Polynucleotides encoding metal ion affinity peptides and related products
View Patent ↗The present invention provides metal ion affinity peptides, fusion proteins comprising metal ion affinity peptides, and polynucleotides encoding the fusion proteins. A feature of the subject invention is that the metal ion affinity peptide has a formula selected from the group consisting of: formula 1: (His-X 1 -X 2 ) n1 -(His-X 3 -X 4 -X 5 ) n2 -(His-X 6 ) n3 , wherein each of X 1 and X 2 is independently an amino acid with an aliphatic or an amide side chain, each of X 3 , X 4 , X 5 is independently an amino acid with a basic side chain (except His) or an acidic side chain, each X 6 is an amino acid with an aliphatic or an amide side chain, n1 and n2 are each independently 1–3, and n3 is 1–5; formula 2: (His-Asn) n , where n=3 to 10; and formula 3: (His-X 1 -X 2 ) n , wherein each of X 1 and X 2 is an amino acid having an acidic side chain, and n=3 to 10. The invention further provides recombinant vectors comprising subject polynucleotides, and host cells comprising the recombinant vectors. The invention further provides methods and kits for purifying a fusion protein comprising a metal ion affinity peptide.
1. An isolated polynucleotide comprising a nucleotide sequence that encodes a metal ion affinity peptide having a formula of
NH 2 -(His-Asn) n or (His-Asn) n -COOH, where n=6 (SEQ ID NO: 15).
2. The polynucleotide according to claim 1 wherein the polynucleotide comprises a nucleotide sequence encoding a fusion protein comprising a polypeptide and the metal ion affinity peptide, wherein the metal ion affinity peptide is fused to an amino- or carboxy-terminal amino acid of the polypeptide.
3. A recombinant vector comprising the polynucleotide according to claim 1 .
4. A recombinant host cell comprising the recombinant vector according to claim 3 .
5. The recombinant host cell according to claim 4 . wherein said cell is a prokaryotic cell.
6. The recombinant host cell according to claim 4 , wherein said cell is a eukaryotic cell.
7. A kit for purifying a protein, comprising:
a) the recombinant vector according to claim 3 ; and
b) a metal ion affinity resin.
8. The kit according to claim 7 , further comprising:
an extraction buffer;
a wash buffer; and
an elution buffer.
9. The kit according to claim 8 , further comprising a column.
10. An isolated polynucleotide comprising a nucleotide sequence that encodes a metal ion affinity peptide having a formula of
NH 2 -(His-Asn) n or (His-Asn) n -COOH, where n=3–5 (SEQ ID NOs: 27–29) or 7–10(SEQ ID NOs:30–33).
11. The polynucleotide according to claim 10 , wherein the polynucleotide comprises a nucleotide sequence encoding a fusion protein comprising a polypeptide and the metal ion affinity peptide, wherein the metal ion affinity peptide is fused to an amino- or carboxy-terminal amino acid of the polypeptide.
12. A recombinant vector comprising the polynucleotide according to claim 10 .
13. A recombinant host cell comprising the recombinant vector according to claim 12 .
14. The recombinant host cell according to claim 13 , wherein said cell is a prokaryotic cell.
15. The recombinant host cell according to claim 13 , wherein said cell is a eukaryotic cell.
16. A kit for purifying a protein, comprising:
a) the recombinant vector according to claim 12 ; and
b) a metal ion affinity resin.
17. The kit according to claim 16 , further comprising:
an extraction buffer;
a wash buffer; and
an elution buffer.
18. The kit according to claim 17 , further comprising a column.