61 results about "Antibody Affinity Chromatography" patented technology

Provided herein are fusion proteins and methods for purifying nucleic acids from a composition. The disclosed compositions and methods allow for faster, more efficient purification of nucleic acids compared to traditional affinity chromatography.

The utility model discloses a kind of device for separating and purifying cosmetic functional raw materials using affinity chromatography technology, it is related to chromatography column field, including fixed plate, chromatography column, heat preservation shell and connecting assembly, chromatography column is by effluent bucket, glass shell and top cover composition, fixed plate fixedly sets in effluent bucket outer wall, effluent bucket and top cover are with glass shell and are formed sealed cavity by thread connection, heat preservation shell is clamped by two half shells and is wrapped in chromatography column outer wall, connecting assembly is installed in fixed plate lower surface, connecting assembly is used for the fixation and separation of two half shells, the utility model is split by heat preservation shell, so that scale can be completely removed in cleaning process, improve heat transfer efficiency, ensure the uniformity of chromatography column internal temperature distribution, conducive to maintaining the stability and separation effect of chromatographic process.

This invention provides a method for screening β-galactosidase inhibitors, belonging to the field of pharmaceutical technology. The first step of this method uses fluorescence sensing technology to rapidly identify natural products with β-Gal inhibitory activity. The principle is based on the fluorescence quenching property of p-nitrophenol (PNP) on sulfur quantum dots (SQDs); β-Gal catalyzes the hydrolysis of p-nitrophenyl-β-D-galactopyranoside (PNPG) to generate the specific product PNP; in the presence of β-Gal, PNPG can be enzymatically catalyzed to generate PNP, quenching the fluorescence of SQDs; when screened for β-Gal inhibitory activity, the fluorescence of SQDs can be restored. The second step uses affinity chromatography to capture components in samples with β-Gal inhibitory activity. This method involves immobilizing β-Gal on a support material, eluting with a solvent to obtain components that can bind to β-Gal, and then using liquid chromatography-mass spectrometry (LC-MS) to identify the components. This method can rapidly and accurately screen β-Gal inhibitors from natural products.

The application provides a high-activity iPSC exosome and a preparation method and application thereof, relates to the technical field of biological medicine, and the preparation method comprises the following steps: obtaining induced pluripotent stem cells; performing flow cytometry sorting to obtain an iPSC subpopulation; performing suspension expansion culture on the iPSC subpopulation to collect a cell culture solution; sequentially performing clarification filtration, ultrafiltration concentration and CD9 antibody affinity chromatography purification, and then eluting and collecting an eluent containing exosomes. The induced pluripotent stem cells overcome donor dependence and batch difference. The specific screening of a cell subpopulation that is positive for CD105, CD73 and CD90 and maintains pluripotency eliminates heterogeneity and guarantees the high activity and uniform efficacy of the exosomes. The use of microcarriers for serum-free suspension culture breaks through the yield bottleneck and realizes large-scale production. Finally, the high-purity and high-activity exosomes are efficiently extracted through ultrafiltration and CD9 antibody affinity chromatography purification.

This invention discloses a method for preparing the multifunctional bioactive peptide Aj-UBL from sea cucumber, relating to the fields of biotechnology and biomedicine. Using the Aj-UBL coding sequence from sea cucumber as a template, the method involves PCR amplification, recombinant expression vector construction, engineered bacterial induction expression, bacterial cell disruption, affinity chromatography purification, and identification to obtain the target peptide possessing triple activities of antibacterial, antiprotozoal, and antitumor activity. This invention employs an optimized coupling process of genetic engineering and affinity chromatography to achieve high-purity, high-quality, and stable product preparation, while being environmentally friendly and free from drug resistance risks. It not only solves the problem of coordinated disease control in sea cucumber farming but also expands the high-value application of sea cucumber resources in the biomedical field, filling the technological gap in the large-scale preparation of multifunctional bioactive peptides from sea cucumber and the biological control of ciliates, possessing significant theoretical and practical application value.

