246 results about "Polyhistidine-tag" patented technology

The invention relates to a recombinant single chain tri-specific antibody, and it is characterized by that it is made by successively connecting antibody of antineoplastic related antigen, FC onnecting peptide, antihuman CD3 antibody, HSA connecting peptide and antihuman CD28 antibody. More specifically, said invention relates to a recombinant single chain tri-specific antibody; CEA-TsAb of anti-CEA, anti-CD3 and anti-CD28. Said antibody is made up by using three antibody fragments (unti-CEA single chain antibody, anti-CD3 single chain antibody and anti-CD28 single chain antibody), utilizing two connecting peptides (FC connecting peptide and HSA connecting peptide) and series-connecting them together, and on its C end C-myc tag and (His)6-tag are added. Said invention also relates to a method for constructing expressing and purifying said antibody, including exonic DNA sequence of said antibody, expression vector and host cell containing said vector.

The invention is related to a recombinant single-chain tri-specific antibody made from anti-Tumor Associated Antigen (TAA) antibody, FC interlinker, anti-CD3 antibody, HSA interlinker and anti-CD28 antibody in turn. Particularly, the invention relates to an anti-CEA, anti-CD3, anti-CD28 recombinant single-chain tri-specific antibody, CEA-scTsAb, which was constructed with three tandem antibody fragments (anti-CEA scFv, anti-CD3 scFv and anti-CD28 single-domain antibody) linked by two interlinkers (FC interlinker, HSA interlinker), and could be appended by C myc tag or histidine tag ((His)6-tag) at the C terminal. It also concerns a method for construction, expression and purification of the antibody. It also offers the encoded DNA sequence of the antibody, expression vectors and host cells for the vectors.

The invention discloses a monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and a method for detecting the nonstructural protein (NSP) antibody of a foot-and-mouth disease virus (FMDV) (FMD NSP B-ELISA); the kit comprises ELISA reaction plates, serum diluent, 25 times concentrated detergent, substrate solution, 100* concentrated ELISA detecting antibody, stop buffer, positive control serum and negative control serum; the ELISA reaction plates are two 96-pore high-affinity ELISA reaction plates, firstly 6* groups of amino acid monoclonal antibody or NSP 2C polyclonal antibody, and then FMDV 3ABC or 2C3AB NSP which is expressed by pronucleus and is provided with 6* groups of amino acid labels is captured through the monoclonal antibody or the polyclonal antibody; and compared with other similar kits, the method has higher coincidence rate and higher positive serum detection rate, and is applicable to detecting the serum of cattle, sheep, pigs and other susceptible animals.

The invention relates to a method for preparing magnetic silicon dioxide microspheres with the surfaces chelated with metal ions; the method of the inventioncomprises the following steps of preparation of the magnetic silicon dioxide microspheres, epoxidation of the magnetic silicon dioxide microspheres, carboxylation of the magnetic silicon dioxide microspheres and chelation technique of metal ions. Compared with the existing preparing method, the method of the invention has the advantages of reasonable design, simple operation and large amount of chelation of metal, etc. The magnetic silicon dioxide microspheres with the surfaces chelated with the metal ions, which are prepared by adopting the method, contain cobalt ions, have stronger bonding force with the modified microspheres so as to lead the cobalt ions to be not easy to leak out, and have higher selectivity and proper bonding strength with histidine on the protein, thus leading the magnetic silicon dioxide microspheres with the surfaces chelated with the metal ions to have high selectivity to hexameric histidine tag protein, and being easy to realize the separation and the purification of the target protein; the magnetic silicon dioxide microspheres with the surfaces chelated with the metal ions, which are prepared by adopting the method, can be used for separating the hexameric histidine tag protein.

The present invention provides a method for purifying recombinant peptides, polypeptides or proteins away from truncated or other full-length forms of these molecules. In particular the invention contemplates a method of purifying a vascular endothelial growth factor (VEGF) molecule by subjecting a biological sample containing the molecule to be purified to affinity chromatography under conditions sufficient for the full length molecules to bind and not the truncated or clipped forms. In the preferred embodiment there are two columns, the first is based on affinity for a poly his tag, the second column based on heparin binding affinity. Particularly preferred VEGF molecules are untagged VEGF-B167, hexa-His-tagged VEGF-B167, hexa-His-tagged VEGF-B186 and hexa-His-tagged VEGF-B10-108.

