Modified TGF-beta superfamily proteins

Abstract

The invention provides modified TGF-β family proteins having altered biological or biochemical properties, and methods for making them. Specific modified protein constructs include TGF-β family member proteins that have N-terminal truncations, “latent” proteins, fusion proteins and heterodimers.

1 . A biologically active TGF-β family member fusion protein competent to refold under suitable refolding conditions, comprising: 
 a TGF-β family protein C-terminal seven cysteine domain, comprising a finger 1 subdomain, a finger 2 subdomain, and a heel subdomain; and    a heterologous leader sequence domain operatively linked to said C-terminal domain.    
     
     
         2 . The fusion protein of  claim 1  wherein said leader sequence is selected from the group consisting of a tissue-targeting domain, a molecular-targeting domain, a metal-binding domain, a protein-binding domain, a ceramic-binding domain, a hydroxyapatite-binding domain, and a collagen-binding domain.  
     
     
         3 . The fusion protein of  claim 2  wherein said tissue-targeting domain binds to a bone matrix protein.  
     
     
         4 . The fusion protein of  claim 2  wherein said tissue-targeting domain binds to a cell surface molecule.  
     
     
         5 . The fusion protein of  claim 4  wherein said cell surface molecule is on an osteoprogenitor cell or a chondrocyte.  
     
     
         6 . A latent TGF-β family member fusion protein competent to refold under suitable refolding conditions, comprising: 
 a TGF-β family protein C-terminal seven cysteine domain, comprising a finger 1 subdomain, a finger 2 subdomain, and a heel subdomain; and    a cleavable leader sequence operably linked to said C-terminal domain wherein said leader sequence inhibits the biological activity associated with said C-terminal domain, and wherein said C-terminal domain becomes active upon cleavage of a part or all of said leader sequence.    
     
     
         7 . The fusion protein of  claim 6  wherein a tissue-targeting domain is embedded within said cleavable leader sequence, whereby cleavage of the leader sequence will not cleave said tissue-targeting domain from said C-terminal domain.  
     
     
         8 . The fusion protein of  claim 1  or  6  wherein said leader sequence is separated from said C-terminal domain by at least seven residues.  
     
     
         9 . The fusion protein of  claim 1  wherein said leader sequence is derived from another TGF-β family protein.  
     
     
         10 . A biologically active TGF-β family member protein mutant competent to refold under suitable refolding conditions, comprising: 
 a TGF-β family member protein C-terminal seven cysteine domain, comprising a finger 1 subdomain, a finger 2 subdomain, and a heel subdomain; and    a leader sequence domain operatively linked to said C-terminal domain, whereby a part or all of said leader sequence is truncated.    
     
     
         11 . The protein mutant of  claim 10  wherein said truncation is carried out by protease cleavage.  
     
     
         12 . The protein mutant of  claim 11  wherein said protease is trypsin.  
     
     
         13 . The protein mutant of  claim 10  wherein said truncation is carried out by chemical cleavage.  
     
     
         14 . The protein mutant of  claim 13  wherein said chemical cleavage is acid cleavage.  
     
     
         15 . The protein mutant of  claim 10  wherein at least one basic residue of said leader sequence is removed.  
     
     
         16 . The protein mutant of  claim 10  wherein said protein mutant consists essentially of amino acid sequence SEQ ID NO. 69.  
     
     
         17 . A biologically active heterodimer of TGF-β family member proteins, comprising: 
 a first subunit being a TGF-β family member fusion protein; and    a second subunit selected from the group consisting of a TGF-β family member fusion protein different from that of the first subunit and a wild type TGF-β family protein.    
     
     
         18 . The heterodimer of  claim 16 , wherein said wild type TGF-β family protein is selected from the group consisting of TGF-β1, TGF-β-2, TGF-β3, TGF-β4, TGF-β5, dpp, Vg-1, Vgr-1, 60A, BMP-2A, BMP-3, BMP-4, BMP-5, BMP-6, Dorsalin, OP-1, OP-2, OP-3, GDF-1, GDF-3, GDF-9, Inhibin α, Inhibin βA and Inhibin βB.  
     
     
         19 . A method of purifying a heterodimer of TGF-β family proteins, said method comprising: 
 (a) providing a first TGF-β family protein subunit;    (b) providing a second TGF-β family protein subunit different from said first subunit;    (c) mixing said first subunit and said second subunit under suitable refolding conditions to generate a mixture comprising 
 (i) a first homodimer comprising two of said first TGF-β family protein subunits;  
 (ii) a second homodimer comprising two of said second TGF-β family protein subunits; and  
 (iii) a heterodimer comprising one of said first TGF-β family subunits and one of said second TGF-β family subunits;  
 wherein said heterodimer is separable from said first homodimer and said second homodimer; and  
   (d) separating said heterodimer from said first homodimer and said second homodimer.