Abstract
Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans. We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in ~9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and ‘Protein S’), from transgenic C. elegans, demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes.
Key points
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This protocol outlines the isolation of membrane protein complexes from transgenic C. elegans
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The primary advantage of the protocol is that it enables isolation of sufficient quantities of low-abundance native membrane protein complexes for use in structural, biochemical or biophysical studies
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Acknowledgements
We thank members of the Gouaux, Aballay and Baconguis laboratories for helpful discussions; S. Petrie and B. Jenkins for help with worm spectral imaging; A. Chinn and M. Frisbie for help with worm growth; R. Hallford for proof reading and M. Freeman for suggesting studies on LPD-3. This work was supported by National Institutes of Health grant 1F32DC017894 to S.C. E.G. gratefully acknowledges J. LaCroute and B. LaCroute for support, and is an investigator of the HHMI.
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S.C., H.J. and A.G. performed the experiments. S.C., H.J., A.G. and Y.K., together with E.G., designed the project and wrote the manuscript. All authors contributed to manuscript preparation.
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Jeong, H. et al. Nature 610, 796–803 (2022): https://doi.org/10.1038/s41586-022-05314-8
Extended data
Extended Data Fig. 1 Comparison of sonication and cryo-bead milling methods for lysis of C. elegans.
Representative FSEC trace of affinity purified TMC-2-mVenus isolated from worms that were either lysed with cryo-bead milling (black traces) or sonication (blue traces). Data from three separate worm harvests are shown to illustrate that cryo-bead milling reproducibly results in a higher ratio of TMC-2 dimer to monomer.
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Clark, S., Jeong, H., Goehring, A. et al. Large-scale growth of C. elegans and isolation of membrane protein complexes. Nat Protoc 18, 2699–2716 (2023). https://doi.org/10.1038/s41596-023-00852-5
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DOI: https://doi.org/10.1038/s41596-023-00852-5
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