Glycoproteins produced in plants and methods of their use

Abstract

Methods of increasing the yield in plant expression of recombinant proteins comprising: engineering glycosylation sites into cloned genes or cDNAs for proteins using codons that drive post-translational modifications in plants; and engineering the cloned genes or cDNAs to contain a plant secretory signal sequence that targets the gene products (protein) for secretion. The methods result in increased recombinant glycosylated protein yields. Proteins produced according to these methods are disclosed.

1 . An injectable pharmaceutical formulation comprising glycosylated human growth hormone, and excluding at least one excipient chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid.  
     
     
         2 . The injectable pharmaceutical formulation according to  claim 1 , wherein the glycosylated human growth hormone comprises a glycomodule chosen from i) X-Pro-Hyp n  (SEQ ID NO: 162), X-Hyp n  (SEQ ID NO: 163) or Hyp n -X (SEQ ID NO: 164), where n is from 6 to about 100, and ii) (X-Hyp) n  (SEQ ID NO: 165) or (Hyp-X) n  (SEQ ID NO: 166), where n is from 6 to about 100; 
 wherein X is any amino acid in the glycomodule X-Pro-Hyp n , and X is chosen from Lys, Ser, Ala, Thr, Gly and Val for the glycomodules X-Hyp n , Hyp n -X, (Hyp-X) n , and (X-Hyp) n .    
     
     
         3 . The injectable pharmaceutical formulation according to  claim 2 , wherein the glycosylated growth hormone comprises (X-Hyp) n  (SEQ ID NO: 167) or (Hyp-X) n  (SEQ ID NO: 168), where n is from 6 to about 20; wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val.  
     
     
         4 . The injectable pharmaceutical formulation according to  claim 3 , wherein X is chosen from Ser, Ala, Thr, and Val.  
     
     
         5 . A lyophilized powder formulation of glycosylated human growth hormone exhibiting a solubility of greater than or equal to about 10 mg/ml, wherein the formulation excludes at least one excipient chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid.  
     
     
         6 . The lyophilized powder formulation according to  claim 5 , wherein the formulation excludes mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, and phospholipid.  
     
     
         7 . A human growth hormone molecule covalently attached to an amino acid sequence comprising at least one glycomodule, wherein the glycomodule is chosen from i) X-Hyp n  (SEQ ID NO: 169) or X-Pro-Hyp n  (SEQ ID NO: 170), where n is from 4 to about 100, ii) Hyp n -X (SEQ ID NO: 171), where n is from 4 to about 100, iii) (Hyp-X) n  (SEQ ID NO: 172), where n is from 4 to about 100, and iv) (X-Hyp) n  (SEQ ID NO: 173), where n is from 4 to about 100; wherein X is any amino acid in the glycomodule X-Pro-Hyp n , and wherein X is chosen from Ser, Ala, Thr, and Val for the glycomodules X-Hyp n , Hyp n -X, (Hyp-X) n , and (X-Hyp) n .  
     
     
         8 . A human growth hormone antagonist molecule covalently attached to an amino acid sequence comprising at least one glycomodule, wherein the glycomodule is chosen from i) X-Hyp n  (SEQ ID NO: 169) or X-Pro-Hyp n  (SEQ ID NO: 170), where n is from 4 to about 100, ii) Hyp n -X (SEQ ID NO: 171), where n is from 4 to about 100, iii) (Hyp-X) n  (SEQ ID NO: 172), where n is from 4 to about 100, and iv) (X-Hyp) n  (SEQ ID NO: 173), where n is from 4 to about 100; wherein X is any amino acid in the glycomodule X-Pro-Hyp n , and wherein X is chosen from Ser, Ala, Thr, and Val for the glycomodules X-Hyp n , Hyp n -X, (Hyp-X) n , and (X-Hyp) n .  
     
     
         9 . A method of treating a patient suffering from growth hormone deficiency or insufficiency comprising administering a therapeutically effective amount of glycosylated human growth hormone.  
     
     
         10 . The method of treating a patient according to  claim 9 , wherein the growth hormone is according to  claim 7 .  
     
     
         11 . A method of treating a patient suffering from excess human growth hormone or growth hormone activity comprising administering a therapeutically effective amount of glycosylated growth hormone antagonist.  
     
     
         12 . The method of treating a patient according to  claim 11 , wherein the growth hormone is according to  claim 8 .  
     
     
         13 . A method of treating a patient suffering from type I or type II diabetes comprising administering a therapeutically effective amount of glycosylated insulin.  
     
     
         14 . The method of treating a patient according to  claim 13 , wherein the glycosylated insulin comprises at least one glycomodule chosen from i) X-Hyp n  (SEQ ID NO: 169) or X-Pro-Hyp n  (SEQ ID NO: 170), where n is from 4 to about 100, ii) Hyp n -X (SEQ ID NO: 171), where n is from 4 to about 100, iii) (Hyp-X) n  (SEQ ID NO: 172), where n is from 4 to about 100, and iv) (X-Hyp) n  (SEQ ID NO: 173), where n is from 4 to about 100; wherein X is any amino acid in the glycomodule X-Pro-Hyp n , and wherein X is chosen from Ser, Ala, Thr, and Val for the glycomodules X-Hyp n , Hyp n -X, (Hyp-X) n , and (X-Hyp) n .