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Production of complex carbohydratesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Compound Containing Saccharide Radical, N-glycosideProduction of complex carbohydrates description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060099691, Production of complex carbohydrates. Brief Patent Description - Full Patent Description - Patent Application Claims
CLAIM OF PRIORITY [0001] This application claims priority under 35 U.S.C. 119(e) from U.S. Provisional Application Ser. No. 60/134,756, filed May 18, 1999, which application is incorporated herein by reference. FIELD OF THE INVENTION [0003] This invention relates to a method for the production of complex carbohydrates on an LPS backbone structure in Gram-negative bacteria BACKGROUND OF THE INVENTION [0004] Complex carbohydrates occur in nature and are involved in a wide array of biological functions, including viral, bacterial and fungal pathogenesis, cell-to-cell and intracellular recognition, binding of hormones and pathogens to cell-surface receptors and in antigen-antibody recognition. The term "complex carbohydrates" embraces a wide array of chemical compounds having the general formula (CH.sub.2O).sub.n where the monomer unit is selected from any of thousands of naturally occurring or synthetic monomers, including, but not limited to, glucose, galactose, mannose, fucose and sialic acid. Saccharides may have additional constituents such as amino, sulfate or phosphate groups, in addition to the carbon-hydrogen-oxygen core. The polymer consisting of two to ten saccharide units is termed an oligosaccharide (OS) and that consisting of more than ten saccharide units is termed a polysaccharide (PS). These monosaccharide building blocks can be linked in at least 10 different ways, leading to an astronomical number of different combinations and permutations. It is found that strains within species and even tissue within an organism differ in complex carbohydrate structure. This high degree of variability, the highly specific composition of naturally occurring complex carbohydrates and the wide range of biological roles make these compounds especially significant. [0005] Gram-negative bacteria contain complex carbohydrates, which are linked to lipids to form lipooligosaccharides (LOS) or lipopolysaccharides (LPS.) The immunogenicity of the LOS and LPS resides in the carbohydrate moiety, while pathogenicity resides in the lipid moiety. For this reason, OS and PS are useful as vaccines against Gram-negative pathogens and for identification of gram-negative bacteria. [0006] U.S. patent application Ser. No. 5,736,533 discloses oligosaccharides useful as therapeutic agents against pathogens that are the causative agents of respiratory infections. It is believed that pathogenic bacteria are able to colonize tissue by binding to carbohydrates on the surface of the tissue and that providing an excess of specific soluble oligosaccharides can result in competitive inhibition of bacterial colonization. [0007] OS and PS from LPS and LOS can be produced by growing the specific bacterial pathogen in culture, with subsequent cleavage of the lipid moiety and purification. However, most pathogenic bacteria are fastidious in their growth requirements and slow growing, making this mode of production impractical. For example, Haemophilus influenzae is known to require a carbon dioxide atmosphere and brain/heart extract for growth Helicobacter pylori grows very poorly in broth cultures required for OS and PS production. In addition, many of these bacterial pathogens (for example, Neisseria meningitidis) can be dangerous to grow in large volumes because of the risk of aerosol and possible infection spread. The ability to produce the OS and PS structures of fastidious bacterial pathogens in bacterial strains such as Escherichia coli and Salmonella minnesota which grow rapidly to high density offers a rapid way to produce these OS and PS from fastidious bacterial pathogens. [0008] Eucaryotic proteins and peptides frequently have carbohydrate moieties on their surfaces, which act as specific binding sites for hormones, which are also glycosylated, that is, have complex carbohydrates linked to the peptide structure. Moreover, in addition to the recognition role, carbohydrates are necessary to the proper three-dimensional folding of polypeptides into functional glycoproteins. Bacteria do not glycosylate peptides and proteins efficiently or in a manner equivalent to that of eucaryotes. For that reason, although bacteria are widely used as production cells for growing eucaryotic peptides and proteins, such useful human glycopeptides such as erythropoetin are grown in mammalian cells. U.S. Pat. No. 4,703,008 discloses a method for the production of erythropoietin, in which cells such as Chinese hamster ovary cells are transfected with the DNA coding for the hormone and grown under a carbon dioxide atmosphere in complex medium. The resulting hormone is sufficiently similar to the naturally occurring hormone to be effective as a therapeutic for human use. [0009] An additional utility for isolated, cell-specific carbohydrates is for competitive inhibition of disease agents in which infection is reliant on surface-recognition glycosylated proteins. For example, the human immunodeficiency virus is known to bind to the surface receptor on T-4 lymphocytes. If an excess of free T-4 receptor carbohydrate is present in the bodily fluids of the patient, the virus will bind to the free carbohydrate and is effectively prevented from infecting the T-4 lymphocyte. [0010] Competitive inhibition of binding of antibodies to cell surfaces by administration of cell-recognition molecules may have therapeutic potential in the treatment of autoimmune diseases such as lupus erythematosus, multiple sclerosis and rheumatoid arthritis. Such molecules may bind to the cell receptor, blocking the binding of the automimmunie antibodies which cause the degeneration seen in such disease states. [0011] U.S. Pat. No. 4,745,051 discloses a method for expressing DNA in an insect cell, a method that has practical application for the production of