Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2652; p. 231
• Main Authors: Dieudonné, Thibaud, Jaxel, Christine, Lejeune, Maylis, Lenoir, Guillaume, Montigny, Cédric
• Format: Journal Article
• Language: English
• Published: United States 2023
• Subjects: Animals; Humans; Mammals - metabolism; Membrane Proteins - metabolism; Phospholipid Transfer Proteins - metabolism; Phospholipids - metabolism; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Yeast; ATP8B1/CDC50A; Flippases; Cholesteryl hemisuccinate; Saccharomyces cerevisiae; Heterologous expression; Membrane proteins
• ISSN: 1940-6029
• DOI: 10.1007/978-1-0716-3147-8_13

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.