ArticleThe Biochemical journal1996
Purification of a rat neurotensin receptor expressed in Escherichia coli.
Article in The Biochemical journal, 1996. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 63 papers.
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63 citing papers in PubMed, 204 citations in OpenAlex.
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- Optimizing the Expression of Human Dopamine Receptors inInternational journal of molecular sciences · 2021Article
- Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling.Chembiochem : a European journal of chemical biology · 2021Article
- Efficient production of a functional G protein-coupled receptor in E. coli for structural studies.Journal of biomolecular NMR · 2021Article
- Biochemical characterization of the mouse ABCF3 protein, a partner of the flavivirus-resistance protein OAS1B.The Journal of biological chemistry · 2019Article
- Mutations in sigma 70 transcription factor improves expression of functional eukaryotic membrane proteins in Escherichia coli.Scientific reports · 2019Article
- Cell-Free Expression and Photo-Crosslinking of the Human Neuropeptide YFrontiers in pharmacology · 2019Article
- In vivo assembly and large-scale purification of a GPCR - Gα fusion with Gβγ, and characterization of the active complex.PloS one · 2019Article
- Characterisation of a cell-free synthesised G-protein coupled receptor.Scientific reports · 2017Article
- A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.Nature protocols · 2016Article
- Large-scale production and protein engineering of G protein-coupled receptors for structural studies.Frontiers in pharmacology · 2015Review
- Effect of C-terminal protein tags on pentitol and L-arabinose transport by Ambrosiozyma monospora Lat1 and Lat2 transporters in Saccharomyces cerevisiae.Applied and environmental microbiology · 2014Article
- Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration.Journal of visualized experiments : JoVE · 2014Article
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- Biophysical characterization of membrane proteins in nanodiscs.Methods (San Diego, Calif.) · 2013Review
- Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.Current protocols in protein science · 2013Article
3 more citing papers are in PubMed but not listed here.
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2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column. This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR. Surprisingly, expression levels varied considerably depending on the C-terminal tag used. Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety. Several affinity chromatography methods were tested for purification. NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail. Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield. Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR. The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor. The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress.
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