37 results about "Recombination Protein A" patented technology

The present invention relates to a recombinant protein A and applications thereof, and particularly discloses a novel recombinant Staphylococcus aureus protein A, which has a structure defined in the specification, wherein the function comprises but is not limited to application as a ligand-specific binding immunoglobulin, and the recombinant protein A can be used as a specific affinity ligand, and has excellent alkali resistance. The invention further relates to a method for constructing the mutant protein.

The invention relates to a method for separating and purifying a recombinant protein A, belongs to a protein separation and purification technology in the technical field of bioengineering and provides a method for separating and purifying a recombinant protein A by combining heating treatment and activated carbon adsorption. The purity of the recombinant protein A obtained by the method through separation and purification can be up to 95% or above, the final protein A recovery rate can be up to 60% or above, the endotoxin content in the recovered protein solution is less than 10 EU/mg, and the endotoxin content can be reduced to 1 EU/mg or below if hydrophobic chromatography is further included in the purification method. The method has the advantages of low cost and high recovery rate, is convenient to realize industrial production and provides a feasible route for the large-scale production of the recombinant protein A.

The present invention discloses a performance improved recombination staphylococcus aureus protein A affinity ligand and a construction method thereof. The present invention adopts a molecular biology method. A sequence B of a nature protein A is selected for molecular transformation. A C-terminal of the sequence B is added with two cysteines, so that the protein A can pass through double-locus coupled chromatography matrix to stabilize the connection. Six glycines are added to the end of a second Loop of the sequence B to increase the length and reduce the binding force with an antibody, so that elution conditions are mild. On this basis, resistance performance to high concentration base of the protein A is transformed. Asparagines and phenylalanine at 23rd and 30th positions of the sequence B are respectively replaced by threonine and alanine to obtain a sequence Z with higher alkaline resistance properties. Then, isocaudarner is used for connecting sequence Zs of different numbers head-to-tail in series. The efficient expression system of e. coli is used for overexpression. The expressed recombination protein A is coupled to agarose matrix preparation affinity chromatography fillers and is used for purifying antibodies. Results show that the recombination protein A affinity ligand prepared by the present invention is good in elution performance and alkali resistance.

The invention discloses a protein A immunosorbent material for adsorbing an antibody, and the protein A immunosorbent material is a polymer material coupled by agarose gel and protein A, wherein the protein A is a recombinant protein A with cysteine. The protein A immunosorbent material is prepared through the following three steps: preparing the recombinant protein A, activating the agarose gel, and synthesizing the immunosorbent material. The protein A immunosorbent material can adsorb the pathogenic antibody related to immunological diseases of the material from body fluid directly, efficiently and selectively, can safely perform extracorporeal circulation, and realizes once one-off perfusion to achieve the aim of removing the pathogenic antibody of a patient.

The invention relates to a separation and purification method of recombinant proteins A, which comprises the steps of: preparing batch IgG Fc segments; directionally immobilizing the Fc segments; and separating and purifying the recombinant proteins A through an affinity chromatography technology by using the Fc segments as aglycones. The method belongs to the technical field of biological engineering. The purification of the proteins A finally obtained and detected by SAS-PAGE and HPLC is more than 95 percent. The method has the advantages of lower cost, high efficiency and simple and convenient operation, and not only can be used for purifying the proteins A in a laboratory scale way, but also can be used for producing industrialized proteins A.

The invention relates to a method for producing recombinant protein A, which belongs to the technical field of biological engineering and provides a technological condition for fermenting and expressing the recombinant protein A in high density by using genetic engineering bacteria, and a method for separating and purifying the recombinant protein A in high purity by using Fc segment of IgG as anaffinity ligand by a one-step affinity chromatography method. By applying the method for producing the recombinant protein A, the culture density of the recombinant bacteria fermented in high densityreaches OD600nm=80-100; the dry weight of the bacteria is 40-50g/L; the expression quantity of the protein A occupies 30% to 50% of total protein of the bacteria; the quantity of the protein A produced by each liter of bacterial liquid reaches 3g; and the purity of the protein A detected by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and HPLC (High Performance Liquid Chromatography) is more than 95%. The method has the advantages of large yield, low cost, high product quality, and the like, and provides a practicable approach for preparing the recombinant protein A.

