Affinity peptides and method for purification of recombinant proteins

Abstract

This invention describes a process for separating a fusion protein or polypeptide in the form of its precursor from a mixture containing said fusion protein and impurities, which comprises contacting said fusion protein with a resin containing immobilized metal ions, said fusion protein covalently operably linked directly or indirectly to an immobilized metal ion-affinity peptide, binding said fusion protein to said resin, and selectively eluting said fusion protein from said resin.

1 . A polypeptide, protein or protein fragment represented by the formula R 1 -Sp 1 -(His-Z 1 -His-Arg-His-Z 2 -His)-Sp 2 -R 2 , wherein (His-Z 1 -His-Arg-His-Z 2 -His) (SEQ ID NO: 24) is a metal ion-affinity peptide, R 1  is hydrogen, a polypeptide, protein or protein fragment, Sp 1  is a covalent bond or a spacer comprising at least one amino acid residue, R 2  is hydrogen, a polypeptide, protein or protein fragment, Sp 2  is a covalent bond or a spacer comprising at least one amino acid residue, Z 1  is an amino acid residue selected from the group consisting of Ala, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z 2  is an amino acid residue selected from the group consisting of Ala, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val. 
     
     
         2 . The polypeptide, protein or protein fragment of  claim 1 , wherein Z 1  is selected from the group consisting of Ala, Asn, Ile, Lys, Phe, Ser, Thr, and Val, and Z 2  is selected from the group consisting of Ala, Asn, Gly, Lys, Ser, Thr and Tyr. 
     
     
         3 . The polypeptide, protein or protein fragment of  claim 1 , wherein Z 1  and Z 2  are selected from the group consisting of:
 (a) Z 1  is Asn and Z 2  is Gly;   (b) Z 1  is Asn and Z 2  is Lys   (c) Z 1  is Lys and Z 2  is Gly.   (d) Z 1  is Lys and Z 2  is Lys.   (e) Z 1  is Ile and Z 2  is Asn;   (f) Z 1  is Thr and Z 2  is Ser;   (g) Z 1  is Ser and Z 2  is Tyr;   (h) Z 1  is Val and Z 2  is Ala; and   (i) Z 1  is Ala and Z 2  is Lys.   
     
     
         4 . The polypeptide, protein or protein fragment of  claim 1 , wherein R 1  or R 2  is hydrogen. 
     
     
         5 . The polypeptide, protein or protein fragment of  claim 1 , wherein R 1  or R 2  is an amino acid residue. 
     
     
         6 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising a proteolytic cleavage site, a fusion protein, a secretion sequence, a leader sequence for cellular targeting an antibody epitope or an internal ribosomal sequences. 
     
     
         7 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising a proteolytic cleavage site. 
     
     
         8 . The polypeptide, protein or protein fragment of  claim 7 , wherein the proteolytic cleavage site is cleaved with enterokinase. 
     
     
         9 . The polypeptide, protein or protein fragment of  claim 1 , wherein any one of Sp 1 , Sp 2 , R 1  and R 2  comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 1-17. 
     
     
         10 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising the enzyme glutathione-S-transferase of the parasite helminth  Schistosoma japonicum.    
     
     
         11 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 15). 
     
     
         12 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising the amino acid sequence DLYDDDDK (SEQ ID NO: 16). 
     
     
         13 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising the amino acid sequence Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 17). 
     
     
         14 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising at least one amino acid residue, said spacer comprising an antigenic domain, wherein the antigenic domain comprises the sequence 
       
         
           
                 
               
                   (SEQ ID NO: 39) 
                 
                   X 20 -(X 1 -Y-K-X 2 -X 3 -D-X 4 ) n -X 5 -(X 1 -Y-K-X 7 -X 8 -D-X 9 -K)-X 21   
                 
             
                
                
               
            
           
         
         where: 
         D, Y and K are their representative amino acids; 
         X 20  and X 21  are independently a hydrogen or a covalent bond; 
         each X 1  and X 4  is independently a covalent bond or at least one amino acid residue selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; 
         each X 2 , X 3 , X 7  and X 8  is independently an amino acid residue selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; 
         X 5  is a covalent bond or a spacer domain, the spacer domain comprising at least one amino acid 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 Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; 
         X 9  is a covalent bond or an aspartate residue; and 
         n is 0, 1 or 2. 
       
