32 results about "Metal affinity chromatography" patented technology

The present invention relates to the purification of polypeptides and the removal of endotoxin via immobilized metal affinity chromatography (IMAC). More specifically, the invention relates to methods for removing bacterial endotoxin in a protein solution. In specific embodiments, the invention relates to the elimination of endotoxin from Moraxella catarrhalis outer membrane proteins.

The present invention provides for a novel method for purification of EGFR family proteins obtained from cultures of insect cells. The process comprises subsequent steps of a) diafiltration and exchange of culture medium with buffer, b) immobilized metal affinity chromatography (IMAC), c) size exclusion chromatography (SEC), and d) anion exchange chromatography (AIE). The method also provides for an immunogenic variant of HER-2 protein for which the purification process has been especially adapted, as well as means for the preparation of the variant.

An immobilized metal affinity chromatography (IMAC) method for separating and / or purifying compounds containing a non-shielded purine or pyrimidine moiety or group such as nucleic acid, presumably through interaction with the abundant aromatic nitrogen atoms in the purine or pyrimidine moiety. The method can also be used to purify compounds containing purine or pyrimidine moieties where the purine and pyrimidine moieties are shielded from interaction with the column matrix from compounds containing a non-shielded purine or pyrimidine moiety or group. Thus, double-stranded plasmid and genomic DNA, which has no low binding affinity can be easily separated from RNA and / or oligonucleotides which bind strongly to metal-charged chelating matrices. IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions. The metal ion affinity of yeast RNA decreases in the order: copper (II), nickel (II), zinc (II), and cobalt (II).

The invention provides a method for simply and rapidly separating exosomes or exosome proteins from plasma on the basis of the immobilized metal affinity chromatography. The method at least comprisesthe following steps: 1), the exosomes are adsorbed by monodispersed immobilized affinity chromatographic microspheres CAE-Ti<4+>-IMAC; 2), the exosomes are eluted by ammonium hydroxide; and 3), phosphate buffer is immediately utilized to replace ammonium hydroxide, and damage of ammonium hydroxide to the exosomes is reduced. The collecting method has the characteristics of being low in cost, simple and fast to operate, time-saving, labor-saving and high in collecting efficiency, and besides, various kinds of subsequent molecular analysis are allowed to be performed on the exosomes with the method and include analysis for genomes, proteomes and the like. The problems of high time consumption, high cost, low purity and the like of current exosome separation and purification can be effectively solved, the feasible technical scheme is provided for research of disease plasma exosomes, and the method has good application prospects in the aspect of discovery of new exosome markers and the like.

The invention relates to an NTA-based (nitrilotriacetate) IMAC (immobilized metal affinity chromatography) filler and a preparation method and an application thereof. The prepared method of the NTA-based IMAC filler includes the following steps of providing a matrix, constructing an aldehyde group on the surface of the matrix to obtain a matrix with the aldehyde group on the surface, converting the aldehyde group to alpha-aminocarboxylic acid to obtain a matrix with the alpha-aminocarboxylic acid on the surface, constructing an NTA group on the surface of the matrix having the alpha-aminocarboxylic acid on the surface to obtain a matrix with the NTA group on the surface, and chelating the NTA group with transition metal ions to obtain the NTA-based IMAC filler. With the above-mentioned preparation method of the NTA-based IMAC filler, the use of high-cost raw materials is avoided, preparation process is clean, and purification process is simple.

The invention belongs to the field of analytical chemistry. A novel phosphate radical containing cotton modification material is prepared by adopting a graft copolymerization method. The phosphorus content of the material can be well controlled by adjusting the content of a monomer VPA, then metal ions are immobilized on the material, and the material can serve as a material for fixing metal affinity chromatography and used for selective enrichment of phosphorylated polypeptides in multiple biological samples. The optimal material CF-NH2-AZO-p(VPA-2)-Ti4+ can be optimized by researching the relation between the content of phosphate radical groups in the material and material extracting capability. The material has the high selectivity and high extraction capability to the phosphorylated polypeptides in a standard polypeptide mixed solution, milk enzymatic hydrolysate, serum and rat brain lysate. Therefore, application of the graft copolymerization method in cellulose post-modification can be promoted, and application of a cellulose adsorbent in bioanalysis is widened.

