Intensive examination of wild type and mutant forms of intact CFTR, and systematic evaluation of its domains will be undertaken by members of the proposed Cystic Fibrosis Research Center at The Hospital for Sick Children. To meet the research needs projected to begin or expand under the CF Research Centers application, the Protein and Expression Core will be essential to insure the availability of CFTR and its various constructs/mutants/fragments, and will be vital to insure the pace of CF protein research within he Center. The global functions of the Core facility will be to provide the CF Center investigators with requisite quantities of wild type and mutant CFTR cDNA constructs; to evaluate and implement expression systems to obtain optimized amounts of purified, reconstituted CFTR proteins; and to prepare the scaled-up amounts of CFTR NBF1 domain, R domain, and transmembrane domain segments that will be required for structural research. Specifically, the Protein and Expression Core will provide materials and services as follows: (1) DNA; Wild type and mutant CFTR cDNA's incorporated into appropriate vectors for mammalian cell systems; the Sf9 insect cell system; and bacterial (E. coli BL21 cells) systems; (2) RNA: Preparation of cRNA's using pBluescript vector; (3) Protein: Purified, reconstituted intact CFTR protein, as obtained from Sf9 cells. (4) CFTR Domains: Purified, solubilized NBF1 domain and R domain fragments of CFTR as obtained from bacterial expression; and (5) Transmembrane Domain Peptide Purification/Characterization: Purification by high performance liquid chromatography, and characterization of synthesized CFTR TM domain peptides corresponding to wild type and CF-causing mutant segments.
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