     
     
         15 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising at least one amino acid residue, said spacer comprising an antigenic domain, wherein the antigenic domain comprises the sequence 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 40) 
                 
                     
                   X 20 -(D-Y-K-X 2 -X 3 -D) n -X 5 -(D-Y-K-X 7 -X 8 -D-X 9 -K)-X 21   
                 
             
                
                
               
            
           
         
         where: 
         D, Y, K are their representative amino acids; 
         X 20  and X 21  are independently a hydrogen or a covalent bond; 
         each X 2 , X 3 , X 7  and X 8  is independently an amino acid residue selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; 
         X 5  is a covalent bond or a spacer domain, the spacer domain comprising at least one amino acid 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 Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; 
         X 9  is a covalent bond or an aspartate residue; and 
         n is at least 2. 
       
     
     
         16 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising at least one amino acid residue, said spacer comprising an antigenic domain, wherein the antigenic domain comprises the sequence 
       
         
           
                 
               
                   (SEQ ID NO: 45) 
                 
                   X 20 -X 10 -(D-Y-K-X 2 -X 3 -D) n -X 5 -(D-Y-K-X 7 -X 8 -D-X 9 -K)-X 21   
                 
             
                
                
               
            
           
         
         where: 
         D, Y, and K are their representative amino acids; 
         X 20  and X 21  are independently a hydrogen or a covalent bond; 
         X 10  is a covalent bond or an amino acid; 
         each X 2 , X 3 , X 7  and X 8  is independently an amino acid residue selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; 
         X 5  is a covalent bond or a spacer domain, the spacer domain comprising at least one amino acid 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 Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; 
         X 9  is a covalent bond or an aspartate residue; and 
         n is at least 2. 
       
     
     
         17 . The polypeptide, protein or protein fragment of  claim 1 , wherein Sp 1  or Sp 2  is a spacer comprising at least one amino acid residue, said spacer comprising an antigenic domain, wherein the antigenic domain comprises the sequence 
       
         
           
                 
               
                   (SEQ ID NO: 42) 
                 
                   X 20 -(D-X 11 -Y-X 12 -X 13 )n-X 14 -(D-X 11 -Y-X 12 -X 13 -D-X 15 -K)-X 21   
                 
             
                
                
               
            
           
         
         where: 
         D, Y and K are their representative amino acids; 
         X 20  and X 21  are independently a hydrogen or a covalent bond; 
         each X 11  is a covalent bond or an amino acid; 
         each X 12  is an amino acid selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; 
         each X 13  is a covalent bond or at least one amino acid selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; 
         X 14  is a covalent bond or a spacer domain, the spacer domain comprising at least one amino acid 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 Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; 
         X 15  is a covalent bond or an aspartate residue; and 
         n is 0 or at least 1. 
       
     
     
         18 . A polypeptide, protein or protein fragment represented by the formula R 1 -Sp 1 -(His-Z 1 -His-Arg-His-Z 2 -His) t -Sp 2 -R 2 , wherein (His-Z 1 -His-Arg-His-Z 2 -His) (SEQ ID NO: 24) is a metal ion-affinity peptide, t is at least 2, R 1  is hydrogen, a polypeptide, protein or protein fragment, Sp 1  is a covalent bond or a spacer comprising at least one amino acid residue, R 2  is hydrogen, a polypeptide, protein or protein fragment, Sp 2  is a covalent bond or a spacer comprising at least one amino acid residue, Z 1  is an amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z 2  is an amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val. 
     
     
         19 . The peptide of  claim 18 , wherein Z 1  and Z 2  are selected from the group consisting of:
 (a) Z 1  is Asn and Z 2  is Lys; and   (b) Z 1  is Lys and Z 2  is Gly.   
     
     
         20 . A polypeptide, protein or protein fragment represented by the formula R 1 -Sp 1 -[(His-Z 1 -His-Arg-His-Z 2 -His)-Sp 2 ] t -R 2 , wherein (His-Z 1 -His-Arg-His-Z 2 -His) (SEQ ID NO: 24) is a metal ion-affinity peptide, t is at least 2, R 1  is hydrogen, a polypeptide, protein or protein fragment, Sp 1  is a covalent bond or a spacer comprising at least one amino acid residue, R 2  is hydrogen, a polypeptide, protein or protein fragment, Sp 2  is a covalent bond or a spacer comprising at least one amino acid residue, Z 1  is an amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z 2  is an amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val; and each Sp 2  of the recombinant polypeptides, proteins or protein fragments may be the same or different. 
     
     
         21 . The peptide of  claim 20 , wherein Z 1  and Z 2  are selected from the group consisting of:
 (a) Z 1  is Asn and Z 2  is Lys; and   (b) Z 1  is Lys and Z 2  is Gly.