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. 2022:2507:201-221.
doi: 10.1007/978-1-0716-2368-8_11.

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

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

Lina Barret et al. Methods Mol Biol. 2022.

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.

Keywords: Isotopic labeling; MAS-NMR; Membrane protein; Multilamellar vesicles; Pichia pastoris; Yeast.

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References

    1. Cheng Y (2018) Membrane protein structural biology in the era of single particle cryo-EM. Curr Opin Struct Biol 52:58–63 - PubMed - PMC - DOI
    1. Reis R, Moraes I (2019) Structural biology and structure-function relationships of membrane proteins. Biochem Soc Trans 47:47–61 - PubMed - DOI
    1. Li F, Egea PF, Vecchio AJ, Asial I, Gupta M, Paulino J, Bajaj R, Dickinson MS, Ferguson-Miller S, Monk BC, Stroud RM (2021) Highlighting membrane protein structure and function: a celebration of the Protein Data Bank. J Biol Chem 17:100557 - DOI
    1. Standfuss J (2019) Membrane protein dynamics studied by X-ray lasers—or why only time will tell. Curr Opin Struct Biol 57:63–71 - PubMed - DOI
    1. Sahu ID, Lorigan GA (2020) Electron paramagnetic resonance as a tool for studying membrane proteins. Biomol Ther 10(5):763

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