Protocol

Cystic Fibrosis

Volume 742 of the series Methods in Molecular Biology pp 335-353

Date:

CFTR Folding Consortium: Methods Available for Studies of CFTR Folding and Correction

  • Kathryn W. PetersAffiliated withDepartment of Cell Biology and Physiology, University of Pittsburgh
  • , Tsukasa OkiyonedaAffiliated withDepartment of Physiology, McGill University
  • , William E. BalchAffiliated withDepartment of Cell Biology, The Scripps Research Institute
  • , Ineke BraakmanAffiliated withCellular Protein Chemistry, Utrecht University
  • , Jeffrey L. BrodskyAffiliated withDepartment of Biological Sciences, University of Pittsburgh
  • , William B. GugginoAffiliated withDepartment of Physiology, Johns Hopkins University
  • , Christopher M. PenlandAffiliated withCystic Fibrosis Foundation
  • , Harvey B. PollardAffiliated withDepartment of Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences
  • , Eric J. SorscherAffiliated withGregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham
    • , William R. SkachAffiliated withDepartment of Biochemistry and Molecular Biology, Oregon Health & Science University
    • , Philip J. ThomasAffiliated withDepartment of Physiology, University of Texas Southwestern Medical Center
    • , Gergely L. LukacsAffiliated withDepartment of Physiology, McGill University
    • , Raymond A. FrizzellAffiliated withDepartment of Cell Biology and Physiology, University of Pittsburgh School of Medicine Email author 

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Abstract

The CFTR Folding Consortium (CFC) was formed in 2004 under the auspices of the Cystic Fibrosis Foundation and its drug discovery and development affiliate, CFF Therapeutics. A primary goal of the CFC is the development and distribution of reagents and assay methods designed to better understand the mechanistic basis of mutant CFTR misfolding and to identify targets whose manipulation may correct CFTR folding defects. As such, reagents available from the CFC primarily target wild-type CFTR NBD1 and its common variant, F508del, and they include antibodies, cell lines, constructs, and proteins. These reagents are summarized here, and two protocols are described for the detection of cell surface CFTR: (a) an assay of the density of expressed HA-tagged CFTR by ELISA and (b) the generation and use of an antibody to CFTR’s first extracellular loop for the detection of endogenous CFTR. Finally, we highlight a systematic collection of assays, the CFC Roadmap, which is being used to assess the cellular locus and mechanism of mutant CFTR correction. The Roadmap queries CFTR structure–function relations at levels ranging from purified protein to well-differentiated human airway primary cultures.

Key words

Protein folding protein degradation antibody generation cell surface protein detection research consortium www.cftrfolding.org