69 results about "Yeast display" patented technology

The present invention relates to the field of protein display libraries and library screening. In preferred embodiments, the present invention provides a three component system for display comprising a cell surface molecule, an adapter molecule and a display molecule.

Provided are protein, nucleic acid, and cellular libraries of multivalent binding proteins (e.g., DVD-Fab or DVD-Ig molecules) and the use of these libraries for the screening of multivalent binding proteins using cell surface display technology (e.g., yeast display).

Provided are methods for protein engineering, such as engineering proteases or kinases. The methods may utilize yeast display and / or ER sequestration of proteins or substrates. In some aspects, TEV proteases with altered substrate specificity, potency, and / or efficiency are provided.

The invention discloses a method for catalytic synthesis of vitamin E succinat by utilizing yeast display lipase, and the method comprises the following steps: dissolving vitamin E and succinic anhydride in an organic solvent, adding the yeast display lipase, reacting at 50-60 DEG C for 10-14 hours under an anaerobic condition, and separating and purifying, thus obtaining the vitamin E succinate.The preparation method of the yeast display lipase comprises the following steps: transforming the recombinant plasmid subjected to linear treatment into a Pichia Pastoris yeast GS115, inoculating the obtained transformants into a BMMY (buffered methanol-complex medium), inducing and culturing for 72-144 hours, carrying out centrifugal collection on thallus, washing, and carrying out organism printing and frozen drying on the washed thallus, thus obtaining the yeast display lipase. Through displaying lipase outside cells, and utilizing the lipase to conduct catalytic synthesis of the vitamin E vitamin E succinate, the conversion efficiency can be improved, the reaction time is shortened, and the production cost is lowered.

The invention discloses a preparation method of a non-fusion anti-desmoglein DSG3 rabbit-derived monoclonal antibody. The preparation method comprises the following steps: 1) selecting a suitable immune animal, and carrying out DSG3 immunization on the animal; 2) extracting successfully immunized animal blood, collecting peripheral blood mononuclear cells, and separating out a B lymphocyte population capable of secreting a DSG3 antibody; 3) establishing a rabbit-derived single-chain antibody yeast display library; 4) constructing a DSG3 rabbit-derived monoclonal antibody expression vector library; 5) constructing a DSG3 rabbit-derived monoclonal antibody yeast expression library: namely converting a pichia pastoris expression vector PZ recombinant plasmid into yeast expression bacteria X33, and screening X33 recombinant transformation monoclonal bacteria capable of secreting a DSG3 antibody; 6) obtaining a X33 recombinant conversion monoclonal strain that can stably secrete a DSG3 antibody; and 7) purifying the obtained DSG3 rabbit-derived monoclonal antibody. The provided preparation method can prepare a stable DSG3 antibody, has industrial repeatability and is suitable for industrial popularization and application.

The invention discloses a method for catalytic synthesis of glucose laurate by utilizing yeast display lipase, and the method comprises the following steps: dissolving glucose and laurate in an organic solvent, adding the yeast display lipase, reacting at 50-60 DEG C for 10-14 hours, and separating and purifying, thus obtaining glucose laurate. The preparation method of the yeast display lipase comprises the following steps: transforming the recombinant plasmid subjected to linear treatment into a Pichia Pastoris yeast GS115, inoculating the obtained transformants into a BMMY (buffered methanol-complex medium), inducing and culturing for 72-144 hours, carrying out centrifugal collection on thallus, washing, and carrying out organism printing and frozen drying on the washed thallus, thus obtaining the yeast display lipase. Through displaying lipase outside cells, and utilizing the lipase to conduct catalytic synthesis of the glucose laurate, the conversion efficiency can be improved, the reaction time is shortened, and the production cost is lowered.

The invention discloses a method for disrupting seaweed cells by using yeast display pectinase cooperated with ultrasonic waves, which comprises the following steps that: dry seaweed powder and yeast display pectinase are added into water, are uniformly mixed, are subjected to enzymatic reaction for 30 to 45min at the temperature of 35 to 37 DEG C, and are disrupted for 65 to 70min by utilizing ultrasonic waves with the power of 730 to 750W at the temperature of 30 to 32 DEG C. The invention also discloses a method for extracting seaweed proteins and porphyra polysaccharides, which is characterized in that: cell disruption liquid obtained after the seaweed cells are disrupted is separated. The invention also discloses a method for preparing seaweed antihypertensive peptides, which is characterized in that: the separated seaweed proteins are subjected to enzymatic reaction for 130 to 133min at the temperature of 36 to 37 DEG C by using protease. In the method, the seaweed cell walls are degraded by yeast display pectinase, and ultrasonic wave treatment is synergically carried out, so that the seaweed cells are efficiently disrupted, thereby the disruption rate of the seaweed cells is improved, and the yields of seaweed polysaccharides, seaweed proteins and seaweed antihypertensive peptides are increased.

