79 results about "Sialyltransferase" patented technology

Disclosed are immortalized human embryonic retina cells, having a nucleic acid sequence encoding an adenoviral E1A protein integrated into the genome of the cells, and further comprising a nucleic acid sequence encoding an enzyme involved in post-translational modification of proteins, such as a sialyltransferase, wherein said nucleic acid sequence encoding the enzyme involved in post-translational modification of proteins is under control of a heterologous promoter. Methods for producing recombinant proteins from such cells and obtaining such recombinant proteins having increased sialylation are provided as are novel compositions of isoforms of erythropoietin .

The present invention relates to eukaryotic host cells which have been modified to produce sialylated glycoproteins by the heterologous expression of a set of glycosyltransferases, including sialyltransferase and / or trans-sialidase, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. Novel eukaryotic host cells expressing a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities (such as those involved in sialylation) to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation.

Disclosed are immortalized human embryonic retina cells, having a nucleic acid sequence encoding an adenoviral E1A protein integrated into the genome of the cells, and further comprising a nucleic acid sequence encoding an enzyme involved in post-translational modification of proteins, such as a sialyltransferase, wherein said nucleic acid sequence encoding the enzyme involved in post-translational modification of proteins is under control of a heterologous promoter. Methods for producing recombinant proteins from such cells and obtaining such recombinant proteins having increased sialylation are provided as are novel compositions of isoforms of erythropoietin.

The present invention relates to a method for the large scale in vivo synthesis of sialylated oligosaccharides, culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as lactose, wherein said microorganism comprises heterologous genes encoding a CMP-Neu5Ac synthetase, a sialic acid synthase, a GlcNAc-6-phosphate 2 epimerase and a sialyltransferase, and wherein the endogenous genes coding for sialic acid aldolase (NanA) and for ManNac kinase (NanK) have been deleted or inactivated. The invention also relates to these micoorganisms which are capable of producing internally activated sialic acid.

This invention provides labeled-substrate conjugates for assaying enzymes, particularly neuraminidases. Also provided are assays that are useful for identifying compounds that inhibit sialyltransferases or neuraminidases and may be useful in treating subjects with influenza. In particular, the present invention relates to methods of using such labeled substrate conjugates to screen for enzyme inhibitors, particularly in a high-throughput format.

This invention provides methods and compositions for treating and preventing atherosclerosis other undesired blood clotting. The methods for treating and preventing atherosclerosis and related conditions involve administering to a mammal an agent that reduces activity of an ST3Gal IV sialyltransferase, which results in enhanced clearance of von Willebrand Factor (vWF) from the mammal.

The present invention relates to eukaryotic host cells which have been modified to produce sialylated glycoproteins by the heterologous expression of a set of glycosyltransferases, including sialyltransferase and / or trans-sialidase, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. Novel eukaryotic host cells expressing a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities (such as those involved in sialylation) to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation.

Provided herein are a methods of reducing a level of activity of a sialyl transferase enzyme, a sialidase enzyme or a combination thereof using inhibitors of these enzymes, for example specific inhibitory compound or antibodies directed against the enzymes. The methods are effective to treat an inflammatory disease or disorder or a primary or metastatic cancer. Also provided is a method for screening potential inhibitors of sialyl transferase enzymes.

The invention discloses a synthetic method of synthesizing double-sialylated tetrasaccharide by a chemically controllable enzyme method. The invention further provides two intermediates, which are used for synthesizing and preparing the double-sialylated tetrasaccharide, are compounds respectively as shown in formulas IV and V. According to the synthetic method disclosed by the invention, the flexibility of a chemical synthetic method and the high-area selectivity and high efficiency of the enzyme synthetic method are combined together, so that synthesis of the double-sialylated tetrasaccharide by using a chemical intervention method is firstly realized, defects such as low substrate reaction activity, multiple synthetic steps and low yield facing in existing chemical synthesis of the double-sialylated tetrasaccharide are solved, as well as a problem that sialyl transferase is difficult to obtain and only glycopeptides is identified in the enzyme method is solved; the synthetic method disclosed by the invention has the advantages of high substrate reaction activity and high yield, and therefore, the synthetic method has important significance in researching biological functions of sialic acid glucoside on molecular level and developing carbohydrate drugs based on the biological functions.

