ArticleBiochimica et biophysica acta2012
Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination.
Article in Biochimica et biophysica acta, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 38 citations in OpenAlex.
- Article
- Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity.PLoS pathogens · 2021Article
- Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies.PloS one · 2021Article
- Membrane proteins in magnetically aligned phospholipid polymer discs for solid-state NMR spectroscopy.Biochimica et biophysica acta. Biomembranes · 2020Article
- Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells.Methods in molecular biology (Clifton, N.J.) · 2020Article
- Article
- ImprovedFrontiers in molecular biosciences · 2017Article
- Current strategies for protein production and purification enabling membrane protein structural biology.Biochemistry and cell biology = Biochimie et biologie cellulaire · 2016Review
- Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy.Protein science : a publication of the Protein Society · 2015Review
- Membrane protein structure from rotational diffusion.Biochimica et biophysica acta · 2015Review
- Large-scale production and protein engineering of G protein-coupled receptors for structural studies.Frontiers in pharmacology · 2015Review
- Amphipols in G protein-coupled receptor pharmacology: what are they good for?The Journal of membrane biology · 2014Review
- Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor.Biochemistry and cell biology = Biochimie et biologie cellulaire · 2014Article
- Detergent optimized membrane protein reconstitution in liposomes for solid state NMR.Biochemistry · 2014Article
- Solid state NMR: The essential technology for helical membrane protein structural characterization.Journal of magnetic resonance (San Diego, Calif. : 1997) · 2014Article
- Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples.Nature protocols · 2013Article
- Structural Characterization of an LPA1 Second Extracellular Loop Mimetic with a Self-Assembling Coiled-Coil Folding Constraint.International journal of molecular sciences · 2013Article
- Article
- Improved 1H amide resonance line narrowing in oriented sample solid-state NMR of membrane proteins in phospholipid bilayers.Journal of magnetic resonance (San Diego, Calif. : 1997) · 2012Article
- M2 protein from influenza A: from multiple structures to biophysical and functional insights.Current opinion in virology · 2012Review
Corrections and comments
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Authors and funding
6 authors at 3 institutions in 2 countries.
Funding
Abstract
The human chemokine receptor CXCR1 is a G-protein coupled receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.