Methods of producing peptides/proteins in plants and peptides/proteins produced thereby

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 . A nucleic acid construct for expression of at least one biologically active protein in plants comprising: a) at least one nucleic acid sequence encoding a glycosylation site and b) at least one nucleic acid sequence encoding a biologically active protein.  
   
   
       2 . A plant-derived biologically active mammalian fusion glycoprotein comprising: a) at least one glycomodule, covalently linked to b) a mammalian biologically active protein.  
   
   
       3 . The plant-derived biologically active mammalian fusion protein according to  claim 2 , wherein the at least one glycomodule is chosen from i) X-Hyp n  (SEQ ID NO: 2) or X-Pro-Hyp n  (SEQ ID NO: 1), where n is from 2 to about 1000, ii) Hyp n -X (SEQ ID NO: 156), where n is from 2 to about 1000, iii) (Hyp-X) n  (SEQ ID NO: 157), where n is from 1 to about 1000, and iv) (X-Hyp) n  (SEQ ID NO: 3), where n is from 1 to about 1000; 
 wherein X is any amino acid.    
   
   
       4 . The plant-derived biologically active mammalian fusion protein according to  claim 3 , wherein 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 .  
   
   
       5 . The plant-derived biologically active mammalian fusion protein according to  claim 4 , wherein X is chosen from Ser, Ala, Thr, and Val.  
   
   
       6 . The plant-derived biologically active mammalian fusion protein according to  claim 3 , wherein the at least one glycomodule is covalently linked at a location chosen from the N-terminus and the C-terminus of the protein.  
   
   
       7 . The plant-derived biologically active mammalian fusion protein according to  claim 3 , wherein the at least one glycomodule is within the interior of the biologically active mammalian protein.  
   
   
       8 . The plant-derived biologically active mammalian fusion protein according to  claim 2 , wherein the biologically active mammalian protein is chosen from growth hormone, growth hormone antagonists, growth hormone releasing hormone, somatostatin, ghrelin, leptin, prolactin, monocyte chemoattractant protein-1, interleukin-10, pleiotropin, interleukin-7, interleukin-8, interferon omega, interferon-Alpha 2, interferon gamma, interleukin-1, fibroblast growth factor 6, IFG-1, insulin-like growth factor I, insulin, erythropoietin, GMCSF, and any humanized monoclonal antibody 
 wherein the glycomodule comprises i) X-Hyp n  (SEQ ID NO: 2), X-Pro-Hyp n  (SEQ ID NO: 1), or Hyp n -X (SEQ ID NO: 156) where n is from 2 to about 1000, and ii) (X-Hyp) n  (SEQ ID NO: 3) or (Hyp-X) n  (SEQ ID NO: 157) where n is from 1 to about 1000; and wherein X is any amino acid.    
   
   
       9 . The plant-derived biologically active mammalian fusion protein according to  claim 8 , wherein 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 .  
   
   
       10 . The plant-derived biologically active mammalian fusion protein according to  claim 9 , wherein X is chosen from Ser, Ala, Thr, and Val.  
   
   
       11 . The plant-derived biologically active mammalian fusion protein according to  claim 8 , wherein the biologically active mammalian protein is a human protein.  
   
   
       12 . The plant-derived biologically active mammalian fusion protein according to  claim 8 , wherein the glycomodule comprises (X-Hyp) n  or (Hyp-X) n , wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val.  
   
   
       13 . The plant-derived biologically active mammalian fusion protein according to  claim 12 , wherein X is chosen from Ser, Ala, Thr, and Val.  
   
   
       14 . The plant-derived biologically active mammalian fusion protein according to  claim 13 , wherein the protein is human growth hormone, and the glycomodule comprises (Ser-Hyp) 10  (SEQ ID NO: 4).  
   
   
       15 . The plant-derived biologically active mammalian fusion protein according to  claim 2 , wherein the fusion glycoprotein is covalently linked to at least one carbohydrate molecule.  
   
   
       16 . A method of increasing the aqueous solubility of a protein molecule, comprising: 
 preparing a nucleic acid sequence encoding: a) at least one glycosylation site and b) at least one protein; and    expressing the nucleic acid construct as a fusion glycoprotein;    wherein carbohydrate component of the glycoprotein accounts for greater than or equal to about 10% of the molecular weight of the glycoprotein.    
   
   
       17 . The method of increasing aqueous solubility of a protein molecule according to  claim 16 , wherein carbohydrate component of the glycoprotein accounts for greater than or equal to about 50% of the molecular weight of the glycoprotein.  
   
   
       18 . The method of increasing aqueous solubility of a protein molecule according to  claim 17 , wherein carbohydrate component of the glycoprotein accounts for greater than or equal to about 75% of the molecular weight of the glycoprotein.  
   
