69 results about "Protein glycosylation" patented technology

The invention relates to novel methods for controlling the glycosylation of a protein and the protein produced thereby. An exemplary method of the invention comprises producing a protein comprising a decreased level of glycosylation, e.g., the protein comprises fewer glycans or fewer saccharides at the glycosylation site, by culturing a host cell expressing the protein in the presence of a glycosylation inhibitor. In an exemplary method of the invention, the biological characteristics of the protein are altered by the decreased level of glycosylation, e.g., the binding of the protein with its target ligand is modified.

The invention provides methods and compositions for the rapid and sensitive detection of post-translationally modified proteins, and particularly of those with post-translational glycosylations. The methods can be used to detect O-GlcNAc posttranslational modifications on proteins on which such modifications were undetectable using other techniques. In one embodiment, the method exploits the ability of an engineered mutant of β-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling detection of the modified protein. The approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Further, the preferred embodiments can be used for detection of certain disease states, such as cancer, Alzheimer's disease, neurodegeneration, cardiovascular disease, and diabetes.

Methods for altering the N-glycosylation pattern of proteins in higher plants are provided. The methods comprise introducing into the plant a recombinant construct that provides for the inhibition of expression of α1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase (XylT) in a plant. Use of these constructs to inhibit or suppress expression of both of these enzymes, and isoforms thereof, advantageously provides for the production of endogenous and heterologous proteins having a “humanized” N-glycosylation pattern without impacting plant growth and development. Stably transformed higher plants having this protein N-glycosylation pattern are provided. Glycoprotein compositions, including monoclonal antibody compositions, having substantially homogeneous glycosylation profiles, and which are substantially homogeneous for the G0 glycoform, are also provided.

The invention discloses a soyabean protein glycosylation graft modification method which comprises the following steps: (1) adding the soyabean protein and sugar with the mass ratio of the soyabean protein and the sugar being 1:1-1:5 into water, adjusting pH value to 6-8 to obtain mixed solution of the soyabean protein and the sugar with the mass percentage of of the soyabean protein and the sugarbeing 0.1%-10%; (2) heating the mixed solution in a sealed container for 0.5-4h under the pressure of 3-13MPa and at the temperature of 80-140 DEG C to obtain reaction liquid; and (3) cooling the reaction liquid to 10-40 DEG C, purifying or not purifying, and drying the reaction liquid to obtain the glycosylation soyabean protein. The method has rapid reaction speed and even reaction, good functional character of reaction products, and industrialized and scaled application value. The functionality of the obtained glycosylation soyabean protein, such as water-solubility, emulsification and antibacterial property is greatly improved, the reaction speed is faster, and the color of the appearance of the products is good.

The invention provides methods and compositions for the rapid and sensitive detection of post-translationally modified proteins, and particularly of those with post-translational glycosylations. The methods can be used to detect O-GlcNAc post-translational modifications on proteins on which such modifications were undetectable using other techniques. In one embodiment, the method exploits the ability of an engineered mutant of β-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling detection of the modified protein. The approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Further, the preferred embodiments can be used for detection of certain disease states, such as cancer, Alzheimer's disease, neurodegeneration, cardiovascular disease, and diabetes.

The present invention relates to methods and genetically engineered methylotrophic yeast strains for producing glycoproteins with mammalian-like glycosylation. The present invention also relates to vectors useful for generating methylotrophic yeast strains capable of producing glycoproteins with mammalian-like glycosylation. Glycoproteins produced from the genetically engineered methylotrophic yeast strains are also provided.

The invention relates to methods for identifying compounds which affect cellular stress. In particular, the method relates to identifying compounds which inhibit protein advanced glycation end product formation, where the compounds are carbonyl scavengers which inhibit the formation. The assay involves combing the substance of interest with histone H1 and ADP-ribose, and then measuring fluorescence and protein cross linking. Various inhibitors ofprotein AGE glycation have been identified, using this assay.

The present invention relates to methods and genetically engineered methylotrophic yeast strains for producing glycoproteins with mammalian-like glycosylation. The present invention also relates to vectors useful for generating methylotrophic yeast strains capable of producing glycoproteins with mammalian-like glycosylation. Glycoproteins produced from the genetically engineered methylotrophic yeast strains are also provided.

Methods for diagnosing pathology of the liver in a subject suspected of having such pathology are disclosed. The methods comprise quantifiably detecting glycosylation, and more specifically fucosylation, on proteins in biological fluids, and comparing the detected glycosylation with reference values for the glycosylation of such proteins in healthy or disease states.

The invention relates to a method for analyzing protein O-glycosylation sites. The method comprises the following steps: (1), sequentially carrying out endonuclease digestion, endoglycosidase and exoglycosidase digestion on a proteome sample or a single protein sample to prepare a peptide and non-glycopeptide protein sample or single protein sample with a GalNAc label; (2) loading the sample on a durian agglutinin chromatographic column and separating to prepare a proteomic sample; (3) separating the single protein sample or the proteomic sample through with a C18 reversed-phase chromatographic column, and then detecting by a high-definition mass spectrometer in a cation mode to obtain a high-definition mass spectrogram; (4) processing the obtained mass spectrometric data and obtaining protein glycosylation site information though the searched result. The site information can be more rapidly, more accurately and more comprehensively obtained through establishing the method for detecting the O-glycosylation sites of glycoproteins in the single protein sample and the proteomic sample.

The present invention relates to methods and compositions for the identification of cancer markers. In particular, the present invention provides methods and compositions for the identification of glycosylated proteins and protein glycosylation patterns. The present invention further provides cancer markers identified using the described methods.

The invention improves glycoprotein production and protein glycosylation engineering in eukaryotes, specifically the production of human-like complex or hybrid glycosylated proteins in lower eukaryotes such as yeasts. The invention provides glycosylation modified eukaryotic host cells capable of producing glycosylation optimized proteins useful as immunoglobulins and other therapeutic proteins, and provides cells capable of producing glycoproteins having glycan structures similar to glycoproteins produced in human cell. The invention further provides proteins with human-like glycan structures and novel compositions thereof producible by these cells.

The invention discloses a kit for detecting protein glycosylation subtype and a detection method thereof. The detection kit comprises agglutinin, confining liquid, a biotin labeled antibody, horse radish peroxidase labeled streptavidin, a 3',3',5',5'-tetramethyl benzidine substrate developing solution, a stop solution and a cleaning buffer solution. The detection method comprises the following steps: fixing the agglutinin onto a polystyrene solid phase carrier to be used for recognizing and binding glycoprotein in the sample based on specific recognition of agglutinin and an oligosaccharide chain, recognizing the glycoprotein needing to be detected by virtue of specific binding of the protein and the antibody thereof, and realizing semi-quantitative analysis of the glycoprotein. According to the detection kit and the detection method disclosed by the invention, semi-quantitative detection of single protein glycosylation subtype can be realized, and the kit is easy and convenient to operate and low in cost.

The invention belongs to the field of biotechnologies, and relates to a method for applying recombinant expression separated and purified human-derived protein Fn3 (fibronectin type III domain) mutants as N-glycosylation efficiency detection model proteins. The method includes steps of constructing Fn3-Gly-loop recombinant protein gene expression vectors of the Fn3 mutants; jointly transforming the constructed expression vectors into Escherichia coli engineering strains CLM37 by means of electric shock processes; screening the expression vectors by the aid of antibiotics to obtain positive clones. Recombinant proteins contain Fn3-Gly-loop proteins which are about to be modified by recombinant glycosyl, and the Fn3-Gly-loop fusion protein glycosylation efficiency can be detected by the aid of Western Blot processes. The method has the advantages that the skeleton proteins Fn3 in Escherichia coli are used as receptor proteins, accordingly, the model receptor proteins suitable for N-glycosylation modification efficiency research can be constructed, and the recombinant protein glycosylation efficiency can be easily, quickly and efficiently detected in the Escherichia coli.

Provided are methods of collecting, detecting and altering cells and molecular entities using glycoprotein micelles and vesicles. Glycoprotein vesicles comprising a glycoprotein micelle, at least a monolayer of lectin and / or a monolayer of biologically active glycoproteins are also provided. The invention further provides methods of detecting protein glycosylation using the vesicles of the invention.

The invention improves glycoprotein production and protein glycosylation engineering in eukaryotes, specifically the production of human-like complex or hybrid glycosylated proteins in lower eukaryotes such as yeasts. The invention provides glycosylation modified eukaryotic host cells capable of producing glycosylation optimized proteins useful as immunoglobulins and other therapeutic proteins, and provides cells capable of producing glycoproteins having glycan structures similar to glycoproteins produced in human cell. The invention further provides proteins with human-like glycan structures and novel compositions thereof producible by these cells.

