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

• US 20060026719A1
• Filed: 01/14/2005
• Published: 02/02/2006
• Est. Priority Date: 01/14/2004
• Status: Abandoned Application

• Current Assignee: Ohio University
• Original Assignee: Ohio University
• Inventors: Kieliszewski, Marcia J., Xu, Jianfeng
• Application Number:
  US11/036,256
• Publication Number:
  US 20060026719A1
• Time in Patent Office: Days
• US Class Current: 800/288
• CPC Class Codes: A61K 38/27 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 : Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/06 : of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/415 : from plants
• CPC Class Codes: C07K 14/56 : IFN-alpha
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 14/765 : Serum albumin, e.g. HSA
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/8257 : for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: C12P 21/005 : Glycopeptides, glycoproteins

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.

Claims

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.