Oligomerization of hepatitis delta antigen

Abstract

This invention relates to HDAg peptides, including mutants, derivatives fragments and fusion molecules, including fusion proteins, coiled-coil oligomers, nucleic acid molecules, vectors comprising HDAg nucleic acid molecules, cells comprising said molecules, methods of multivalent expression and association of binding moieties of HDAg fusion molecules, and methods of use involving the molecules. The molecules are particularly useful as a framework for multivalent display via formation of C-terminal and/or N-terminal fusion proteins or via chemical coupling to chemically reactive sidechains, e.g. cysteine residues.

1 . An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: 
 a) a nucleotide sequence depicted in  FIG. 9 , nucleotides 37-150 of  FIG. 9 , nucleotides 37-186 of  FIG. 9 ,  FIG. 10 , nucleotides 1421-1566 of  FIG. 10 , nucleotides 1457-1566 of  FIG. 10 ,  FIG. 15  and  FIG. 16 ;    b) a complementary strand of the sequence of a);    c) DNA sequences that hybridize to the sequence of a) or b); and    d) RNA sequences transcribed from the sequences of a), b) or c), or a fragment or mutation thereof, which encodes a coiled-coil oligomer.    
     
     
         2 . An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: 
 a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence depicted in a row of  FIG. 1 , amino acids 12-48 of a row of  FIG. 1 , the top row of  FIG. 3C ,  FIG. 9 , amino acids 12-48 of a row of  FIG. 9 ,  FIG. 10 , amino acids 12-88 of  FIG. 10 ,  FIG. 11  and  FIG. 17 ;    b) the complementary strand of the sequence of a);    c) RNA sequences transcribed from the sequences of a) or b), or a fragment or mutation thereof, which encodes a coiled-coil oligomer.    
     
     
         3 . An isolated nucleic acid molecule encoding a fusion molecule comprising a fusion molecule comprising HDAg and at least one binding moiety.  
     
     
         4 . A fusion gene comprising a nucleic acid molecule of  claim 1  operably linked to a nucleic acid molecule encoding a heterologous peptide.  
     
     
         5 . A fusion gene comprising a nucleic acid molecule of  claim 2  operably linked to a nucleic acid molecule encoding a heterologous peptide.  
     
     
         6 . A vector comprising a nucleic acid molecule which encodes a subunit of an HDAg coiled-coil octamer.  
     
     
         7 . A vector comprising a nucleic acid molecule of  claim 1 .  
     
     
         8 . A vector comprising a nucleic acid molecule of  claim 2 .  
     
     
         9 . A vector comprising a nucleic acid molecule of  claim 3 .  
     
     
         10 . A vector comprising a nucleic acid molecule encoding a fusion molecule comprising HDAg and at least one binding moiety.  
     
     
         11 . A vector comprising a nucleic acid molecule encoding HDAg and at least one multiple cloning site.  
     
     
         12 . The vector of  claim 11  wherein at least one multiple cloning site is located 3′ to the nucleic acid molecule encoding HDAg.  
     
     
         13 . The vector of  claim 11  wherein at least one multiple coding site is located 5′ to the nucleic acid molecule encoding HDAg.  
     
     
         14 . The vector of  claim 11  wherein there are at least two multiple coding sites, wherein at least one multiple coding site is located in a flanking region 3′ to the nucleic acid molecule encoding HDAg and at least one multiple coding site is located in a flanking region 5′ to the nucleic acid molecule encoding HDAg.  
     
     
         15 . A vector comprising a nucleic acid molecule of  claim 1  and at least one multiple cloning site.  
     
     
         16 . A vector comprising a nucleic acid molecule of  claim 2  and at least one multiple cloning site.  
     
     
         17 . The vector of  claim 16  further comprising a nucleic acid molecule encoding a nuclear localization signal.  
     
     
         18 . The vector for expression of the fusion molecule comprising HDAg and at least one binding moiety wherein a first heterologous gene encodes a first binding moiety and a second heterologous gene encodes a second binding moiety.  
     
     
         19 . A vector of  claim 11  which further comprises a nucleic acid molecule which encodes a heterologous gene.  
     
     
         20 . A host cell which comprises a nucleic acid molecule which encodes a fusion molecule comprising HDAg and at least one binding moiety.  
     
     
         21 . A host cell which comprises a nucleic acid molecule of  claim 1 .  
     
     
         22 . A host cell which comprises a nucleic acid molecule of  claim 2 .  
     
     
         23 . A method of manufacturing a host cell comprising a nucleic acid molecule encoding a fusion molecule comprising HDAg and at least one binding moiety comprising introducing a vector of  claim 10  into the host cell.  
     
     
         24 . A method of expressing a high valency display of at least one binding moiety comprising introducing into a cell a vector comprising a nucleic acid molecule encoding HDAg and a nucleic acid molecule encoding the binding moiety and culturing the cell under conditions sufficient to permit expression of a fusion molecule comprising the binding moiety and HDAg.  
     
     
         25 . A method for delivering molecules to a cell comprising contacting them with a fusion molecule comprising HDAg and at least one binding moiety.  
     
     
         26 . The method of  claim 25  wherein the binding moiety is an oligonucleotide.  
     
     
         27 . The method of  claim 25  wherein the oligonucleotide hybridizes to a nucleic acid molecule in the cell.  
     
     
         28 . The method of  claim 26  wherein said fusion molecule further comprises a double-stranded nuclease.  
     
     
         29 . The method of  claim 25  wherein the fusion molecule comprises a first binding moiety and a second binding moiety wherein the first binding moiety interacts with a binding partner and the second binding moiety functions as an effector.  
     
     
         30 . The method of  claim 29  wherein the first binding moiety interacts with a cell surface receptor and the second binding moiety can kill the cell.  
     
     
         31 . A method of amplifying a signal in a solid phase assay comprising coupling an HDAg octamer with at least one copy of a domain which interacts with a ligand and at least two copies of a label.  
     
     
         32 . A method of  claim 31  wherein the label is selected from the group consisting of alkaline phosphatase, a radiolabel, streptadavin and green fluorescent protein.  
     
     
         33 . The method of  claim 31  wherein the solid phase assay is an ELISA assay.  
     
     
         34 . A method of facilitating exchange of substrates and products comprising coupling an HDAg oligomer to at least two enzymes which function in a linked pathway.  
     
     
         35 . A method of enhancing a reaction between binding partners comprising coupling the binding partners to an HDAg oligomer.  
     
     
         36 . A method of enhancing a reaction between two binding partners comprising coupling one binding partner to an HDAg oligomer and contacting the oligomer to a second binding partner.