Almost five years have elapsed since the CFTR gene was identified and sequenced. Yet, investigators have been unable to produce sufficient amounts of native CFTR so that its function(s) can be precisely defined and extensively studied in vitro. For this reason, our molecular understanding of CFTR, an integral membrane protein, lies far behind established areas of membrane biology. To help resolve this rather serious problem, this project will focus first on several novel approaches to produce large amounts of native CFTR and then utilize the preparation(s) obtained to define its ATP-dependent function(s) in a pure liposomal membrane system.
SPECIFIC AIMS ARE FIVE FOLD AND WILL BE TO: 1. Coexpress CFTR in a baculoviral/insect system with one or more molecular chaperons in order to facilitate the abundant production of properly folded molecules. 2. Employ the use of one or more rapidly proliferating parasitic organisms, which we have shown to contain a CFTR-like protein, as an alternative eukaryotic overexpression system. 3. Utilization those expression systems which have resulted very recently in the large scale production in E. coli, in functional form, of two other membrane proteins, the complete ATP synthase, and an anion carrier. 4. Incorporate those pure CFTR preparations obtained, which exhibit significant secondary structure, into liposomal vesicles and assess their capacity to catalyze chloride permeability in the presence of ATP and Mg++. 5. Establish the extent to which phosphorylation, ATP hydrolysis, and counterion flow are required for CFTR to facilitate chloride permeability, and whether these or other conditions favor a channel or pump-like function. This work is both necessary and fundamental to our understanding of the vectorial nature and hence the molecular function and dysfunction of the CFTR molecule, and should be invaluable to other members of the SCOR team in helping interpret data derived in vivo.
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