Novel complex-forming proteins

Abstract

The invention relates to a complex of specifically complex-forming proteins which are not naturally occurring, comprising the following components: a) at least one ligand specific for a target structure, b) at least one protein comprising a mutated dimerization domain, the mutated dimerization domain having been derived by mutation of a naturally occurring dimerization domain, it being possible for this mutated dimerization domain to interact specifically with component c) and the component b) being connected covalently to the component a), c) at least one protein comprising a mutated dimerization domain, the mutated dimerization domain having been derived by mutation of a naturally occurring dimerization domain, it being possible for this mutated dimerization domain to interact specifically with component b) and the component c) is linked covalently to the component d), and d) at least one effector. In addition, the invention relates to the use and preparation of these complexes, and to nucleic acid constructs coding for the proteins mentioned and use thereof.

1 . A complex comprising the following components: 
 a) at least one ligand specific for a target structure;    b) at least one protein comprising a mutated dimerization domain obtained by mutation of a naturally occurring dimerization domain, wherein said mutated dimerization domain binds specifically with component c) and said component b) is covalently bonded to said component a);    c) at least one protein comprising a mutated dimerization domain obtained by mutation of a naturally occurring dimerization domain, wherein said mutated dimerization domain binds specifically with said component b) and said component c) is covalently bonded to component d); and    d) at least one effector;    wherein said components b) and c) are not naturally occurring proteins.    
     
     
         2 . The complex as claimed in  claim 1 , wherein said component a) is replaced by said component d).  
     
     
         3 . The complex as claimed in  claim 1 , wherein said component d) is replaced by said component a).  
     
     
         4 . The complex as claimed in  claim 1 , which further comprises a fusogenic peptide or a translocalization peptide.  
     
     
         5 . The complex as claimed in  claim 1 , which further comprises a cleavage sequence for a protease between said components c) and d) or a) and b).  
     
     
         6 . The complex as claimed in  claim 1 , wherein said component a) is selected from the group consisting of: a growth factor, a cytokine, TNF, a chemokine, a peptide hormone, a mediator, a steroid hormone, a vitamin, a complement factor, a clotting factor, a kinin system factor, a fibrinolysis system factor, a plasmatic enzyme, a cell enzyme, a plasmatic enzyme inhibitor, a cell enzyme inhibitor, a virus coat protein, a cell receptor for the afore-mentioned molecules, an antibody, an antibody cleavage product, a DNA binding protein, a DNA binding domain of a transcription factor and an activation domain of a transcription factor.  
     
     
         7 . The complex as claimed in  claim 1 , wherein said components b) and c) are obtained by mutating the naturally occurring dimerization domains of proteins which bind naturally to one another.  
     
     
         8 . The complex as claimed in  claim 7 , wherein said naturally occurring dimerization domains of components b) and c) are selected from the group of naturally dimerizing partners consisting of:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                     
                 
                     
                   Fos 
                   Jun or Jun B or Jun D; 
                 
                     
                   FRAU-1 
                   Jun or Jun B or Jun D; 
                 
                     
                   FRAU-2 
                   Jun or Jun B or Jun D; 
                 
                     
                   FOS-B 
                   Jun or Jun B or Jun D; 
                 
                     
                   OCT-1 
                   Jun or Jun B or Jun D; 
                 
                     
                   NFKB (p 65) 
                   IKB; 
                 
                     
                   Ras 
                   RAF; 
                 
                     
                   CD4 
                   p56LcK; 
                 
                     
                   bcl-2 
                   bad or bax; 
                 
                     
                   Cyclin A 
                   cdkl; 
                 
                     
                   E2F 
                   DP; 
                 
                     
                   CD40 
                   CD40L; 
                 
                     
                   Myc 
                   Max; 
                 
                     
                   Myc N 
                   Max; 
                 
                     
                   Myc L 
                   Max; 
                 
                     
                   p 105 (Rb1) 
                   AFF-2, E1A; 
                 
                     
                   p 107 (Rb2) 
                   E7, E2F or Myc; 
                 
                     
                   p 130 
                   E7, E2F or Myc; 
                 
                     
                   CBL 
                   Gag; 
                 
                     
                   TRP 
                   TRP; 
                 
                     
                   Met 
                   Met; 
                 
                     
                   Myb 
                   p 67 or p 160; 
                 
                     
                   VAV 
                   p 67 or p 160; 
                 
                     
                   APC 
                   α- or β-Catenin; 
                 
                     
                   APC 
                   APC; 
                 
                     
                   VD receptor 
                   h-(Retinoid x-receptor) RXR 
                 
                     
                     
                   α or β; 
                 
                     
                   T3 receptor 
                   HRXR α or β; 
                 
                     
                   MyoD 
                   E12; 
                 
                     
                   E12 
                   Id; 
                 
                     
                   E47 
                   Id; 
                 
                     
                   HHSP90 
                   Progesterone receptor; 
                 
                     
                   HHSP90 
                   Glucocorticoid receptor; 
                 
                     
                   HHSP90 
                   Mineralocorticoid receptor; 
                 
                     
                   HHSP90 
                   Dioxin receptor; 
                 
                     
                   Dioxin receptor 
                   Arnt; 
                 
                     
                   HPS90 
                   FKBP59; 
                 
                     
                   HSP90 
                   Cyclosporin-binding protein; 
                 
                     
                   HPS90 
                   Pp60 V-src   
                 
                     
                   HSP70 
                   HSF1 heat shock factor 1; and 
                 
                     
                   HSP70 
                   HSF2 heat shock factor 2. 
                 
