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  Glycoproteins produced in plants and methods of their useUSPTO Application #: 20060148680Title: 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. (end of abstract) Agent: Calfee Halter & Griswold, LLP - Cleveland, OH, US Inventors: Marcia J. Kieliszewski, Jianfeng Xu, John J. Kopchick, Shigeru Okada USPTO Applicaton #: 20060148680 - Class: 514008000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Glycoprotein (carbohydrate Containing) The Patent Description & Claims data below is from USPTO Patent Application 20060148680. Brief Patent Description - Full Patent Description - Patent Application Claims
DESCRIPTION OF THE INVENTION [0001] This application claims priority to U.S. Provisional Application Nos. 60/536,486, filed Jan. 14, 2004, and 60/582,027, filed Jun. 22, 2004, and 60/602,562, filed Aug. 18, 2004, the entire disclosure of each of which is incorporated by reference herein. FIELD OF THE INVENTION [0003] The present invention relates to novel methods of producing fusion peptides, polypeptides, and proteins in plants, the nucleic acid constructs used in these methods, and the products produced according to these methods. The methods generally involve expressing the peptide, polypeptide, or protein as fusion proteins, which are glycosylated by the plant. In some embodiments, a plant-based signal peptide is expressed as part of the fusion protein. According to the present invention, novel glycoproteins are presented. BACKGROUND OF THE INVENTION [0004] Support of young growing plant tissues depends largely on the turgidity of cells restrained by an elastic cell wall comprised of three interpenetrating networks, namely, cellulosic-xyloglucan, pectin, and hydroxyproline-rich glycoproteins (HRGPs). When these networks are loosened, turgor drives cell extension. Significantly, HRGPs have no animal homologs, thus emphasizing a plant-specific function. [0005] Quantitatively, most of the cell surface HRGPs (extensins) form a covalently cross-linked cell wall network. Unlike extensins, another set of HRGPs, arabinogalactan-proteins (AGPs) occur as monomers that are hyperglycosylated by arabinogalactan polysaccharides. AGPs are initially tethered to the plasma membrane by a lipid anchor whose cleavage results in their movement from the periplasm through the cell wall to the exterior. Although implicated in diverse aspects of plant growth and development, the precise functions of AGPs remain unclear. SUMMARY OF THE INVENTION [0006] The present invention provides novel methods of producing glycoproteins in plants. The glycoproteins include a glycosylation site element and a core protein element. In some embodiments, the core protein element can be of mammalian (including human) origin, and in some embodiments, the core protein element can be a biologically active protein. In some cases, the protein can be an FDA-approved recombinant protein that is used therapeutically, e.g. recombinant human growth hormone ("hGH"). The glycosylation site is an amino acid sequence that acts as a target for glycosylation by the plant. [0007] One feature of the present method is an increase in yield in protein production. By including a glycosylation site(s) and a signal peptide sequence in the expressed protein, recombinant protein yield considerably increases in comparison to expression of the same protein in plants without the glycosylation site and signal peptide sequence. [0008] Glycoproteins produced according to the method exhibit additional advantages over their wild-type counterparts, including increased solubility, increased resistance to proteolytic enzymes, and increased stability. Another important feature includes increased biological half-life as compared to wild-type proteins. [0009] Additional features and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The features and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. [0010] The present invention provides nucleic acid constructs for expression of at least one biologically active protein in plants comprising: a) at least one nucleic acid sequence encoding a glycosylation site utilized in plants and b) at least one nucleic acid sequence encoding a biologically active protein. [0011] The invention also provides plant-derived biologically active fusion proteins comprising: a) at least one glycomodule covalently linked to b) a biologically active protein. In some embodiments, the at least one glycomodule comprises a glycosylation site chosen from i) X-Pro-Hyp.sub.n, where n is from 2 to about 1000, ii) X-Hyp.sub.n, where n is from 2 to about 1000, and iii) (X-Hyp).sub.n, where n is from 1 to about 1000; wherein X is chosen from Lys, Ser, Ala, Thr, Gly, and Val, but is more preferably selected from Ser, Thr, Val, and Ala. In some embodiments, the at least one glycomodule is covalently linked at a location chosen from the N-terminus and/or the C-terminus of the protein. In some embodiments, the at least one glycomodule is within the interior of the biologically active mammalian protein. While Lys, Ser, Thr, Val, Gly, and Ala, are specifically identified above as corresponding to X, it is believed that any amino acid can serve that purpose, and that the motif will be glycosylated in plants. [0012] The biologically active mammalian protein can be selected from a group including 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 2a and 2b, interferon gamma, interleukin-1, fibroblast growth factor 6, IFG-1, insulin-like growth factor I, insulin, erythropoietin, GMCSF, and any humanized monoclonal antibody or monoclonal antibody, wherein the glycomodule comprises a glycosylation site chosen from i) X-Pro-Hyp.sub.n, where n is from 2 to about 1000, ii) X-Hyp.sub.n, where n is from 2 to about 1000, and iii) (X-Hyp).sub.n, where n is from 1 to about 1000; and wherein X is selected from Lys, Ser, Ala, Thr, Gly, and Val, and is preferably Ser, Ala, Thr, and Val. In some embodiments, the glycomodule comprises (X-Hyp).sub.n, X is selected from Lys, Ser, Ala, Thr, Gly and Val, more preferably Ser, Ala, Thr, and Val, and n=1-1000. In some embodiments, the protein is human growth hormone, and the glycomodule