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A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids

Nature Protocols volume 9, pages 2256–2266 (2014)Cite this article

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Abstract

Interactions between lipids and proteins in the aqueous phases of cells contribute to many aspects of cell physiology. Here we describe a detailed protocol to systematically characterize in vivo–assembled complexes of soluble proteins and lipids. Saccharomyces cerevisiae strains expressing physiological amounts of a protein of interest fused to the tandem-affinity purification (TAP) tag are first lysed in the absence of detergent to capture intact protein-lipid complexes. The affinity-purified complexes (typically 30–50 kDa) are subjected to analytical size-exclusion chromatography (SEC) to remove contaminating lipids that elute at the void volume (>600 kDa), in order to achieve sufficient signal-to-background lipid ratios. Proteins in the SEC fractions are then analyzed by denaturing gel electrophoresis. Lipidomics techniques such as high-performance thin-layer chromatography or gas or liquid chromatography–mass spectrometry can then be applied to measure the elution profiles of lipids and to pinpoint the true interactors co-eluting with the TAP fusions. The procedure (starting from cell lysis) requires 2 d, and it can easily be adapted to other organisms.

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Figure 1: Overview of the procedure to capture protein-lipid complexes assembled in vivo in Saccharomyces cerevisiae.
Figure 2: Purification of the Osh4/Kes1-ergosterol complex.
Figure 3: Performance of the protocol for lipid-transfer proteins (LTPs).

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Acknowledgements

We are grateful to E.M. Vilalta, F. O'Reilly, A. Tarafder and the Protein Expression and Purification Core Facility (V. Rybin) for expert help. We also thank other members of A.-C.G.'s groups for continuous discussions and support. K.M. is supported by the Danish Natural Science Research Council (09-064986/FNU). This work is partially funded by the Deutsche Forschungsgemeinschaft (DFG) in the framework of the Cluster of Excellence, CellNetworks Initiative of the University of Heidelberg.

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Authors and Affiliations

  1. Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Kenji Maeda, Mattia Poletto, Antonella Chiapparino & Anne-Claude Gavin

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  1. Kenji Maeda
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  2. Mattia Poletto
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  3. Antonella Chiapparino
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  4. Anne-Claude Gavin
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Contributions

K.M. and A.-C.G. designed the protocol. K.M. and M.P. were responsible for the development and optimization of the protocol. A.C. supported the protocol optimization and A.-C.G. provided valuable advice.

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Correspondence to Anne-Claude Gavin.

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The authors declare no competing financial interests.

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Maeda, K., Poletto, M., Chiapparino, A. et al. A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids. Nat Protoc 9, 2256–2266 (2014). https://doi.org/10.1038/nprot.2014.148

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