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Cell-Free Expression for the Study of Hydrophobic Proteins: The Example of Yeast ATP-Synthase Subunits

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Membrane Protein Structure and Function Characterization

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1635))

Abstract

Small hydrophobic membrane proteins or proteins with hydrophobic domains are often difficult to produce in bacteria. The cell-free expression system was found to be a very good alternative for the expression of small hydrophobic subunits of the yeast ATP-synthase, such as subunits e, g, k, i, f and the membrane domain of subunit 4, proteins that are suspected to play a role in the stability of ATP-synthase dimers. All of these proteins could be produced in milligrams amounts using the cell-free “precipitate mode” and were successfully solubilized in the presence of lysolipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1′-rac-glycerol. Purified proteins were also found suitable for structural investigations. An example is given with the NMR backbone assignment of the isotopically labeled subunit g. Protocols are also described for raising specific polyclonal antibodies against overexpressed cell-free proteins.

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Acknowledgment

We would like to thank Franck Bernhard (Institute of Biophysical Chemistry, Goethe-Universität Frankfurt am Main) for helpful advises concerning the CF-expression system. This work was supported by the F1Fo-Struct ANR grant (ANR-12-BSV8-024). We thank D. Brèthes for a careful reading of the manuscript.

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Authors and Affiliations

  1. SysTEMM, CNRS, UMR5095, IBGC, CS 61390, 1 rue Camille Saint-Saëns, 33077, Bordeaux, France

    Isabelle Larrieu, Corinne Sanchez, Edmond Yazo Kone, Alexandre Barras & Marie-France Giraud

  2. Université de Bordeaux, Campus Carreire, 146 Rue Léo Saignat, 33000, Bordeaux, France

    Isabelle Larrieu, Corinne Sanchez, Edmond Yazo Kone & Marie-France Giraud

  3. CNRS, UMR 5248, CBMN, 33600, Pessac, France

    James Tolchard, Alexandre Barras & Benoît Odaert

  4. Université de Bordeaux, Campus de Pessac, Allée Geoffroy Saint-Hilaire, 33600, Pessac, France

    James Tolchard, Alexandre Barras & Benoît Odaert

  5. CNRS, CRPP, UPR 8641, F-33600, Pessac, France

    Claire Stines-Chaumeil

  6. Université de Bordeaux, CRPP, UPR 8641, F-33600, Pessac, France

    Claire Stines-Chaumeil

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  1. Isabelle Larrieu
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  2. James Tolchard
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  7. Benoît Odaert
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  8. Marie-France Giraud
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Correspondence to Marie-France Giraud .

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  1. Département de Chimie, CNRS UMR 7203 LBM, Sorbonne Universités—UPMC Univ Paris 06, École Normale Supérieure—PSL Research University, Paris, France

    Jean-Jacques Lacapere

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Larrieu, I. et al. (2017). Cell-Free Expression for the Study of Hydrophobic Proteins: The Example of Yeast ATP-Synthase Subunits. In: Lacapere, JJ. (eds) Membrane Protein Structure and Function Characterization. Methods in Molecular Biology, vol 1635. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7151-0_4

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  • DOI: https://doi.org/10.1007/978-1-4939-7151-0_4

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