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Expression and characterization of the cystic fibrosis transmembrane conductance regulator

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Abstract

CYSTIC fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion1,2, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl− channel in the apical surface3–5. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR)6–8. Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.

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Authors and Affiliations

  1. Genzyme Corporation, One Mountain Road, Framingham, Massachusetts, 01701, USA

    Richard J. Gregory, Seng H. Cheng, John Marshall, Sucharita Paul & Kathleen Hehir

  2. IG laboratories Inc., One Mountain Road, Framingham, Massachusetts, 01701, USA

    Katherine W. Klinger

  3. Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa, College of Medicine, Iowa City, Iowa, USA

    Devra P. Rich, Lynda Ostedgaard & Michael J. Welsh

Authors
  1. Richard J. Gregory
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  2. Seng H. Cheng
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  4. John Marshall
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  6. Kathleen Hehir
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  7. Lynda Ostedgaard
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  8. Katherine W. Klinger
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  9. Michael J. Welsh
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Gregory, R., Cheng, S., Rich, D. et al. Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 347, 382–386 (1990). https://doi.org/10.1038/347382a0

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  • DOI: https://doi.org/10.1038/347382a0

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