The invention provides immobilized transaminase. The immobilized transaminase is obtained by fixing transaminase, with histidine tag, derived from Mycobacterium vanbaalenii PYR-1 onto an enzyme immobilization carrier; the enzyme immobilization carrier is obtained by derivatization of epoxy resin through IDA (imino diethyl acetic acid) and cobalt chloride or derivatization of ionic chelate resin through cobalt chloride. The immobilized transaminase has the advantages of firmness in combination, low enzyme activity loss, simplicity in separation and high reusability; low cost of an asymmetric conversion process, mild reaction conditions, environment friendliness, simplicity and convenience in operation, easiness in industrial expanding and promising industrial application prospect are realized.

The invention provides a Beta-mannose which has amino acid sequences shown in SEQ ID: NO.2, SEQ ID: NO.4, SEQ ID: NO.6 and SEQ ID: NO.8. Or substitution, depletion or addition of one or multiple amino acids is carried out to the amino acid sequences shown in the SEQ ID: NO.2, SEQ ID: NO.4, SEQ ID: NO.6 or SEQ ID: NO.8 to obtain amino acid sequences of Beta-mannose with the same activity. The invention also provides a gene for coding the Beta-mannose, a recombinant vector containing the gene and a host cell containing the recombinant vector. The invention further provides a preparation method of the Beta-mannose, including the culture of the host cell provided by the invention. The Beta-mannose provided by the invention has heat-resisting property and acid resistance and a prokaryotic expression system established by the invention can be utilized for production. Furthermore, six histidine tags can be utilized for purification.

The invention relates to a novel immobilized metal ion affinity chromatograph (IMAC) material, and a preparation and application thereof. According to the novel IMAC material, the surface of the traditional IMAC material is subjected to coating modification, the naked tail end of protein enters the coating through the selectivity of the surface coating pore sieving action to be captured by metal ions, and other integrated protein has high mass transfer resistance and enters the coating difficultly. The material combines the strong affinity of the IMAC material and the high selectivity of the surface coating pore sieving action, and is further applied to specific capture, release and purification of histidine label protein in the technical fields of biological analysis, biological analysis and biology.

The invention discloses a simple and convenient method for recombinase immobilization. According to the method, the characteristic that a histidine label of recombinant protein and metal ion affinity chromatography resin are combined specifically is utilized, and therefore recombinase is immobilized on the resin, and the immobilized recombinase is prepared. Immobilized carbonyl reductase is used for continuous production of chiral alcohol, and the space time yield is greatly increased.

The invention discloses a nucleic acid aptamer capable of identifying HCV E1E2 (hepatitis C virus E1E2), which is a DNA fragment having a sequence shown in any one of SEQ ID No.1 to SEQ ID No.5. Derivatives of the nucleic acid aptamer include nucleotide-substituted RNA nucleic acid aptamers, skeleton-modified derivatives, peptide nucleic acid derivatives and derivatives of nucleic acid aptamer conjugated with radioactive molecules. A method for screening the nucleic acid aptamer comprises the following steps: firstly preparing HCV E1E2 proteins with histidine tags and then preparing pET200 / D / LacZ proteins with histidine tags, then immobilizing the proteins and designing a random nucleic acid library, and finally screening nucleic acid aptamers. Specifically, the screening step comprises the following steps: pre-treating the DNA library, performing reverse screening, performing forward screening, and repeatedly screening, to obtain the nucleic acid aptamer with strong competitiveness and optimized sequence length. The nucleic acid aptamer and derivatives thereof can be applied to preparing HCV E1E2 protein detection kits or diagnostic kits as well as agents for suppressing HCV infection in liver cells.

The invention discloses a nucleic acid aptamer molecular beacon probe for detecting histidine-tag recombinant proteins and a direct general method for detecting the histidine-tag recombinant proteins in cell lysates by the probe. The nucleic acid aptamer probe comprises sequences shown in SEQIDNO.1 and SEQIDNO.2; the end 3' of the sequence shown in the SEQIDNO.1 is bridged with the end 5' of the sequence shown in the SEQIDNO.2 through PEG36; the end 5' of the sequence shown in the SEQIDNO.1 is modified with fluorescein FAM; and the end 3' of the sequence shown in the SEQIDNO.2 is modified with a quenching group Dabcy1. According to the nucleic acid aptamer molecular beacon probe, the expression of the histidine-tag recombinant proteins in the cell lysates can be quickly and quantificationally analyzed without protein tagging, and the method disclosed by the invention has the advantages of universality and low cost, and the advantage that the histidine-tag recombinant proteins can be simply, quickly and quantificationally analyzed.

