Cysteine variants of alpha interferon-2
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. An isolated cysteine variant of alpha interferon-2 of SEQ ID NO:3, wherein a cysteine residue is substituted for an amino acid selected from the group consisting of D2, L3, Q5, S8, N45, Q101, G104, T106, E107, S163, K164, and E165, wherein said variant has alpha interferon activity in vitro comparable to alpha interferon wild-type activity in vitro as measured using an alpha interferon-dependent cell assay.
2. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of D2, L3, Q5, and S8.
3. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for D2.
4. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for L3.
5. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for Q5.
6. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for S8.
7. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for N45.
8. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of Q101, G104, T106, and E107.
9. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for Q100.
10. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for G104.
11. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for T106.
12. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for E107.
13. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of S163, K164, and E165.
14. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for S163.
15. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for K164.
16. The isolated cysteine variant according to claim 1 , wherein a cysteine residue is substituted for E165.
17. The isolated cysteine variant according to any one of claims 3 - 5 , 6 , 13 , 14 - 16 , 8 , 9 , 10 - 12 , 1 , or 7 , wherein the substituted cysteine residue is modified with a cysteine-reactive moiety.
18. The isolated cysteine variant according to any one of claims 3 - 5 , 6 , 13 , 14 - 16 , 8 , 9 , 10 - 12 , 1 , or 7 , wherein the substituted cysteine residue is modified with polyethylene glycol.
19. The isolated cysteine variant according to any one of claims 3 - 5 , 6 , 13 , 14 - 16 , 8 , 9 , 10 - 12 , 1 , or 7 , wherein the substituted cysteine residue is modified with at least one polyethylene glycol.