98 results about "Sialic acid aldolase" 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.

Methods for manipulating carbohydrate processing pathways in cells of interest are provided. Methods are directed at manipulating multiple pathways involved with the sialylation reaction by using recombinant DNA technology and substrate feeding approaches to enable the production of sialylated glycoproteins in cells of interest. These carbohydrate engineering efforts encompass the implementation of new carbohydrate bioassays, the examination of a selection of insect cell lines and the use of bioinformatics to identify gene sequences for critical processing enzymes. The compositions comprise cells of interest producing sialylated glycoproteins. The methods and compositions are useful for heterologous expression of glycoproteins.

Culture media comprising manganese and methods of culturing cells to improve sialylation and glycosylation of glycoproteins are provided.

The present invention describes methods and processes for the production of proteins, particularly glycoproteins, by animal cell or mammalian cell culture, illustratively, but not limited to, fed-batch cell cultures. The methods comprise feeding the cells with D-galactose, preferably with feed medium containing D-galactose, preferably daily, to sustain a sialylation effective level of D-galactose in the culture for its duration, thus increasing sialylation of the produced proteins. The methods can also comprise at least two temperature shifts performed during the culturing period, in which the temperature is lower at the end of the culturing period than at the time of initial cell culture. The cell culture processes of the invention involving two or more temperature shifts sustain a high cell viability, and can allow for an extended protein production phase. The methods can also comprise the delayed addition of polyanionic compound at a time after innoculation. Supplementation of the cultures with D-galactose, preferably in a feed medium, to sustain galactose at sialylation effective levels in the cultures until the end of a culture run reverses a decline in sialylation that accompanies culture scale up, and is advantageous for large scale culturing processes.

Two Hepatitis C Virus envelope proteins (E1 and E2) are expressed without sialylation. Recombinant expression of these proteins in lower eukaryotes, or in mammalian cells in which terminal glycosylation is blocked, results in recombinant proteins which are more similar to native HCV glycoproteins. When isolated by GNA lectin affinity, the E1 and E2 proteins aggregate into virus-like particles.

An unbranched polylactosamine comprising at least 6 monosaccharides and having terminal alpha 2->3 sialylation and internal alpha 1->3 fucosylation at various N-acetylglucosamine residues except for solely at the penultimate N-acetylglucosamine residue.

Methods of isolating highly sialylated recombinant vitamin K dependent proteins, particularly Factor IX, by chromatographic methods are described. The highly sialylated recombinant proteins are characterized. The improved Factor IX has at least 62% N-glycosylation with 3 or 4 sialic acid residues and improved bioavailability and pharmokinetic properties.

Methods are provided for the prognosis, diagnosis and treatment of various pathological states, including cancer, chemotherapy resistance and dementia associated with Alzheimer's disease. The methods provided herein are based on the discovery that various proteins with a high level of sialylation are shown herein to be associated with disease states, such as, cancer, chemotherapy resistance and dementia associated with Alzheimer's disease. Such methods provide a lysosomal exocytosis activity profile comprising one or more values representing lysosomal exocytosis activity. Also provided herein, is the discovery that low lysosomal sialidase activity is associated with various pathological states. Thus, the methods also provide a lysosomal sialidase activity profile, comprising one or more values representing lysosomal sialidase activity. A lysosomal sialidase activity profile is one example of a lysosomal exocytosis activity profile. As such, the level of lysosomal exocytosis activity and/or lysosomal sialidase activity is predictive of a diagnosis and/or prognosis of cancer, chemotherapy resistance or dementia associated with Alzheimer's disease.

The present invention provides compositions and methods for the production of glycoproteins with enhanced sialylation. In particular, the invention provides cell lines comprising disrupted sialidase expression and methods of using the cell lines to produce glycoproteins with enhanced sialylation.

The invention provides an FSH preparation having a high degree of sialylation, and showing increased efficacy.

The use of anthranilic acid (2-AA) to label N-glycans prior to separation using a reversed-phase liquid chromatography (RP-LC) column under acidic conditions using formic acid is described herein. Negatively charged 2-AA offers stronger retention on the reversed phase column than 2-aminobenzamide (2-AB) in RP-LC and allows efficient ionization and detection of 2-AA labeled N-glycans. The acidic conditions used for the RP-LC leads to an efficient separation of acidic 2-AA N-glycans carrying terminal sialylation without the need for an ion-pairing reagent. The method and compositions described herein may be used with RP-nano-LC-MS and a 96-well plate sample preparation, which allows attomolar sensitivity and high throughput.

The properties of an Fc-containing protein, for example, an antibody, are controlled by altering the sialylation of the oligosaccharides in the Fc region. The modified Fc-containing proteins have therapeutic utility in diseases or conditions in which it is desirable to control the affinity for one or more of the FcgRI, FcgRIIA, and FcgRIIIA receptors, ADCC activity, macrophage or monocyte activation, serum half-life, and avidity.

Changes in sialylation of cell surface or plasma proteins are often associated with various cancers and other disease conditions Provided are methods of detecting biomarkers of conditions associated with a change of sialylation status. Sialylated peptides are first isolated from biological and other samples by loading onto titanium dioxide (TiO2) or zirconium dioxide (ZrO2) stationary phase under acidic conditions in a solution comprising at least 20% organic phase and at least about 6.5 mM of substituted aromatic carboxylic acid, or, alternatively, at least about 1 mM short chain, non-aromatic, hydroxylated carboxylic acid. Sialic acid containing proteins can then be eluted from loaded stationary phase material by exposure to an alkaline solution having pH of 9.0 or greater, preferably at least 10.5. Sialylated peptides isolated by the methods provided can then be analysed by mass spectrometry to identify patterns of sialylation across a sialiome (the entire complement of sialic acid containing peptides in a biological sample) and/or to identify proteins in a sample that are sialylated or that show changes in sialylation status between two or more different samples. In preferred embodiments, sialylated peptides from control and patient samples can be differentially isotopically labelled and compared in a single mass sprectrometry experiment. Also provided are specific biomarkers of bladder cancer. The methods for isolating and analysing sialylated proteins can also by applied to phosphorylated proteins.

The invention discloses a synthesis method of oligosaccharides of a Lewis x unimer, a dimer and saliva acidifying derivatives thereof. The method comprises the steps that core framework compounds (V)to (IX) are built by enzymic method modular assembly 1 and enzymic method modular assembly 2; a Lex unimer and a dimer (I) to (II) are assembled by enzymic method modular assembly 3; finally, an sLexunimer and A dimer (III) to (IV) are built by enzymic method modular assembly 4. High region selectivity and high efficiency of the enzymic method synthesis are combined; the target molecule synthesisis realized at higher yield. Glycosyltransferase, riboside generation enzyme and hexokinase are from prokaryotic organisms; the advantages of high protein expression, wide substrate adaptability, high catalysis efficiency and the like are realized; the synthesis method can be used for large-scale preparation.