The invention relates to a histidine tag protein affinity purification material. The histidine tag protein affinity purification material is prepared from a molecular imprinting material based on a metal chelation function, an immobilized metal chelation affinity chromatography material (IMAC) utilized as a matrix material and a histidine tag utilized as a template module by using a molecular imprinting technique. The histidine tag protein affinity purification material combines the good affinity of the metal chelation function and the high selectivity of the molecular imprinting material, and can be further applied to high-purity purification of the histidine tag protein.

The invention discloses a method for purifying and directionally immobilizing histidine tag protein and application. According to the method disclosed by the invention, tannic acid, metal ions and a hydrophilic anti-protein adhesion polymer are self-assembled and modified on the surface of a carrier to obtain a material surface which is efficiently chelated with cobalt ions, so that one-step purification and immobilization of the histidine tag protein is realized; a ligand of trivalent cobalt has inertia and divalent cobalt ions are oxidized into trivalent cobalt ions so that the stability of the immobilized protein can be improved. The method disclosed by the invention has the advantages of simple process and low price; a separation and purification or immobilization operation process for the histidine tag protein of the method is simple and effective, so that the technology has a wide application prospect.

The invention discloses an expressing carrier of black porgy antibiotic peptide Hepcidin, expressing product and preparing method, which is characterized by the following: incorporating E. coliTrc promoter, protein project improving CKS gene and procaryotic expressing carrier pTrc-CKS of histidine tag (His-Tag); connecting to black porgy Hepcidin gene; constructing pTrc-CKS / hepcidin expressing plasmid; possessing P3C enzyme cutting site; fusing six histidine on C end; getting CKS-hepcidin; purifying through C end His-Tag affinity chromatography; getting the purified product; cutting CKS with P3C enzyme in fuse protein; getting the purified Hepcidin.

The invention relates to a method of producing cysteine containing polypeptides in fusion proteins by recombinantly expressing in a host cell sequences encoding an antifungal polypeptide, a maltose binding protein, and a histidine tag. The method is carried out in the presence of a reducing agent to prevent misfolding of the fusion proteins.

The invention provides a protein chip. The affinity of histidine labels and cations is utilized to fix protein to be fixed on a chip solid phase carrier on a chip carrier. The combined mode basically does not generate influences to functions of protein; meanwhile the carrying of the histidine labels simplifies the separation purification of protein as well as a fixing method of an SPR chip; the preparation of the protein chip is simple and is easy to implement; and the protein chip is low in cost, and high in throughput.

The invention discloses a method for preparation of GLP-1 polypeptide or an analogue thereof through MFH fusion protein and application of the GLP-1 polypeptide or the analogue thereof, and belongs to the field of biotechnology. The structural formula of the MFH fusion protein of the GLP-1 polypeptide or the analogue thereof is MFH-DP-H6-E7-PEP, wherein the MFH is a protein fusion vector, DP is a formic acid hydrolysis site, H6 is a histidine label, E7 is a special protease cutting site and PEP is the GLP-1 polypeptide or the analogue thereof. The DNA of the code DP-H6-E7-PEP is connected to pMFH plasmid to obtain pMFH-PEP plasmid and then the pMFH-PEP plasmid is transferred into escherichia coli, MFH-DP-H6-E7-PEP fusion protein is obtained through prokaryotic expression, and formic acid hydrolysis, special protease hydrolysis and affinity chromatography are performed, so that GLP-1 polypeptide or the analogue thereof is obtained. According to the invention, the purposes of large-scale efficient expression of polypeptide, less steps of a peptide release process, mild condition and low production cost are achieved, and the GLP-1 polypeptide or the analogue thereof is suitable for industrial production.

A substrate includes: a base material; and chelators which are three dimensionally covalently bound to the base material at a density of 7.8×1015 / mm3 or greater and 4.5×1017 / mm3 or less, or two dimensionally covalently bound to the base material at a planar density of 2.0×1011 / mm2 or greater and 1.0×1013 / mm2 or less. Metal ions are coordinately bound to the chelators, and a bioactive substance having histidine tags are coordinately bound to the metal ions. A blocking agent having ligand sets are coordinately bound to the metal ions to which the bioactive substance is not coordinately bound.