                     
                     
                 
                     
                     
                 
             
                
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         9 . The complex as claimed in  claim 8 , wherein said naturally dimerizing partners belong to the helix-loop-helix/leucine zipper family of proteins.  
     
     
         10 . The complex as claimed in one of  claim 7 , wherein said mutated dimerization domains are obtained by inserting: 
 3-18 cysteines in said naturally occurring dimerization domain in at least one of said proteins of component b) and in at least one of said proteins of component c);    3-24 basic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component b) and 3-24 acidic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component c);    3-24 basic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component c) and 3-24 acidic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component b);    3-24 hydrophobic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component b) and 3-24 aromatic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component c);    3-24 hydrophobic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component c) and 3-24 aromatic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component b); and/or    3-24 aromatic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component b) and 3-24 aromatic amino acids in said naturally occurring dimerization domain in at least one of said proteins of component c).    
     
     
         11 . The complex as claimed in  claim 10 , in which said components b) and c) are mutated binding domains of c-fos and c-jun, comprising the following mutations:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                     
                 
                     
                   c-fos amino acid 
                   167 E → K 
                 
                     
                     
                   172 E → K 
                 
                     
                     
                   181 E → K 
                 
                     
                   c-jun amino acid 
                   283 K → E 
                 
                     
                     
                   288 K → E 
                 
                     
                     
                   302 K → E 
                 
                     
                     
                 
                     
                     
                 
             
                
                
               
               
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         12 . The complex as claimed in  claim 1 , wherein said component d) is selected from the group consisting of: inhibitors of cell proliferation, apoptosis-inducing proteins, cytostatic proteins, cytotoxic proteins, coagulation-inducing factors, angiogenesis-inducing factors, angiogenesis-inhibiting factors, growth factors, cytokines, chemokines, interleukins, interferons, complement factors, clotting factors, fibrinolysis-inducing proteins, peptide hormones, mediators, bacterial proteins, receptors, viral antigens, parasitic antigens, tumor antigens, autoantigens, tissue antigens, adhesion molecules, antibodies, antibody cleavage products, enzymes for reacting with a signal-emitting component, enzymes for converting a precursor of an active substance into an active substance, fluorescent dyes, isotopes, metal-binding proteins, and a DNA-binding domain.  
     
     
         13 . A method for treating diseased cells, wherein said disease results from inflammation, autoimmune diseases, defective formation of blood cells, nervous system damage, blood-clotting system disorders, blood circulation system disorders, tumor formation, viral infections, or bacterial infections, comprising administering said complex of  claim 1  to said cells.  
     
     
         14 . A method for introducing a vector into a cell of an organism or cell culture, comprising binding said complex of  claim 1  to a viral or nonviral vector and introducing said vector into said cell of an organism or a cell culture in a cell-specific manner.  
     
     
         15 . A method of detecting the presense or amount of a reactant in vitro or in vivo, comprising contacting said complex of  claim 1  with said reactant, either in vivo or in vitro, wherein said component a) binds to said reactant and said component d) emits a signal, such that the presence or amount of said reactant is detected by measuring the amount of said emitted signal.  
     
     
         16 . A nucleic acid construct coding for a protein of  claim 1 .  
     
     
         17 . The nucleic acid construct as claimed in  claim 16 , coding for an activator subunit of an activator-responsive promoter unit.  
     
     
         18 . A host cell comprising said nucleic acid construct as claimed in  claim 16 .  
     
     
         19 . The host cell as claimed in  claim 18 , selected from the group consisting of a bacterium, a yeast and a mammalian cell.  
     
     
         20 . A method of expressing a protein of  claim 1 , comprising expressing a nucleic acid encoding said protein in a host cell under conditions such that said protein is expressed in a detectable amount.  
     
     
         21 . A method of producing a complex between said component b) and said component c) as claimed in  claim 1 , comprising: 
 (a) expressing said at least one protein of said component b) by translating a nucleic acid construct encoding said protein;    (b) expressing said at least one protein of said component c) by translating a nucleic acid construct encoding said protein;    (c) isolating said proteins of steps (a) and (b);    (d) contacting said at least one protein of step a) with said at least one protein of step b) under conditions such that said proteins bind to one another.    
     
     
         22 . The complex as claimed in  claim 6 , wherein said antibody cleavage product is selected from the group consisting of: F(ab) 2 , a single-chain Fv, a single-chain, a double antigen-binding molecule, and an Fc fragment.  
     
     
         23 . The complex as claimed in  claim 12 , wherein said receptor is selected from the group consisting of receptors for: growth factors, cytokines, chemokines, interleukins, interferons, complement factors, clotting factors, fibrinolysis-inducing proteins, peptide hormones, steroid hormones, mediators, and virus coat proteins.  
     
     
         24 . The complex as claimed in  claim 12 , wherein said antibody cleavage product is selected from the group consisting of: F(ab) 2 , Fab, single-chain Fv, and single-chain double antigen-binding proteins.  
     
     
         25 . A vaccine comprising the complex as claimed in  claim 1 .  
     
     
         26 . A complex comprising the following components: 
 a) at least one ligand specific for a target structure;    b) at least one protein comprising a mutated dimerization, wherein said mutated dimerization domain binds specifically with component c) and said component b) is covalently bonded to said component a);    c) at least one protein comprising a mutated dimerization domain, wherein said mutated dimerization domain binds specifically with said component b) and said component c) is covalently bonded to component d); and    d) at least one effector;    wherein said components b) and c) are not naturally occurring proteins.    
     
     
         27 . The complex as claimed in  claim 1 , wherein at least one protein of component b) binds specifically with at least one protein of component c) with a binding constant of at least a K M  of 10 −7  mol I −1 .