Loop-Variant Pdz Domains As Biotherapeutics, Diagnostics And Research Reagents

Abstract

The present invention provides polypeptides that contain one or more PDZ loop-variants and are useful in the detection of pathogens and disease-associated molecules. The polypeptides of the invention are also useful in the diagnosis, treatment, and prevention of diseases. Also provided are methods of preparing polypeptides of the invention.

1 . A polypeptide comprising at least one PDZ loop-variant, said PDZ loop-variant comprising one or more amino acids inserted or substituted in one or more of loops β12, α2β6, β23, or β5α2. 
     
     
         2 . A polypeptide comprising at least one PDZ loop-variant, said PDZ loop-variant comprising one or more amino acids inserted or substituted in one or more of loops β23 or β5α2. 
     
     
         3 . A polypeptide comprising at least one PDZ loop-variant, said PDZ loop-variant comprising one or more amino acids inserted or substituted in loop β23. 
     
     
         4 . A polypeptide comprising at least one PDZ loop-variant, said PDZ loop-variant comprising one or more amino acids inserted in loop β23. 
     
     
         5 . A polypeptide comprising at least one PDZ loop-variant, said PDZ loop-variant comprising one or more amino acids substituted in loop β23. 
     
     
         6 . A polypeptide of any one of  claims 1 ,  2 ,  3 ,  4 , or  5 , wherein said polypeptide has binding affinity for a target. 
     
     
         7 . A polypeptide of  claim 6 , wherein said target is associated with a pathogen or disease. 
     
     
         8 . A polynucleotide encoding the polypeptide of any one of  claims 1 ,  2 ,  3 ,  4 , or  5 . 
     
     
         9 . A host cell containing the polynucleotide of  claim 8 . 
     
     
         10 . A method of preparing a polypeptide according to any one of  claims 1 ,  2 ,  3 ,  4 , or  5 , comprising:
 a) creating a library of polypeptide PDZ loop-variants;   b) identifying one or more polypeptides from said library having binding affinity for a target.   
     
     
         11 . The method of  claim 10 , wherein said target is associated with a pathogen or disease. 
     
     
         12 . A method of detecting the presence of a pathogen or disease in a patient comprising:
 a) administering a polypeptide of any one of  claims 1 ,  2 ,  3 ,  4 , or  5  to said patient; and   b) detecting binding or absence of binding of said polypeptide to a target which is associated with said pathogen or disease in said patient, wherein detection of binding indicates the presence of said pathogen or disease and wherein absence of binding indicates absence of said pathogen or disease.   
     
     
         13 . A method of detecting the presence of a pathogen or disease in a sample comprising:
 a) contacting a polypeptide of any one of  claims 1 ,  2 ,  3 ,  4 , or  5  with said sample; and   b) detecting binding or absence of binding of said polypeptide to a target which is associated with said pathogen or disease in said sample, wherein detection of binding indicates the presence of said pathogen or disease and wherein absence of binding indicates absence of said pathogen or disease.   
     
     
         14 . A method of treating a disease, comprising administering to a patient afflicted with or likely to be afflicted with said disease a therapeutically effective amount of a polypeptide of any one of  claims 1 ,  2 ,  3 ,  4 , or  5  capable of binding to a target associated with said disease. 
     
     
         15 . The method of  claim 14  wherein said disease is associated with a pathogen. 
     
     
         16 . A library of polypeptide PDZ loop-variants. 
     
     
         17 . A library of polynucleotides encoding the library of polypeptides of  claim 16 . 
     
     
         18 . An isolated polypeptide of any one of  claims 1 ,  2 ,  3 ,  4 , or  5 , that is bivalent. 
     
     
         19 . A method of detecting the presence of a pathogen or disease in a patient comprising:
 a) administering a polypeptide of  claim 18  to said patient; and   b) detecting binding or absence of binding of said polypeptide to a target which is associated with said pathogen or disease in said patient, wherein detection of binding indicates the presence of said pathogen or disease and wherein absence of binding indicates absence of said pathogen or disease.   
     
     
         20 . A method of detecting the presence of a pathogen or disease in a sample comprising:
 a) contacting a polypeptide of  claim 18  with said sample; and   b) detecting binding or absence of binding of said polypeptide to a target which is associated with said pathogen or disease in said sample, wherein detection of binding indicates the presence of said pathogen or disease and wherein absence of binding indicates absence of said pathogen or disease.   
     
     
         21 . A method of treating a disease, comprising administering to a patient afflicted with or likely to be afflicted with said disease a therapeutically effective amount of a polypeptide of  claim 18  capable of binding to a target associated with said disease. 
     
     
         22 . The method of  claim 21  wherein said disease is associated with a pathogen. 
     
     
         23 . A polynucleotide encoding a polypeptide comprising SEQ ID NO: 21, or encoding a polypeptide comprising a region at least about 90% identical to SEQ ID NO: 21. 
     
     
         24 . A library of polynucleotides wherein members of said library encode polypeptides comprising SEQ ID NO: 21 or encode polypeptides comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 21. 
     
     
         25 . A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 22. 
     
     
         26 . A library of polynucleotides wherein members of said library comprise the nucleotide sequence of SEQ ID NO 22.