Methods of producing peptides in plants and peptides 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 are present. The methods result in increased recombinant glycosylated protein yields. Proteins produced according to these methods are also present.

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 plant hydroxyproline O-glycosylation site b) at least one nucleic acid sequence encoding a biologically active vertebrate protein and c) an operatively linked plant promoter; wherein the at least one biologically active protein in plants exhibits a pharmacokinetic plasma half-life that is increased as compared with the same biologically active protein in plants that lacks a plant hydroxyproline O-glycosylation site. 
     
     
         2 . The nucleic acid construct according to  claim 1 , wherein the biologically active vertebrate protein is a biologically active mammalian protein. 
     
     
         3 . The nucleic acid construct according to  claim 2 , wherein the biologically active mammalian protein is a biologically active human protein. 
     
     
         4 . The nucleic acid construct according to  claim 2 , wherein the biologically active mammalian protein is selected from monocyte chemoattractant protein-1, interleukin-10, pleiotropin, interleukin-7, interleukin-8, interferon omega, interferon-Alpha 2a and 2b, interferon gamma, interleukin-1, fibroblast growth factor 6, IGF-1, insulin-like growth factor I and II, adrenocorticotropic hormone, beta-amyloid, amylin, atrial natriuretic polypeptide, bombesin, bradykinin, brain natriuretic peptide, calcitonin, calcitonin gene related peptide, corticotropin releasing factor, dynorphin, endorphin, endothelin, enkephalin, epidermal growth factor, gastric inhibitory peptide, gastrin, gastrin releasing peptide, growth hormone releasing hormone, HIV-1 envelope proteins, katacalcin, luteinizing hormone-releasing hormone, neurokinins, neuromedins, neuropeptide Y, neurotensin, oxytocin, pancreatic polypeptide, pancreatic polypeptide, pancreastin, pancreastatin, parathyroid hormone, secretin, somatostatin, substance P, transforming growth factor, vasoactive intestinal peptide, vasopressin, vasotocin, glucagon and the glucagon-like peptides, erythropoietin, granulocyte colony stimulating factor, PORF-1 and -2 (preoptic regulatory factors), PYY 3-36, insulin, ghrelin, leptin, adiponectin and resistin. 
     
     
         5 . A nucleic acid construct for expression of at least one biologically active protein in plants comprising: a) at least one nucleic acid encoding at least one hydroxyproline O-glycosylation plant glycomodule, b) at least one nucleic acid sequence encoding a biologically active vertebrate protein, and c) an operatively linked plant promoter; wherein the at least one biologically active protein in plants exhibits a pharmacokinetic plasma half-life that is increased as compared with the same biologically active protein in plants that lacks at least one hydroxyproline O-glycosylation plant glycomodule. 
     
     
         6 . The nucleic acid construct according to  claim 5 , 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; and wherein X is any amino acid. 
     
     
         7 . The nucleic acid construct according to  claim 6 , 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 . 
     
     
         8 . The nucleic acid construct according to  claim 7 , wherein X is chosen from Ser, Ala, Thr, and Val. 
     
     
         9 . The nucleic acid construct according to  claim 7 , wherein the at least one glycomodule is covalently linked at a location chosen from the N-terminus and the C-terminus of the biologically active vertebrate protein encoded by the at least one nucleic acid sequence. 
     
     
         10 . The nucleic acid construct according to  claim 7 , wherein the at least one glycomodule is within the interior of the biologically active vertebrate protein encoded by the at least one nucleic acid sequence. 
     
     
         11 . The nucleic acid construct according to  claim 6 , wherein the at least one nucleic acid sequence encoding a biologically active vertebrate protein is a biologically active mammalian protein selected from monocyte chemoattractant protein-1, interleukin-10, pleiotropin, interleukin-7, interleukin-8, interferon omega, interferon-Alpha 2a and 2b, interferon gamma, interleukin-1, fibroblast growth factor 6, IGF-1, insulin-like growth factor I and II, adrenocorticotropic hormone, beta-amyloid, amylin, atrial natriuretic polypeptide, bombesin, bradykinin, brain natriuretic peptide, calcitonin, calcitonin gene related peptide, corticotropin releasing factor, dynorphin, endorphin, endothelin, enkephalin, epidermal growth factor, gastric inhibitory peptide, gastrin, gastrin releasing peptide, growth hormone releasing hormone, HIV-1 envelope proteins, katacalcin, luteinizing hormone-releasing hormone, neurokinins, neuromedins, neuropeptide Y, neurotensin, oxytocin, pancreatic polypeptide, pancreatic polypeptide, pancreastin, pancreastatin, parathyroid hormone, secretin, somatostatin, substance P, transforming growth factor, vasoactive intestinal peptide, vasopressin, vasotocin, glucagon and the glucagon-like peptides, erythropoietin, granulocyte colony stimulating factor, PORF-1 and -2 (preoptic regulatory factors), PYY 3-36, insulin, ghrelin, leptin, adiponectin and resistin; and
 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; and wherein X is any amino acid.   
     
     
         12 . The nucleic acid construct according to  claim 11 , 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 . 
     
     
         13 . The nucleic acid construct according to  claim 12 , wherein X is chosen from Ser, Ala, Thr, and Val. 
     
     
         14 . The nucleic acid construct according to  claim 11 , wherein the encoded biologically active mammalian protein is a biologically active human protein. 
     
     
         15 . The nucleic acid construct according to  claim 11 , wherein the glycomodule comprises (X-Hyp) n  or (Hyp-X) n , wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val. 
     
     
         16 . The nucleic acid construct according to  claim 15 , wherein X is chosen from Ser, Ala, Thr and Val.