The invention relates to preparation of high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts, which belongs to a method for carrying out site directed mutagenesis wild phospholipids enzyme D by recombinant deoxyribonucleic acid (DNA) for improving the activity, connecting the mutated gene with yeast show carriers pPIC9K-Flo and efficiently displaying the mutated gene on the surface of pichia pastoris cells, and relates to a preparation method of the high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts. The method has the advantages that the activity of the wild phospholipids enzyme D is improved and is shown on the surfaces of the pastoris cells, the stability is improved,and the advantages of immobilized enzymes are realized. The method has the technical scheme that wild phospholipids enzyme D genes are separated from microbes, particularly streptomyces aureofuscus, the mutation is carried out on the amino acid residue of Glu69 and Ser285, through the efficient expression on the surfaces of the pichia pastoris cells, the enzyme activity of the high-activity phospholipids enzyme is improved by 11 percent than the wild type phospholipids enzyme D, and the enzyme activity of the recombination strains GS115 / pPIC9K-Flo-pldm prepared through high-density fermentation is 120U / (g. stem cells).

The invention discloses a method for catalytic synthesis of L-ascorbic acid linoleate by utilizing yeast display lipase, and the method comprises the following steps: dissolving L-ascorbic acid and linoleic acid in an organic solvent, adding the yeast display lipase, reacting at 50-60 DEG C for 4-8 hours under an anaerobic condition, and separating and purifying, thus obtaining the L-ascorbic acid linoleate. The preparation method of the yeast display lipase comprises the following steps: transforming the recombinant plasmid subjected to linear treatment in a Pichia Pastoris yeast GS115, inoculating the obtained transformants into a BMMY (buffered methanol-complex medium), inducing and culturing for 72-144 hours, carrying out centrifugal collection on thallus, washing, and carrying out organism printing and frozen drying on the thallus, thus obtaining the yeast display lipase. Through displaying lipase outside cells, and utilizing the lipase to conduct catalytic synthesis of the L-ascorbic acid L-ascorbic acid linoleate, the conversion efficiency can be improved, the reaction time is shortened, and the production cost is lowered.

The invention discloses a method for producing cream-aroma selenium-enriched bread from yeast display lipase. The method includes displaying rice lipase on the surface of pichia pastoris, performing multiplication culture for 48-96 hours, enabling yield of the rice lipase to sharply increase with yield of yeast, adding 1-4 parts of saccharomycetes displayed with the rice lipase into 200-300 parts of cream liquid for esterlysis reaction at the temperature of 35-55 DEG C for 2-6 hours, subjecting the mixture to filtering separation, washing the yeast to allow the same to be reusable, adding filtrate into 1000 parts, by weight, of flour, then adding 1-4 parts of selenium-enriched yeast and other raw materials, and performing dough kneading, fermentation, forming and baking to obtain the delicious healthcare bread with rich cream aroma and rich organic selenium.

The invention discloses a method for synthesizing L-ascorbyl palmitate by catalysis of yeast display lipase. The method comprises the following steps of: dissolving L-ascorbic acid and palmitic acid into an organic solvent, adding the yeast display lipase, reacting for 4 to 8 hours in the absence of oxygen at the temperature of between 50 and 60 DEG C, and performing separation and purification to obtain the L-ascorbyl palmitate, wherein the yeast display lipase is prepared by the following steps of: transferring linearly treated recombinant plasmids to pichia pastoris GS115, inoculating the obtained transformant to a buffered methanol complex (BMMY) medium, performing induced culture for 72 to 144 hours, performing centrifugal collection on bacteria, and performing flushing, biological imprinting and freeze drying on the bacteria to obtain the yeast display lipase. The lipase is displayed outside cells, and the L-ascorbyl palmitate is synthesized by catalysis of the enzyme preparation, so that the transformation efficiency is improved, the reaction time is shortened and the production cost is reduced.