Cell
Volume 68, Issue 4, 21 February 1992, Pages 809-818
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Article
Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR)

https://doi.org/10.1016/0092-8674(92)90155-6Get rights and content

Abstract

Circumstantial evidence has accumulated suggesting that CFTR is a regulated low-conductance Cl channel. To test this postulate directly, we have purified to homogeneity a recombinant CFTR protein from a high-level baculovirus-infected insect cell line. Evidence of purity included one- and two-dimensional gel electrophoresis, N-terminal peptide sequence, and quantitative amino acid analysis. Reconstitution into proteoliposomes at less than one molecule per vesicle was accomplished by established procedures. Nystatin and ergosterol were included in these vesicles, so that nystatin conductance could serve as a quantitative marker of vesicle fusion with a planar lipid bilayer. Upon incorporation, purified CFTR exhibited regulated chloride channel activity, providing evidence that the protein itself is the channel. This activity exhibited the basic biophysical and regulatory properties of the type of Cl channel found exclusively in CFTR-expressing cell types and believed to underlie cAMP-evoked secretion in epithelial cells.

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