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Mol Biotechnol. 2015 May;57(5):391-405. doi: 10.1007/s12033-014-9830-5.

A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.

Author information

1
Department of Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430.
2
Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.
3
Department of Biochemistry & Biophysics, University of North Carolina, Chapel Hill, NC 27599.
4
Department of Chemistry, University of Connecticut, Storrs, CT 06269.
5
Department of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294.
6
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294.
7
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294.
8
Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
9
Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, AL 35233.
#
Contributed equally

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.

PMID:
25577540
PMCID:
PMC4405497
DOI:
10.1007/s12033-014-9830-5
[Indexed for MEDLINE]
Free PMC Article

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