Fusion protein having enhanced in vivo erythropoietin activity
Provided is a fusion protein comprising, at its carboxy terminal of human erythropoietin (EPO), a mutant having one to four amino acid substitutions in the carboxy terminal peptide (CTP) fragment of a human chorionic gonadotropin (HCG) β subunit, for increasing an in vivo half-life activity of EPO. The in vivo half-life can be greatly elongated while retaining the intrinsic activity of the EPO, without increasing the sugar chain content.
1. A fusion protein comprising human erythropoietin (EPO) having a carboxy terminal and a mutant linked to the carboxy terminal, said mutant being the carboxy terminal peptide (CTP) fragment of a human chorionic gonadotropin (HCG) β subunit having serine residues at positions 121, 127, 132, and 138 of the HCG β subunit replaced with alanine, wherein the CTP fragment has 34 amino acids corresponding to amino acid positions 112–145 of the HCG β subunit or 28 amino acids corresponding to amino acid positions 118–145 of the HCG β subunit.
2. The fusion protein according to claim 1 , wherein the CTP fragment has the amino acid sequence set forth in SEQ ID NO. 11.
3. A nucleic acid encoding the fusion protein as claimed in claim 1 .
4. The nucleic acid according to claim 3 , the nucleic acid having the nucleotide sequence set forth in SEQ ID NO. 12.
5. A method for preparing a fusion protein having an enhanced in vivo EPO activity comprising culturing a cell line transfected with a recombinant vector containing the nucleic acid as claimed in claim 3 .
6. A method for preparing a fusion protein having an enhanced in vivo EPO activity comprising culturing a cell line transfected with a recombinant vector containing the nucleic acid as claimed in claim 4 .