Cysteine variants of erythropoietin
The growth hormone supergene family comprises greater than 20 structurally related cytokines and growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the proteins or substituting cysteine residues for non-essential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue. Disclosed herein are preferred sites for adding cysteine residues or introducing cysteine substitutions into the proteins, and the proteins and protein derivatives produced thereby.
1. A cysteine variant of erythropoietin corresponding to at least amino acids 1-165 of SEQ ID NO: 2, wherein a cysteine residue is substituted for an amino acid selected from the group consisting of: R110, Q115, and E117; wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to erythropoietin.
2. The cysteine variant according to claim 1 wherein a cysteine residue is substituted for R110.
3. The cysteine variant according to claim 1 wherein a cysteine residue is substituted for Q115.
4. The cysteine variant according to claim 1 wherein a cysteine residue is substituted for E117.
5. The cysteine variant according to any one of claims 1 , 2 , 3 or 4 , wherein the substituted cysteine residue is modified with a cysteine-reactive moiety.
6. The cysteine variant according to any one of claims 1 , 2 , 3 or 4 , wherein the substituted cysteine residue is modified with polyethylene glycol.
7. The cysteine variant according to any one of claims 1 , 2 , 3 or 4 , wherein said cysteine variant is modified with at least one polyethylene glycol.