The application discloses a kind of bionic transmembrane protein affinity chromatography column and its preparation method and application.The system (iSTAC) realizes the in-situ construction of transmembrane protein in highly bionic dynamic microenvironment by integrating cell-free protein synthesis, functional mesoporous silica modification and amphiphilic (AH) peptide stabilized planar lipid bilayer technology.The core is to use long-chain PEG-24 crosslinking agent to retain lipid bilayer on the surface of silica gel, provide sufficient conformational dynamic space for multi-transmembrane protein, and introduce AH peptide to repair membrane defects, ensure that the receptor realizes directional embedding while maintaining natural functional attributes.The preparation cycle is shortened from 168 hours to 5 hours, which significantly improves the efficiency of targeted drug screening and in-situ analysis of binding sites.Using this platform, 5-HT 1A Receptor agonists crocin I and crocin II with anti-insomnia and neuroprotective effects are successfully screened from saffron, and the action site is accurately depicted.

The application belongs to the field of marine biotechnology, and specifically discloses a preparation method of a long oyster insulin-like peptide receptor (ILPR) polyclonal antibody and application thereof. The method is characterized by the following steps: specific antigen epitopes of the long oyster ILPR protein are screened through bioinformatics analysis, a pET32a-ILPR recombinant plasmid is constructed and induced to express in Escherichia coli; the purified recombinant protein is used as an immunogen to immunize New Zealand white rabbits, and rabbit antisera are collected after four times of booster immunization; specific polyclonal antibodies are purified by using antigen affinity chromatography technology, and can specifically recognize the ILPR protein in long oyster tissues. The antibody prepared by the application has high specificity and sensitivity, and provides an important biological tool for studying the insulin signal transduction pathway, growth regulation and energy metabolism mechanism of long oysters.

The application provides a method for separating nucleic acid binding proteins in solid tissue cells, and the method comprises the following steps: step S1, preparing solid tissue into a cell suspension 1; step S2, cross-linking the cell suspension 1 by using a cross-linking agent to form a cell suspension 2 with nucleic acid-NABPs complexes; step S3, lysing red blood cells in the cell suspension 2 of step S2 to remove high-abundance protein interference; step S4, after the cells treated in step S3 are lysed by using an organic reagent, the nucleic acid-NABPs complexes in the solid tissue cells are selectively enriched by using affinity chromatography; and step S5, after the nucleic acid-NABPs complexes obtained in step S4 are de-cross-linked, nucleic acid binding proteins (NABPs) in the solid tissue cells are obtained.

This invention relates to a high-titer prealbumin antibody purification method and its application, belonging to the field of biotechnology. The method utilizes Protein A affinity chromatography to capture total IgG from antiserum, followed by purification using an affinity chromatography column prepared by targeted conjugation of a specific sequence peptide. The antibody purified by this method achieves a titer of 1:256,000 in ELISA detection, with a half-maximal effective concentration (EC50). 50 The antibody concentration was 3.2 ng / mL, exhibiting extremely high affinity and reactivity. When used in a double-antibody sandwich chemiluminescent immunoassay, it achieved a detection limit of 0.08 µg / mL, a wide dynamic range (0.08–400 µg / mL), and a titer retention rate as high as 88% after accelerated storage at 37°C for 14 days, demonstrating significantly better stability than antibodies prepared by traditional methods. This invention provides a highly active and stable core antibody raw material for the preparation of high-performance immunodiagnostic reagents.

The application discloses a method for removing chaperone and application thereof. The method for removing chaperone comprises the following steps: after a sample containing a target recombinant protein is loaded on an affinity chromatography column, a washing liquid containing urea and KCl is used to flush the affinity chromatography column, so as to remove the chaperone combined with the target recombinant protein. The method of the application can effectively remove the chaperone of about 60 kDa from an escherichia coli host bacterium by combined washing of urea and KCl, and has the advantages of simple operation, low cost, good chaperone removal effect for various recombinant proteins with different molecular weights, wide universality, and the like, and can significantly improve the purity and quality of the recombinant protein.

