2209 results about "Trypsin" patented technology

Recombinant protein complexes having human Factor VIII:C activity are expressed in a eukaryotic host cell by transforming the host cell with first and second expression cassettes encoding a first polypeptide substantially homologous to human Factor VIII:C A domain and a second polypeptide substantially homologous to human Factor VIII:C C domain, respectively. In the present invention, the first polypeptide may be extended having at its C-terminal a human Factor VIII:C B domain N-terminal peptide, a polypeptide spacer of 3-40 amino acids, and a human Factor VIII:C B domain C-terminal peptide. Expression of the second polypeptide is improved by employing an .alpha..sub.1 -antitrypsin signal sequence.

The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of diseases and conditions associated with alpha-1 antitrypsin (AAT) allelic variants that respond to the modulation of gene expression and / or activity. The present invention also concerns compounds, compositions, and methods relating to diseases and conditions associated with alpha-1 antitrypsin (AAT) allelic variants that respond to the modulation of expression and / or activity of genes involved in alpha-1 antitrypsin (AAT) gene expression pathways or other cellular processes that mediate the maintenance or development of alpha-1 antitrypsin (AAT) diseases and conditions such as liver disease, lung disease, and any other diseases or conditions that are related to or will respond to the levels of an alpha-1 antitrypsin (AAT) variant protein in a cell or tissue, alone or in combination with other therapies. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against the expression disease related genes or alleles having alpha-1 antitrypsin (AAT) sequences.

The present invention provides methods and compositions for generating novel nucleic acid molecules through targeted spliceosomal mediated RNA trans-splicing. The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with a SERPINA1 target precursor messenger RNA molecule (target pre-mRNA) and mediate a trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (chimeric RNA). In particular, the PTMs of the present invention include those genetically engineered to interact with SERPINA1 target pre-mRNA so as to result in correction of SERPINA1 genetic defects responsible for AAT deficiency. The PTMs of the invention may also comprise sequences that are processed out of the PTM to yield duplex siRNA molecules directed specifically to mutant SERPIN A1 mRNAs. Such duplexed siRNAs are designed to reduce the accumulation of toxic AAT protein in liver cells. The methods and compositions of the present invention can be used in gene therapy for correction of SERPINA1 disorders such as AAT deficiency.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a nucleic acid encoding a mutant α-antitrypsin protein to correct a point mutation associated with a disease or disorder, e.g., with chronic obstructive pulmonary disease (COPD) disease. The methods provided are useful for correcting an α-antitrypsin point mutation within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Compounds of formula (I) are useful as inhibitors of trypsin like serine protease enzymes such as thrombin, factor VIIa, factor Xa, TF / FVIIa, and trypsin. These compounds could be useful to treat and / or prevent clotting disorders, and as anticoagulating agents.

The present invention demonstrated the potential use of Lactobacillus johnsonii D115 as a probiotic, as a prophylactic agent or as a surface treatment of materials against human and animal pathogens such as Brachyspira pilosicoli, Brachyspira hyodysenteriae, Shigella sonnei, Vibrio cholera, Vibrio parahaemolyticus, Campylobacter jejuni, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Yersinia enterocolitica, Escherichia coli, Klebbsiella pneumoniae, Staphylococcus aureus, Salmonella spp., Bacillus cereus, Aspergillus niger and Fusarium chlamydosporum. The proteineous antimicrobial compound was partially characterized and found to be heat tolerant up to 121° C. for 15 min, and acid tolerant up to pH1 for 30 min at 40° C. The compound is also stable to enzymatic digestion, being able to retain more than 60% antimicrobial activity when treated with pepsin and trypsin.

A protein expression vector containing (a) a nucleotide sequence encoding an IgG(κ) or a trypsin secretory signal peptide, (b) a nucleotide sequence encoding a polyhistidine amino acid sequence, (c) a nucleotide sequence encoding an amino acid sequence comprising amino acid residues 36–40 of SEQ ID NO:19 (Asp-Asp-Asp-Asp-Lys), which is cleavable by an enterokinase, and (d) a cloning site into which a nucleotide sequence encoding a target protein can be inserted, wherein (a), (b), (c) and (d) are assembled within the vector in the order recited.

