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Characterisation of the salmon cystic fibrosis transmembrane conductance regulator protein for structural studies

1 Faculty of Life Sciences, University of Manchester, Manchester, M13 9PL, U.K.;
2 Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149, Genova, Italy;
3 U.O.C di Genetica Molecolare, Istituto Giannina Gaslini, 16147, Genova, Italy

Special Issues: Structure and function of membrane proteins: channels and transporters

The cystic fibrosis transmembrane conductance regulator protein (CFTR) is a chloride channel highly expressed in the gills of Salmo salar, with a role in osmoregulation. It shares 60% identity with the human CFTR channel, mutations to which can cause the common genetic disorder cystic fibrosis CF. The expression and localisation of salmon CFTR have been investigated, but the isolated protein has not been extensively characterised. Here we present a protocol for the purification of recombinant salmon CFTR, along with biophysical and structural characterisation of the purified protein. Salmon CFTR was overexpressed in Saccharomyces cerevisiae, solubilised in the detergent LPG-14 and chromatographically purified by nickel-affinity and size-exclusion chromatography methods. Prior to size-exclusion chromatography samples of salmon CFTR had low purity, and contained large quantities of aggregated protein. Compared to size-exclusion chromatography profiles of other orthologues of CFTR, which had less evidence of aggregation, salmon CFTR appeared to have lower intrinsic stability than human and platypus CFTR. Nonetheless, repeated size-exclusion chromatography allowed monodisperse salmon CFTR to be isolated, and multi-angle light scattering was used to determine its oligomeric state. The monodispersity of the sample and its oligomeric state were confirmed using cryo-electron microscopy and small-angle X-ray scattering (SAXS). These data were also processed to calculate a low-resolution structure of the salmon CFTR, which showed similar architecture to other ATP-binding cassette proteins.
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Keywords CFTR; CFTR purification; salmon; osmoregulation; Saccharomyces cerevisiae; heterologous overexpression; membrane protein purification; cryo-electron microscopy; small-angle X-ray scattering (SAXS)

Citation: Naomi L. Pollock, Oscar Moran, Debora Baroni, Olga Zegarra-Moran, Robert C. Ford. Characterisation of the salmon cystic fibrosis transmembrane conductance regulator protein for structural studies. AIMS Molecular Science, 2014, 1(4): 141-161. doi: 10.3934/molsci.2014.4.141

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