A process for the extraction, purification and enzymatic modification of β-conglycinin α′ subunit, wherein β-conglycinin is selectively extracted from ground, defatted soy, then precipitated by treatment with aqueous ethanol; the enriched fraction is then subjected to Metal Affinity Chromatography (MAC) in denaturant conditions to obtain the α′ subunit, which is treated with chymotrypsin, then subjected to a further MAC to recover the amino-terminal region of this polypeptide (MW 28,000 Da).

The invention discloses a method for evaluating the stability of metal ion bonding on a fixed metal affinity chromatographic column, which includes the preparation of epoxy silica gel, the preparation of aminocarboxylic chelating agents, and the measurement of complex stability constant and saturated adsorption capacity. Connect the aminocarboxylic chromatography column to the chromatography system, and use cutting-edge chromatography to bond different concentrations of metal ions to the aminocarboxylic chelating agent in different buffer systems. When the bonding reaches equilibrium, respective breakthrough curves are obtained. Use Peakfit software calculates their respective breakthrough times; calculates the apparent molar adsorption capacity based on the breakthrough time; substitute the metal ion concentration and apparent molar adsorption capacity into the Langmuir adsorption model to measure the fixed metal ions on the aminocarboxyl chelating column The complexation stability constant and saturation adsorption capacity. The method is fast and simple, can measure multiple adsorption parameters at the same time, and more truly and accurately reflects the adsorption stability of fixed metal ions on the aminocarboxyl chelating agent.

Different from the conventional method for purifying human serum albumin from human plasma, the invention provides a simple method for separating and purifying the human serum albumin from fermentation liquor or a mammal cell culture solution. By an immobilized metal affinity chromatography (IMAC) step, the method is suitable for purifying recombinant proteins at various salt concentrations, fermentation supernate at high salt concentration is allowed to be directly loaded, the total volume of loading buffer is effectively reduced and the cost is reduced. The method is stable and high-efficiency, and is suitable for laboratories, pilot scale tests and industrial production scale-up.

The invention relates to a high carrying capacity metal chelating affinity chromatography medium. Silica gel microspheres are adopted as a core; pores are formed inside the silica gel microspheres; carboxymethyl chitosan oligosaccharide molecules are adhered to the surfaces and the pores of the silica gel microspheres; a part of the carboxymethyl chitosan oligosaccharide molecules are complexed with first metal ions to form a first layer of metal chelating; the rest part of the carboxymethyl chitosan oligosaccharide molecules are bonded with allyl glycidyl ether; the allyl glycidyl ether is connected with aglycone; and the aglycone is complexed with second metal ions to form a second layer of metal chelating. By adopting the medium, double metal layers are successfully immobilized on the surface of the medium, the carrying capacity of the metal affinity chromatography medium is effectively increased, and the medium is simple in improvement method, low in production cost and beneficialto large-scale popularization and application.

The invention relates to the field of biomedicine and particularly relates to a method for preparing a recombinant GPx7 (glutathione peroxidase) protein. The invention discloses a method for preparing a GPx7 recombinant protein. The method comprises the steps of constructing a GPx7 recombinant protein prokaryotic expression plasmid and strain; expressing the GPx7 recombinant protein; extracting the GPx7 recombinant protein and purifying the GPx7 recombinant protein. The method provided by the invention has the advantages of simple steps, low cost and high degree of purification; the prepared protein has good activity. The method is far superior to a metal affinity chromatography used in the prior art.

A method for purification of a sulfatase using metal chelating chromatography without using tags such as His-tag, etc. is disclosed. An embodiment provides a method for purifying a sulfatase including the steps of: (a) providing a sulfatase-containing solution comprising one or a plurality of impurities; (b) performing a first chromatographic separation of the sulfatase-containing solution using a metal affinity chromatography resin; (c) performing a second chromatographic separation using a cation exchange chromatography resin; and (d) performing a final chromatographic separation using an anion exchange chromatography resin, wherein the impurities are removed thereby.

