Materials and methods for detecting interaction of CFTR polypeptides
AssigneeThe Florida State University Research Foundation, Inc.
InventorJohn L. Teem
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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.
Claims24
A method of identifying a compound that facilitates 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, and 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 host cell further comprises said detectable gene wherein transcription of said detectable gene is under control of said transcriptional activator, wherein said first and second CFTR polypeptides comprise a mutation that reduces or prevents interaction of said fusion proteins;
- (b) expressing said polynucleotide encoding said first CFTR polypeptide and said second CFTR polypeptide under conditions in which said detectable gene is expressed when said first CFTR polypeptide and 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.
A method for treating a person afflicted with cystic fibrosis, said method comprising providing or administering to said person an effective amount of a compound that restores or enhances dimerization of CFTR polypeptide or the exit of CFTR polypeptide from endoplasmic reticulum of a cell.
A method for detecting or determining the interaction of a first CFTR polypeptide with a second CFTR polypeptide, said method comprising:
- (a) providing a first polynucleotide encoding a fusion protein comprising all or a portion of a first CFTR polypeptide 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 said NBD1;
- (b) providing a second 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 said detectable gene, wherein said second CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1;
- (c) incorporating said first and second polynucleotide into a host cell comprising said detectable gene wherein transcription of said detectable gene is under control of said transcriptional activator;
- (d) 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
- (e) detecting transcription of said detectable marker gene or expression of the gene product of said detectable gene,
- wherein one of said CFTR polypeptides contains a ΔF508 mutation and the other of said CFTR polypeptides contains a mutation other than ΔF508.
A method for detecting or determining the interaction of a first CFTR polypeptide with a second CFTR polypeptide, said method comprising (a) providing 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 marker gene, wherein said first CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1;
- (b) providing a second 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 the detectable marker gene, wherein said second CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1;
- (c) contacting said first fusion protein and said second fusion protein under conditions where if said NBD1 of said first fusion protein and said NBD1 of said second fusion protein interact then said interaction causes said transcriptional activation domain to activate transcription of said detectable marker gene;
- and (d) detecting transcription of said detectable marker gene or expression of the gene product of said detectable marker gene, wherein one of said CFTR polypeptides contains a ΔF508 mutation and the other of said CFTR polypeptides contains a mutation other than ΔF508.
A host cell comprising 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 a polynucleotide encoding a fusion protein comprising all or a portion of a second CFTR protein and a transcriptional activation domain of a transcriptional activator that can activate transcription of said detectable gene, wherein said second CFTR polypeptide comprises a first nucleotide binding domain (NBD1) of a CFTR polypeptide, or a functional fragment of said NBD1, wherein one of said CFTR polypeptides contains a ΔF508 mutation and the other of said CFTR polypeptides contains a mutation other than ΔF508.
Dependencies are inferred from the claim text.