Chimeric proteins

Abstract

This invention relates to modular proteins that interact with one or more target molecules. The chimeric proteins comprise two or more repeat domains, such as tetratricopeptide repeat domains; inter-repeat loops linking the repeat domains; and one or more peptide ligands. Each peptide ligand is located in an inter-repeat loop or at the N or C terminus of the chimeric protein. The peptide ligands may include heterologous peptidyl binding motifs, such as short linear motifs (SLiMs). Chimeric proteins with various configurations and methods for their production and use are provided.

1 - 15 . (canceled) 
     
     
         16 . A chimeric protein comprising two or more repeat domains linked by inter-repeat loops; and one or more heterologous peptide ligands that bind to a target molecule, each said peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein. 
     
     
         17 . A chimeric protein according to  claim 16  wherein the repeat domains are helix-turn-helix repeat domains. 
     
     
         18 . A chimeric protein according to  claim 17  wherein the repeat domains are tetratricopeptide (TPR) repeat domains. 
     
     
         19 . A chimeric protein according to  claim 18  wherein the repeat domains have the amino acid sequence Y-X 1 X 2 X 3 X 4 ;
 wherein Y is an amino acid sequence shown in any of Tables 4 to 6 or a variant thereof and X 1 , X 2 , X 3 , X 4  are independently any amino acid. 
 
     
     
         20 . A chimeric protein according to  claim 19  wherein the repeat domains have the amino acid sequence; 
       
         
           
                 
                 
               
                     
                   AEAWYNL GNAYYKQGDYQKAIEYYQKALEL-X 1 X 2 X 3 X 4 ; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   AEALNNLGNVYREQGDYQKAIEYYQKALEL-X 1 X 2 X 3 X 4   
                 
                     
                   or 
                 
                     
                     
                 
                     
                   AEAWYNLGNAYYRQGDYQRAIEYYQRALEL-X 1 X 2 X 3 X 4 ; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   AEALNNLGNVYREQGDYQRAIEYYQRALEL-X 1 X 2 X 3 X 4 ; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   AEALRNLGRVYRRQGRYQRAIEYYRRALEL-X 1 X 2 X 3 X 4   
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein X 1 , X 2 , X 3 , X 4  are independently any amino acid, and optionally wherein X 1  is D and/or wherein X 2  is P. 
       
     
     
         21 . A chimeric protein according to  claim 16  comprising 2-5 repeat domains. 
     
     
         22 . A chimeric protein according to  claim 16  wherein the peptide ligands are locating in one or more inter-repeat loops, and optionally are connected to the inter-repeat loops by a linker, and optionally wherein the peptide ligands are non-hydrophobic. 
     
     
         23 . A chimeric protein according to  claim 16  wherein a peptide ligand is located at the N terminus, the C terminus or at both the N and C termini, thereby to provide an N terminal peptide ligand and/or a C terminal peptide ligand. 
     
     
         24 . A chimeric protein according to  claim 23  wherein the N terminal peptide ligand, the C terminal peptide ligand or both of the N and C terminal peptide ligands comprises an α helix. 
     
     
         25 . A chimeric protein according to  claim 24  wherein the N terminal peptide ligand comprises the sequence Xn-XYXXXIXXYXXXLXX-X 1 X 2 XX, where residues denoted by X are independently any amino acid, X1 and X2 are independently any amino acid and n is 0 or any number, and optionally wherein X1 is D and/or wherein X2 is P. 
     
     
         26 . A chimeric protein according to  claim 24  wherein the C terminal peptide ligand comprises the sequence X1X2XX-XXAXXXLXX[AV]XXXXX-Xn, where residues denoted by X are independently any amino acid, X 1 , and X 2  are independently any amino acid n is 0 or any number, and optionally wherein X 1  is D and/or wherein X 2  is P. 
     
     
         27 . A chimeric protein according to  claim 16  wherein the target molecule is β-catenin, KRAS, tankyrase, c-myc, n-myc, ras, notch and aurora A, α-synuclein, β-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, or oncogenic histone methyltransferase. 
     
     
         28 . A chimeric protein according to  claim 16  comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds an E3 ubiquitin ligase. 
     
     
         29 . A chimeric protein according to  claim 28  comprising
 (i) an N terminal peptide ligand that binds a target protein and a C terminal peptide ligand that binds an E3 ubiquitin ligase, or 
 (ii) an inter-repeat peptide ligand that binds a target protein and a C terminal peptide ligand that binds an E3 ubiquitin ligase, or 
 (iii) an inter-repeat peptide ligand that binds a target protein and an N terminal peptide ligand that binds an E3 ubiquitin ligase, or 
 (iv) a C terminal domain that binds a target protein and an N terminal peptide ligand that binds an E1 ubiquitin ligase, or 
 (v) an inter-repeat binding domain that binds an E3 ubiquitin ligase and an N terminal binding domain that binds a target protein, or 
 (vi) an inter-repeat binding domain that binds an E3 ubiquitin ligase and a C terminal binding domain that binds a target protein. 
 
     
     
         30 . A method of producing a library of chimeric proteins comprising;
 (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising
 (i) two or more repeat domains 
 (ii) inter-repeat loops linking said repeat domains; and 
 (iii) one or more binding domains, each said binding domain being located in an inter-repeat loop or comprise the N and/or C terminus of the chimeric protein, 
   
       wherein the binding domains in said population are diverse, and
 (b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins. 
 
     
     
         31 . A method of screening a library comprising;
 (a) providing a library produced according to the method of  claim 30 , wherein at least one amino acid residue in the binding domains in said library is diverse,   (b) screening the library for chimeric proteins which display a binding activity, and   (c) identifying one or more chimeric proteins in the library which display the binding activity.