The present invention relates to a method for the large scale in vivo synthesis of sialylated oligosaccharides, culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as lactose, wherein said microorganism comprises heterologous genes encoding a CMP-Neu5Ac synthetase, a sialic acid synthase, a GlcNAc-6-phosphate 2 epimerase and a sialyltransferase, and wherein the endogenous genes coding for sialic acid aldolase (NanA) and for ManNac kinase (NanK) have been deleted or inactivated. The invention also relates to these micoorganisms which are capable of producing internally activated sialic acid.

The invention relates to a fusion protein comprising a bifunctional sialytransferase and a poly-sialytransferase and methods to use the fusion proteins for production of poly-sialylated end products, e.g. oligosaccharides and glycoproteins.

The invention discloses an IgG antigen epitope and an application thereof as a target. The IgG antigen epitope is a CH1 structural domain of non-B-cell-derived IgG, and an Asn162 site of the structural domain is modified by N-glycosylated sialic acid, and realization of an antigenic function needs to depend on the sialylation of the site. The invention also provides an application of the IgG antigen epitope as a drug action target in preparation of drugs for diagnosis and / or treatment of epithelial tumors. In addition, research results show that the function of the antigen as the drug target depends on the sialylation of the Asn162 site, the sialylation of the Asn162 site also needs to depend on a sialic acid transferase ST3GAL4, the enzyme is suggested to be used as a drug action target for preparation of tumor therapy drugs. In addition, integrin beta 4 is co-expressed and co-localized with IgG containing the antigen epitope. In view of the function of IgG, the IgG antigen epitope can be used as a marker for preparation of drugs for aided detection of epithelial tumors.

The present invention relates to eukaryotic host cells which have been modified to produce sialylated glycoproteins by the heterologous expression of a set of glycosyltransferases, including sialyltransferase and / or trans-sialidase, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. Novel eukaryotic host cells expressing a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities (such as those involved in sialylation) to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation.

The present invention provides an extremely useful and novel β-galactoside-α2,6-sialyltransferase having an optimum reaction pH in a neutral to alkaline range, and a nucleic acid encoding the sialyltransferase. The present invention further provides a vector carrying a nucleic acid encoding the sialyltransferase, and a host cell transformed with the vector, as well as a method for producing a recombinant β-galactoside-α2,6-sialyltransferase.

The present invention provides modified Campylobacter sialyltransferase proteins that exhibit enhanced expression as compared to its unmodified form. Nucleic acids that encode the sialyltransferase proteins are also included, as are methods to produce and use the sialyltransferase proteins.

The invention discloses a method for preparing sialyllactose, and relates to the technical field of preparation of sialyllactose. The method for preparing sialyllactose disclosed by the invention comprises the steps of adding kinase to a reaction system containing a substrate, and performing an enzyme catalysis reaction to prepare the sialyllactose, wherein the kinase comprises CMP kinase, polyphosphate sialic acid synthase and sialytransferase; and the substrate comprises CMP, polyphosphate, sialic acid and lactose. The method adopts various kinase namely the CMP kinase, the polyphosphate kinase, the CMP-sialic acid synthetase and sialytransferase to catalyze the corresponding substrate to prepare the sialyllactose. The method has the characteristics of being high in yield, low in cost, short in cycle and the like.

The present invention is directed to sialytransferases, such as SiaA sialytransferases isolated from Haemophilus influenzae. Further provided herein are methods for producing sialylated lipooligosaccharides, vaccines, and host cells and systems for the production of sialylated lipooligosaccharides.

The invention discloses a synthesis method of a disialic acid tetrasaccharide MAG antagonist by a chemical enzyme process. The invention also discloses two intermediates for synthesizing and preparing a tetrasaccharide MAG antagonist, which are respectively compounds disclosed as general formulae IV and V. By combining the flexibility of the chemical synthesis process and the regioselectivity and high efficiency of the enzyme synthesis process, the method overcomes the defects of low substrate reaction activity, multiple synthesis steps and low yield in the chemically synthesized disialic acid tetrasaccharide MAG antagonist at present, and the defects of difficulty in obtaining sialyl transferase, only recognition of glycopeptide and the like in the enzyme process, and has the advantages of high substrate reaction activity and high yield. Therefore, the method has important meanings in researching interactions between the sialic acid antagonist and the myelin related glycoprotein on the molecular level.

