title: A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.
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.
authors.affiliation: Department of Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430.
authors.forename: Ellen
authors.initials: E
authors.lastname: Hildebrandt
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Alok
authors.initials: A
authors.lastname: Mulky
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Haitao
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Qun
authors.initials: Q
authors.lastname: Dai
authors.affiliation: Department of Biochemistry & Biophysics, University of North Carolina, Chapel Hill, NC 27599.
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Aleksandrov
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269.
authors.forename: Bekim
authors.initials: B
authors.lastname: Bajrami
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269.
authors.forename: Pamela Ann
authors.initials: PA
authors.lastname: Diego
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Xing
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Ray
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Anjaparavanda P
authors.initials: AP
authors.lastname: Naren
authors.affiliation: Department of Biochemistry & Biophysics, University of North Carolina, Chapel Hill, NC 27599.
authors.forename: John R
authors.initials: JR
authors.lastname: Riordan
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269.
authors.forename: Xudong
authors.initials: X
authors.lastname: Yao
authors.affiliation: Department of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: DeLucas
authors.affiliation: Department of Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430.
authors.forename: Ina L
authors.initials: IL
authors.lastname: Urbatsch
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.|Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294.|Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.|Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, AL 35233.
authors.forename: John C
authors.initials: JC
authors.lastname: Kappes
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C505032:CFTR protein, human; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator