Methods of producing glycosylated proteins

Abstract

Described herein are methods of producing glycosylated proteins in vitro and in vivo. The methods include using host cells to produce glycosylated proteins. Also described herein are glycosylated proteins produced using such methods and uses thereof.

1 . A host cell comprising (i) a nucleic acid that encodes a target protein comprising an N-glycosylation consensus sequence; (ii) a nucleic acid that encodes an N-glycosyltransferase (NGT) that adds glucose to an amino acid residue present in said N-glycosylation consensus sequence, and (iii) a nucleic acid that encodes a glycosyltransferase that catalyzes addition of a monosaccharide to said glucose. 
     
     
         2 . The host cell of  claim 1 , wherein said target protein is heterologous to the host cell. 
     
     
         3 . The host cell of  claim 1 , wherein said NGT is heterologous to the host cell. 
     
     
         4 . The host cell of  claim 1 , wherein said glycosyltransferase that catalyzes addition of a monosaccharide to said glucose is heterologous to the host cell. 
     
     
         5 . The host cell of  claim 1 , wherein said NGT is the NGT of  Actinobacillus  pleuropneumonias, the NGT of a species of  Haemophilus , the NGT of a species of  Mannheimia , the NGT of a species of  Bibersteinia , or the NGT of a species of  Yersinia.    
     
     
         6 . The host cell of  claim 1 , wherein said glycosyltransferase that catalyzes addition of a monosaccharide to said glucose is a galactosyltransferase. 
     
     
         7 . The host cell of  claim 6 , wherein said galactosyltransferase is LgtB of  N. meningitidis , the LgtB of  N. gonorrhoeae , LgtE of  N. meningitidis , CgtB of  C. jejuni , WaaX of  E. coli , HP0826 of  Helicobacter pylori , or a eukaryotic β4Gal-T1. 
     
     
         8 .- 20 . (canceled) 
     
     
         21 . The host cell of  claim 1 , wherein said host cell is a prokaryotic host cell. 
     
     
         22 . The host cell of  claim 21 , wherein said host cell is  E. coli.    
     
     
         23 . The host cell of  claim 21 , wherein said host cell is an  Escherichia  species,  Shigella  species,  Klebsiella  species,  Xhantomonas  species,  Salmonella  species,  Yersinia  species,  Lactococcus  species,  Lactobacillus  species,  Pseudomonas  species,  Corynebacterium  species,  Streptomyces  species,  Streptococcus  species,  Staphylococcus  species,  Bacillus  species, or  Clostridium  species. 
     
     
         24 . The host cell of  claim 1 , wherein said host cell is a eukaryotic host cell. 
     
     
         25 . The host cell of  claim 24 , wherein said host cell is a yeast cell, a plant cell, an insect cell, A kinetoplastida cell, or a mammalian cell. 
     
     
         26 . The host cell of  claim 1 , wherein said target protein is a bacterial protein. 
     
     
         27 . The host cell of  claim 1 , wherein said target protein is a eukaryotic protein. 
     
     
         28 . The host cell of  claim 1 , wherein said target protein is a therapeutic protein. 
     
     
         29 . The host cell of  claim 28 , wherein said therapeutic protein is an enzyme, a cytokine, a hormone, a growth factor, an inhibitor protein, a protein receptor, a ligand that binds a protein receptor, or an antibody. 
     
     
         30 . The host cell of  claim 29 , wherein said enzyme or inhibitor is Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor VIIa, Antithrombin III (AT-III), Protein C, Tissue plasminogen activator (tPA) and tPA variants, Urokinase, Hirudin, Streptokinase, Glucocerebrosidase, Alglucosidase-α, Laronidase (α-L-iduronidase), Idursulphase (Iduronate-2-sulphatase), Galsulphase, Agalsidase-β (human α-galactosidase A), Botulinum toxin, Collagenase, Human DNAse-I, Hyaluronidase, Papain, L-Asparaginase, Uricase (Urate oxidase), glutamate carboxypeptidase (glucarpidase), α1 Protease inhibitor (α1 antitrypsin), Lactase, Pancreatic enzymes (lipase, amylase, protease), or Adenosine deaminase. 
     
