Secreted soluble alpha2delta-2, alpha2delta-3, or alpha2delta-4 calcium channel subunit polypeptides and screening assays using same

Abstract

Soluble α 2 δ sub-type polypeptides. Methods for cloning, expression and purification of freely soluble α 2 δsubtype polypeptides. A method for the screening of ligands which bind to soluble α 2 δ subtype polypeptides.

1 - 7 . (canceled)  
     
     
         8 . A method for the amplification of a nucleic acid encoding a mammalian secreted soluble cerebral cortical voltage-dependent calcium channel α 2 δ-n subunit polypeptide wherein n is 2, 3 or 4, said method comprising the steps of: 
 (a) contacting a test sample suspected of containing the target secreted soluble α 2 δ-n subunit nucleic acid, or a sequence complementary thereto, with an amplification reaction reagent comprising a pair of amplification primers located on either side of the α 2 δ-n subunit nucleic acid region to be amplified, and    (b) optionally, detecting the amplification products.    
     
     
         9 - 12 . (canceled)  
     
     
         13 . A purified or isolated recombinant polypeptide comprising the amino acid sequence of a secreted soluble α 2 δ-2, α 2 δ-3 or α 2 δ-4 subunit polypeptide.  
     
     
         14 . A recombinant polypeptide according to  claim 14 , having at least 80% amino-acid identity with a polypeptide comprising: 
 from amino acid 1 to between amino acids 1027 and 1062 of the amino acid sequence of SEQ ID No20, or    from amino acid 1 to between amino acids 1019 and 1079 of the amino acid sequence of SEQ ID No22.    
     
     
         15 . A recombinant polypeptide according to  claim 14 , wherein said recombinant polypeptide is selected from the group consisting of the amino acid sequences of SEQ ID NO:4 SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 10 SEQ ID NO:11. SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18  
     
     
         16 . A method for the screening of ligands which bind a cerebral cortical voltage-dependent calcium channel α 2 δ-n subunit wherein n is 2, 3 or 4, said method comprising the steps of: 
 contacting a secreted soluble recombinant calcium channel α 2 δ-n subunit polypeptide with: 
 a ligand of interest; and  
 a labelled compound which binds the α 2 δ-n subunit; and  
   measuring the level of binding of the labelled compound to the α 2 δ-n subunit.    
     
     
         17 . A method according to  claim 16 , wherein said method is a scintillation proximity assay.  
     
     
         18 . A method according to  claim 16 , wherein said method is a flashplate assay.  
     
     
         19 . A method according to  claim 16 , wherein said method is a filter binding assay.  
     
     
         20 . A method according to  claim 16 , wherein said secreted soluble recombinant calcium channel α 2 δ-n subunit polypeptide is selected from polypeptides having at least 80%, preferably 90%, more preferably 95%, and most preferably 98 or 99% amino-acid identity with the polypeptide comprising from amino acid 1 to between amino-acids 984 and 1063, preferably between amino-acids 994 and 1054, and most preferably between amino-acids 1019 and 1054 of SEQ ID NO:5 or SEQ ID NO:16.  
     
     
         21 . A method according to  claim 16 , wherein said secreted soluble recombinant calcium channel α 2 δ-n subunit polypeptide is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, with the polypeptides of SEQ ID NO: 9 and SEQ ID NO: 15 being most preferred.  
     
     
         22 . A kit for the screening of ligands which bind bind a cerebral cortical voltage-dependent calcium channel α 2 δ-n subunit wherein n is 2, 3 or 4, said kit comprising: 
 a secreted soluble recombinant calcium channel α 2 δ-n subunit; and    a labelled compound which binds to the α 2 δ-n subunit.