Herein is reported a method for separating full AAV particles from empty AAV particles, the method comprising the steps of applying a solution comprising full, empty and partially filled AAV particles to an AAV affinity column comprising an AAV affinity material and thereby binding the AAV particles to the AAV affinity material, wherein the solution has a pH in the range of 7.4 to pH 4,5 eluting full AAV particles from the AAV affinity column by applying a pH gradient from the pH of the solution of the first step to a pH in the range of 4 to 2.5, wherein the pH of the applying step and the pH of the eluting step differ in a least 0.5 pH units, and recovering full AAV particles from the eluate, and thereby separating full AAV particles from empty and partially filled AAV particles.

This invention relates to the field of recombinant protein technology, specifically to an α-hemolysin recombinant protein and its prokaryotic expression method. A method for preparing a high-purity, tag-free α-hemolysin monomer is as follows: an expression vector encoding the fusion protein of SEQ ID NO.7 is constructed and transformed into competent cells to obtain engineered bacteria; after culturing and inducing expression, the bacterial cells are lysed and centrifuged to obtain the supernatant; the fusion protein eluate is obtained by a first affinity chromatography on a nickel column, dialyzed, and digested with HisSUMO protease; a second nickel column chromatography is performed, and the flow-through is collected to obtain a high-purity, tag-free α-hemolysin monomer. This technical solution can solve the technical problems of existing prokaryotic expression of α-hemolysin recombinant proteins, such as severe inclusion body formation, complex purification processes, insufficient product activity, and large batch-to-batch variability, thereby obtaining a high-purity, highly active, and structurally intact α-hemolysin monomer, which has ideal application value.

The application provides a preparation method of a Rhodobacter sphaeroides CsCobT and CsCobN protein complex in vitro, and steps are as follows: constructing an expression vector pET-28a(+)‑Cs CobN , pET-28a(+)‑SUMO‑Cs CobT ; expressing and purifying His-CsCobN protein; preparing a non-tagged CsCobT protein; mixing the purified His-CsCobN protein and the non-tagged CsCobT protein to form a protein complex; separating the complex through affinity chromatography; and refining the complex through gel filtration chromatography. The application adopts a strategy of double vectors, step-by-step purification, in-vitro assembly and multi-dimensional verification, can obtain a CsCobT and CsCobN protein complex with high purity and uniform conformation, and the experimental process can be accurately controlled, the result is specific, and provides high-quality protein materials for subsequent molecular mechanism research and application development of cobalt-chelating enzyme.

The application discloses a method for preparing a reproductive vaccine based on a novel carrier protein coupled GnRH polypeptide and a product, wherein the amino acid sequence of the carrier protein is shown as SEQ ID NO.1, the gene sequence for coding the carrier protein is shown as SEQ ID NO.2, the gene for coding the carrier protein is subjected to prokaryotic expression, and then subjected to affinity chromatography, anion exchange chromatography purification, TEV enzyme cutting to remove a tag protein, and then subjected to affinity chromatography and molecular sieve to obtain the purified carrier protein, and finally covalently coupled with a GnRH polypeptide to obtain the reproductive vaccine, wherein the reproductive vaccine prepared by the application has a contraceptive effect, and high-quality, low-cost recombinant protein vaccines can be obtained on a large scale for commercialization.

This invention relates to the field of biotechnology, and more particularly to recombinant rBoNT protein, its precursor, preparation methods, and applications. The recombinant protein (rBoNT) obtained by deleting variable structural regions (1-15 amino acids from the C-terminus of the light chain and 1-3 amino acids from the N-terminus of the heavy chain) exhibits extremely strong biological activity. The recombinant strain constructed by this invention can achieve soluble expression of rBoNT, and it can be specifically activated by enzymatic cleavage, is not easily affected by protease cleavage similar to precursor toxin hydrolases, significantly improving the purity and uniformity of the target precursor polypeptide. This invention combines membrane ultrafiltration with hydrophobic and ion exchange, which, compared to traditional affinity chromatography, not only simplifies the process and reduces costs while ensuring the yield and purity of the recombinant protein, but also significantly reduces biosafety risks, facilitating the scale-up of botulinum toxin industrial production and providing a solid foundation for the large-scale production of botulinum toxin.