The invention relates to a mass spectrometry method for a complete glycosylated peptide fragment based on quasi-multistage spectrum and an application thereof in early diagnosis of liver cancer. The method mainly comprises the following five steps: 1) enrichment of a glycosylated peptide fragment; 2) sample preparation of a deglycosylated peptide fragment-complete glycosylated peptide fragment; 3)mass spectrometry of the deglycosylated peptide fragment-complete glycosylated peptide fragment; 4) spectrogram data retrieval of the deglycosylated peptide fragment-complete glycosylated peptide fragment; and 5) based on the above, through combination of glycosylation site and complete glycopeptide quantification technology, realizing glycosylation refined difference analysis of a cancer patientand a normal person and screening potential disease markers. The method realizes refined analysis of human protein glycosylation, and has important application potential in screening the tumor markers.

The disclosure relates to an oligosaccharyltransferase polypeptide and the production of glycosylated recombinant protein in a microbial host cell; and including vaccines comprising glycosylated recombinant antigens.

The invention discloses a protein glycosylation detection kit and a detection method, and an application. Specific protein targeting, an HCR-based non-enzymatic amplification strategy, and the proteinglycosylation detection kit and detection method for detecting and imaging specific protein glycosylation on a cell membrane surface in living cells and living bodies by using the HCR-based non-enzymatic amplification strategy, and detection applications in various application occasions such as an in vitro application occasion and an in vivo application occasion are achieved by using technical means such as modification of azido glycosyl on the surface of a protein by a glycosyl-labelled nucleic acid sequence, introduction of a protein probe nucleic acid sequence, and introduction of a hybridization chain reaction reagent.

The present invention relates, in general, to methods for detecting and quantitating plasma-derived protein and recombinant protein in a sample based on the difference in protein glycosylation, when the plasma protein and the recombinant protein are essentially the same protein.

The invention discloses a method for preparing beta-carotene uniform emulsion from whey separation protein glycosylation reaction products and the uniform emulsion. The method comprises the followingsteps: 1) preparing the whey separation protein glycosylation reaction products; 2) preparing a glycosylated protein-polyphenol compound; and 3) preparing the beta-carotene uniform emulsion. The goodemulsifying property and interface property of the glycosylated protein-polyphenol nano-compound effectively inhibit the coalescence phenomenon among oil drops, the emulsion drops show good uniformity, and the emulsion drop uniformity is directly related to the storage stability. Meanwhile, the glycosylation reaction enables protein to form a denser and thicker interface layer outside the oil drops loaded with active substances, so that the interaction between the active substances and an oxidant (metal ions, free radicals and dissolved oxygen) is inhibited.

The invention discloses a vaccine for preventing and treating diseases caused by Acinetobacter baumannii and a preparation method thereof. The vaccine for preventing and treating the diseases caused by the Acinetobacter baumannii provided by the invention has an active component which is an Acinetobacter baumannii glycoprotein obtained through glycosylation of a protein composed of bases from site20 to site 156 of SEQ ID No. 1 or bases from site 1 to site 156 of SEQ ID No. 1. According to the invention, Acinetobacter baumannii glycoprotein modified rCTB4573 prepared by a genetic engineering method is used for preparation of an Acinetobacter baumannii glycoprotein protein conjugate vaccine; uniformity of the vaccine can be improved; production efficiency of the vaccine is improved; the cost is reduced; extensive application prospect is achieved; and the vaccine can be used for preventing the diseases caused by pathogenic Acinetobacter baumannii.

The present invention relates to methods for glycosylating a protein in which the protein is modified to include an alkyne and / or an azide group. The invention further relates to a protein glycosylated by these methods.

The present invention relates to pancreatic cancer markers. In particular, the present invention provides methods and compositions for the identification of protein glycosylation patterns associated with pancreatic cancer.

Methods for producing isotopically-labelled peptides are provided. Aspects of the method include: contacting a sample including a metabolically tagged protein with a cleavable probe to produce a probe-protein conjugate; separating the probe-protein conjugate from the sample; digesting the probe-protein conjugate to produce a probe-peptide conjugate; and cleaving a cleavable linker to release an isotopically labelled peptide. The method may further include: identifying a predetermined isotopic pattern in a mass spectrum; determining an amino acid sequence of the isotopically labelled peptide; and identifying the site of protein glycosylation based on the determined amino acid sequence. Also provided are cleavable probes for practicing the subject methods, described by the Formula: A-L-(M-Z) where A is an affinity tag, L is a cleavable linker, M is an isotopic label and Z is a chemoselective tag capable of cross-linking a metabolically tagged protein. Compositions and kits for practicing the subject methods are also provided.