ArticleJournal of biomolecular NMR2011
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Article in Journal of biomolecular NMR, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 45 citations in OpenAlex.
- A random fractionalMagnetic resonance letters · 2026Article
- Solid-State NMR Spectroscopy on Microbial Rhodopsins.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Production and Preparation of Isotopically Labeled Human Membrane Proteins in Pichia pastoris for Fast-MAS-NMR Analyses.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions.Biomolecules · 2021Article
- Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 inBiomolecules · 2020Article
- Improved strategy for isoleucineJournal of biomolecular NMR · 2019Article
- Isotopic Labeling of Eukaryotic Membrane Proteins for NMR Studies of Interactions and Dynamics.Methods in enzymology · 2019Article
- Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system.Journal of biomolecular NMR · 2018Article
- On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies.Journal of biomolecular NMR · 2018Review
- Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the AJournal of the American Chemical Society · 2018Article
- GdJournal of biomolecular NMR · 2017Article
- Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.Journal of biomolecular NMR · 2016Article
- Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy.Protein science : a publication of the Protein Society · 2015Review
- Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.Journal of biomolecular NMR · 2015Article
- Advances in NMR structures of integral membrane proteins.Current opinion in structural biology · 2013Review
- Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra.Journal of biomolecular NMR · 2013Article
- Enhanced sensitivity by nonuniform sampling enables multidimensional MAS NMR spectroscopy of protein assemblies.The journal of physical chemistry. B · 2012Article
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4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly (13)C,(15)N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution (13)C and (15)N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.
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