Cysteine variants of interferon gamma
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 recombinant cysteine variant of gamma interferon, comprising at least one cysteine residue substituted for at least one amino acid located in the C-D loop of gamma interferon, wherein said cysteine variant has gamma interferon biological activity in vitro comparable to gamma interferon wild-type activity in vitro.
2. The cysteine variant of claim 1 , wherein said variant further comprises at least one cysteine residue substituted for at least one amino acid located in the region preceding helix A of gamma interferon.
3. The cysteine variant of claim 1 , wherein said variant further comprises at least one cysteine residue substituted for at least one amino acid located in the B-C loop of gamma interferon.
4. The cysteine variant of claim 1 , wherein said variant further comprises at least one cysteine residue substituted for at least one amino acid located in the D-E loop of gamma interferon.
5. The recombinant cysteine variant of claim 1 , wherein said cysteine variant is modified with a cysteine-reactive moiety.
6. The recombinant cysteine variant of claim 1 , wherein said variant is modified with a cysteine-reactive polyethylene glycol.
7. The recombinant cysteine variant of claim 1 , wherein said variant is modified with polyethylene glycol.
8. The cysteine variant of claim 2 , wherein said cysteine variant is modified with a cysteine-reactive moiety.
9. The cysteine variant of claim 2 , wherein said variant is modified with a cysteine-reactive polyethylene glycol.
10. The cysteine variant of claim 2 , wherein said variant is modified with polyethylene glycol.
11. The cysteine variant of claim 3 , wherein said cysteine variant is modified with a cysteine-reactive moiety.
12. The cysteine variant of claim 3 , wherein said variant is modified with a cysteine-reactive polyethylene glycol.
13. The cysteine variant of claim 3 , wherein said variant is modified with polyethylene glycol.
14. The cysteine variant of claim 4 , wherein said cysteine variant is modified with a cysteine-reactive moiety.
15. The cysteine variant of claim 4 , wherein said variant is modified with a cysteine-reactive polyethylene glycol.
16. The cysteine variant of claim 4 , wherein said variant is modified with polyethylene glycol.