Peptide Purification By Means Of Metal Ion Affinity Chromatography

Abstract

A polymer substrate functionalized with a functionality comprising at least one cyclic, metal ion coordinating ligand group, the cyclic ligand group comprising at least 3 metal ion coordinating donor atoms independently selected from the group consisting of N, O and S.

1 . A histidine-containing oligopeptide comprising an amino acid sequence selected from the group consisting of: 
       
         
           
                 
                 
                 
                 
               
                     
                   HHHNSWDHDINR 
                   (SEQ. ID No. 1) 
                     
                 
                     
                     
                 
                     
                   HTNIHQDQHNHFHR 
                   (SEQ. ID No. 2) 
                 
                     
                     
                 
                     
                   HAMLDRAHDHGTR 
                   (SEQ. ID No. 3) 
                 
                     
                     
                 
                     
                   SLHEHHSGDNLR 
                   (SEQ. ID No. 4) 
                 
                     
                     
                 
                     
                   THYNAVHSHNTLQ 
                   (SEQ. ID No. 5) 
                 
                     
                     
                 
                     
                   DIHHWTDHLQSSTH 
                   (SEQ. ID No. 6) 
                 
                     
                     
                 
                     
                   LYNHYSHTAHVNHL 
                   (SEQ. ID No. 7) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         2 . A histidine-containing oligopeptide according to  claim 1 , said amino acid sequence having an N-terminal extension comprising the amino acid sequence Met-Xaa, wherein Xaa is an amino acid residue other than a proline residue. 
     
     
         3 . A histidine-containing oligopeptide according to  claim 1 , said amino acid sequence having an N-terminal extension comprising an amino acid sequence with at most ten amino acid residues. 
     
     
         4 . A polypeptide which is a fusion protein comprising a protein molecule of interest fused at its amino terminus or carboxy terminus to at least one oligopeptide according to  claim 3 . 
     
     
         5 . A polypeptide according to  claim 4 , wherein the amino terminus and the carboxy terminus of the amino acid sequence of said oligopeptide are each fused to a protein molecule of interest. 
     
     
         6 . A polypeptide according to  claim 5 , wherein the amino acid sequence of said oligopeptide is flanked by cleavage sites selected from the group consisting of enzymatic and chemical cleavage sites. 
     
     
         7 . A polypeptide according to  claim 5 , wherein said two protein molecules of interest are molecules of the same protein. 
     
     
         8 . A polypeptide according to  claim 5 , wherein said two protein molecules of interest are different. 
     
     
         9 . A polypeptide obtainable by cultivating a host cell comprising a polynucleotide construct encoding a polypeptide according to  claim 4  in an appropriate growth medium under conditions allowing expression of said polypeptide, and recovering said polypeptide from the culture medium. 
     
     
         10 . A polypeptide according to  claim 9 , wherein said host cell is a strain of  Escherichia coli.    
     
     
         11 . A polynucleotide construct encoding a polypeptide according to  claim 4 . 
     
     
         12 . A polynucleotide construct according to  claim 11 , which is a vector. 
     
     
         13 . A host cell comprising a polynucleotide construct according to  claim 11 . 
     
     
         14 . A host cell according to  claim 13 , which is a strain of  Escherichia coli.    
     
     
         15 . A method for producing a polypeptide according to  claim 4 , the method comprising cultivating a host cell containing a polynucleotide construct encoding the polypeptide of  claim 4  in an appropriate growth medium under conditions allowing expression of said polypeptide, and recovering said polypeptide from the culture medium. 
     
     
         16 . A method for purifying a protein of interest, the method comprising the steps of:
 contacting a protein sample containing a polypeptide according to  claim 4 , comprising said protein of interest, together with other proteins with a metal ion-containing functionalized polymer substrate under conditions whereby said polypeptide binds to said metal ion-containing functionalized polymer substrate so as to form a complex therewith;   washing the complex with a buffer solution to remove said other proteins; and   eluting the bound polypeptide from the washed complex.   
     
     
         17 . A method according to  claim 16 , further comprising a step wherein said oligopeptide is cleaved from said polypeptide. 
     
     
         18 . A method according to  claim 17 , wherein said oligopeptide is cleaved from said polypeptide by enzymatic means. 
     
     
         19 . A method according to  claim 18 , wherein an aminopeptidase, such as a dipeptidyl aminopeptidase, is employed to cleave said oligopeptide from said polypeptide. 
     
     
         20 . A purified protein obtainable by a method according to  claim 17 .