The present disclosure is directed to affinity chromatography devices including a fibrillated polymer membrane that contains inorganic particles having a spherical shape and a particle size distribution that has a D90 / D10 less than or equal to 3. A blend or a combination of spherical inorganic particles may be utilized. A nominal particle size of the spherical inorganic particles is from about 5 microns to about 20 microns. An affinity ligand may be bonded to the spherical inorganic particles and / or to the fibrillated polymer membrane. Also, the affinity chromatography devices have a hydraulic permeability from about 100 (X 10-12 cm2) to about 500 (X 10-12 cm2). Additionally, the affinity chromatography devices have a cycling durability of at least 100 cycles without exceeding an operating pressure of 0.3 MPa. Manifolds containing multiple affinity chromatography devices in a parallel configuration and multiple manifolds in a parallel configuration are also disclosed.

A method for isolating mRNA molecules by a combination of magnetic particles and positive pressure affinity chromatography is provided. mRNA molecules can be obtained from in vitro transcription with high yield and purity by using a combination of magnetic particles and positive pressure affinity chromatography. In particular, the method can be adapted to an automated process to obtain high quality mRNA molecules with a high-throughput simplified workflow.

This invention discloses a mutant of the RspCas12a_1 protein and its applications. The mutant is obtained by replacing at least one amino acid from D163R, G549R, G609A, and S846L in the wild-type RspCas12a_1 protein (amino acid sequence shown in SEQ ID NO.1). This invention also discloses the nucleic acid molecule encoding the mutant, a vector containing the nucleic acid molecule, and a host cell. A mutant expression vector is constructed through homologous recombination, and the target protein is obtained after induced expression and purification by nickel column affinity chromatography. This mutant can specifically recognize target DNA by binding to crRNA and, combined with RPA amplification technology, can be used in nucleic acid detection scenarios such as meat species identification. It has the advantages of high detection sensitivity, high specificity, and convenient operation, and can be widely applied in fields such as nucleic acid detection and food safety testing.

The application discloses a method for expressing BirA enzyme by using prokaryotic Escherichia coli and purifying the BirA enzyme, and relates to the technical fields of genetic engineering and protein engineering. A recombinant expression vector for expressing the BirA enzyme is a pET-28A-BirA plasmid which is constructed by inserting a BirA enzyme coding gene into a multiple cloning site of a pET-28A vector. An 8× histidine tag coding nucleotide sequence is fused to a C terminal of the BirA enzyme coding gene. The nucleotide sequence of the BirA enzyme coding gene is shown as SEQ ID NO:1. The prokaryotic expression system is simple in culture condition, short in cycle, and low in cost, and is suitable for large-scale preparation. The 8× histidine tag is introduced into the C terminal of the BirA enzyme gene, so that the expression product can be purified by one-step nickel column affinity chromatography, and the purification process is simple and high in efficiency.

This invention discloses a marine-derived superoxide dismutase (MhSOD), its preparation method, and its applications. The invention provides a superoxide dismutase MhSOD derived from the hydrothermal bacterium *Marinithermus hydrothermalis*, and establishes an efficient preparation method for MhSOD. Through systematic optimization of expression conditions, the optimal expression conditions were determined: TB medium, a final IPTG concentration of 0.7 mM, and an induction temperature of 30°C. Under these conditions, MhSOD is efficiently expressed in a soluble form. After one-step purification by nickel column affinity chromatography, a single target band free of contaminating proteins is obtained, indicating that this purification process can effectively enrich the target protein.

This invention discloses an earthworm protein peptide with in vitro thrombin-inhibiting activity and its preparation method, belonging to the field of functional food technology. The method first pre-treats earthworm raw materials to obtain earthworm protein; then, it uses pepsin and alkaline protease for stepwise enzymatic hydrolysis under varying temperature and pH, and ultrafiltration to collect short peptide solutions with a molecular weight below 3 kDa; the short peptide solution is specifically captured using an affinity chromatography material coupled with bovine thrombin, followed by elution, nanofiltration desalting concentration, and spray drying to obtain the final product. This invention fully releases the peptides through a dual enzymatic hydrolysis process and utilizes thrombin affinity chromatography to directionally enrich peptides with specific inhibitory activity, effectively solving the problems of low content and poor targeting of active ingredients in earthworm protein peptides in existing technologies, and significantly improving the purity and in vitro anticoagulant activity of the product.

