Purification of recombinant proteins fused to multiple epitopes

Abstract

The present invention provides novel identification polypeptides containing multiple copies of an antigenic domain joined in tandem to provide increased sensitivity for the detection and purification of target peptides, a cleavable linking sequence and optionally a spacer domain. Further provided are hybrid polypeptide molecules composed of an identification polypeptide and a target peptide which are produced by recombinant DNA technology and purified using affinity chromatography using one or more ligands. Accordingly, also provided are DNA expression vectors containing DNA encoding for identification polypeptides and methods for using such identification polypeptides for the purification of target peptides. Also provided are methods of constructing DNA vectors encoding the novel identification polypeptides and DNA expression vectors encoding the identification polypeptides linked to a target peptide.

1 . An identification polypeptide for use in purifying a target peptide wherein said identification polypeptide comprises: 
 a. multiple copies of an antigenic domain joined together in tandem, each of the antigenic domains comprising no more than twenty amino acid residues with at least two different amino acid residues; and    b. a linking sequence between the multiple copies of the antigenic domain and the target peptide molecule, the linking sequence comprising a cleavable site wherein the cleavable site is not duplicated within or interposed between the multiple copies of the antigenic domain.    
     
     
         2 . The identification polypeptide of  claim 1  wherein the amino acid sequence of each such antigenic domain comprises at least one-half hydrophilic amino acid residues.  
     
     
         3 . The identification polypeptide of  claim 2  wherein the amino acid sequence of each such antigenic domain further comprises at least three-fourths hydrophilic amino acid residues.  
     
     
         4 . The identification polypeptide of  claim 1  wherein the amino acid sequence of each such antigenic domain comprises at least one amino acid selected from the group of hydrophilic amino acid residues and at least one amino acid selected from the group of aromatic amino acid residues.  
     
     
         5 . The identification polypeptide of  claim 4  wherein the amino acid sequence of each such antigenic domain comprises no more than ten amino acid residues with at least two different amino acid residues.  
     
     
         6 . The identification polypeptide of  claim 4  wherein the amino acid sequence of each such antigenic domain comprises no more than six amino acid residues with at least two different amino acid residues.  
     
     
         7 . The identification polypeptide of  claim 1  wherein the amino acid sequence of each such antigenic domain comprises a plurality of amino acids of the group consisting of arginine, proline, glutamic acid, aspartic acid, phenylalanine and isoleucine.  
     
     
         8 . The identification polypeptide of  claim 1  wherein said cleavable site comprises an amino acid sequence being cleavable by a sequence-specific proteolytic agent at a specific amino acid residue adjacent to the target peptide molecule, wherein the sequence-specific proteolytic agent is selected from the group consisting of enterokinase, Factor Xa and thrombin.  
     
     
         9 . The identification polypeptide of  claim 8  wherein said cleavable site is an enterokinase recognition site.  
     
     
         10 . The identification polypeptide of  claim 1  further comprising a spacer domain comprising at least one amino acid residue interposed between any two or more antigenic domains of said multiple copies of the antigenic domain or between the multiple copies of the antigenic domain and the linking sequence.  
     
     
         11 . The identification polypeptide of  claim 10  wherein the amino acid sequence of said spacer domain is selected from the group consisting of hydrophilic amino acid residues.  
     
     
         12 . The identification polypeptide of  claim 10  wherein said spacer domain further comprises at least one histidine residue, at least one glycine residue or a combination of multiple or alternating histidine residues, said combination comprising His-Gly-His or -(His-X) m —, wherein m is 1 to 6 and X is selected from the group consisting of Gly, His, Tyr, Lys, Phe, Met, Ala, Glu, Ile, Thr, Asp, Asn, Gln, Arg, Cys, and Pro.  
     
     
         13 . The identification polypeptide of  claim 10  wherein the amino acid sequence of said spacer domain comprises isoleucine.  
     
     
         14 . The identification polypeptide of  claim 1  further comprising a metal chelating sequence joined to said multiple copies of the antigenic domain, wherein the metal chelating sequence comprises at least one histidine residue, at least one glycine residue or a combination of multiple or alternating histidine residues, said combination comprising His-Gly-His or -(His-X) m —, wherein m is 1 to 6 and X is selected from the group consisting of Gly, His, Tyr, Trp, Met, Asp, Asn, Gln, Arg, Cys, and Pro.  
     
     
         15 . The identification polypeptide of  claim 1  further comprising a multiple cloning site comprising multiple restriction enzyme recognition ssites.