18 results about "Marker enzymes" patented technology

The invention belongs to the technical field of biology, and particularly relates to a nano antibody-proximity marker enzyme fusion protein and application thereof. Specifically, the present disclosure provides a fusion protein for proximity labeling wherein the fusion protein is operably linked together by (a) a nanobody and (b) a proximity labeling enzyme; optionally, a peptide linker is also included between the nano antibody and the proximity marker enzyme. The nano antibody-proximity marker enzyme fusion protein disclosed by the invention is obtained through in-vitro expression and purification, target protein is targeted by using an antibody, and the nano antibody-proximity marker enzyme fusion protein does not depend on an overexpression system and has compatibility to various cell types, tissues and clinical fixed samples; proteins with post-translational modification and various organelles, such as membraneless organelles, can be targeted; and the positioning accuracy rate reaches 100%.

The invention relates to the technical field of chemiluminescence immunoassay, and discloses a horse radish peroxidase-labeled secondary antibody compound and a preparation method and application thereof, the horse radish peroxidase-labeled secondary antibody compound comprises a chitosan compound skeleton, and horse radish peroxidase and a secondary antibody carried on the chitosan compound skeleton; the chitosan compound skeleton is a macromolecular skeleton compounded by a chitosan spherical cross-linking body and dopamine. Through optimization and improvement of a polymer skeleton, the problems of large steric hindrance, low sensitivity and non-specific staining of a traditional polymer can be solved, and further improvement of the sensitivity of a DAB staining method for immunoassay detection is facilitated; the adopted chitosan compound skeleton has abundant amino groups, can bear more labeled enzymes and secondary antibodies, reduces steric hindrance, increases the penetrating power to a cell nuclear membrane, and improves the detection sensitivity.

The invention relates to the field of gene engineering, in particular to a proximity marker enzyme mutant and application thereof. The proximity marker enzyme mutant takes ProtA-Turbo as a starting sequence, and one or more sites in K71, K240, K335, K345 and K351 are mutated into non-polar hydrophobic amino acid, polar neutral amino acid, acidic amino acid, basic amino acid or non-natural amino acid. The proximity marker enzyme mutant overcomes the influence of self biotinylation on the proximity marker activity, can continuously and stably mark the proximity proteome of the target protein on the immobilized cell, and obviously improves the sensitivity of identifying the proximity interacting protein of the target protein.

The invention discloses an enzyme-labeled secondary antibody polymer for immunohistochemical staining. The enzyme-labeled secondary antibody polymer is formed by combining a secondary antibody with a polymer backbone, the polymer is labeled with a marker enzyme, the polymer backbone is polymethacrylate-2-hydroxyethyl ester, and the marker enzyme is connected to polymethacrylate-2-hydroxyethyl ester through a coupling agent. The present invention utilizes polymethacrylate-2-hydroxyethyl ester to modify proteins, reduce immunogenicity and antigenicity, and can also weaken the hydrolysis of proteases and increase the solubility of protein molecules. The enzyme-labeled secondary antibody polymer is applied to immunohistochemical staining and has high specificity and sensitivity.

The invention relates to the technical field of biology, and provides a method for constructing a bacterial mutant library and application thereof, and the method comprises the following steps: (1) based on a mutation site of a target protein, carrying out splitting treatment on coding nucleic acid of the target protein, and constructing a saturated mutation site fragment for coding the target protein; (2) constructing a first vector which contains an expression signal marker and an SUMO-FutC enzyme fragment and does not contain an IIS type restriction enzyme digestion site; (3) connecting the saturated mutation site fragment with a first vector to obtain a saturated mutation vector library; and (4) transferring the vectors in the saturated mutation vector library into bacteria to obtain the bacterial mutant library. The bacterial mutant library can be combined with a microfluidic technology to screen key functional sites and corresponding strains of a target protein, for example, a target strain with high yield of 2 '-fucosyllactose can be efficiently obtained, the yield of the target strain is 4.7-7.6 times that of a wild strain, meanwhile, the time, instrument and reagent cost is saved, and the operation requirement is low.

