Methods of assessing wolframin protein activity
AssigneePharmacia & Upjohn Company
InventorRonald R. Hiebsch
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Abstract
The present invention relates to a method of assaying modulators of the interaction between Wolframin protein and its identified cellular binding partner.
Claims135
A method for identifying agents that increase the propensity of a Wolframin protein polypeptide having at least 80% identity to SEQ ID NO:2 to associate with a carboxypeptidase E binding partner polypeptide having at least 75% identity to SEQ ID NO:4 comprising:
- (a) contacting said Wolframin protein polypeptide and said carboxypeptidase E binding partner polypeptide in the presence of a test agent;
- and
- (b) determining the propensity of said Wolframin protein polypeptide to associate with said binding partner polypeptide and
- (c) determining whether the propensity of said Wolframin protein polypeptide to associate with said carboxypeptidase E binding partner polypeptide is increased in the presence of the test agent.
. A method according to claim 1, wherein the Wolframin protein polypeptide is an isolated polypeptide comprising the amino acid sequence identified in SEQ ID NO:10.
A method of identifying agents which increase the propensity of a Wolframin protein polypeptide having at least 80% identity to SEQ ID NO:2 to associate with a carboxypeptidase E binding partner polypeptide having at least 75% identity to SEQ ID NO:4 comprising the steps of:
- (a) contacting a cell with a test compound, wherein the cell comprises:
- i) a first fusion protein comprising a DNA binding domain and said Wolframin protein polypeptide
- ii) a second fusion protein comprising a transcriptional activating domain and said carboxypeptidase E binding partner polypeptide wherein the interaction of said Wolframin protein polypeptide with said carboxypeptidase E binding partner polypeptide reconstitutes a sequence specific transcriptional activating factor;
- and
- iii) a reporter gene comprising a DNA sequence to which the DNA binding domain of the first fusion protein specifically binds;
- and
- (b) measuring the expression of the reporter gene.
A method of identifying agents which increase the propensity of a Wolframin protein polypeptide having at least 80% identity to SEQ ID NO:2 to associate with a carboxypeptidase E binding partner polypeptide having at least 75% identity to SEQ ID NO:4 comprising the steps of:
- (a) contacting a cell with a test compound, wherein the cell comprises:
- i) a first fusion protein comprising a transcriptional activating domain and said Wolframin protein polypeptide
- ii) a second fusion protein comprising a DNA binding domain and said carboxypeptidase E binding partner polypeptide;
- wherein the interaction of said Wolframin protein polypeptide with said carboxypeptidase E binding partner polypeptide reconstitutes a sequence specific transcriptional activating factor;
- and
- iii) a reporter gene comprising a DNA sequence to which the DNA binding domain of the second fusion protein specifically binds;
- and
- (b) measuring the expression of the reporter gene.
103 The method of claim 93 wherein the first fusion protein comprises about the first 200 amino terminal amino acids of SEQ ID NO:8.
109 The method of claim 93 wherein the first fusion protein comprises the first 242 amino terminal amino acids of SEQ ID NO:8.
Dependencies are inferred from the claim text.