The invention provides a method for removing endotoxin in a recombinant protein A solution. The method is good in endotoxin removal effect, high in the recovery rate of recombinant protein A and greatly reduced in cost. The method comprises the following steps: (1) heating a bacterial suspension containing recombinant protein A with a histidine tag so as to fully fragment bacteria; (2) adding polyethyleneimine and successively carrying out uniform mixing and centrifugation so as to obtain supernatant containing the recombinant protein A, wherein a weight ratio of the bacterial suspension containing recombinant protein A to polyethyleneimine is 10000: 1 to 100: 1; (3) allowing the supernatant to pass through a metal ion chelated chromatography column and flushing a medium by using a PBS buffer solution so as to obtain a pretreated recombination protein A solution; (4) removing a salt so as to obtain a desalted recombination protein A solution; and (5) purifying the desalted recombination protein A solution with an affinity chromatographic filling material so as to obtain the recombinant protein A solution without endotoxin, wherein the affinity chromatographic filling material can specifically bind to endotoxin.

The invention provides functionalized bacterium magnetic particles for expressing specific recombinant proteins. The bacterium magnetic particle membrane protein is fused with a flexible polypeptide chain to express the specific recombinant proteins. The specific recombinant proteins are Z structure field dimers improved on the basis of recombinant protein A or C structure field dimers improved on the basis of recombinant protein G, and the gene sequences are particularly modified, so that the prepared functionalized bacterium magnetic particles have excellent IgG combining capacity.

The invention belongs to the field of biological separation engineering and relates to a protein A immune adsorption material used for purifying blood and a preparation method thereof. The invention discloses a biopolymer separating material coupled with agarose gel microspheres and recombinant protein A. An immune adsorption material with high protein A absorption performance is prepared according to the following steps: taking agarose gel as a carrier, activating with allyl glycidyl ether, coupling with sulfydryl on recombinant protein A amino terminal (C terminal) cysteine, and finally, blocking the terminal, thereby fixing the C terminal of the protein A on the agarose gel microspheres at a fixed point. The material has the advantages that the recombinant protein A on the agarose gel microspheres is uniform in structure and fixed in sequence, so that the adsorption capacity of the adsorption material for an antibody is promoted, and the adsorption material can be used for clinical immune adsorption treatment.

The invention, pertaining to the field of genetic engineering techniques, relates to an artificially reconstituted tetrad protein A and a constructing method and usage thereof. The artificially reconstituted tetrad protein A of the invention has an amino acid sequence shown in SEQ ID NO.1 of the sequence listing. The activity of the reconstituted protein A of the invention exceeds that of the reconstituted protein A reported and is slightly lower than the level of natural protein A with high production and relatively simple purification technique, thus avoiding the danger of pathogenic bacteria in naturally extracted proteins and being able to produce in a large scale.

The invention relates to high-stability Recomb Protein A having an antibody binding capacity and preparation thereof. The invention further provides affinity chromatography columns which contain the high-stability Recomb Protein A having an antibody binding capacity. Compared with wide-type Protein A, the high-stability Recomb Protein A having an antibody binding capacity is greatly improved on stability and has a higher antibody binding capacity.

The invention provides a recombinant protein A gene with a nucleotide sequence expressed as SEQ ID No: 1 and recombinant protein A coded by the gene. The invention also provides a recombinant expression vector PET32a-P for expressing the recombinant protein A gene, escherichia coli BL21/DE3 transformed by the recombinant expression vector and a method for preparing the recombinant protein A. The recombinant protein A provided by the invention is used for antibody detection, separation and purification, and the recombinant protein A and a chromatographic medium vector can form a compatible chromatographic medium. The recombinant protein A produced by the invention has the advantages of strong protein binding specificity, high affinity and great improvement on purification efficiency; and compared with the commercial ProteinA Sepharose 4Fast Flow, the recombinant protein A has obviously improved dynamic adsorption capacity.

