Aldehyde Tags, Uses Thereof in Site-Specific Protein Modification

Abstract

The invention features compositions and methods for site-specific modification of proteins by incorporation of an aldehyde tag. Enzymatic modification at a sulfatase motif of the aldehyde tag through action of a formylglycine generating enzyme (FGE) generates a formylglycine (FGly) residue. The aldehyde moiety of FGly residue can be exploited as a chemical handle for site-specific attachment of a moiety of interest to a polypeptide.

1 .- 22 . (canceled) 
     
     
         23 . A recombinant nucleic acid comprising:
 an expression cassette comprising:
 a first nucleic acid comprising an aldehyde tag-encoding sequence; and 
 a restriction site positioned 5′ or 3′ of the aldehyde tag-encoding sequence, which restriction site provides for insertion of a second nucleic acid encoding a polypeptide of interest; and 
   a promoter operably linked to the expression cassette to provide for expression of an aldehyde tagged-polypeptide produced by insertion of the second nucleic acid encoding the polypeptide of interest into the restriction site.   
     
     
         24 . (canceled) 
     
     
         25 . The recombinant nucleic acid of  claim 23 , wherein the aldehyde tag-encoding sequence encodes a heterologous sulfatase motif having a 2-formylglycine residue, wherein the heterologous sulfatase motif is less than 13 amino acid residues and contains a contiguous sequence of the formula: 
       
         
           
                 
                 
               
                     
                   (I) 
                 
                     
                   (SEQ ID NO: 1) 
                 
                     
                   X 1 (FGly)X 2 Z 2 X 3 R 
                 
             
                
                
                
               
            
           
         
       
       wherein
 FGly is a 2-formylglycine residue; 
 Z 2  is a proline or alanine residue; and 
 X 1 , X 2  and X 3  are each independently any amino acid. 
 
     
     
         26 . The recombinant nucleic acid of  claim 25 , wherein
 the heterologous sulfatase motif is positioned at a C-terminus of the polypeptide;   the heterologous sulfatase motif is present in a terminal loop of the polypeptide;   the heterologous sulfatase motif is, when the polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop;   the heterologous sulfatase motif is present at an internal site or at an N-terminus of the polypeptide, and is solvent-accessible when the polypeptide is folded; and/or   the heterologous sulfatase motif is present at a site of post-translational modification.   
     
     
         27 . The recombinant nucleic acid of  claim 25 , wherein X 1 , X 2 , and X 3  are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid. 
     
     
         28 . The recombinant nucleic acid of  claim 25 , wherein X 2  and X 3  are each independently S, T, A, V, G, or C. 
     
     
         29 . The recombinant nucleic acid of  claim 25 , wherein X 1  is L, M, V, S or T and X 2  and X 3  are each independently S, T, A, V, G or C. 
     
     
         30 . The recombinant nucleic acid of  claim 25 , wherein X 1  is L, M, V, S or T. 
     
     
         31 . The recombinant nucleic acid of  claim 30 , wherein the heterologous sulfatase motif is L(FGly)TPSR (SEQ ID NO: 62). 
     
     
         32 . The recombinant nucleic acid of  claim 30 , wherein the heterologous sulfatase motif is selected from M(FGly)TPSR (SEQ ID NO: 63), V(FGly)TPSR (SEQ ID NO: 64), L(FGly)SPSR (SEQ ID NO: 65), L(FGly)APSR (SEQ ID NO: 66), L(FGly)VPSR (SEQ ID NO: 67), and L(FGly)GPSR (SEQ ID NO: 68). 
     
     
         33 . The recombinant nucleic acid of  claim 26 , wherein the heterologous sulfatase motif is positioned at an internal sequence of the polypeptide. 
     
     
         34 . The recombinant nucleic acid of  claim 26 , wherein the heterologous sulfatase motif is positioned at a terminal loop, a C-terminus, or an N-terminus of the polypeptide. 
     
     
         35 . The recombinant nucleic acid of  claim 26 , wherein the heterologous sulfatase motif is positioned on a solvent-accessible region of the polypeptide when folded. 
     
     
         36 . The recombinant nucleic acid of  claim 26 , wherein the heterologous sulfatase motif is positioned at a site of post-translational modification of the polypeptide that is native or non-native to the amino acid sequence of the polypeptide. 
     
     
         37 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide comprises an Fc fragment. 
     
     
         38 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide comprises an Fc polypeptide. 
     
     
         39 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide is an antibody. 
     
     
         40 . The recombinant nucleic acid of  claim 39 , wherein the antibody is an IgG antibody. 
     
     
         41 . The recombinant nucleic acid of  claim 39 , wherein the antibody is a humanized antibody. 
     
     
         42 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide comprises an antigen-binding fragment of an antibody. 
     
     
         43 . The recombinant nucleic acid of  claim 42 , wherein the polypeptide comprises a Fab or Fv. 
     
     
         44 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide comprises a single chain antibody. 
     
     
         45 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide is a blood factor. 
     
     
         46 . The recombinant nucleic acid of  claim 45 , wherein the blood factor is Factor VIII. 
     
     
         47 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide is a fibroblast growth factor. 
     
     
         48 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide is a protein vaccine. 
     
     
         49 . The recombinant nucleic acid of  claim 25 , wherein the polypeptide is an enzyme. 
     
     
         50 . The recombinant nucleic acid of  claim 25 , wherein the heterologous sulfatase motif is less than 12 amino acid residues. 
     
     
         51 . The recombinant nucleic acid of  claim 25 , wherein the heterologous sulfatase motif is less than 11 amino acid residues. 
     
     
         52 . The recombinant nucleic acid of  claim 25 , wherein the heterologous sulfatase motif is less than 10 amino acid residues. 
     
     
         53 . The recombinant nucleic acid of  claim 25 , wherein the heterologous sulfatase motif is less than 9 amino acid residues. 
     
     
         54 . The recombinant nucleic acid of  claim 25 , wherein the heterologous sulfatase motif is less than 8 amino acid residues. 
     
     
         55 . The recombinant nucleic acid of  claim 25 , wherein the heterologous sulfatase motif is less than 7 amino acid residues.