METHODS FOR MAKING PROTEINS CONTAINING FREE CYSTEINE RESIDUES
The present invention relates to novel methods of making soluble proteins having free cysteines in which a host cell is exposed to a cysteine blocking agent. The soluble proteins produced by the methods can then be modified to increase their effectiveness. Such modifications include attaching a PEG moiety to form pegylated proteins.
1 . A method for obtaining a soluble protein having a free cysteine comprising the step of:
(a) obtaining a host cell capable of expressing the soluble protein;
(b) exposing the host cell to a cysteine blocking agent; and
(c) isolating the soluble protein from the host cell.
2 . The method of claim 1 , further comprising disrupting the host cell in the presence of the cysteine blocking agent and isolating the protein from the soluble fraction of the disrupted host cell.
3 . The method of claim 1 , wherein exposing the host cell to a cysteine blocking agent occurs before, during or after synthesis of the soluble protein by the host cell and wherein the soluble protein is secreted from the host cell.
4 . The method of claim 1 , wherein said host cell is a bacteria, yeast, insect or mammalian cell.
5 . The method of claim 1 , wherein said host cell is a bacteria cell.
6 . The method of claim 5 , wherein said host cell is E. coli.
7 . The method of claims claim 1 , wherein said soluble protein is a recombinant protein.
8 . The method of claim 7 , wherein said recombinant protein is a cysteine mutein of a member of the growth hormone supergene family, a derivative or an antagonist thereof.
9 . The method of claim 8 , wherein said member is growth hormone.
10 . The method of claim 8 , wherein said member is erythropoietin.
11 . The method of claim 8 , wherein said interferon is alpha interferon alpha (IFN-α).
12 . The method of claim 8 , wherein said alpha interferon is interferon alpha 2 (IFN-α2).
13 . The method of claim 7 , wherein said recombinant protein is a cysteine mutein of a member of the TGF-beta superfamily, platelet derived growth factor-A, platelet derived growth factor-B, nerve growth factor, brain derived neurotophic factor, neurotrophin-3, neurotrophin-4, vascular endothelial growth factor, or a derivative or an antagonist thereof.
14 . The method of claim 7 , wherein said recombinant protein is a cysteine mutein of a heavy or light chain of an immunoglobulin or a derivative thereof.
15 . The method of claim 1 , wherein said cysteine blocking agent is a thiol-reactive compound.
16 . The method of claim 15 , wherein said thiol-reactive compound is cystine, cystamine, dithioglycolic acid, oxidized glutathione, iodine, hydrogen peroxide, dihydroascorbic acid, tetrathionate, O-iodosobenzoate or oxygen in the presence of a metal ion.
17 . The method of claim 15 , wherein said thiol-reactive compound is cystine.
18 . The method of claim 1 , further comprising attaching a cysteine-reactive moiety to said isolated protein to form a cysteine modified protein.
19 . The method of claim 1 , further comprising attaching a polyethylene glycol to said isolated protein to form a pegylated protein.
20 . A pegylated human growth hormone (hGH) or a derivative thereof having an EC 50 of less than about 110 ng/ml.
21 .- 31 . (canceled)