34 results about "Protease digestion" patented technology

The invention discloses a preparation method and application of GHK oligopeptide, and belongs to the technical field of biology. According to the method, a nucleotide sequence of the GHK polypeptide is obtained by designing tandem repeat GHK polypeptide ([GHK] 63) and combining preferred codons of bacillus subtilis to optimize a polypeptide coding gene. The GHK oligopeptide is subjected to recombinant expression in bacillus subtilis by utilizing a genetic engineering technology, the obtained recombinant protein is not easy to form an inclusion body and does not contain endotoxin, and the high-yield GHK oligopeptide is obtained after separation, purification and protease digestion treatment. According to the GHK oligopeptide preparation method provided by the invention, extra optimization treatment does not need to be carried out on tandem repeat GHK polypeptides, the yield of the GHK oligopeptide is improved, the whole production process is simplified, the cost is low, and the method is suitable for large-scale production of blue copper peptides and has wide application prospects in the fields of medicines, medical beauty and the like.

Methods are provided for the isolation and analysis of circular DNA from complex samples, based on the topology of the DNA molecule. A sample comprising DNA species is combined with a chaotropic dense salt solution. A fraction containing the circular DNA of interest is isolated and dialyzed to remove excess salt. In some embodiments salt gradients are generated by ultracentrifugation in the absence of intercalating dyes, e.g. ethidium bromide; and in the absence of protease digestion. The circular DNA thus isolated is substantially pure, e.g. greater than about 75%, greater than about 80%, greater than about 90%, greater than about 95% of DNA in the isolated fraction is comprised of circular DNA.

This invention discloses a method for preparing and applying plant sulfonated small peptide PSK based on synthetic biology, belonging to the field of biotechnology. Based on the design concept of synthetic biology, this invention constructs an orthogonal synthetic system composed of genetic code expansion technology and SUMO fusion protease digestion technology. A specific PSK dotted vector and a pUltra-sY vector are co-transformed into *E. coli* to construct a genetically engineered strain. The sulfonated small peptide PSK precursor is expressed in this genetically engineered strain, and mature plant sulfonated small peptide PSK is obtained through specific hydrolysis by SUMO protease. This method significantly improves the synthesis efficiency and purity of sulfonated small peptides and has broad application prospects in agricultural production and biotechnology.

This invention provides a method for forming a mammalian acoustic extracellular matrix (ECM) hydrogel without the use of acidic or alkaline solutions or protease digestion, as well as the resulting acoustic ECM hydrogel. [Solution] A method for producing an extracellular matrix (ECM) hydrogel is provided, comprising using ultrasonic frequencies to solubilize mammalian ECM in a liquid to produce a liquid-phase acoustic ECM hydrogel.

A method of pretreating a blood sample for measuring ATP of a pathogenic microorganism in blood, comprising: preparing a pellet of platelets and the pathogenic microorganism from the blood sample; and subjecting the pellet of platelets and the pathogenic microorganism to the following steps (A) to (C) in any order (including multiple simultaneous steps). A method of pretreating a cultured blood sample, wherein the method is a simplified method, does not comprise the following step (A), and comprises the steps (B) and (C) only simultaneously performed. (A) digesting cell membrane proteins of the platelets with a protease; (B) swelling the platelets in a hypotonic solution; and (C) disrupting cell membranes of the platelets with a detergent solution under a condition of suppressing an effect on the pathogenic microorganism.

The application discloses a DNA / RNA co-extraction method and kit based on a double-water-phase temperature-induced phase separation, which utilizes polyethylene glycol-sodium citrate to form a primary double-water-phase system at 18-25 DEG C, and by adding an ionic liquid modified temperature-sensitive polymer (IL-NIPAM) with a LCST of 32 DEG C plus or minus 1 DEG C, the IL-NIPAM selectively binds with RNA, and the separation of RNA and DNA in a biological sample is realized by temperature regulation. The DNA / RNA co-extraction kit includes a double-water-phase premix, and the IL-NIPAM is added in the double-water-phase premix in advance or during use, and the use concentration of the IL-NIPAM is 0.5-1.5% w / w. The method does not need proteinase K digestion and does not need to add organic solvents, and can quickly and efficiently recover DNA, mRNA and miRNA, and is suitable for the co-extraction of nucleic acids from trace samples such as fingertip blood, dry blood spots and FFPE sections, and is suitable for automation and large-scale production.

