Cysteine substitution variants of beta interferon
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 beta interferon of SEQ ID NO: 5, wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of: Q23, N25, G26, N80, E109, D110, F111, T112, R113, G114, K115, and N166; wherein said variant has biological activity in vitro as measured by inhibition of proliferation of a cell line whose proliferation is inhibited in response to beta interferon.
2. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for N166.
3. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for Q23.
4. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for R113.
5. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for E109.
6. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for D110.
7. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for F111.
8. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for T112.
9. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for G114.
10. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for N25.
11. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for G26.
12. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for N80.
13. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for K115.
14. The cysteine variant of claim 1 , wherein a non-cysteine amino acid is substituted for C17 of beta interferon.
15. The cysteine variant of claim 14 , wherein the non-cysteine amino acid substituted for C17 is selected from the group consisting of serine and alanine.
16. The cysteine variant of claim 1 , wherein the substituted cysteine residue is modified with a cysteine-reactive moiety.
17. The cysteine variant of claim 16 , wherein a non-cysteine amino acid is substituted for C17.
18. The cysteine variant of claim 17 , wherein the non-cysteine amino acid substituted for C17 is selected from the group consisting of serine and alanine.
19. The cysteine variant of claim 1 , wherein the substituted cysteine residue is modified with polyethylene glycol.
20. The cysteine variant of claim 19 , wherein a non-cysteine amino acid is substituted for C17.
21. The cysteine variant of claim 20 , wherein the non-cysteine amino acid substituted for C17 is selected from the group consisting of serine and alanine.
22. The cysteine variant of any one of claims 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , or 10 , wherein said cysteine variant is modified with polyethylene glycol.
23. The cysteine variant of claim 15 , wherein the non-cysteine amino acid substituted for C17 is a serine.
24. The cysteine variant of claim 18 , wherein the non-cysteine amino acid substituted for C17 is a serine.
25. The cysteine variant of claim 21 , wherein the non-cysteine amino acid substituted for C17 is a serine.