAs, and to Rosa López-Marqués (University of Copenhagen, Denmark) who kindly gave us the W303.1b/Δpep4 strain. This work was supported by Marie Sklodowska-Curie individual fellowship from the European Commission to T.D., an ANR grant (ANR-14-CE09-0022) to G.L., the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05) and by the Centre National de la Recherche Scientifique (CNRS).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dieudonné, T., Jaxel, C., Lejeune, M., Lenoir, G., Montigny, C. (2023). Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase. In: Sousa, Â., Passarinha, L. (eds) Advanced Methods in Structural Biology. Methods in Molecular Biology, vol 2652. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3147-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3147-8_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3146-1
Online ISBN: 978-1-0716-3147-8
eBook Packages: Springer Protocols