This invention belongs to the field of genetic engineering and relates to a method for preparing amylase derived from halophilic archaea and its application; based on halophilic archaea... Haloarcula sp. ZJK10-12、 Haladaptatus The genes for α-amylase, maltose amylase, and glucoamylase were obtained from the whole genome sequence of sp. ZJK29-3. Recombinant plasmids were constructed using primer design and molecular cloning, and transformed into prokaryotic host bacteria to obtain the corresponding recombinant strains. The bacteria were enriched and cultured in Hv-YPC liquid medium, and the cells were collected and lysed by sonication. α-amylase, maltose amylase, and glucoamylase were obtained using nickel column affinity chromatography. The amylases exhibit excellent enzymatic properties and outstanding salt tolerance. Their application in the fermentation of high-salt, dilute soy sauce can improve raw material utilization and product quality, demonstrating good industrial applicability and promotional value.

This invention relates to the fields of enzyme engineering and bioactive peptide preparation technology, and discloses a method for preparing equine cardiac muscle peptides with antioxidant and antithrombotic effects, comprising the following steps: S1: Pre-treating equine heart tissue to obtain equine cardiac muscle plasma; S2: Compound enzymatic hydrolysis and directing agent regulation: Adding a compound enzyme system and an antithrombotic directing agent to the equine cardiac muscle plasma; S3: Affinity chromatography enrichment. This invention constructs a synergistic process of compound enzymatic hydrolysis, directing regulation, and affinity enrichment, specifically addressing the technical problems of incomplete tissue fragmentation, low enzymatic hydrolysis efficiency, insufficient yield of active peptides, and easy oxidative inactivation in equine cardiac muscle due to the high proportion of type IIa myofibrous fibers and dense connective tissue. Furthermore, it employs a specific ratio of compound enzyme system adapted to the characteristics of equine cardiac muscle, significantly improving enzymatic hydrolysis efficiency; introduces a cationic directing agent to directionally increase the proportion of antithrombotic peptides generated; and combines thrombin affinity chromatography to achieve efficient enrichment of target peptides, significantly enhancing antithrombotic function while ensuring high antioxidant activity.

Affinity chromatography devices that include a fibrillated heat treated polymer membrane that contains inorganic particles having a spherical shape and a particle size distribution that has a D90 / D10 less than or equal to 3 are disclosed. A blend or combination of spherical inorganic particles having a nominal particle size from about 5 microns to about 20 microns may be utilized. Also, the devices have a hydraulic permeability from about 200 (X 10-12 cm2) to about 700 (X 10-12 cm2). The affinity chromatography devices have a dynamic binding capacity (DBC) greater than 35 mg / ml at 10% breakthrough at a residence time of 20 seconds. Additionally, the affinity chromatography devices have a cycling durability of at least 100 cycles without exceeding an operating pressure of 0.3 MPa. Manifolds containing multiple affinity chromatography devices and manifolds in a parallel configuration are also disclosed.

The present application belongs to the technical field of blood product preparation, and particularly relates to a preparation method of intravenous human immunoglobulin, comprising the following steps which are sequentially performed: Cohn FIII supernatant is pretreated to obtain a sample I to be loaded; the sample I to be loaded is subjected to first anion exchange chromatography to obtain a first chromatography product; the first chromatography product is pretreated to obtain a sample II to be loaded; the sample II to be loaded is subjected to second anion exchange chromatography to obtain a second chromatography product; the second chromatography product is pretreated to obtain a sample III to be loaded; the sample III to be loaded is subjected to heparin affinity chromatography to obtain a third chromatography product; and the third chromatography product is configured into human immunoglobulin finished product. The technical scheme can solve the technical problems of the existing preparation method of intravenous human immunoglobulin, such as introduction of new materials, poor effect of impurity protein precipitation, high content of anti-A and anti-B, and the like, and has an ideal application prospect.