Methods for making proteins containing free cysteine residues

Abstract

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 .- 31 . (canceled) 
     
     
         32 . An isolated, disulfide-linked growth hormone protein dimer comprising two growth hormone protein monomers, wherein each growth hormone protein monomer in the dimer comprises at least one added cysteine residue, wherein at least one of the added cysteine residues participates in a disulfide bond joining the two growth hormone protein monomers together in the dimer, and wherein the protein dimer stimulates proliferation of a cell line that proliferates in response to growth hormone. 
     
     
         33 . The isolated growth hormone protein of  claim 32 , where the growth hormone protein is human Growth Hormone. 
     
     
         34 . The isolated human Growth Hormone protein of  claim 33 , wherein said human Growth Hormone protein comprises a cysteine residue added preceding the F1 amino acid of the mature human Growth Hormone protein. 
     
     
         35 . The isolated human Growth Hormone protein of  claim 33 , where said human Growth Hormone protein comprises a cysteine residue added following the terminal F191 amino acid of the mature human Growth Hormone protein. 
     
     
         36 . The isolated human Growth Hormone protein of  claim 33 , where said human Growth Hormone protein comprises a cysteine residue substituted for at least one amino acid selected from the group consisting of P2, T3, P5, A34, K38, Q40, S43, S55, S57, A98, N99, S100, L101, V102, Y103, A105, S132, P133, R134, T135, Q137, K140, Q141, Y143, S144, D147, T148, N149, E186, and G187. 
     
     
         37 . The isolated human Growth Hormone protein of  claim 33 , where said human Growth Hormone protein comprises a cysteine residue substituted for T3. 
     
     
         38 . The isolated human Growth Hormone protein of  claim 33 , where said human Growth Hormone protein comprises a cysteine residue substituted for A105. 
     
     
         39 . The isolated human Growth Hormone protein of  claim 33 , where said human Growth Hormone protein comprises a cysteine residue substituted for T135. 
     
     
         40 . The isolated human Growth Hormone of  claim 33 , wherein said isolated Growth Hormone protein is modified with polyethylene glycol. 
     
     
         41 . The isolated human Growth Hormone protein of  claim 33 , wherein said isolated Growth Hormone protein is modified with a cysteine-reactive polyethylene glycol. 
     
     
         42 . The isolated growth hormone protein dimer of  claim 32 , where said growth hormone protein dimer is a recombinant protein. 
     
     
         43 . The isolated growth hormone protein dimer of  claim 32 , wherein the dimer is a homodimer. 
     
     
         44 . A method for obtaining an isolated, disulfide-linked growth hormone protein dimer comprising two growth hormone protein monomers, wherein each growth hormone protein monomer in the dimer comprises at least one added cysteine residue, and wherein the at least one added cysteine residue participates in a disulfide bond joining the two growth hormone protein monomers together in the dimer, the method comprising:
 (a) obtaining a host cell capable of expressing the growth hormone protein comprising at least one added cysteine residue;   (b) causing the host cell to express the growth hormone protein comprising at least one added cysteine residue in a soluble form;   (c) exposing the growth hormone protein comprising at least one added cysteine residue to a cysteine blocking agent prior to step (d); and   (d) isolating the growth hormone protein dimer by column chromatography, wherein the buffers used to elute the growth hormone protein from the column do not contain a cysteine blocking agent.   
     
     
         45 . The method of  claim 44 , wherein the column chromatography method of step (d) is selected from the group consisting of ion-exchange column chromatography, hydrophobic interaction column chromatography, metal chelation affinity column chromatography, size exclusion column chromatography, and combinations thereof. 
     
     
         46 . The method of  claim 44 , where the column chromatography method of step (d) is ion-exchange column chromatography. 
     
     
         47 . The method of  claim 44 , wherein the cysteine blocking agent in step (c) is selected from the group consisting of cystine, oxidized glutathionine, and cystamine. 
     
     
         48 . The method of  claim 44 , wherein the cysteine blocking agent in step (c) is cystine. 
     
     
         49 . A method of treating a disease treatable with growth hormone, comprising administering to a patient in need thereof an effective amount of a composition comprising an isolated, disulfide-linked growth hormone protein dimer comprising two growth hormone protein monomers, wherein each growth hormone protein monomer in the dimer comprises at least one added cysteine residue, and wherein the at least one added cysteine residue participates in a disulfide bond joining the two growth hormone protein monomers together in the dimer. 
     
     
         50 . The method of  claim 49 , wherein the disease is selected from the group consisting of: growth hormone deficiency, short stature resulting from growth hormone deficiency, short stature resulting from Turner's Syndrome, short stature resulting from renal failure, and cachexia. 
     
     
         51 . The method of  claim 49 , wherein the composition is administered to the patient by a route selected from the group consisting of intravenous administration, intraperitoneal administration, and subcutaneous administration.