The present invention relates to a high-load agarose chromatography media comprising a shell and an inner core, the shell portion is covalently bonded with linear macromolecules such as glucan and the like, the shell portion and the inner core portion are coupled with ligands, and ligand species include metal ions, hydrophobic ligands, ion exchange groups and proteins, and the like. The present invention also includes a preparation method of the high-load agarose chromatography media. For example, the load of histidine tagged (His-tagged) recombinant protein of a metal chelated chromatography media prepared by the method is about 2.5 times of that of conventional media. The high-load agarose chromatography media has good prospects in the fields of biological separation and purification.

The invention belongs to the field of genetic engineering and particularly relates to a single-domain heavy chain antibody specific to a histidine label, having an amino acid sequence shown as in SEQ ID NO: 1 and applicable to the fields such as immunodetection and antigen enrichment and purification. The amino acid sequence provided herein may function as a precursor, and by modifying through random or fixed-point mutation technology, it is possible to obtain a mutant with better properties (affinity, specificity, stability and the like), and the mutant is useful to develop proteins or polypeptides applied to medicine, industry and agriculture.

The invention provides metal chelating magnetic microspheres and a preparation method thereof. According to the metal chelating magnetic microspheres, superparamagnetic nano ferroferric oxide is takenas a core, the surface of the superparamagnetic nano ferroferric oxide is wrapped with a layer of silicon dioxide, and then a ligand formed by a silane coupling agent, epoxy chloropropane and iminodiacetic acid is adopted to modify the surface of silicon dioxide. The silane coupling agent and the epoxy chloropropane in the ligand can be used as connecting arms to prolong the distance between metal ions and a magnetic core, nonspecific adsorption is reduced, and thus specific adsorption of the magnetic beads to label proteins is improved. Space hindrance of combination of the target protein and the magnetic beads is reduced, so that relatively high protein extraction efficiency is achieved. The iminodiacetic acid in the ligand can chelate the metal ions with strong binding force with histidine tag protein, so that the metal chelating magnetic microspheres disclosed by the invention have relatively high capability for selectively separating proteins.

The invention provides recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase as well as a construction method and application thereof and belongs to the technical field of genetic engineering. The invention provides a recombinant plasmid pMD-GDH containing FAD-dependent glucose dehydrogenase genes. After the FAD-dependent glucose dehydrogenase genes are subjected to codon preference adjustment, EcoRI restriction sites and NotI restriction sites are added at two ends of the sequence, and six histidine tags are added at the 3' end. The recombinant pichiapastoris takes Pichia pastoris as an expression host and comprises the recombinant plasmid. The invention further provides application of the recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase in fermentation production of the FAD-dependent glucose dehydrogenase. The embodiments show that when the recombinant pichiapastoris is subjected to induction culture for 136 hours during culture in a 10L of fermentation tank, the enzyme activity reaches 257600U / L and is 5.3 times that of the enzyme activity reported in the prior art.

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 relates to an antigen determinant molecularly imprinted material capable of carrying out selective recognition on a target protein and a preparation process of the antigen determinant molecularly imprinted material. The molecularly imprinted material is prepared by the following steps: by utilizing a section of specific peptide fragment corresponding to the target protein as a template molecule, fixing the specific peptide fragment on a matrix material by employing a histidine tag as a connecting arm; and polymerizing on the matrix material through a functional monomer. The imprinted material is applied to selective recognition of a standard protein.

The invention relates to a clostridium septicum alpha toxin recombinant subunit vaccine and a production method thereof. According to the production method, 54-site cysteine of wild clostridium septicum alpha mature toxin is mutated into leucine, 264-site asparaginate is mutated into alanine, 269-site histidine is mutated into alanine, and 310-site tryptophan is mutated into alanine, so that an alpha toxin mutant which is nontoxic to animals is obtained; and 6 groups of histidine tags are added to a C end of a mutant protein so as to obtain an antigen for preparing vaccines. The invention simultaneously discloses a recombinant expression vector and a recombinant host cell which contain an encoding gene of a nontoxic alpha toxin mutant of clostridium septicum. The efficacies of the preparedclostridium septicum alpha toxin recombinant subunit vaccine are far higher than the efficacies of existing vaccines, and the bio-safety risk in the production process of the vaccine is greatly reduced. Besides, by virtue of the superiority that a semi-finished product of the vaccine is high in protein concentration, a mixed vaccine can be prepared without increasing the dosage of the vaccine.

