ArticleProtein expression and purification2010
Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation.
Article in Protein expression and purification, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 32 citations in OpenAlex.
- Article
- Special issue for Klaus Gawrisch.Biophysical journal · 2023Article
- COGRIMEN: Coarse-Grained Method for Modeling of Membrane Proteins in Implicit Environments.Journal of chemical theory and computation · 2022Article
- Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture.Scientific reports · 2020Article
- Expression and Preparation of a G-Protein-Coupled Cannabinoid Receptor CBCurrent protocols in protein science · 2019Article
- Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CBProtein expression and purification · 2017Article
- Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II.Methods in enzymology · 2017Article
- Global fold of human cannabinoid type 2 receptor probed by solid-state 13C-, 15N-MAS NMR and molecular dynamics simulations.Proteins · 2014Article
- Use of tandem affinity chromatography for purification of cannabinoid receptor CB₂.Methods in molecular biology (Clifton, N.J.) · 2014Article
- Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2.Biochimica et biophysica acta · 2013Article
- Application of HaloTag technology to expression and purification of cannabinoid receptor CB2.Protein expression and purification · 2013Article
- Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra.Journal of biomolecular NMR · 2013Article
- Advances in methods to characterize ligand-induced ionic lock and rotamer toggle molecular switch in G protein-coupled receptors.Methods in enzymology · 2013Article
- Methods for recombinant expression and functional characterization of human cannabinoid receptor CB2.Computational and structural biotechnology journal · 2013Review
- Mistic and TarCF as fusion protein partners for functional expression of the cannabinoid receptor 2 in Escherichia coli.Protein expression and purification · 2012Article
- Recombinant cannabinoid type 2 receptor in liposome model activates g protein in response to anionic lipid constituents.The Journal of biological chemistry · 2012Article
- Large multiple transmembrane domain fragments of a G protein-coupled receptor: biosynthesis, purification, and biophysical studies.Biopolymers · 2012Article
- Tolerance to changes in membrane lipid composition as a selected trait of membrane proteins.Biochemistry · 2011Review
- Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.Journal of biomolecular NMR · 2011Article
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Authors and funding
6 authors at 4 institutions in 2 countries.
Funding
Abstract
We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and (15)N(2)-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2mg of purified, labeled CB2/L of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two (15)N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of (15)N chemical shifts strongly suggests that the (15)N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation.
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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.