A method of isolating epithelial cells from a human skin tissue or internal organ tissue using trypsin and ethylenediamine tetraacetic acid (EDTA) simultaneously with the application of magnetic stirring, a method of preconditioning isolated biological cells by the application of physical stimulus, i.e., strain, are provided. Epithelial cells can be isolated by the method with increased yield, colony forming efficiency (CFE), and colony size. Also, the increased percentage of stem cells in isolated cells is advantageous in therapeutic tissue implantation by autologous or allogeneic transplantation. In skin cells preconditioned by the application of strain, cell division is facilitated, and the secretion of extracellular matrix components and growth factors and the activity of matrix metalloproteinases (MMPs) are improved. When preconditioned cells are implanted by autologous or allogeneic transplantation to heal a damaged tissue, the improved cell adhesion, mobility, and viability provides a biological adjustment effect against a variety of stresses or physical stimuli which the cells would undergo after implantation, with improved capability of integration into host tissue, thereby markedly improving the probability of success in skin grafting.

Embodiments herein illustrate methods and compositions for treating medical disorders. In certain embodiments, compositions and methods relate to reducing, inhibiting or treating graft rejection, transplant rejection or diabetes in a subject. Other embodiments herein relate to compounds including naturally occurring and synthetic mutant compositions of alpha-1 antitrypsin, wherein the alpha-1 antitrypsin has no significant serine protease inhibitor activity.

A novel method of treating and preventing diseases is provided. In particular, compositions and methods of blocking diseases associated with aberrant levels of nitric oxide and facilitated by a serine proteolytic (SP) activity are disclosed, which consist of administering to a subject a therapeutically effective amount of a compound having a serine protease inhibitory activity. Among effective compounds are alpha1-antitrypsin and synthetic drugs mimicking some or all of the actions of alpha1-antitrypsin.

The invention discloses a comprehensive extraction process of holothurian polypeptides and polysaccharides. The invention is characterized in that: fresh holothurians are taken as raw materials, gelatinized and hydrolyzed under the effect of composite trypsinases; precipitation and supernatant are respectively collected through centrifugation of the holothurians; ethanol is added into the supernatant for centrifugation; supernatant obtained is mixture of the holothurian polypeptides; centrifugal precipitations obtained for two times are synthesized, and A.S1398 purified neutral proteases are added for hydrolysis and centrifugation; ethanol is added into the supernatant for centrifugation; supernatant obtained for two times is synthesized into holothurian polypeptide mixture, and precipitation is crude polysaccharides; the holothurian polypeptide mixture is dissolved in water, and ultrafiltration membranes with different apertures are used for separation and purification of subsections of different molecular weights; holothurian polypeptide powders of different molecular weight sections are obtained after debitterizing, decolorization and freeze drying; crude polysaccharides are mixed into 5 percent aqueous solution; after centrifugation, decolorization and repeated centrifugation, precipitation is colleted and washed, and aqueous solution is mixed and dialyzed; ethanol is added for repeated precipitation for at least three times, and purified holothurian polysaccharides are obtained. The invention simultaneously prepares the holothurian polypeptides with different molecular weight sections and the holothurian polysaccharides, and has the advantages of high yield, high purity, high safety and mild reaction conditions.

The present invention comprises a method of protecting organs or tissue susceptible to reperfusion-induced dysfunction after ischemia. The method comprises parenterally administering to a patient a therapeutical composition containing natural alpha-1 acid glycoprotein, natural alpha-1 antitrypsin or a mixture thereof. Alternatively, organ or tissue transplants can be contacted with natural alpha-1 acid glycoprotein, natural alpha-antitrypsin or mixtures by perfusing or flushing them with a solution containing natural alpha-1 acid glycoprotein, natural alpha-1 antitrypsin or mixtures thereof in a concentration of 0.1 to 5 g / l.

A sensitive bioluminescent assay to detect proteases including caspases, trypsin and tryptase is provided.

