101 results about "Fucosyltransferase" patented technology

The present invention provides a process for producing an antibody composition using a cell, which comprises using a cell into which an RNA having activity of suppressing the function of an enzyme relating to the modification of a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is introduced; the RNA used in the production process; a DNA corresponding to the RNA; a cell in which the RNA or DNA is introduced or expressed; a process for producing the cell; and a method for suppressing the enzyme.

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 method and application for screening an ultralow fucose cell line. The invention provides a method for constructing fucose deficit type host cells capable of expressing an antibody and an IgG-FC fusion protein, a detection method for the fucose activity of the antibody and the IgG-Fc fusion protein, and concrete application of the cell lines. The method provided by the invention is realized by highly-efficient knockout of fucose-based transferase (FUT8) gene in an engineering cell producing the antibodies and the IgG-Fc fusion protein through a TALEN (and/or CRISPR) technology; and through lens culinaris lectin (LCA) pressurizing, gene sequencing and a flow cytometry screening process, the host cells with highly-efficiently knocked-out fucose is obtained. Meanwhile, fucose deficit CHOK1 host cell lines are constructed into stable engineering cell lines capable of expressing antibody proteins; and after the antibody proteins are obtained, glycoform analysis is performed; and results show that fucose knockout efficiency reaches to 99% or above.

The invention provides recombinant bacillus subtilis for synthesizing 2'-fucosyllactose and an establishment method and application of recombinant bacillus subtilis. Recombinant bacillus subtilis is obtained in the mode that a lactose transport enzyme gene is subjected to reinforcement expression in a gene group of bacillus subtilis 168, and exogenous fucosyltransferase, fucokinase and gtp-mannose-1-phosphate guanylyltransferase genes are expressed. According to recombinant bacillus subtilis, the lactose transport enzyme gene is subjected to reinforcement expression on the gene group, expression of the lactose transport enzyme is effectively reinforced, the efficiency of transferring exogenous lactose into cytoplasm is improved, the concentration of lactose in cytoplasm is increased, and synthesis of 2'-fucosyllactose is promoted. The establishment method of recombinant bacillus subtilis is simple and convenient to use and has good application prospects.

The present invention discloses a construction method of escherichia coli for synthesizing fucosylated oligosaccharide by fermentation. The construction method includes the steps: (1) overexpressing at least one gene for encoding enzymes required for denovo synthesizing GDP-L-fucose in a prokaryotic host cell; (2) expressing an exogenous gene for encoding a fucosyltransferase in the prokaryotic host cell; (3) reducing or eliminating the activity of GDP-mannanohydrolase in the prokaryotic host cell; and (4) reducing or eliminating the activity of beta-galactosidase in the prokaryotic host cell.According to the present invention, the escherichia coli is constructed by using the method, and the escherichia coli may be used for preparing the fucosylated oligosaccharide. Shown by a result generated by performing fermentation production verification on the fucosylated oligosaccharide (with 2'-fucosyllactose as an example) in a 5L tank by adopting an engineering strain constructed by the present invention, the highest production level of the 2'-fucosyllactose (2'-FL) may reach 50 g/L.

The invention provides compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.

The invention discloses a method for increasing the yield of 2'-fucosyllactose in recombinant escherichia coli, and belongs to the technical field of gene engineering. According to the method, four different protein labels are fused at the N ends of alpha-1, 2-fucosyltransferase FutC respectively by adopting flexible Linker, and the constructed fusion protein FP-futC can improve the yield of 2'-FLcatalyzed and synthesized by alpha-1,2-fucosyltransferase to different extents. The yield of the 2'-FL synthesized by adopting the TrxA-futC fusion protein is the highest and reaches 2.94 g/L, the consumption of substrate lactose reaches 3.55 g/L, and the lactose conversion rate is 0.58 mol/mol. In the control group, the yield of the 2'-FL synthesized by the recombinant escherichia coli obtainedby plasmid pMA09-futC constructed without protein tags is only 1.71 g/L, the lactose consumption is 2.88 g/L, and the lactose conversion rate is only 0.42 mol/mol. Furthermore, the TrxA-futC fusion protein gene is integrated to a yjiP site on an escherichia coli MG1655 genome to obtain a plasmid-free 2'-FL genetically engineered strain MG-26 delta yjiP:: trxA-futC, the yield of the 2'-FL after shake-flask fermentation reaches 3.85 g/L, and the lactose conversion rate is 0.68 mol/mol.

The invention discloses fucosyltransferase, genetically engineered bacteria thereof and an application, belongs to the technical field of gene engineering, and relates to nucleic acid and amino acid sequences from Sideroxydans lithotrophicus ES-1. Codes represent novel alpha-1, 2-fucosyltransferase SlFucT, the fucosyltransferase SlFucT obtained by separation constructs an expression vector pET15b-slfuct, and the expression vector is converted into an Escherichia coli expression strain BL21 (DE3) to realize efficient expression. The invention further provides a method for generating fucosylation oligose by alpha-1, 2-fucosyltransferase, and particularly provides an application and a method of discovering the fucosylation oligose in breast milk. The application and the method have application prospects in the field of food and medical industry.

