Abstract
The study of membrane proteins is a difficult task due to their natural embedding in hydrophobic environment made by lipids. Solubilization and purification from native membranes or overexpressed system involves the use of detergent to make them soluble while maintaining their structural and functional properties. The choice of detergent is governed not only by their ability to reach these goals, but also by their compatibility with biochemical and structural studies. A different detergent can be used during purification, and characterization of the detergent amounts present in each purification step is crucial. To address this point, we developed a colorimetric method to measure detergent content in different preparations. We analyzed detergent present in the collected fractions from the purification of the recombinant membrane translocator protein (RecTSPO). We followed detergent removal during the reconstitution of RecTSPO in liposomes and observed by electron microscopy the formation of proteoliposomes. We addressed the RecTSPO functionality by testing its ability to bind high affinity drug ligand [3H]PK 11195. We described the different parameters that should be controlled in order to optimize the measurement of this ligand binding using a filtration procedure. These protocols are useful to characterize functionality and detergent content of membrane protein, both key factors for further structural studies.
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Acknowledgment
The authors would like to thank Professor V. Papadopoulos for the generous gift of TSPO plasmids, G. Péranzi and A. Letort for their contribution in preliminary experiments. This work was supported by CNRS (Centre National de la Recherche Scientifique) and ANR (Agence National pour la Recherche) Grant 06-Blan-0190-01 to JJL.
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Ostuni, M.A., Iatmanen, S., Teboul, D., Robert, JC., Lacapère, JJ. (2010). Characterization of Membrane Protein Preparations: Measurement of Detergent Content and Ligand Binding After Proteoliposomes Reconstitution. In: Lacapère, JJ. (eds) Membrane Protein Structure Determination. Methods in Molecular Biology, vol 654. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-762-4_1
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DOI: https://doi.org/10.1007/978-1-60761-762-4_1
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Publisher Name: Humana Press, Totowa, NJ
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