Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase - Archive ouverte HAL
Article Dans Une Revue Methods in Molecular Biology Année : 2023

Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase

Résumé

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.
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Dates et versions

hal-04238569 , version 1 (12-10-2023)

Identifiants

Citer

Thibaud Dieudonné, Christine Jaxel, Maylis Lejeune, Guillaume Lenoir, Cédric Montigny. Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase. Methods in Molecular Biology, 2023, Advanced Methods in Structural Biology, 2652, pp.231 - 246. ⟨10.1007/978-1-0716-3147-8_13⟩. ⟨hal-04238569⟩
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