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Production and Preparation of Isotopically Labeled Human Membrane Proteins in Pichia pastoris for Fast-MAS-NMR Analyses

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Heterologous Expression of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2507))

Abstract

Membrane proteins (MPs) comprise about one-third of the human proteome, playing critical roles in many physiological processes and associated disorders. Consistently, they represent one of the largest classes of targets for the pharmaceutical industry. Their study at the molecular level is however particularly challenging, resulting in a severe lack of structural and dynamic information that is hindering their detailed functional characterization and the identification of novel potent drug candidates.

Magic Angle Spinning (MAS) NMR is a reliable and efficient method for the determination of protein structures and dynamics and for the identification of ligand binding sites and equilibria. MAS-NMR is particularly well suited for MPs since they can be directly analysed in a native-like lipid bilayer environment but used to require aggravating large amounts of isotope enriched material. The frequent toxicity of human MP overexpression in bacterial cultures poses an additional hurdle, resulting in the need for alternative (and often more costly) expression systems. The recent development of very fast (up to 150 kHz) MAS probes has revolutionized the field of biomolecular solid-state NMR enabling higher spectral resolution with significant reduction of the required sample, rendering eukaryotic expression systems cost-effective.

Here is presented a set of accessible procedures validated for the production and preparation of eukaryotic MPs for Fast-MAS 1H-detected NMR analysis. The methodology is illustrated with the human copper uptake protein hCTR1 recombinantly produced and 13C-15N uniformly labeled with the versatile and affordable Pichia pastoris system. Subsequent purification procedures allow the recovery of mg amounts that are then reconstituted into liposome formulations compatible with solid-state NMR handling and analysis.

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Acknowledgments

The authors warmly thank Dr. Heddy Soufari (NovAliX, Illkirch, France) for the cryo-TEM analysis of the MLV material. This work was supported by the CNRS, by the University of Strasbourg, by the European Research Council (ERC-2015-CoG GA 648974 to G.P.), and by the EC (project iNext Discovery GA 871037). Access to high-field NMR was cofunded by the CNRS (IR-RMN FR3050). L.G. is supported by a PhD fellowship from ANRT (CIFRE N°2018/1643).

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Authors and Affiliations

  1. Biotechnology and Cell Signalling, IMPReSs Protein Facility, UMR7242 CNRS-University of Strasbourg, Illkirch, France

    Lina Barret, Valérie Kugler, Lucile Guyot & Renaud Wagner

  2. Centre de Résonance Magnétique Nucléaire à Très Hauts Champs de Lyon (UMR 5082-CNRS, Université Claude Bernard Lyon 1, École Normale Supérieure Lyon), Université de Lyon, Villeurbanne, France

    Lina Barret, Tobias Schubeis & Guido Pintacuda

  3. NovAliX, Illkirch, France

    Lucile Guyot

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  1. Lina Barret
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  2. Tobias Schubeis
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  3. Valérie Kugler
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  4. Lucile Guyot
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  5. Guido Pintacuda
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  6. Renaud Wagner
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Correspondence to Renaud Wagner .

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  1. Université Côte d’Azur, CNRS UMR7275, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France

    Isabelle Mus-Veteau

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Barret, L., Schubeis, T., Kugler, V., Guyot, L., Pintacuda, G., Wagner, R. (2022). Production and Preparation of Isotopically Labeled Human Membrane Proteins in Pichia pastoris for Fast-MAS-NMR Analyses. In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology, vol 2507. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2368-8_11

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  • DOI: https://doi.org/10.1007/978-1-0716-2368-8_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2367-1

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