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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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  • C12N9/1048—Glycosyltransferases (2.4)
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/13—Dipeptidases (3.4.13)
  • C12Y304/13009—Xaa-Pro dipeptidase (3.4.13.9) i.e. prolidase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2800/00—Nucleic acids vectors
  • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/485—Exopeptidases (3.4.11-3.4.19)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
  • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
  • C12Y114/11002—Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase

Definitions

• the invention relates to collagen 7, collagen 7 related nucleic acids and cells, and related methods.
• Collagens are a family of proteins that strengthen and support connective tissues, such as the skin, tendons, ligaments, and bones. Collagen 7, as the major component of anchoring fibrils, functions in strengthening and stabilizing various tissues, including the skin (Ricard- Blum, Cold Spring Harb Perspect Biol 3(l):a004978 (2011)).
• the disclosure features, a method of making collagen 7, or a functional fragment thereof.
• the method comprises:
• the method further comprises purifying collagen 7, or functional fragment thereof, from said cultured cell.
• the disclosure features, a vector described herein.
• the disclosure features, a cell, or isolated preparation of cells, described herein. In another aspect, the disclosure features, a high glycine optimized sequence encoding collagen 7 described herein.
• the disclosure features an isolated preparation of cells described herein which can further comprise any of culture medium, and collagen 7, or functional fragment thereof, produce by said cell.
• polypeptides e.g., one or more polypeptides that increase collagen 7 production in the cell (e.g., prolidase and/or prolyl hydroxylase);
• the cell is recombinantly manipulated to express collagen 7, or a functional fragment thereof, and the cell is recombinantly manipulated to express one or more polypeptides, e.g., that increase the expression of collagen 7 in the cell.
• the cell is recombinantly manipulated to express collagen 7, or a functional fragment thereof before said cell is recombinantly manipulated to express one or more polypeptides, e.g., that increase the expression of collagen 7 in the cell, e.g., one or more of prolidase, prolyl hydroxylase, glycosyl transferase, and functional fragments thereof.
• the cell is recombinantly manipulated to express collagen 7, or a functional fragment thereof after said cell is recombinantly manipulated to express one or more polypeptides, e.g., that increase the expression of collagen 7 in the cell, e.g., one or more of prolidase, prolyl hydroxylase, glycosyl transferase, and functional fragments thereof.
• one or more polypeptides e.g., that increase the expression of collagen 7 in the cell, e.g., one or more of prolidase, prolyl hydroxylase, glycosyl transferase, and functional fragments thereof.
• the cell is recombinantly manipulated to express collagen 7, or a functional fragment thereof, at the same time said cell is recombinantly manipulated to express one or more polypeptides, e.g., that increase the expression of collagen 7 in the cell, e.g., one or more of prolidase, prolyl hydroxylase, glycosyl transferase, and functional fragments thereof.
• one or more polypeptides e.g., that increase the expression of collagen 7 in the cell, e.g., one or more of prolidase, prolyl hydroxylase, glycosyl transferase, and functional fragments thereof.
• the invention features, collagen 7, or a functional fragment thereof, made by a method described herein.
• the invention features, a purified or isolated preparation of collagen 7, or functional fragment thereof, wherein at least 30, 40, 50, 60, 70, 80, 90 or 95 % of said collagen 7, or functional fragment thereof, is incorporated into homotrimers.
• Recombinant in reference to a protein or polypeptide molecule, pertains to a protein or polypeptide molecule expressed utilizing isolated nucleic acid molecules or recombinant nucleic acid molecules.
• the less common glycine codon is a less common glycine codon for a cell referred to herein, e.g., a CHO or HEK cell.
• the sequence includes at least one, and in embodiments, at least 10, 20 or 30, less common glycine codons that is not present in the native human sequence for collagen 7. In an embodiment at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the glycine codons are less common glycine codons.
• amino acid sequence for human collagen 7 is as follows:
• Nucleotide sequence for human ST6 beta-galactosamide alpha-2,6-sialyltransferase 1 (St6gall), transcript variant 1 (rST6Gall) can be optimized.
• Host cells of the current invention may be recombinantly manipulated to express the human PDI, before, after, or simultaneously with manipulating said host cell to recombinantly express collagen 7 or collagen 7 and prolidase.
• Oxoglutarate carrier is a mitochondrial resident protein which transports the a- ketoglutarate across the inner membrane of the mitochondria and facilitates the coupling of decarboxylated a-ketoglutarate to proline.
• Host cells may be recombinantly manipulated to express the human OGC, before, after, or simultaneously with manipulating said host cell to recombinantly express collagen 7 or collagen 7 and prolidase.
• compositions of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 70%, 85%, 90%, 95% identical or higher to the sequence specified.
• substantially identical is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
• amino acid sequences that contain a common structural domain having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 are termed substantially identical.
• NBLAST NBLAST
• sequences that hybridize under low, medium or high stringency to a recited nucleic acid can be used.
• the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing.
• Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used.
• Proteins produced by recombinant methods described herein may be recovered from the host cell culture system according to standard protocols known in the art (e.g., precipitation, centrifugation, etc). Recombinant collagen 7 described herein may be secreted into the host cell medium and recovered by ammonium sulfate precipitation and subsequent centrifugation; as demonstrated in the following reference, (Chen et al. J Bio Chem 277(18): 2118-2124 (2002)).
• DMEM/F12 (1: 1) with L-glutamine and L sodium bicarbonate (Mediatech, Inc., DMEM prepares 10 L at 13.48 g/L Cat. No. 50-003-PB and Ham's F-12 Medium prepares 10 L at 10.64 g/L Cat. No. 50-040-PB), 10% FBS (Omega Scientific Cat# FB-02) and 200 ⁇ ascorbic acid (Sigma CAT# A4544) (1 ml stock solution of 100 mg/ml added to 500 ml medium).
• Serum free medium DMEM/F12 without serum and ascorbic acid.

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