     
         31 . The host cell of  claim 29 , wherein said cytokine is Interferon-α (INF-α), Interferon-β(INF-β), Interferon-γ (INF-γ), Interleukin-2 (IL2), Chimeric diphteria toxin-IL-2 (Denileukin diftitox), Interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), or Tumor necrosis factor alpha (TNF-α). 
     
     
         32 . The host cell of  claim 29 , wherein said antibody is adalimumab (Humira) and Remicade (Infliximab); ReoPro (Abciximab); Rituxan (Rituximab); Simulect (Basiliximab); Synagis (Palivizumab); Herceptin (Trastuzumab); Mylotarg (Gemtuzumab ozogamicin); Campath (Alemtuzumab); Zevalin (Ibritumomab tiuxetan); Xolair (Omalizumab); Bexxar (Tositumomab-I-131); Erbitux (Cetuximab); Avastin (Bevacizumab); Tysabri (Natalizumab); Actemra (Tocilizumab); Vectibix (Panitumumab); Lucentis (Ranibizumab); Soliris (Eculizumab); Cimzia (Certolizumab pegol); Simponi (Golimumab); Ilaris (Canakinumab); Stelara (Ustekinumab); Arzerra (Ofatumumab); Prolia (Denosumab); Numax (Motavizumab); ABThrax (Raxibacumab); Benlysta (Belimumab); Yervoy (Ipilimumab); Adcetris (Brentuximab Vedotin); Perjeta (Pertuzumab); Kadcyla (Ado-trastuzumab emtansine); or Gazyva (Obinutuzumab). 
     
     
         33 . The host cell of  claim 29 , wherein said hormone or growth factor Insulin, Pramlintide, Growth hormone (GH), Insulin-like growth factor (IGF1), Human parathyroid hormone, Calcitonin, Glucagon-like peptide-1 agonist (GLP-1), Glucagon, Growth hormone-releasing hormone (GHRH), Secretin, Thyroid stimulating hormone (TSH), Human bone morphogenic protein 2 (hBMP2), Human bone morphogenic proetin 7 (hBMP7), Gonadotropin releasing hormone (GnRH), Keratinocyte growth factor (KGF), Platelet-derived growth factor (PDGF), Fibroblast growth factor 7 (FGF7), Fibroblast growth factor 20 (FGF20), Fibroblast growth factor 21 (FGF21), Epidermal growth factor (EGF), Vascular endothelial growth factor (VEGF), Neurotrophin-3, Human follicle-stimulating hormone (FSH), Human chorionic gonadotropin (HCG), Lutropin-α, Erythropoietin, Granulocyte colony-stimulating factor (G-CSF), or Granulocyte-macrophage colony-stimulating factor (GM-CSF). 
     
     
         34 . The host cell of  claim 1 , wherein said host cell does not comprise an oligosaccharyltransferase (OST). 
     
     
         35 . A method for producing a glycosylated target protein that comprises a glucose assembled at an amino acid residue present in an N-glycosylation consensus sequence; wherein said glucose is linked to a monosaccharide, said method comprising (i) culturing the host cell of  claim 1  under conditions suitable for protein production and (ii) isolating said target protein. 
     
     
         36 . The method of  claim 35 , wherein said glycosylated target protein is N-glycosylated. 
     
     
         37 . A method for producing a sialylated target protein, comprising (i) culturing the host cell of  claim 36  under conditions suitable for protein production and (ii) isolating said sialylated target protein. 
     
     
         38 . A method for producing a polysialylated target protein, comprising (i) culturing the host cell of  claim 36  under conditions suitable for protein production and (ii) isolating said polysialylated target protein. 
     
     
         39 . (canceled) 
     
     
         40 . A composition comprising proteins produced by the method of  claim 37 , wherein said at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of said proteins in said composition are sialylated or polysialylated. 
     
     
         41 .- 53 . (canceled) 
     
     
         54 . A method of treating a disease or disorder in a subject in need thereof, comprising administering the composition of  claim 40 . 
     
     
         55 .- 65 . (canceled) 
     
     
         66 . A method for producing a glycosylated recombinant target protein in a host cell, wherein said method does not comprise use of an oligosaccharyltransferase (OST) or chemical coupling in said cell. 
     
     
         67 . A method for producing a glycosylated recombinant target protein in a host cell, wherein said method comprises culturing a target protein and an N-glycosyltransferase (NGT) in said cell. 
     
     
         68 .- 72 . (canceled)