The present invention relates to the field of biotechnology, and in particular to the preparation and application of a CD56 protein-specific nanoantibody-HRP fusion protein. The present application discloses a CD56 protein-specific nanoantibody-HRP fusion protein and a preparation method thereof, as well as the application of the fusion protein in preparing a product for detecting CD56 protein-specific antibodies in serum. The HRP-fused nanoantibody prepared in the present application constructs a fusion protein of a primary antibody and a labeled HRP through genetic engineering, converting two molecular hybridizations into one molecular hybridization, which can greatly shorten the duration of enzyme-linked immunosorbent assay and improve the accuracy. Compared with traditional antibodies, nanoantibodies are easier to genetically manipulate and can be coupled with a variety of labeled enzymes.

The invention relates to a method for marking neurons and application of the neurons to unicellular reconstruction of the neurons. The method comprises the following steps: expressing a proximity marker enzyme with enhanced solubility in a target neuron, and providing biotin to the target neuron so as to form biotinylated protein in the neuron; carrying out integral sample dyeing on the biotinylated protein by using monovalent streptavidin coupled with a detectable marker; imaging the dyed sample to obtain a neuron structure image; and reconstructing the morphological structure of the single neuron based on the image data.

The invention discloses a polymer enzyme-labeled secondary antibody compound, a preparation method and an immunodetection kit, and relates to the technical field of immunodetection. The complex comprises a polymer enzyme cluster and a secondary antibody, the polymer enzyme cluster comprises at least one multi-arm polyethylene glycol skeleton, and the arm tail end of the multi-arm polyethylene glycol skeleton is covalently connected with a plurality of labeled enzyme molecules; the secondary antibody is covalently linked with the polymer enzyme cluster through a polyethylene glycol connecting bridge molecule, or covalently linked with an activating group of the polymer enzyme cluster through a surface functional group of the secondary antibody; the chain length of the polyethylene glycol connecting bridge molecule is smaller than that of the arm forming the multi-arm polyethylene glycol skeleton. According to the invention, PEG chains with different lengths are used for connecting the labeled enzyme and the antibody, so that the contradiction that high signal amplification and high enzyme activity retention, high sensitivity and low background, and large-size compounds and good tissue penetrability are difficult to consider at the same time in the prior art is solved; and in the immunodetection, the excellent comprehensive performance of high sensitivity, strong specificity and low background staining is shown.

Compounds useful for biotin-free proximity labeling are provided. Also provided are methods for biotin-free labeling of biomolecules in proximity to a target in a cell, which involve attaching an engineered labeling enzyme to the target; and contacting the cell to a compound as disclosed, which is a substrate of the engineered labeling enzyme and which includes a label moiety, wherein the biomolecules in proximity to the target are labeled.

The invention belongs to the technical field of cellular neurobiology, and relates to a method for capturing a dynamic interaction group of fusion protein and intracellular amyloid protein and application of the method. In the capturing method, the fusion protein is formed by fusing tandem repeat amyloid protein and proximity marker enzyme through flexible connecting peptide; through the design of the tandem repeat sequence of the amyloid A beta, the flexible connecting peptide, the proximity marker enzyme and the like, the local multivalence of the protein is remarkably improved, the saturation concentration required by phase separation is further reduced exponentially, more and more lasting liquid condensates can be stably formed in cells, and the phase separation efficiency is improved. Thus, the metastable oligomer droplets formed by the liquid aggregate are stabilized under physiological conditions. Fluorescence bleaching recovery verifies that the agglomerate generated by the method has pathological liquid-solid phase change characteristics (no fluorescence recovery), and successfully simulates the aging process of amyloid protein. The invention provides a powerful tool for researching a phase separation mediated pathogenic mechanism in amyloid protein related neurodegenerative diseases (such as Alzheimer's disease) and screening a therapeutic drug for agglutinate aging or protein hijacking.

The present invention develops a method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy technology. β-galactosidase is a marker enzyme for identifying senescent cells. The technical scheme includes: 1) preparing nano-Venus, 2) preparing a two-dimensional array of monolayer, self-assembled nano-Venus and labeled molecules of para-mercaptobenzoic acid, 3) preparing a label, and 4) combining the two-dimensional array with the label through lactose to form a functionalized gold plate as a detection platform. When β-galactosidase acts on the substrate, the two-dimensional array can be separated from the label by decomposing the β-galactosidic bond in lactose, and the data spectrum is collected by means of a laser Raman spectrometer, and the new signal 1437cm generated by PATP on the label when irradiated with laser ‑1 As the marker peak, the ultrasensitive detection of serum β-galactosidase content was achieved. When detecting the activity of the enzyme, the method ‑3 ‑0.5U·mL ‑1 and 0.5-100 U·mL ‑1 The detection limit can reach 4.79×10 ‑5 U·mL ‑1 .