The invention provides compositions comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto, characterized in that said glycans comprise LewisX structures and on average at least 6 sialic acid moieties per EPO molecule. The invention further provides methods for obtaining a composition comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto wherein said glycans comprise on average at least 6 sialic acids per EPO molecule and from 0 to 2 Lewis x structures, said method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and further containing a nucleic acid encoding an EPO in expressible format, wherein said cell further contains a nucleic acid sequence encoding a sialyltransferase, preferably an alpha-2,6-sialyltransferase or an alpha-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing said cell in a serum-free culture medium and allow expression of an EPO in said cell; c) harvesting the expressed EPO from said cell and / or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions which have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more iso forms of an EPO comprising glycans linked thereto wherein said glycans comprise on average at least 6 sialic acids per EPO molecule and from 0 to 2 Lewis x structures.

The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. It was found that the combination of two different truncation variants of human β-galactoside-α-2,6-sialyltransferase I (hST6Gal-I) exhibited different specific sialyltransferase enzymatic activities. In one example, under conditions wherein the first variant Δ89 hST6Gal-I catalyzed formation of bi-sialylated target molecules the second variant Δ108 hST6Gal-I catalyzed formation of mono-sialylated target molecules. Thus, disclosed are variants of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variants of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Provided are compositions comprising one or more isoforms of an erythropoietin (“EPO”) comprising glycans linked thereto, wherein the glycans have Lewis x structures and on average at least six sialic acid moieties per EPO molecule. Further provided are methods for obtaining a composition comprising one or more isoforms of EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures, the method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and a nucleic acid encoding EPO in expressible format, wherein the cell further contains a nucleic acid sequence encoding a sialyltransferase, e.g., an α-2,6-sialyltransferase or an α-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing the cell in a serum-free culture medium and allowing expression of EPO in the cell; c) harvesting the expressed EPO from the cell and / or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions that have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more isoforms of an EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures.

The invention provides compositions comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto, characterized in that said glycans comprise LewisX structures and on average at least 6 sialic acid moieties per EPO molecule. The invention further provides methods for obtaining a composition comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto wherein said glycans comprise on average at least 6 sialic acids per EPO molecule and from 0 to 2 Lewis x structures, said method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and further containing a nucleic acid encoding an EPO in expressible format, wherein said cell further contains a nucleic acid sequence encoding a sialyltransferase, preferably an alpha-2,6-sialyltransferase or an alpha-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing said cell in a serum-free culture medium and allow expression of an EPO in said cell; c) harvesting the expressed EPO from said cell and / or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions which have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more iso forms of an EPO comprising glycans linked thereto wherein said glycans comprise on average at least 6 sialic acids per EPO molecule and from 0 to 2 Lewis x structures.

The invention discloses a construction method for an engineered Escherichia coli strain capable of producing sialyllactose. The construction method comprises the following steps: knocking out a polysialytransferase gene (neuS) and beta-galactosidase gene (lacZ) in Escherichia coli so as to obtain a Delta neuS-Delta lacZ double-defective strain; knocking out a gene cluster (nanKETA) related to decomposition of N-acetylneuraminic acid so as to obtain a Delta neuS-Delta lacZ-Delta nanKETA defective strain; and transferring a recombinant plasmid pXC1k-Nst into the Delta neuS-Delta lacZ-Delta nanKETA defective strain so as to obtain a K1-KETA-pNst strain. The Escherichia coli strain K1-KETA-pNst provided by the invention is preserved in China General Microbiological Culture Collection Center, No. 3, Yard 1, West Beichen Road, Chaoyang District, Beijing City, China, on October 16, 2017, with an accession number of CGMCC No. 14825. The construction method has the advantages that introductionof genes for synthesis and activation of sialic acid is not needed; the resources of cells are directly used; and the method is simple and practical to implement, low in cost and high in yield.

The invention discloses a preparation method of sialyl enteromorpha oligosaccharide promoting intestinal probiotics proliferation. Enteromorpha is dried and subjected to superfine grinding, and by means of an ethanol reflux extraction technology, the dried and degreased enteromorpha powder is obtained; the enteromorpha oligosaccharide is obtained through microwave-assisted extraction, deproteinization and polysaccharide and pigment removing; sialyltransferase is added into a reaction system containing enteromorpha oligosaccharide, a glycosylation reaction is conducted, and the sialyl enteromorpha oligosaccharide is prepared. The oligosaccharide can significantly increase the number of intestinal probiotics and can be used for preparing drugs or health-care products which improve intestinal flora.