A method of in vitro fucosylation of selectin ligands on cord blood-derived hematopoietic stem cells for bone marrow transplantation is disclosed. In this method, an effective amount of an α1,3-fucosyltransferase, e.g., α1,3-fucosyltransferase VI, is used in vitro to treat cord blood-derived hematopoietic stem cells to convert non-functional PSGL-1 or other ligands on the cell surface into functional forms that bind selectins, especially P-selectin or E-selectin. The treated cells have enhanced effectiveness in reconstituting bone marrow in patients in need of such therapy.

The invention provides an alpha-1, 2-fucosyltransferase and an application thereof in a milk powder and belongs to the technical field of gene engineering. The invention relates to a nucleic acid andan amino acid sequence from thermophilic synechococcus (Thermosynechococcus sp. Nk 55a); the alpha-1, 2-fucosyltransferase is encoded; and obtained groups are separated to construct an expression vector pET15b-Tnfuct and converted into Escherichia coli strain BL21(DE3) to realize efficient expression. A method for generating fucosylated oligosaccharide, glycopeptide and glycoprotein by using the alpha-1, 2-fucosyltransferase has an application prospect in the industrial fields of infant milk powder, foods and medicines.

The invention relates to a molecular kit and detection method for accurately and quickly detecting the resistance of pig Escherichia coli F18. By using 7 kinds of working solutions and the detection method which are provided by the invention, on the basis of analyzing the polymorphism of the alpha (1, 2) fucosyltransferase (FUT1) gene M307, the resistance of the pig Escherichia coli F18 can be simply, conveniently and accurately determined, thereby realizing the large-scale detection on samples. The invention also provides a positive control which can achieve the aim of identifying the resistance of the pig Escherichia coli F18 at high sensibility and specificity, has simple and convenient operation, high accuracy and strong repeatability, and has significance for resistance of diarrhea of weanling pigs, breeding for hydropsy resistance and research and diagnosis of epidemiology.

The invention relates to modified CHO cells and application thereof. The modified CHO cells can realize stable and high-density growth under condition of serum-free suspension culture, and preferably,the modified CHO cells do not express functional glutamine synthetase (GS) and fucosyltransferase 8 (FUT8). The modified CHO cells are particularly applicable to high-level eukaryotic expression of recombinant proteins (especially antibodies), and thus have good application prospects in the fields of genetic engineering and protein engineering.

The invention provides a recombinant escherichia coli for synthesizing 2'-fucosyllactose and construction method thereof, and the recombinant Escherichia coli is obtained by knocking out a beta-galactosidase gene in the genome of Escherichia coli, synthesizing a key enzyme gene of colanic acid from guanosine diphosphate fucose, enhancing expression key enzyme genes of lactose permease genes and a guanylate diphosphate fucose denovo synthesis route and expressing exogenous fucosyltransferase genes. According to the scheme, lactose hydrolysis can be reduced, and the rate of transferring exogenous lactose into cells is increased, so that the concentration of lactose in the cells is effectively increased; meanwhile, the consumption of the guanosine diphosphate fucose is reduced, and the concentration of the guanosine diphosphate fucose in cells is effectively improved; through codon optimization, the expression of an exogenous fucosyltransferase gene is improved, and synthesizing of the 2'-fucosyllactose is effectively promoted.

The invention discloses an optimized saccharomyces cerevisiae engineering strain capable of highly producing 2'-fucosyllactose. Through modification by metabolic engineering, the saccharomyces cerevisiae can absorb lactose and takes GDP-mannose as a precursor in a cell to synthesize GDP-fucose, and alpha-1,2-fucosyltransferase derived from bacillus cereus is heterologously expressed. The 2'-fucosyllactose with a high yield can be obtained by the obtained recombinant strain, and the 2'-fucosyllactose yield can be increased to about 3.8 g/L through subsequent overexpression of the GDP-mannose synthesis related enzyme. Therefore, an important reference is provided for industrial production of the 2'-fucosyllactose from the saccharomyces cerevisiae.

An object of the present invention is to provide a means for producing O-fucosylated protein in large quantities, a means for searching for new genes associated with the synthesis system of O-fucosylated protein or the proteins expressed by the genes, and a means for elucidating the functions thereof. According to the present invention, a yeast transformant characterized in that the genes associated with the synthesis system of O-fucosylated protein are a GDP-fucose synthase gene, a GDP-fucose transporter gene, a fucosyltransferase gene, and a fucose receptor gene is provided.

This invention provides nucleic acid and amino acid sequences of fucosyltransferases from Helicobactor pylori. The invention also provides methods to use the fucosyltransferases to synthesize oligosaccharides, glycoproteins, and glycolipids.

Disclosed are a recombinant Corynebacterium glutamicum (C. glutamicum) for producing fucosyllactose which is transformed to express α-1,2-fucosyltransferase, GDP-D-mannose-4,6-dehydratase (Gmd), GDP-L-fucose synthase (WcaG) and lactose permease (LacY), wherein the Corynebacterium glutamicum has phosphomannomutase and GTP-mannose-1-phosphate guanylyltransferase, and a method for producing fucosyllactose using the same. According to the recombinant Corynebacterium glutamicum and the method for producing fucosyllactose according to the present invention, with use of a GRAS Corynebacterium glutamicum strain, which is safer than conventional Escherichia coli, 2′-fucosyllactose can be produced at a high concentration while overcoming drawbacks of conventional methods associated with industrial inapplicability resulting from low production concentrations.