comprises (Ser-Hyp).sub.10. While Lys, Ser, Thr, Val, Gly, and Ala, are specifically identified as corresponding to X, it is believed that any amino acid can serve that purpose, and that the motif will be glycosylated in plants. [0013] In some embodiments, the plant-derived biologically active mammalian fusion glycoproteins of the invention are covalently linked to at least one carbohydrate molecule. In some embodiments, the carbohydrate is an arabinogalactan moiety, and in some it is an arabinosyl moiety. [0014] The invention also provides methods of increasing the aqueous solubility of a protein molecule, wherein one: prepares a nucleic acid construct encoding a) at least one glycosylation site and b) at least one peptide or protein; and expressing the nucleic acid construct as a glycoprotein; wherein carbohydrate component of the glycoprotein accounts for greater than or equal to about 10% of the molecular weight of the glycoprotein. The carbohydrate component of the glycoprotein can account for greater than or equal to about 50%, about 75%, or about 90% of the molecular weight of the glycoprotein. [0015] The invention also provides methods of producing a biologically active fusion glycoprotein, comprising: expressing in a plant at least one nucleic acid construct 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, 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. In some embodiments, the molecular weight of the glycoprotein is greater than or equal to about 35 kD, about 40 kD, about 45 kD, about 50 kD, or about 55 kD. In some embodiments, the pharmacokinetic half-life of the glycoprotein is greater than the pharmacokinetic half-life of a corresponding wild-type protein. In some embodiments, the at least one glycosylation site is chosen from i) X-Pro.sub.n, where n is from 2 to about 1000, and ii) (X-Pro).sub.n, where n is from 2 to about 1000; wherein X is any amino acid or is selected from Lys, Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr, and Val. Of course, n can range from 4 to 200 or from 6 to 100 or from 8 to 50 or from 10 to 25, or any number in between or any combination thereof. In some embodiments, the biologically active protein is human growth hormone and the glycoprotein comprises (Ser-Hyp).sub.10, and in some embodiments, the (Ser-Hyp).sub.10 is covalently attached to the C-terminus of the human growth hormone protein. [0016] The invention also provides injectable pharmaceutical formulations comprising glycosylated human growth hormone, and excluding additional excipients normally required for solvating or increasing the solubility of proteins. In some embodiments, the formulation excludes at least one excipient chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid. In some embodiments, the glycosylated human growth hormone comprises a glycomodule chosen from i) X-Pro-Hyp.sub.n, where n is from 2 to about 100, and wherein X is any amino acid, or is chosen from Lys, Ser, Ala, Thr, Gly and Val, or more preferably chosen from Ser, Ala, Thr, and Val, ii) X-Hyp.sub.n, where n is from 2 to about 100, and wherein X is any amino acid, or is chosen from Lys, Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr, and Val, and iii) (X-Hyp.sub.n, where n is from 1 to about 100; wherein X is any amino acid or is selected from Lys, Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr, and Val. The glycosylated growth hormone can comprise X-Hyp.sub.n, where n is from 2 to about 20; wherein X is selected from Lys, Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr, and Val. [0017] The invention also provides lyophilized powder formulations of glycosylated human growth hormone exhibiting a solubility of greater than or equal to about 10 mg/ml, wherein the formulation excludes additional excipients required for peptide solubility. In some embodiments, the excipient is chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid. [0018] The invention still further provides methods of increasing the yield in plant production of a protein, comprising: preparing a nucleic acid construct comprising: a) at least one nucleic acid sequence encoding a secretory signal peptide, b) at least one nucleic acid sequence encoding a glycosylation site, and c) at least one nucleic acid sequence encoding a protein; and expressing the nucleic acid construct as a glycoprotein in plants or plant cell cultures. In some embodiments, the at least one HRGP glycosylation site is chosen from i) X-Pro.sub.n, where n is from 2 to about 1000, and ii) (X-Pro).sub.n, where n is from 1 to about 1000; wherein X is any amino acid, or is chosen from Lys, Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr, and Val. The nucleic acid construct can also include or exclude a nucleic acid sequence encoding green fluorescent protein. The invention also provides proteins produced according to these methods. [0019] The invention also provides growth hormone molecules covalently attached to an amino acid sequence comprising a glycomodule, wherein the glycomodule is chosen from i) X-Pro-Hyp.sub.n, where n is from 2 to about 100, ii) X-Hyp.sub.n, where n is from 2 to about 100, and ii) (X-Hyp).sub.n, where n is from 1 to about 100; wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr, and Val. [0020] The invention also provides growth hormone antagonist molecules covalently attached to an amino acid sequence comprising a glycomodule, wherein the glycomodule is chosen from i) X-Pro-Hyp.sub.n, where n is from 2 to about 100, ii) X-Hyp.sub.n, where n is from 2 to about 100, and ii) (X-Hyp).sub.n, where n is from 1 to about 100; wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr, and Val. [0021] Also provided are methods of treating a patient suffering from growth hormone deficiency or insufficiency comprising administering a therapeutically effective amount of glycosylated human growth hormone. Continue reading... Full patent description for Glycoproteins produced in plants and methods of their use Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Glycoproteins produced in plants and methods of their use patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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