We used Accutase™, a commercially available cell detachment solution, for single cell propagation of pluripotent hESCs. Unlike trypsin dissociation, Accutase treatment does not significantly affect the plating efficiency of hESC dissociation into single cells. Cultures dissociated with Accutase to single cells at each passage maintain a higher proportion of pluripotent cells as compared to collagenase-passaged hESCs. Accutase-treated hESCs can be grown to a high density as monolayers, and yet retain their pluripotency.

An amino acid composition comprising the following amino acids at the following molar ratio: proline (12.6 to 23.4), alanine (8.4 to 15.6), glycine (13.3 to 24.9), valine (8.2 to 15.4), threonine (5.0 to 9.4), eucine (4.3 to 8.1), histidine (1.8 to 11.9), serine (1.7 to 3.3), lysine (6.0 to 11.2), isoleucine (3.1 to 5.9), glutamic acid (2.2 to 10.4), arginine (2.4 to 4.6), phenylalanine (2.6 to 5.0), tyrosine (4.2 to 7.8) and trypsin (1.5 to 2.9). The composition supplements blood amino acids reduced during hard exercise and shows effects to improve motor function, to reduce fatigue after exercise and to help recovery from the fatigue.

The invention discloses a method and process for extracting animal micro-molecule polypeptides and amino acids by utilizing a complex enzyme. The method comprises the following steps: (1) crushing animal tissues so that the animal tissues can pass through a sieve of 80-200 meshes, and adding water to prepare a suspension with the concentration of 8-30 percent; (2) raising the temperature to be 35-40 DEG C, regulating the pH value to be 8.0-9.0 by using sodium carbonate, adding a complex enzyme I accounting for 0.5-2.0 percent of the animal tissues, performing ultrasonic enzymolysis at 14-16 kHz for 1-2 hours so that the proteins between cells are subjected to full enzymolysis, regulating the pH value to be 6.5-7.5, adding a complex enzyme II accounting for 0.5-3.0 percent of the animal tissues, fully and uniformly stirring, controlling the temperature of the water bath to be 37-45 DEG C, and performing ultrasonic enzymolysis at 15-17 kHz for 1-6 hours; (3) raising the temperature of the enzymolysis solution to be 90-100 DEG C to inactivate the enzyme for 20-30 minutes after the enzymolysis is finished; (4) filtering the solution subjected to enzymolysis through a 0.22mu m microfiltration membrane; and (5) concentrating the filtrate to the relative density of 1:1.0-1:1.2 to prepare the concentrated solution, wherein the complex enzyme II consists of Alcalase, Flavourzyme, Protamex, neutral protease and papain. According to the method, the enzymolysis efficiency can be improved, and the production cost can be reduced.

Compounds of formula (I) are useful as inhibitors of trypsin like serine protease enzymes such as thrombin, factor VIIa, factor Xa, TF / FVIIa, and trypsin. These compounds could be useful to treat and / or prevent clotting disorders, and as anticoagulating agents.

The invention discloses a method for extracting polysaccharide from a cordyceps militaris medium. The cordyceps militaris is crushed and mixed with water, is extracted in an enzyme liquor and an alkali liquor under 30 DEG C to 80 DEG C, and an obtained solution is centrifugalized. An insoluble substance is extracted repeatedly, and supernatants are combined. Amylase enzymolysis is performed on the obtained product under 50 DEG C to 75 DEG C. After centrifugation, a supernatant is condensed to be with a sugar content of 1.5 percent to 3.0 percent. The supernatant is added with ethanol and alcohol precipitation is performed on the obtained product under 0 DEG C to 4 DEG C, a product is obtained by drying the obtained product after centrifugation. After deproteinization and dialysis, crude cordyceps polysaccharide is obtained. A single enzyme method, a double enzyme method, a compound enzyme method and a supersonic wave enzyme method can be adopted, and protease, trypsin, etc. can be adopted. The method optimizes the technology route for extracting the cordyceps polysaccharide, enhances an obtaining rate and an extraction efficiency of the cordyceps polysaccharide. Extraction, separation and purification of the cordyceps polysaccharide are carried out effectively. The method provides a solution approach for the utilization of agricultural wastes, and provides a technology base for further exploitation of the cordyceps polysaccharide.

The invention discloses a porcine epidemic diarrhea virus, and a culture method and an application thereof. The porcine epidemic diarrhea virus is called as coronaviridae coronavirus porcine epidemic diarrhea virus GDSJ / 2012, is preserved in China center for type culture collection on May 15th, 2013, and has the preservation number of CCTCC NO:V201309. The culture of the virus strain requires a serum-free DMEM culture solution containing trypsin and magnesium chloride. The virus strain can be used for preparation of a PEDV diagnostic kit and a PEDV vaccine, has good immunogenicity, and can make up for the deficiency of few conventional vaccine types.

The invention discloses a giant salamander active peptide and an application. The giant salamander active peptide is prepared by the following steps: conducting enzymatic hydrolysis on giant salamander meat which serves as a raw material by virtue of a complex enzyme of marine alkaline protease and papain; separating an enzymatic hydrolysate by virtue of a trypsin immobilized ultra-filtration membrane separator; and then conducting separation and purification by virtue of Sephadex LH-20 molecular sieve chromatography and high performance liquid chromatography, so that the giant salamander active peptide is obtained. The giant salamander active peptide is capable of effectively scavenging free radicals and boosting body immunity, and meanwhile, the active peptide can also promote proliferation of skin fibroblasts; therefore, the giant salamander active peptide has a broad application prospect in the fields of food, medicines and cosmetics.

The invention relates to a method for processing soybean isolated protein by utilizing a biological enzyme technology. The method comprises the following steps of: during preparation, selecting high-quality soy protein isolate, and dissolving; performing thermal denaturation; adjusting the pH value to 8.0 to 9.0; adding composite hydrolase of alkali protease, neutral protease, papain and trypsin, and reacting for 2 to 3 hours; adjusting the pH value to 4.0 to 6.0; inactivating enzyme; when cooling to a temperature below 50 DEG C, adding filter aid enzyme; collecting filtrate to dissolve filter residues; adding composite hydrolase of acidic protease, neutral protease, papain and trypsin, and reacting for 1 to 2 hours; inactivating the enzyme; when cooling to temperature of below 50 DEG C, adding the filter aid enzyme; filtering by a paperboard; performing ultrafiltration; drying by microwaves under vacuum; and crushing and sieving. The soybean polypeptide powder prepared by the method can improve the solubility of albumen powder to a maximum limit and keep the color and luster of appearance, mouthfeel, flavors and the bioactivity of active ingredients, offers a good mouthfeel, is easy to absorb and can be widely applied to industry of dairy products, drinks and health-care products.

The present invention is separating and culturing process of human amnion mesenchyme stem cell and its medical composition. The separating and culturing process includes digesting human amnion successively with trypsin, collagenase and deoxyribonuclease, and filtering to prepare single cell suspension; culturing in DMEM / F12 culture medium with VDMEM and VF12 in the equal ratio and containing ox embryo blood serum in 10-20 vol% and basic fibroblast growth factor of ultimate concentration 10-20 ng / ml inside a culture box at 37 deg.c, saturated humidity and CO2 in 5 vol%; and replacing liquid and culture passage to proliferate and purify human amnion mesenchyme stem cell. The process has wide material source no ethnic limitation and wide application foreground. The medical composition may be used in various kinds of treatment.

The invention relates to a quantitative detection method for thermal-denaturation and non-denaturation bovine alpha-lactalbumin in milk and milk products by applying an enzymolysis-liquid chromatography and mass spectrometry combination technology. The quantitative detection method comprises the steps as follows: taking a certain amount of milk or milk samples, dissolving and diluting the milk or milk samples in water to obtain solution with total protein content being about 1mg / mL; after volume metering, correctly sucking 500 mu L, adding an internal standard substance, reacting disulfide bond with dithiothreitol (DTT), alkylating to protect sulfydryl produced in reaction by iodoacetamide (IAA), and then conducting constant-temperature and constant-time enzymolysis with trypsin; and separating enzymolysis products by reversed phase liquid chromatography, conducting detection with a mass spectrum multiple reaction monitoring (MRM) manner, and calculating the result by an internal standard method. The quantitation limit of the method is 0.001g / 100g; when adding amount is 0.2, 1.7 and 5.0g / 100g, the recovery rate is 98.9-110.8% (n is equal to 6) and repeatability: RSD (Relative Standard Deviation) is smaller than 7.6%; and the quantitative detection method can be applicable to the quantitative detection of samples with different contents of bovine alpha-lactalbumin.

The invention discloses lactobacillus plantarum with broad-spectrum antifungal properties. The lactobacillus plantarum is named lactobacillus plantarum AB-1 and is preserved in China General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms with a preservation number of CGMCC No.9653 on September 15th, 2014. The lactobacillus plantarum shows relatively strong broad-spectrum antifungal properties in whole-milk culture mediums and participant compounded and fermented yoghourt; when the lactobacillus plantarum participates in the fermentation of the yoghourt, the flavor features of the fermented yoghourt can be effectively enhanced, and supernatant of the compounded and fermented yoghourt can still show relatively good broad-spectrum bacteriostasis after being treated by pepsin and trypsin. The relatively high survival rate of the lactobacillus plantarum in gastrointestinal fluid further proves that the lactobacillus plantarum has excellent probiotic properties and relatively strong survival ability in human bodies. Besides, the lactobacillus plantarum has relatively good bacteriostasis for fusariwn oxysporum and phytophthora drechsleri.

The present invention relates to a method for isolating and culturing adult stem cells derived from human amniotic membrane in high yield, and more particularly to a method for obtaining a large amount of adult stem cells, the method comprising obtaining amniotic epithelial cells from human amniotic tissue in high yield by treatment with dithiothreitol (DTT) and a low concentration of trypsin and culturing the amniotic epithelial cells in a medium containing a Rho-associated kinase inhibitor. The human amniotic epithelial cell-derived stem cells are easily extracted compared to existing therapeutic stem cells such as umbilical cord blood stem cells and bone marrow stem cells, the yield and proliferation thereof are significantly increased by DTT treatment, the addition of the ROCK inhibitor or the replacement of medium. Thus, the method can be used to efficiently prepare adult stem cells.

The invention relates to a method for producing a soybean peptide. In the method, fermented soybean meal is taken as a raw material and then animal-derived protease is utilized to produce the soybean peptide. The method comprises the following steps of: 1) enzymolyzing liquid, namely mixing the fermented soybean meal and water in a weight ratio of 1:(4-8), adding trypsinase, and preserving heat and enzymolyzing; 2) killing enzyme at high temperature; 3) filtering, namely filtering and separating enzymatic hydrolysate to obtain crude soybean protein; 4) decolorizing and deodorizing; 5) refining; and 6) spray-drying, and sterilizing to obtain a finished product, wherein the decolorization and deodorization are realized through filtration by a pretreated macroporous resin. The method has theadvantages that: 1) the fermented soybean meal is taken as the raw material, so that a protease inhibitor in the raw material is eliminated; 2) the prehydrolysis of soybean protein in the fermented soybean meal can be completed; and 3) the amount of the additional enzyme is reduced greatly, so that the cost is effectively reduced and the higher hydrolysis rate is obtained.

The invention discloses a method for extracting an anti-gastric cancer active component from water chestnut shells. The method sequentially includes the following steps that: (1) water chestnut shell powder is added with buffer solution with a pH value being equal to 2 and pepsin, kept still for extraction under 37DEG C of water bath and centrifugated; (2) filter residue obtained in step 1 is added with buffer solution with a pH value being equal to 8 and trypsin, kept still for extraction under 37DEG C of water bath and centrifugated; and (3) supernate obtained in step 2 is centrifugated at 12000 to 14000 rotations per minute for 25 to 35min, and after centrifugation, the obtained liquid is lyophilized, so that the anti-gastric cancer active component is obtained. The anti-gastric cancer active component extracted by adopting the method is characterized by high activity.