The present invention provides a method for making a transparent stained specimen of medium and large adult fish individuals, belonging to the field of specimen making. The method includes sample pretreatment, specimen making, and specimen preservation; the specimen making includes scaling and skinning, fixing with absolute ethanol, bone staining, pretreatment for clearing, and clearing; the present invention adopts the method of precise targeted intramuscular injection of trypsin digestion solution, which can accelerate the digestion process and thus shorten the production process; the present invention immerses the digested specimen successively in two potassium hydroxide-water-glycerol-diethylene glycol systems, and then transfers it to diethylene glycol-glycerol to complete the clearing, which can not only make the clearing faster and more complete, but also exclude the interference of histamine, pigments, etc., so that the spatial structure of the internal bones of the fish body can be observed through the cleared soft tissues without removing the soft tissues, and a transparent bone stained specimen of adult fish individuals with a body length of 10-50 cm and a body weight of 0-1 kg is successfully made.

In the analysis method according to the present invention, a predetermined peptide is separated by immunoprecipitation using an antibody which specifically binds to either an N-terminus or a C-terminus of the predetermined peptide. The separated predetermined peptide is digested with a protease to prepare peptide fragments, and among the peptide fragments, a peptide fragment at a terminus opposite to a terminus binding to the antibody is mass-spectrometrically detected.

The invention provides a method for efficiently preparing high-purity NudCD3 protein in an in-vitro eukaryotic cell. The method comprises the following steps: synthesizing a target gene; carrying out enzyme digestion on the target gene and the pLEXmMBP vector, and connecting to obtain a recombinant vector; transforming the recombinant vector into competent cells, culturing, and extracting plasmids; transfecting an HEK 293F cell, culturing, and collecting the cell; resuspending the cells in a lysis buffer solution, carrying out ultrasonication and centrifugation, and taking a supernatant; and carrying out affinity chromatography column purification, Prescission protease digestion and molecular sieve purification to finally obtain the NudCD3 protein with the purity of more than 90%. The problems that in the prior art, NudCD3 protein is difficult to express in quantity and insufficient in purity are solved, high-purity protein is provided for structural biology research, drug screening and antibody preparation, and the method has the advantages of being easy to operate, high in yield, high in protein stability and the like.

This document discloses polypeptides comprising an extended recombinant polypeptide (XTEN) containing multiple overlapping sequence motifs and one or more barcode fragments, which are releasable upon protease digestion and detectable from all other protease-releasable fragments. Some embodiments of these polypeptides also comprise a bioactive polypeptide, wherein advantageous embodiments include a protease-cleavable releasable segment capable of cleaving the link between the XTEN polypeptide and the bioactive polypeptide. Methods for preparing and using the polypeptides are also disclosed.

A derivative of insulin aspart and a method for preparing the same, the derivative comprising a fusion protein of a green fluorescent protein folding unit and an insulin aspart precursor or an active fragment thereof. The fusion protein is expressed in a significantly increased amount, the insulin aspart precursor protein in the fusion protein is folded correctly and has biological activity. Furthermore, the green fluorescent protein folding unit in the fusion protein can be digested by a protease into small fragments, and has a large difference in molecular weight compared to the target protein, and is easy to separate. A method for preparing insulin aspart and an intermediate using the fusion protein is also provided.

A method for quantifying active orexin A in a specimen, comprising: a step of contacting the specimen with a monoclonal antibody that recognizes the C-terminal side of orexin A to separate orexin A species; a step of digesting the separated orexin A species with a protease to obtain a peptide consisting of an amino acid sequence of SEQ ID NO: 1; and a step of performing mass spectrometry on the peptide.

The invention belongs to the technical field of biological tissue engineering, and discloses a fat acellular extracellular matrix hydrogel preparation method, which comprises: (1) cutting cleaned fresh fat into pieces, and repeatedly freezing and thawing with a hypertonic salt solution; (2) homogenizing the frozen and thawed fat at low temperature, and collecting and precipitating; (3) re-suspending the precipitate by using PBS, oscillating in a shaking table, centrifuging at room temperature, and collecting the precipitate again; (4) carrying out degreasing and decellularization treatment on the collected precipitate by using a composite eluent; and (5) washing the precipitate with isopropanol for 24 hours, finally repeatedly washing the precipitate with distilled water, and collecting the precipitate, namely the fat acellular matrix. And (6) digesting the fat extracellular matrix with pepsin hydrochloride, and adjusting the pH value to be neutral with sodium hydroxide to obtain the fat extracellular matrix hydrogel. According to the preparation method of the fat extracellular matrix hydrogel, biological enzyme and high-speed centrifugation are not needed in the decellularization stage, the technology is simple, time consumption is short, the yield is high, the fat extracellular matrix prepared through the method is easy to digest, and the prepared hydrogel solution is uniform, good in injectability and capable of being used for production of a filling material for injection.

