Materials and methods for detecting interaction of CFTR polypeptides
First Claim
1. A method of determining whether a compound restores or enhances interaction of CFTR polypeptides, said method comprising:
- (a) contacting a host cell with said compound, wherein said host cell comprises a polynucleotide encoding a fusion protein comprising all or a portion of a first CFTR protein and a DNA binding domain of a transcriptional activator that can bind to a site on a detectable gene, wherein said first CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1, and said host cell comprises a polynucleotide encoding a fusion protein comprising all or a portion of a second CFTR polypeptide and a transcriptional activation domain of a transcriptional activator that can activate transcription of a detectable gene, wherein said second CFTR polypeptide comprises an NBD1 of a CFTR polypeptide, or a functional fragment of said NBD1, and wherein said host cell further comprises said detectable gene wherein transcription of said detectable gene is under control of said transcriptional activator, wherein one or both of said first and second CFTR polypeptides comprise a mutation in said NBD1 that reduces or prevents interaction of said fusion proteins;
(b) expressing said polynucleotide encoding said first CFTR polypeptide and expressing said polynucleotide encoding said second CFTR polypeptide under conditions in which said detectable gene is expressed when said NBD1 of said first CFTR polypeptide and said NBD1 of said second CFTR polypeptide interact; and
,(c) determining whether said detectable gene is expressed in said host cell at a level greater than the level of expression observed in said host cell in the absence of said compound.
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Abstract
The subject invention concerns materials and methods for detecting the interaction of CFTR proteins. In one embodiment, the method can be used to determine whether one CFTR polypeptide interacts with a second CFTR polypeptide. The subject invention also concerns materials and methods for screening for drugs or compositions that can restore or enhance interaction of CFTR proteins containing mutation(s) that reduce or prevent dimerization of the proteins. The assay of the present invention can be used to screen a large number of compounds in a high throughput format. The subject invention also pertains to host cells useful in the methods of the invention. The subject invention also concerns compositions and methods for treating patients afflicted with cystic fibrosis.
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Citations
19 Claims
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1. A method of determining whether a compound restores or enhances interaction of CFTR polypeptides, said method comprising:
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(a) contacting a host cell with said compound, wherein said host cell comprises a polynucleotide encoding a fusion protein comprising all or a portion of a first CFTR protein and a DNA binding domain of a transcriptional activator that can bind to a site on a detectable gene, wherein said first CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1, and said host cell comprises a polynucleotide encoding a fusion protein comprising all or a portion of a second CFTR polypeptide and a transcriptional activation domain of a transcriptional activator that can activate transcription of a detectable gene, wherein said second CFTR polypeptide comprises an NBD1 of a CFTR polypeptide, or a functional fragment of said NBD1, and wherein said host cell further comprises said detectable gene wherein transcription of said detectable gene is under control of said transcriptional activator, wherein one or both of said first and second CFTR polypeptides comprise a mutation in said NBD1 that reduces or prevents interaction of said fusion proteins; (b) expressing said polynucleotide encoding said first CFTR polypeptide and expressing said polynucleotide encoding said second CFTR polypeptide under conditions in which said detectable gene is expressed when said NBD1 of said first CFTR polypeptide and said NBD1 of said second CFTR polypeptide interact; and
,(c) determining whether said detectable gene is expressed in said host cell at a level greater than the level of expression observed in said host cell in the absence of said compound. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification