Two helix binders

Abstract

Provided herein are isolated polypeptides derived from the staphylococcal protein A protein B domain comprising a pair of anti-parallel alpha helices that are capable of binding a target. Also provided are nucleic acid sequences encoding such two helix binders, vectors containing the nucleic acid sequences encoding for two helix binders, and host cells transformed with vectors containing the nucleic acid sequences encoding for the two-helix binders. Also provided are methods of using the two helix binders.

1 . A method of binding a target in a sample comprising:
 applying an isolated polypeptide comprising a two helix segment derived from the Z domain of protein A that is capable of binding the Fc region of IgG and wherein the isolated polypeptide comprises a disulfide bond between two members of a thiol-containing pair of residues, wherein both members of the disulfide pair are positioned outside of the binding interface of the polypeptide and wherein the first amino acid of the binding interface is positioned eleven amino acids in an N-terminal direction relative to a conserved proline, wherein the conserved proline is an internal residue within the isolated polypeptide and wherein the isolated polypeptide comprises more than eleven amino acids in the N-terminal direction relative to the conserved proline, wherein the isolated polypeptide comprises a variant of SEQ ID NO. 4 modified by a substitution that comprises substituting two amino acids with the thiol-containing residues and wherein Xaa is any naturally occurring amino acid except cysteine to a sample containing the target.   
     
     
         2 . The method of  claim 1 , wherein X 1  of SEQ ID No. 4 is Glu or Asp 
     
     
         3 . The method of  claim 1 , wherein the first member of the disulfide pair is located at one of residue 1 through residue 8 and the second member of the disulfide pair is located at one of residue 36 through residue 39 of SEQ ID NO. 4. 
     
     
         4 . The method of  claim 1 , wherein the first member of the disulfide pair is located at one of residue 1 through residue 8 and the second member of the disulfide pair is located at appended residue 40 of SEQ ID NO. 4. 
     
     
         5 . The method of  claim 1 , wherein the first member of the disulfide pair is located at one of residue 4 through residue 6 and the second member of the disulfide pair is located at residue 39 of SEQ ID NO. 4. 
     
     
         6 . The method of  claim 1 , wherein the first member of the disulfide pair is located at residue 5 and the second member of the disulfide pair is located at residue 39 or appended residue 40 of SEQ ID NO. 4. 
     
     
         7 . The method of  claim 1 , the sequence of which consists of SEQ ID NO. 7. 
     
     
         8 . The method of  claim 1 , the sequence of which consists of SEQ ID NO. 8. 
     
     
         9 . The method of  claim 1 , the sequence of which consists of SEQ ID NO. 9. 
     
     
         10 . The method of  claim 1 , the sequence of which consists of SEQ ID NO. 10 or conservative variants thereof. 
     
     
         11 . The method of  claim 1 , comprising a signal generator attached to the polypeptide. 
     
     
         12 . The method of  claim 11 , wherein the signal generator comprises a radioisotope. 
     
     
         13 . The method of  claim 11 , wherein the signal generator comprises a fluorophore. 
     
     
         14 . The method of  claim 1 , comprising a tag attached to the polypeptide. 
     
     
         15 . A method of forming a two-helix binder comprising:
 providing a nucleotide encoding a polypeptide comprising a two helix segment derived from the Z domain of protein A that is capable of binding the Fc region of IgG and wherein the isolated polypeptide comprises a disulfide bond between two members of a thiol-containing pair of residues, wherein both members of the disulfide pair are positioned outside of the binding interface of the polypeptide and wherein the first amino acid of the binding interface is positioned eleven amino acids in an N-terminal direction relative to a conserved proline, wherein the conserved proline is an internal residue within the isolated polypeptide and wherein the isolated polypeptide comprises more than eleven amino acids in the N-terminal direction relative to the conserved proline, wherein the isolated polypeptide comprises a variant of SEQ ID NO. 4 modified by a substitution that comprises substituting two amino acids with the thiol-containing residues and wherein Xaa is any naturally occurring amino acid except cysteine;   causing the nucleotide to express the polypeptide in an organism; and   isolating the polypeptide.   
     
     
         16 . The method of  claim 15 , wherein X 1  of SEQ ID No. 4 is Glu or Asp 
     
     
         17 . The method of  claim 15 , wherein the first member of the disulfide pair is located at one of residue 1 through residue 8 and the second member of the disulfide pair is located at one of residue 36 through residue 39 of SEQ ID NO. 4. 
     
     
         18 . The method of  claim 15 , wherein the first member of the disulfide pair is located at one of residue 1 through residue 8 and the second member of the disulfide pair is located at appended residue 40 of SEQ ID NO. 4. 
     
     
         19 . The method of  claim 15 , wherein the first member of the disulfide pair is located at one of residue 4 through residue 6 and the second member of the disulfide pair is located at residue 39 of SEQ ID NO. 4. 
     
     
         20 . The method of  claim 15 , wherein the first member of the disulfide pair is located at residue 5 and the second member of the disulfide pair is located at residue 39 or appended residue 40 of SEQ ID NO. 4.