Disclosed herein are polypeptides comprising an extended recombinant polypeptide (XTEN) comprised of a plurality of overlapping sequence motifs and one or more barcode fragments releasable upon protease digestion and detectable from ail other proteolytic-ally releasable fragments. Certain embodiments of these polypeptides further comprise a biologically active polypeptide, wherein advantageous embodiments thereof comprise a releasable segment capable of proteolytic cleavage that cleaves the linkage between the XTEN polypeptide and the biologically active polypeptide. Methods of making and methods of using said polypeptides are also disclosed.

The invention belongs to the field of medicines, and particularly relates to a CD71-targeting ROS responsive micelle as well as a preparation method and application thereof. In order to improve the efficacy of an anti-tumor drug in treating AML patients and reduce toxic and side effects, the invention provides a nano drug delivery system, namely a CD71-targeted ROS responsive micelle, which comprises the following components: amphiphilic molecules with ROS response, DSPE-PEG-DT7 and an anti-tumor drug. The micelle provided by the invention adopts a small molecule polypeptide ligand, so that competition with endogenous Tf is avoided, the micelle has protease digestion resistance, and the targeting property in blood and the in-vivo stability are improved; an amphiphilic block containing ROS responsiveness is introduced as a micelle skeleton, so that specific drug release in AML cells is realized, the curative effect is enhanced, and the side effect is reduced; aiming at AML (acute myelogenous leukemia) cells with ROS (reactive oxygen species) rise caused by FLT3-ITD mutation, the micelle disclosed by the invention can be used for effectively releasing drugs and generating a stronger drug effect reaction.

The invention relates to the technical field of borosilicate glass, in particular to a sample DNA (deoxyribonucleic acid) rapid extraction method which comprises the following steps: step S1, sample pretreatment: taking 20-100 mu L of liquid sample or 25-50 mg of solid sample, adding 100-200 mu L of pretreatment liquid with the pH value of 7.5-8.0, and uniformly mixing by vortex for 10-30 seconds; s2, synergistic cracking and adsorption: adding 150-200 [mu] L of a composite cracking solution and 10-20 [mu] g of modified diatomite into the pretreatment solution, incubating at 55-60 DEG C for 3-4 min, and reversing and uniformly mixing twice during incubation; s3, impurity removal: adding 150-200 [mu] L of a washing liquid I, standing at room temperature for 1 min, and pouring to remove a supernatant; then adding 200 [mu] L of a washing liquid II, standing for 30 s, and dumping to remove the supernatant. By optimizing the composite lysate, the modified diatomite and reaction conditions, rapid DNA extraction of which the whole process is less than or equal to 8 minutes without protease K digestion and high-speed centrifugation is realized, the method has wide sample adaptability, high automation compatibility and stable DNA yield and purity, the extraction efficiency is greatly improved, and the operation cost and threshold are reduced.

An automatic extraction device for large-volume free nucleic acid through a centrifugal column method comprises a back plate, a shifting mechanism and a reagent filling module, the shifting mechanism and the reagent filling module are sequentially and fixedly arranged on the back plate, and a centrifugal column filtering module with a negative-pressure liquid pumping module is arranged at the bottom of the reagent filling module. Through a centrifugal column specifically combined with nucleic acid and a special reagent filling module, after a sample is subjected to cracking and protease K digestion treatment, nucleic acid in the sample is rapidly adsorbed and rapidly washed through a filter membrane, so that protein, pigments, lipids and other impurities are removed to the maximum extent; the obtained total nucleic acid can be stored in an eluent for a long time to be used for a next experiment. The whole device can realize centrifugal column method large-volume free nucleic acid automatic extraction for extracting, enriching and purifying the free total nucleic acid from fresh or frozen serum, plasma, lymph and other cell-free body fluids.

Provided is a novel antibody analysis method that does not require capture or immobilization through an Fc structure. Specifically provided is a method for preparing a peptide fragment, the method comprising: a substrate immobilization step in which a target substrate antibody 125 in a biological sample is immobilized in a pore 129 of a porous body 120 that has affinity for a Fab domain of the substrate antibody 125; and a digestion step in which the porous body 120 on which the substrate antibody 125 has been immobilized is brought into contact with fine particles 110 each having a surface on which a protease 115 has been immobilized, whereby the digestion of the substrate antibody 125 with the protease is performed and a peptide fragment of the Fab domain is produced. In the substrate immobilization step, the Fab domain of the substrate antibody 125 is immobilized in the pore 129 of the porous body 120, and the average particle diameter D11 of the fine particles 110 is larger than the average pore diameter D12 of the porous body 120.

The invention provides a designable fusion protein for expression of small-molecule collagen peptide as well as a recombinant vector and a recombinant expression method thereof, and belongs to the technical field of recombinant protein expression. The fusion protein for collagen peptide expression comprises the following connected fragments: a soluble tag, a purification tag, a connecting peptide, a protease digestion recognition sequence and a designed target collagen peptide, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO: 1. And the recombinant fusion protein is subjected to purification, enzyme digestion treatment, re-purification, refining and separation to obtain a peptide fragment with a target molecular weight. Experiments show that the method can obtain the small molecular collagen peptide with small molecular weight, designable sequence and yield reaching the level of gram per liter on the premise of not depending on chemical synthesis and subsequent random enzymolysis, and the scheme is simple and easy to implement, stable in preparation process, high in purity and suitable for industrial large-scale production.

We disclose a composition comprising a plurality of plant derived peptides obtained by protease digestion prepared from a plant of the genus Brassicae and its use in the enhancement of cellular metabolism and in the treatment of diseases, for example diseases associated with muscle atrophy such as muscular dystrophy or sarcopenia.

The invention provides a method for detecting bacteria. The method utilises a peptide that forms a complex with a conjugated reporter polymer and is susceptible to cleavage by one or more proteases on the surface of a bacteria. Presence or absence of the bacteria can be determined by assessing the optical absorption and / or colour and / or photoluminescence (e.g. fluorescence) of the conjugated reporter polymer, which may undergo a conformational change after binding with the to the cleaved peptide substrate. Specifically, the peptide substrate may comprise a cleavage site for digestion by the protease, and the protease may be an omptin protease. The conjugated reporter polymer may be selected from a polythiophene, a poly(1,4-phenylene vinylene) (PPV), a poly(1,4-phenylene) (PPP), a polyfluorenes (PFO), a nitrogen-containing polymer such as polyquinoline, poly(2,5-pyridinevinylene) (PPyV), 1,3,4-oxadiazole, and poly(9-vinylcarbazole) (PVK), and a polypyrrole. The method may be used to detect contamination in food or water, or as a clinical and / or diagnostic test.

The present invention discloses polypeptides for promoting osteoblast proliferation and differentiation and their applications, relating to the field of biotechnology. Using gelatin from tilapia fish scales and fish skin as raw materials, through means such as protease digestion technology, LC-MS / MS peptide identification, computer simulation screening combined with molecular docking-assisted screening, three polypeptides GM-15, AG-17, and GN-18 with the ability to promote osteoblast proliferation and improve differentiation activity are provided. These three polypeptides have relatively high cell proliferation-promoting activity, and the maximum proliferation rate can reach 124%. At the same time, these three polypeptides exhibit a strong effect on promoting osteoblast differentiation and mineralization. The three polypeptides provided by the present invention have the characteristics of good water solubility, non-toxicity, non-allergy, and strong activity in promoting osteoblast proliferation and differentiation, and have good application prospects in the fields of orthopedic drugs, health products, functional foods, etc. related to promoting osteoblast proliferation.

The present invention relates to barcoded XTEN polypeptides and compositions thereof and methods of making and using the same, specifically disclosing polypeptides comprising an extended recombinant polypeptide (XTEN) comprising a plurality of overlapping sequence motifs and one or more barcode fragments that are releasable upon protease digestion and detectable from all other proteolytically releasable fragments. Certain embodiments of these polypeptides further comprise a biologically active polypeptide, with advantageous embodiments thereof comprising a proteolytic cleavage releasable segment capable of cleaving the linkage between the XTEN polypeptide and the biologically active polypeptide. Methods of making the polypeptides and methods of using the polypeptides are also disclosed.

Methods are provided for the isolation and analysis of circular DNA from complex samples, based on the topology of the DNA molecule. A sample comprising DNA species is combined with a chaotropic dense salt solution. A fraction containing the circular DNA of interest is isolated and dialyzed to remove excess salt. In some embodiments salt gradients are generated by ultracentrifugation in the absence of intercalating dyes, e.g. ethidium bromide; and in the absence of protease digestion. The circular DNA thus isolated is substantially pure, e.g. greater than about 75%, greater than about 80%, greater than about 90%, greater than about 95% of DNA in the isolated fraction is comprised of circular DNA.