The present disclosure relates to a method for preparing recombinant glycoproteins with high sialic acid content. More specifically, for UDP-GlcNAc 2-epimerase / ManNAc kinase (GNE / MNK) enzyme where point mutation was induced by substituting arginine at position 263 by leucine only or by further substituting arginine at position 266 by glutamine, epimerase activity is constantly maintained, and overexpressed cells thereof experience an increase in intracellular cytidine monophosphate (CMP)-sialic acid content, irrespective of CMP-sialic acid concentration.,Particularly, since in an glycoprotein(such as, erythropoietin and thrombopoietin)-producing host cell where point mutationinduced GNE / MNK, human alpha-2,3-sialyltransferase and a CMP-sialic acid transporter gene are simultaneously overexpressed, intracellular content of CMP-sialic acid and sialic acid in glycoprotein increases in cells, overexpression in a host cell producing a sialylated recombinant glycoprotein the three genes above may be useful for preparing glycoprotein with increased sialic acid content.

The present invention relates to new methods to promote sialylation of glycoconjugates, including recombinant glycoproteins, in glycoconjugate production systems. The invention relates to methods to promote efficient glycoconjugate sialylation in recombinant expression systems, by providing simpler and more economical ways to produce large intracellular pools of sialic acid precursors. The invention is directed to nucleic acids, vectors, and cells harboring vectors comprising nucleic acids encoding enzymes involved in the synthesis of sialic acid precursors, and cells harboring these nucleic acids in combination with nucleic acids encoding glycosyltransferases, including sialyltransferases, to facilitate the production of humanized recombinant glycoproteins in bacterial, fungal, plant, and animal cell expression systems. The engineered cells can be used to produce glycosylated proteins in virally-infected, transiently-transformed, or stably-transformed host cells, including lepidopteran insects and cultured cell lines derived from Spodoptera frugiperda, Trichoplusia ni, and Bombyx mori that can be infected by baculovirus expression vectors.

Disclosed are methods, genetically engineered cells, sialyltransferases and nucleic acid molecules encoding said sialyltransferases for producing sialylated oligosaccharides as well as the use of saidsialylated oligosaccharides for providing nutritional compositions.

The present invention relates to new methods to promote sialylation of glycoconjugates, including recombinant glycoproteins, in glycoconjugate production systems. The invention relates to methods to promote efficient glycoconjugate sialylation in recombinant expression systems, by providing simpler and more economical ways to produce large intracellular pools of sialic acid precursors. The invention is directed to nucleic acids, vectors, and cells harboring vectors comprising nucleic acids encoding enzymes involved in the synthesis of sialic acid precursors, and cells harboring these nucleic acids in combination with nucleic acids encoding glycosyltransferases, including sialyltransferases, to facilitate the production of humanized recombinant glycoproteins in bacterial, fungal, plant, and animal cell expression systems. The engineered cells can be used to produce glycosylated proteins in virally-infected, transiently-transformed, or stably-transformed host cells, including lepidopteran insects and cultured cell lines derived from Spodoptera frugiperda, Trichoplusia ni, and Bombyx mori that can be infected by baculovirus expression vectors.

The present invention provides a novel β-galactoside-α2,6-sialyltransferase having high productivity and / or high activity, and a nucleic acid encoding the sialyltransferase. The present invention also provides a microorganism producing the sialyltransferase. The present invention further provides a vector carrying a nucleic acid encoding the sialyltransferase, and a host cell transformed with the vector, as well as a method for producing a recombinant β-galactoside-α2,6-sialyltransferase.

Disclosed are methods for the fermentative production of a sialylated saccharide and genetically engineered microbial cells for use in said method, wherein the genetically engineered microbial cells comprise (i) a sialic acid biosynthesis pathway comprising a glucosamine-6-phosphate N-acetyltransferase, (ii) a cytidine 5'-monophospho- (CMP)-N- acetylneuraminic acid synthetase; and (iii) a sialyltransferase, for producing sialylated saccharides, as well as the use of said sialylated oligosaccharides for providing nutritional compositions.