The invention discloses a preparation method of nano magnetic beads for purifying histidine tagged protein. The method comprises the following steps: taking sodium alginate, FeCl3.6H2O, NaAc.3H2O andPEG as raw materials, taking ethylene glycol and deionized water as solvent, reacting through a solvothermal method to generate monodispersed sodium alginate / Fe3O4 nano magnetic beads, subsequently further carrying out an epoxy chloropropane cross-linking reaction and activation on the nano magnetic beads and the newly added sodium alginate, then introducing an iminodiacetic acid (IDA) chelating agent so as to obtain water-stable nano magnetic beads efficiently chelated with metal nickel or cobalt ions; and efficient, rapid, simple and low-cost separation and purification of the histidine tagged protein are realized. The method disclosed by the invention is simple, rapid and stable to operate and low in cost; the synthesized nano magnetic beads are large in specific surface area and stablein a water phase and not prone to agglomeration and precipitation; the purification efficiency of the histidine tagged protein is greatly improved; and the large-batch industrial production of the nano magnetic beads can be realized.

The invention provides a recombinant vector for expressing a histidine-tag-fused foreign gene in a Kluyveromyces marxianus nutritional deficient strain, and a preparation method and application of the recombinant vector. The recombinant vector sequentially comprises an ampicillin resistance gene, a PKD1 vector sequence, an inulase promotor, multiple cloning sites, an inulase terminator, a nutritional gene promotor and a nutritional gene open reading frame, and further comprises histidine tags located at C ends or N ends of the multiple cloning sites. The recombinant vector provided by the invention can be used for building a transformant to realize expression of the foreign gene.

The invention discloses a novel preparation technology of targeting antitumor fusion protein (lipid peroxidation). The novel preparation method comprises the steps of building a fusion expression protein which takes the glutathione S-transferase A (TrxA)-His-tag (His6-Tag)-SUMO protease recognition substrate as a protein soluble expression auxiliary fragment and takes LHRH (luteinizing hormone releasing hormone)-PEA (phenylethylamine) trans-cell penetrating peptides-ONC (LPO for short) as a target segment, connecting the two proteins with each other by a correct read frame and carrier positive sequence and converting to enter into expression bacteria, finally building fusion protein which is connected with six expression substances in series, crudely extracting, carrying out metal chelating medium purification in the presence of imidazole, and carrying out SUMO protease digestion. Compared with the LPO prepared by a conventional method, the LPO prepared by the novel preparation technology disclosed by the invention can obviously improve the inhibiting effect on the tumor cell lines such as colon cancer HT-29 cells, ovarian cancer OVCAR3 cells, cervical adenocarcinoma HeLa cells and liver cancer HepG-2 cells.

The invention relates to composite magnetic nanoparticles Fe3O4 / MPS / PAA / NTA-Ni<2+> and a preparation method and an application thereof in separation and purification of histidine-tagged proteins, and belongs to the technical fields of nano magnetic materials and biological analysis. Fe3O4 magnetic nanoparticles with an average hydration particle size of 100-400 nm are used as a core, MPS surface modification is adopted for making the surface rich in double bonds, polycarboxyl-structure magnetic nanoparticles with an average hydration particle size of 100-600 nm are obtained through PAA coating, and then furthermore, the polycarboxyl-structure magnetic nanoparticles are coupled with NTA-Ni<2+> to obtain the composite magnetic nanoparticles Fe3O4 / MPS / PAA / NTA-Ni<2+> having fast magnetic field response. The prepared composite magnetic nanoparticles enable the thickness of a coating layer to be controlled in nanometer scale; the prepared composite magnetic nanoparticles have the advantages of uniform size distribution, strong magnetic and low cost, have higher ability of enrichment and separation purification of the histidine-tagged proteins, and have the purification efficiency of the histidine-tagged proteins as high as 40-70 [mu]g / mg composite magnetic nanoparticles; and the composite magnetic nanoparticles can be repeatedly used.