Scaffolded fusion polypeptides and compositions and methods for making the same

Abstract

The present invention provides soluble forms of integral membrane proteins, or domains or portions thereof, that retain the biological activity of the integral membrane protein, domain or portion from which they are designed or derived and that can readily be expressed in high yield.

1 . A scaffolded fusion polypeptide comprising a functional polypeptide domain fused to a scaffold domain, wherein the functional polypeptide domain corresponds to a soluble loop of an integral membrane protein.  
     
     
         2 . A polymeric scaffolded fusion polypeptide comprising a plurality of functional polypeptide domains wherein, each functional polypeptide domain corresponds to a soluble loop of an integral membrane protein and is fused to a scaffold domain.  
     
     
         3 . The scaffolded fusion polypeptide according to  claim 1  wherein the scaffold domain is a metal chelating motif.  
     
     
         4 . The scaffolded fusion polypeptide according to  claim 3  wherein the metal chelating motif is capable of chelating a zinc ion.  
     
     
         5 . The scaffolded fusion polypeptide of  claim 4  wherein the metal chelating motif comprises a sequence of amino acids corresponding to SEQ ID NO:3 and a sequence of amino acids corresponding to SEQ ID NO:4.  
     
     
         6 . The scaffolded fusion polypeptide of  claim 2  in which a first scaffold domain is linked to a second scaffold domain via a polypeptide linker.  
     
     
         7 . The scaffolded fusion polypeptide of  claim 2  which corresponds to an extracellular domain of a naturally occurring receptor.  
     
     
         8 . The scaffolded fusion polypeptide of  claim 7  which corresponds to the extracellular domain of CCR5.  
     
     
         9 . A nucleic acid encoding the scaffolded fusion polypeptide of  claim 1 .  
     
     
         10 . A vector cassette for the expression of a scaffolded fusion polypeptide comprising an expression region operably linked to a promoter, said expression region comprising a plurality of cassettes, each of which encodes a module, domain or strand of the scaffolded fusion protein.  
     
     
         11 . A vector comprising the nucleic acid of  claim 9 .  
     
     
         12 . A host cell comprising the nucleic acid of  claim 9 .  
     
     
         13 . A host cell comprising the vector cassette of  claim 10 .  
     
     
         14 . A host cell comprising the vector of  claim 11 .  
     
     
         15 . A method of making a scaffolded fusion polypeptide comprising: 
 (a) expressing from a host cell the polypeptide of  claim 1;  and    (b) recovering said polypeptide.    
     
     
         16 . The polypeptide produced by the method of  claim 15 .  
     
     
         17 . A method of screening molecules that bind a scaffolded fusion polypeptide comprising: 
 (a) expressing from a host cell the polypeptide of  claim 1;  and    (b) identifying a molecule that binds to the polypeptide.    
     
     
         18 . A method of screening molecules that activate a scaffolded fusion polypeptide comprising: 
 (a) expressing from a host cell the polypeptide of  claim 1;  and    (b) identifying a molecule that binds to the polypeptide.    
     
     
         19 . A method of screening molecules that inhibit a scaffolded fusion polypeptide comprising: 
 (a) expressing from a host cell the polypeptide of  claim 1;  and    (b) identifying a molecule that binds to the polypeptide.    
     
     
         20 . A method of screening molecules that modulate a scaffolded fusion polypeptide comprising: 
 (a) expressing from a host cell the polypeptide of  claim 1;  and    (b) identifying a molecule that binds to the polypeptide.