Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase.

Autor:	Dieudonné T; Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France. tdieu@mbg.au.dk.; DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. tdieu@mbg.au.dk., Jaxel C; Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France., Lejeune M; Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France.; Institut Pasteur, Université de Paris, CNRS UMR3528, Structural Bioinformatics Unit, Paris, France., Lenoir G; Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France. guillaume.lenoir@i2bc.paris-saclay.fr., Montigny C; Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France. cedric.montigny@i2bc.paris-saclay.fr.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2023; Vol. 2652, pp. 231-246.
DOI:	10.1007/978-1-0716-3147-8_13
Abstrakt:	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. (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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