Provided herein are methods and systems for the simultaneous targeted detection and sequencing of DNA, RNA, and Protein. In typical embodiments, the DNA, RNA, and Proteins are detected, characterized, and sequenced using just a single mammalian cell. One embodiment of detecting and characterizing DNA, RNA, or protein from a mammalian cell includes encapsulating a single cell in a drop and performing a protease digest on the encapsulated cell drop, performing a reverse transcriptase reaction; performing a droplet merger with barcoding PCR reagents and barcoding beads; performing a PCR reaction to attach the cell barcodes to the DNA targeted amplicons, RNA targeted amplicons, and protein tag amplicons, where all amplicons from the same emulsion contain the same cell barcode; and detecting and characterizing a DNA, RNA, or protein amplicon by sequencing the cell barcode incorporated into each amplicon.

Provided herein is a sensitive, quantitative, and scalable targeted proteomics assay of Alzheimer's Disease biomarkers representing neuronal, glial, vasculature and metabolic pathways. The biomarkers are protease-digested peptides selected from biological samples of individuals having normal Aβ and Tau levels (AT−) and from symptomatic and asymptomatic individuals having low Aβ and high Tau levels (AT+). The assay uses selective reaction monitoring-based mass spectrometry (SRM-MS) of peptides in the biological samples after digestion.

Provided is a culture method for high-purity Muse cells. The culture method comprises the following steps: S1, incubating mesenchymal stem cells with an SSEA3 primary antibody, performing centrifugation, removing a supernatant, adding immunomagnetic beads for incubation, and performing magnetic separation to obtain Muse cells; S2, culturing the Muse cells obtained in step S1 and a microcarrier in a culture medium under stirring at a rate of 40-60 r / min, performing digestion and passaging by means of using a pancreatic enzyme or protease, and replenishing the culture medium and the microcarrier; and S3, centrifugally washing the Muse cells cultured to third to sixth generations, detecting SSEA3 and CD105 phenotypes, performing cell counting, and performing cryopreservation. By means of combining the magnetic separation and microcarrier suspension culture, i.e. using the magnetic separation to improve the purity of Muse cells at the beginning, and then using the microcarrier to organically combine 3D spheroid culture and microsurface adherent culture, the present invention achieves amplification of 10 folds or more simple in about ten days, and the purity is higher than 80%. Therefore, the present invention can obtain high-purity Muse cells of clinical order of magnitudes in short time.

Provided are a somatostatin derivative and a method for preparing the same. Specifically, provided is a fusion protein comprising a green fluorescent protein folding unit and a somatostatin precursor or an active fragment thereof, and the expression amount of the fusion protein is significantly improved. Also, the green fluorescent protein folding unit in the fusion protein can be digested into small fragments by a protease, and the molecular weight difference is large compared to the target protein, and it is easy to separate. Also provided is a method for preparing somatostatin and an intermediate using the fusion protein.

To provide a bispecific T cell engager (TCE) cancer therapeutic agent having an increased therapeutic index.SOLUTION: (a) an extended recombinant polypeptide (XTEN), wherein the XTEN comprises a barcode fragment (BAR) that is releasable from the polypeptide upon digestion by a protease; And (c) a polypeptide having N - and C-terminal amino acids comprising a release segment (RS) located between the XTEN and the diabody construct, wherein the diabody construct specifically binds to CD3 and comprises CDR - L1, CDR - L2 and CDR - L3, and CDR - H1, CDR - H2 and CDR - H3, wherein the CDR - H3 comprises a specific amino acid sequence, wherein the first antigen-binding fragment (AF1) specifically binds to human epidermal growth factor-receptor 2 (). HER2 AF2.SELECTED DRAWING: None

The present invention provides a method for simultaneously enriching intrinsically disordered proteins and their bound three-dimensional genomic structures, which relates to the technical field of molecular biology. The method of the present invention comprises the following steps: after crosslinking cells with formaldehyde and disuccinimidyl glutarate (DSG), performing digestion treatment, end repair and proximity ligation with the restriction endonuclease Dpn II; after ligation, ultrasonically fragmenting the cell nucleus to obtain an ultrasonically fragmented product, dividing the ultrasonically fragmented product into two parts, biotinylating isoxazole to capture the DNA and protein complex with one part of the product, digesting the protein with protease, extracting and purifying the DNA, and constructing a library and sequencing the purified DNA; treating the other part of the product with nuclease, extracting the protein, and then performing proteomic analysis. The present invention can achieve non-destructive detection of intrinsically disordered proteins and their bound chromatin three-dimensional structures throughout the genome, providing an effective way for their structural analysis.