The invention discloses a recombinant protein A efficiently combined with IgG (Immunoglobulin G) and a construction method of an engineering bacterium thereof. The method comprises the following steps of: designing a recombinant protein A primer for IgG antibody combining areas E, D, A, B, C of a staphylococcal protein A (SPA) according to a gene sequence announced by the NCBI (National Center For Biotechnology Information), and adding a cysteine codon into an end C primer; amplifying by taking the genome of a staphylococcus aureus as a template to obtain a gene (SPA) of an encoding recombinant protein A antibody combining area, connecting the gene (SPA) with a plasmid pMD18-T, transforming competent E.coil JM109 to obtain a large quantity of recombinant plasmids pMD18-T-spa, storing the gene and increasing the copy numbers of the gene; and extracting the recombinant plasmids, performing double digestion on the recombinant plasmids and a plasmid pET-28a by using Noc I and BamH I, connecting a recombinant plasmid pET-28a-spa, transforming competent E.coil BL21, adding IPTG (Isopropyl beta-D-Thiogalactoside) of which the finial concentration is 1mmol/L for inducing expression at the temperature 30 DEG C, and identifying whether the expression is successful by using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electropheresis).

The invention provides a fixed-point immobilization method for recombinant protein A affinity ligands. According to the method, an FGE (formylglycine generating enzyme) identification gene sequence is inserted at the tail end of a gene sequence of coding target protein with a genetic engineering means, an expression vector of the target protein is constructed, then the target protein and FGE are co-expressed in escherichia coli, and intracellular aldehyde catalytic conversion of the target protein is realized; then fixed-point immobilization is realized through selective reactions of hydrazide groups on the surface of a medium and aldehyde groups on the surface of the protein. With the adoption of the method, the target recombinant protein can be directly fixed in a broken supernatant of a cell, conditions are mild, steps are simple, the immobilization time is greatly shortened, and the immobilization cost is greatly reduced.

The invention relates to a test paper for detecting deer epizootic haemorrhagic disease virus, comprising a soleplate (7), and a sample absorption pad (1), a nitrocellulose membrane (3) and a water absorption pad (6) which are sequentially fixed on the soleplate, wherein the sample absorption pad comprises a colloid metal pad (2) which is porous fiber absorbed with colloid metal labeling of monoclonal antibodies, the nitrocellulose membrane is provided with parallel detection lines (4) and comparison lines (5), and the detection lines are coated or sprayed with deer epizootic haemorrhagic disease virus monoclonal antibodies, and the comparison lines are coated or sprayed with recombination protein. The invention also provides a preparation method of the test paper which can directly and rapidly detect the deer epizootic haemorrhagic disease virus monoclonal antigen, and has strong idiosyncracy, high sensitivity and rapid detection.

The invention discloses composite filler. The composite filler contains a compound substrate mainly prepared from agarose and cellulose and composite ligands crosslinked with the composite substrate,wherein the composite ligands comprise ion exchange ligands and affinity ligands, the ion exchange ligands are sodium metabisulfite and aminobenzene, and the affinity ligands are hydroxyapatite, protein A or recombinant protein A. The composite filler integrates characters of multiple substrates and multiple ligands, has the characteristic of high protein loading capacity, and can increase workingflow velocity, thereby effectively improving purification efficiency and effect of protein.

The invention belongs to the filed of biological pharmacy, more particularly, the invention discloses a method for preparing an affinity chromatography medium in a reaction kettle. The method is characterized in that gel activating reaction, coupling reaction of activated gel and recombinant protein A, pretreatment before reaction and after-treatment after reaction of reactant are all carried out in the same reaction kettle. The method reduces the inconvenience of using more containers in the process that reactant needs transferring or processing before or after reaction, and simultaneously also shortens the transferring and processing time to have the effects of saving time, labor and material. The invention further discloses the use of affinity chromatography medium purified antibody protein prepared by using the method.