A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface

Academic Article

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 His 10 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.
  • Published In

    Digital Object Identifier (doi)

    Author List

  • Hildebrandt E; Ding H; Mulky A; Dai Q; Aleksandrov AA; Bajrami B; Diego PA; Wu X; Ray M; Naren AP
  • Start Page

  • 391
  • End Page

  • 405
  • Volume

  • 57
  • Issue

  • 5