   
       19 . The method of increasing aqueous solubility of a protein molecule according to  claim 18 , wherein carbohydrate component of the glycoprotein accounts for greater than or equal to about 90% of the molecular weight of the glycoprotein.  
   
   
       20 . A method of producing a biologically active fusion protein, comprising: 
 expressing in a plant at least one nucleic acid sequence encoding: a) at least one glycosylation site and b) at least one biologically active protein, as a glycoprotein;    wherein the molecular weight of the glycoprotein is greater than or equal to about 10 kD and wherein the carbohydrate component of the glycoprotein accounts for greater than or equal to about 10% of the molecular weight of the glycoprotein.    
   
   
       21 . The method of producing a biologically active fusion protein according to  claim 20 , wherein the at least one biologically active protein is selected from insulin, insulin-like growth factor, somatostatin, growth hormone releasing hormone, ghrelin, prolactin, placental lactogen, growth hormone, and growth hormone antagonist.  
   
   
       22 . The method of producing a biologically active fusion protein according to  claim 20 , wherein the molecular weight of the glycoprotein is greater than or equal to about 35 kD.  
   
   
       23 . The method of producing a biologically active fusion protein according to  claim 22 , wherein the molecular weight of the glycoprotein is greater than or equal to about 40 kD.  
   
   
       24 . The method of producing a biologically active fusion protein according to  claim 23 , wherein the molecular weight of the glycoprotein is greater than or equal to about 45 kD.  
   
   
       25 . The method of producing a biologically active fusion protein according to  claim 20 , wherein the pharmacokinetic half-life of the glycoprotein is greater than the pharmacokinetic half-life of a corresponding wild-type protein.  
   
   
       26 . The method of producing a biologically active fusion protein molecule according to  claim 20 , wherein the at least one glycosylation site is chosen from i) X-Pro n  (SEQ ID NO: 158) or Pro n -X (SEQ ID NO: 159), where n is from 6 to about 100, and ii) (X-Pro) n  (SEQ ID NO: 160) or (Pro-X) n  (SEQ ID NO: 161), where n is from 6 to about 100; wherein X is any amino acid.  
   
   
       27 . The method of producing a biologically active fusion protein molecule according to  claim 26 , wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val.  
   
   
       28 . The method of producing a biologically active fusion protein according to  claim 27 , wherein X is chosen from Ser, Ala, Thr, and Val.  
   
   
       29 . The method of producing a biologically active fusion protein according to  claim 20 , wherein the biologically active protein is growth hormone and the glycoprotein comprises (Ser-Hyp) 10  (SEQ ID NO: 4).  
   
   
       30 . The method of producing a biologically active fusion protein according to  claim 29 , wherein the (Ser-Hyp) 10  (SEQ ID NO: 4) is covalently attached to the C-terminus of the growth hormone.  
   
   
       31 . The method of producing a biologically active fusion protein according to any of claims  29  or  30 , wherein the growth hormone is human growth hormone.  
   
   
       32 . A method of increasing the yield in plant production of a protein, comprising: 
 preparing a nucleic acid construct comprising: a) at least one signal peptide nucleic acid coding sequence, b) at least one glycosylation site nucleic acid coding sequence, and c) at least one protein nucleic acid coding sequence; and    expressing the nucleic acid construct as a glycoprotein.    
   
   
       33 . The method according to  claim 32 , wherein the at least one glycosylation site is chosen from i) X-Pro n  (SEQ ID NO: 5) or Pro n -X (SEQ ID NO: 96), where n is from 2 to about 1000, and ii) (X-Pro) n  (SEQ ID NO: 11) or (Pro-X) n  (SEQ ID NO: 94), where n is from 1 to about 1000; wherein X is any amino acid.  
   
   
       34 . The method according to  claim 33 , wherein X is chosen from Gly, Lys, Ser, Thr, Ala, and Val.  
   
   
       35 . The method according to  claim 34 , wherein X is chosen from Ser, Ala, Thr, and Val.  
   
   
       36 . The method according to  claim 32 , wherein the nucleic acid construct excludes a nucleic acid sequence coding for green fluorescent protein.  
   
   
       37 . A protein produced according to the method according to  claim 36 .  
   
   
       38 . A method of preventing allergic immune response in a mammal comprising performing at least one administration to the animal of a plant-derived fusion glycoprotein comprising: a) at least one glycomodule, covalently linked to b) a biologically active protein, wherein the at least one glycomodule is chosen from X-Hyp n  (SEQ ID NO: 2) and X-Pro-Hyp n  (SEQ ID NO: 1), where n is from 2 to about 1000, and wherein X is any amino acid.  
   
   
       39 . The method of preventing allergic immune response according to  claim 38 , wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val.