glycosylated peptides and proteins. However, the glycosylation resulting is that native to the insect, consisting of higher levels of mannose than are typical of mammalian cells. [0012] Practical production of peptides and polypeptides in bacterial production cells is well established. Chemical and enzymatic means for glycosylating peptides and proteins are well known in the art. For example, U.S. Pat. No. 5,370,872 discloses a method for coupling PS through a carboxyl or hydroxyl group to a protein. Classic organic syntheses of complex carbohydrates have long been known, but with limited practical application. In addition to the difficulties inherent in the complexity of the glycopolymer molecule, many glycosidic bonds are labile and must be protected and deprotected during chemical synthesis, adding to the difficulty of synthesis and reducing the yield of product. [0013] Because of the drawbacks of organic synthesis, enzymatic synthesis has been devised. It is known that glycosylation proceeds by the step-wise addition of monomers through the action of such enzymes as glycotransferases. The reaction products can be further modified by lyases, acetylases, sulftases, phosphorylases, kinases, epimerases, methylases, transferases and the like. U.S. Pat. No. 5,308,460 discloses such a step-wise synthesis on an immobilized matrix. [0014] A need remains for a more efficient and practical method for the production of complex carbohydrates, and glycoproteins and glycopeptides containing complex carbohydrates specific to a species or tissue. SUMMARY OF THE INVENTION [0015] The present invention is directed to the production of complex carbohydrates in a production cell. It is here disclosed that certain bacteria, such as Escherichia coli Strain K-12, have a core liposaccharide with a terminal heptose. A suitable production cell also contains an enzyme which catalyzes the transfer to the terminal heptose of an acceptor molecule, such as N-acetylglucosamine, to form a "scaffold" upon which glycotransferases add other saccharide monomers to form complex carbohydrates. If an otherwise suitable production cell lacks such an enzyme, the DNA encoding the gene rfe (UDP-GlcNAc:Undecaprenol GlcNAc-1 phosphate transferase) of Haemophilus influenzae may be inserted into the production cell. Preferably, production of rfe is enhanced by the presence of the gene products of the gene lsgG. By inserting genes encoding glycotransferases into the production cell, the complex carbohydrates specific to bacteria such as Haemophilus influenzae, Neisseria spp, Salmonella spp and Escherichia coli are produced. Mammalian complex carbohydrate such as polysialyl can also be produced. [0016] Accordingly, the invention provides a process for the production of a complex carbohydrate which comprises the steps of: (a) inoculating transformed production cells into a culture medium capable of supporting the growth of said production cells wherein said production cells are prepared by transforming bacteria comprising (i) a core lipid structure containing a terminal heptose molecule and (ii) an enzyme capable of adding an acceptor molecule to said heptose molecule by inserting an isolated DNA sequence encoding glycotransferase synthesizes a complex carbohydrate into said bacteria to yield transformed production cells; (b) allowing growth of said transformed production cells; and (c) recovering said complex carbohydrate from the culture medium. [0017] The invention also provides a process for the production of an oligosaccharide which comprises the steps of: (a) transforming gram-negative bacteria comprising (i) a core lipid structure containing a terminal heptose and (ii) an enzyme that adds a galactose molecule to said heptose wherein said transformed gram-negative bacteria are prepared by constructing a vector comprising an isolated DNA sequence coding for a glycotransferase that synthesizes an oligosaccharide; (b) inoculating said transformed gram-negative bacteria into a culture medium capable of supporting the growth of said transformed bacteria; (c) allowing growth of said inoculated gram-negative bacteria; and (d) recovering said oligosaccharide from the culture medium. [0018] Using methods disclosed in this application, a production cell suitable for the practical production of other complex carbohydrates can be identified. Such a suitable production cell will have an acceptor molecule specific to the carbohydrate to be synthesized, or a site that can be modified to add such a specific acceptor molecule. The production cell will contain the initiating IsgG or IsgF to form the appropriate acceptor. DNA coding for the glycotransferases of other species, strains, tissues, hormones, receptors or other cell-surface carbohydrates can then be inserted into such a production cell, with the resultant production of oligosaccharides or polysaccharide specific to that species, strain, tissue, hormone, receptor or other cell-surface carbohydrate. The nucleotide sequences for the genes rfc and lsG are on file in the H. influenzae Rd database available from TIGR (Bethesda, Md.). Sequences for glycotransferases are available from the references herein disclosed. [0019] Also provided are methods for separating and purifying the product. [0020] The invention also provides a process for the production of a complex carbohydrate, comprising culturing production cells comprising a chimeric DNA sequence encoding a glycotransferase, so as to yield production cells comprising an altered level of complex carbohydrate, wherein the production cells are bacteria comprising a core lipid structure containing a terminal heptose molecule and encoding an enzyme capable of adding an acceptor molecule to the heptose molecule. The invention also provides a process further comprising recovering the complex carbohydrate. BRIEF DESCRIPTION OF THE DRAWINGS Continue reading about Production of complex carbohydrates... Full patent description for Production of complex carbohydrates Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Production of complex carbohydrates patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. 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