NH2-terminal glutamine modified cysteine variants of interferon gamma
Disclosed is a method for refolding a protein or peptide that does not contain essential disulfides and that contains at least one free cysteine residue. Also disclosed are polymer IFN-γ conjugates that have been created by the chemical coupling of polymers such as polyethylene glycol moieties to IFN-γ, particularly via a free cysteine in the protein. Also disclosed are analogs of bioactive peptides that may be used to create longer acting versions of the peptides, including analogs of glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, Gastric inhibitory peptide (GIP), PYY, exendin, ghrelin, gastrin, amylin, and oxyntomodulin.
1. An isolated cysteine variant of human interferon-gamma (IFN-γ) (SEQ ID NO:1), wherein a cysteine residue is substituted for at least one amino acid selected from the group consisting of: D63, Q64, S65, I66, Q67, V99, V100, T101, D102 and L103; wherein Q1 is deleted or substituted by a non-glutamine amino acid; and wherein the cysteine variant has IFN-γ biological activity in vitro comparable to IFN-γ wild-type activity in vitro.
2. The cysteine variant of claim 1 , wherein Q1 is deleted.
3. The cysteine variant of claim 1 , wherein Q1 is substituted by a non-glutamine amino acid.
4. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for an amino acid selected from the group consisting of V99, V100, T101, D102, and L103.
5. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for L103.
6. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for an amino acid selected from the group consisting of D63, Q64, S65, I66, and Q67.
7. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for Q67.
8. The cysteine variant of claim 1 wherein said variant is modified with a cysteine reactive moiety.
9. The cysteine variant of claim 8 , wherein said cysteine reactive moiety is a polyethylene glycol.
10. The cysteine variant of claim 1 , wherein said variant is a recombinant protein.
11. A method for treating a patient with a disease treatable with IFN-gamma, comprising administering to said patient a therapeutically effective amount of a cysteine variant of IFN-gamma of claim 1 .
12. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for D63.
13. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for Q64.
14. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for S65.
15. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for I66.
16. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for V99.
17. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for V100.
18. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for T101.
19. The cysteine variant of claim 1 , wherein a cysteine residue is substituted for D102.