Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase
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...
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Published in | Methods in molecular biology (Clifton, N.J.) Vol. 2652; p. 231 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
2023
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Subjects | |
Online Access | Get more information |
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Summary: | 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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ISSN: | 1940-6029 |
DOI: | 10.1007/978-1-0716-3147-8_13 |