Affinity peptides and method for purification of recombinant proteins
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 recombinant vector comprising a vector and a DNA sequence coding for a polypeptide, protein or protein fragment comprising 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, and wherein the recombinant vector is capable of directing expression of the DNA sequence in a compatible unicellular host organism.
2. The recombinant vector of claim 1 , wherein Z 1 is Asn and Z 2 is Lys.
3. A host cell comprising the recombinant vector as set forth in claim 1 .
4. The host cell of claim 3 , wherein the recombinant vector comprises the DNA sequence wherein Z 1 is Lys and Z 2 is Gly.
5. The host cell of claim 4 , wherein said host cell is E. coli , yeast, insect cells, mammalian cells, or plant.
6. The recombinant vector 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 formula
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.
7. The recombinant vector 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 formula
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.
8. The recombinant vector 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 formula
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.
9. The recombinant vector of claim 8 , wherein the antigenic domain comprises the 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).
10. The recombinant vector 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 formula
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 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 15 is a covalent bond or an aspartate residue; and
n is 0 or at least 1.
11. The recombinant vector 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 DYKDDDDK (SEQ ID NO: 15).
12. The recombinant vector 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 DLYDDDDK (SEQ ID NO: 16).
13. A host cell comprising the recombinant vector as set forth in claim 6 .
14. A host cell comprising the recombinant vector as set forth in claim 7 .
15. A host cell comprising the recombinant vector as set forth in claim 8 .
16. A host cell comprising the recombinant vector as set forth in claim 10 .
17. The recombinant vector 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.
18. The host cell of claim 3 , wherein the recombinant vector comprises the DNA sequence 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.
19. The recombinant vector of claim 6 , 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.
20. The recombinant vector of claim 7 , 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.
21. The recombinant vector of claim 8 , 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.
22. The recombinant vector of claim 10 , 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.
23. The recombinant vector of claim 19 wherein Z 1 is selected from the group consisting of Asn and Lys and Z 2 is selected from the group consisting of Gly and Lys.
24. The recombinant vector of claim 20 wherein Z 1 is selected from the group consisting of Asn and Lys and Z 2 is selected from the group consisting of Gly and Lys.
25. The recombinant vector of claim 21 wherein Z 1 is selected from the group consisting of Asn and Lys and Z 2 is selected from the group consisting of Gly and Lys.
26. The recombinant vector of claim 22 wherein Z 1 is selected from the group consisting of Asn and Lys and Z 2 is selected from the group consisting of Gly and Lys.
27. The recombinant vector of claim 19 wherein Z 1 is Asn and Z 2 is Lys.
28. The recombinant vector of claim 20 wherein Z 1 is Asn and Z 2 is Lys.
29. The recombinant vector of claim 21 wherein Z 1 is Asn and Z 2 is Lys.
30. The recombinant vector of claim 22 wherein Z 1 is Asn and Z 2 is Lys.
31. The host cell of claim 3 , wherein the recombinant vector comprises the DNA sequence wherein Z 1 is Asn and Z 2 is Lys.
32. A process for producing a polypeptide, protein or protein fragment comprising 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, the process comprising
(a) transforming a host cell with a recombinant vector encoding the polypeptide, protein or protein fragment comprising the formula R 1 -Sp 1 -(His-Z 1 -His-Arg-His-Z 2 -His)-Sp 2 -R 2 ;
(b) culturing the host cell under conditions which permit the expression of the polypeptide, protein or protein fragment;
(c) lysing the host cell; and
(d) purifying the polypeptide, protein or protein fragment or a portion thereof by metal ion affinity chromatography.
33. The process of claim 32 , wherein the recombinant vector comprises a DNA sequence coding for the polypeptide, protein or protein fragment, wherein the recombinant vector is capable of directing expression of the DNA sequence in a compatible host cell.
34. The process of claim 33 , wherein the recombinant vector comprises the DNA sequence wherein Z 1 is Asn and Z 2 is Lys or wherein Z 1 is Lys and Z 2 is Gly.
35. The process of claim 34 , wherein the host cell is E. coli , yeast, insect cells, mammalian cells, or plants.
36. The process of claim 32 , 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.