ArticleMolecular biotechnology2015
A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.
Article in Molecular biotechnology, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 17 papers.
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The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
17 citing papers in PubMed.
- Allosteric inhibition of CFTR gating by CFTRinh-172 binding in the pore.Nature communications · 2024Article
- Peptide Tags and Domains for Expression and Detection of Mammalian Membrane Proteins at the Cell Surface.Methods in molecular biology (Clifton, N.J.) · 2022Article
- A topological switch in CFTR modulates channel activity and sensitivity to unfolding.Nature chemical biology · 2021Article
- Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity.The Journal of biological chemistry · 2020Article
- Ubiquitination of disease-causing CFTR variants in a microsome-based assay.Analytical biochemistry · 2020Article
- Growth hormone (GH) receptor (GHR)-specific inhibition of GH-Induced signaling by soluble IGF-1 receptor (sol IGF-1R).Molecular and cellular endocrinology · 2019Article
- Domain-interface dynamics of CFTR revealed by stabilizing nanobodies.Nature communications · 2019Article
- The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity.Nucleic acids research · 2018Article
- Potentiation of the cystic fibrosis transmembrane conductance regulator by VX-770 involves stabilization of the pre-hydrolytic, OBritish journal of pharmacology · 2018Article
- Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1.Biochimica et biophysica acta. Biomembranes · 2018Article
- Substitution of Yor1p NBD1 residues improves the thermal stability of Human Cystic Fibrosis Transmembrane Conductance Regulator.Protein engineering, design & selection : PEDS · 2017Article
- Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers.Journal of virology · 2017Article
- Improved fluorescence assays to measure the defects associated with F508del-CFTR allow identification of new active compounds.British journal of pharmacology · 2017Article
- Specific stabilization of CFTR by phosphatidylserine.Biochimica et biophysica acta. Biomembranes · 2017Article
- Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.PLoS pathogens · 2017Article
- Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.Protein expression and purification · 2015Article
- Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140.Journal of virology · 2015Article
Corrections and comments
- Erratum issued
Authors and funding
15 authors.
Funding
Abstract
Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTR(FLAG)-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTR(FLAG)-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment future CF drug discovery efforts, including biophysical and structural studies.
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Registered trials
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.