Purification and biophysical characteristics of human F508del CFTR for screening directly binding-F508del CFTR correctors

  • Xiaomeng Wang

    Student thesis: Unknown


    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (ABCC7) is the only ion channel in one of the largest membrane proteins families in human systems (the ATP-binding cassette proteins). Mutations in the gene encoding CFTR cause a lethal genetic disease called Cystic Fibrosis (CF). Among these mutants, F508del CFTR occupies the largest proportion however little progress has been seen in studying its structure and characteristics due to its high vulnerability to degradation by endoplasmic reticulum (ER) components. In order to better understand the F508del CFTR-induced defect and the biophysical properties of F508del CFTR, and also to generate assays for screening directly binding F508del CFTR correctors, human F508del and wild-type CFTR with a fused C-terminal GFP+His tag were optimally expressed in yeast (S. cerevisiae) and then purified in the detergent dodecyl maltoside (DDM). The thermal stability of purified CFTR was profiled using the thermal gel analysis assay and the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)-phenyl] maleimide (CPM) labelling assay. The structural dynamics of CFTR were probed using a limited proteolysis assay, which revealed a surprisingly small difference between wild-type and F508del CFTR. However data obtained in the CPM assay and thermal gel analysis assay implied less thermal stability for F508del CFTR compared to wild-type CFTR. The F508del CFTR corrector, VX809, was tested in the CPM assay on the purified CFTR and revealed that VX809 directly interacted with F508del CFTR, and rescued the thermal stability of F508del CFTR to close to that of wild-type at a low concentration (0.08μM).
    Date of Award1 Aug 2015
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorRobert Ford (Supervisor)

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