The invention belongs to the technical field of biology. The invention relates to a recombinant fibronectin with anti-wrinkle repairing functions. The recombinant fibronectin comprises at least one RGD structural domain capable of being combined with a cytointegrin receptor, and at least one sodium ion channel binding peptide fragment capable of acting on a sodium ion channel, the RGD structural domain and the sodium ion channel binding peptide fragment are connected through a polypeptide connecting fragment containing 0-300 amino acid residues, the capability of combining a sodium ion channeland an integrin receptor is realized, the effects of promoting skin repair and metabolic renewal and enhancing freckle fading and wrinkle reducing are achieved, and the process for the preparation thereof, high-density fermentation, super-tolerant metal affinity chromatography purification, specific protease label excision and ion column refining purification methods are adopted. The preparationmethod utilizes high-density fermentation, super-tolerant metal affinity chromatography purification, specific protease excision label and ion column refining purification method. The obtained recombinant fibronectin rFN does not contain a purification label, is high in purity, high in specific activity, simple and efficient in preparation process, low in cost and suitable for large-scale production, and further relates to an application of the recombinant fibronectin rFN in in-vitro adherent cell culture, skin injury repair, wrinkle removal and spot fading.

The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

The invention relates to a phosphorylated peptide adsorbent and its preparation and application. Specifically, trimethylolpropane trimethacrylate (Trimethylolpropane trimethacrylate, TRIM) and allyl glycidyl ether (Allyl glycidyl ether, AGE) are used as functional monomers, and the polymerization with epoxy functional groups is prepared by suspension polymerization. and then modified with O‑phospho‑L‑serine (O‑phospho‑L‑serine) to introduce phosphate groups directly on the surface of the microspheres, and finally with Ti 4+ After ion chelation, it can be used as an adsorbent for immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides in complex biological samples. The preparation method is simple, the raw materials are readily available, the cost is low, the reaction conditions are mild, and the method is suitable for large-scale preparation. The obtained material has stable properties, strong alkali resistance, and high enrichment efficiency and specific selectivity for phosphopeptides in biological samples.

The present invention provides improved methods for the purification of factor XIII. In particular, the methods provide compositions containing 5% or less contaminating proteins. In particular embodiments of the present invention the methods provide purified factor XIII compositions comprising less than 1% activated factor XIII, less than 2% protein aggregates, and / or less than 5% charge isomers of factor XIII. The methods do not require the use a precipitation or crystallization step common to prior methods of isolating factor XIII. Instead, the method uses immobilized metal affinity chromatography to remove various contaminants common to recombinant expression of factor XIII. Further, a combination of various chromatography methods including ion exchange chromatography, hydrophobic affinity chromatography, and immobilized metal affinity chromatography comprise a simple and less expensive method to produce a pharmaceutical grade factor XIII product at high yield.

The invention discloses a hyperbranched fixed metal affinity chromatography stationary phase represented by the general structural formula (I), which uses a hyperbranching method on the surface of the chromatography stationary phase to modify the material surface, thereby increasing the density of functional groups on the surface of the material and improving the Protein has a higher adsorption capacity. The chromatography stationary phase prepared by the invention has metal chelation chromatography separation performance. When it is used to separate lysozyme in egg white, good separation effect and high protein recovery rate can be achieved. .

The invention belongs to the field of analytical chemistry. A novel phosphate radical containing cotton modification material is prepared by adopting a graft copolymerization method. The phosphorus content of the material can be well controlled by adjusting the content of a monomer VPA, then metal ions are immobilized on the material, and the material can serve as a material for fixing metal affinity chromatography and used for selective enrichment of phosphorylated polypeptides in multiple biological samples. The optimal material CF-NH2-AZO-p(VPA-2)-Ti4+ can be optimized by researching the relation between the content of phosphate radical groups in the material and material extracting capability. The material has the high selectivity and high extraction capability to the phosphorylated polypeptides in a standard polypeptide mixed solution, milk enzymatic hydrolysate, serum and rat brain lysate. Therefore, application of the graft copolymerization method in cellulose post-modification can be promoted, and application of a cellulose adsorbent in bioanalysis is widened.