Disclosed are compositions comprising a ligandable tag, a linker, and a labeling enzyme, wherein the linker attaches the ligandable tag to the labeling enzyme. Disclosed are nucleic acid sequences capable of encoding one or more of the disclosed protein based compositions. Disclosed are compositions comprising a ligand conjugated to a molecule of interest. Disclosed are systems comprising a labeling composition and a targeting composition, wherein the labeling composition comprises a ligandable tag, a linker, and a labeling enzyme, wherein the linker attaches the ligandable tag to the labeling enzyme, wherein the targeting composition comprises a ligand conjugated to a molecule of interest, wherein the ligand of the targeting composition is a ligand for the ligandable tag of the labeling composition. Disclosed are methods of using the disclosed compositions and systems.

The invention provides a Cdc42 active probe based on a self-labeling enzyme TMP-tag3 and an application of the Cdc42 active probe. According to the invention, the monitoring of the Cdc42 activity on the living cell population level is realized, and the dynamic visualization of the Cdc42 activity on the single cell level and the subcellular level with high temporal-spatial resolution is realized. On the living cell population level, spectrum displacement which is 12 nm different from that of a positive control group and about 4 times of light intensity change are achieved, response is very obvious, and after Cdc42 specific GEFs and RhoGDI are added for regulation and control, the spectrum of the probe moves in the corresponding expected direction, which indicates that related signal channels in cells are not damaged due to introduction of exogenous Cdc42. Clear division of the Cdc42WT and the positive control group Cdc42CA is realized on the single cell level. On the subcellular level, the probe can detect different subcellular localization Cdc42 molecular activity in long-term imaging.

Compositions, kits, and systems that can include one or more self-labeling enzymes. The self-labeling enzymes can be utilized with a fluorogenic substrate to allow for turn-on fluorescence.

The present disclosure describes fluorescent probes for cations. Such probes remain responsive to their corresponding analyte after binding to a self-labeling enzyme, thus allowing the detection of changes of metal ion concentration at particular intracellular locations to which the self-labeling enzyme has been expressed or in specific cells expressing the enzyme in a co-culture.

The present invention relates to the field of genetic engineering, and in particular to a proximity marker enzyme mutant and its application. The proximity marker enzyme mutant uses ProtA-Turbo as the starting sequence, and mutates one or more sites among K71, K240, K335, K345 and K351 to non-polar hydrophobic amino acids, polar neutral amino acids, acidic amino acids, basic amino acids or non-natural amino acids. The proximity marker enzyme mutant of the present invention overcomes the influence of self-biotinylation on its proximity marker activity, and can continuously and stably mark the proximity protein group of the target protein on the fixed cell, significantly improving the sensitivity of identifying the proximity interacting proteins of the target protein.

The invention belongs to the technical field of biology, and discloses a capturing method of ready-to-use RNA (Ribonucleic Acid) in-situ interaction protein. The method comprises the following steps: S1, adding a biotin-labeled FISH probe into a cell suspension; s2, then adding streptavidin to be labeled with horse radish peroxidase (Streptavidin-HRP), and carrying out enzyme deactivation on the horse radish peroxidase; s3, washing off non-specific binding; and S4, adding biotin phenol and a hydrogen peroxide substrate to trigger a biotinylation labeling reaction. According to the method, a ready-to-use RNA in-situ proximity biotinylation labeling technology without genetic manipulation is established through fusion RNA-FISH in-situ hybridization and proximity labeling technologies, proximity labeling enzyme can be directly guided to interested RNA through an RNA hybridization probe in a fixed cell through the technology, then a proximity biotinylation labeling reaction is carried out, and the RNA in-situ proximity biotinylation labeling technology is established. The method comprises the following steps of: adding a biotin label to interacting protein adjacent to RNA (Ribonucleic Acid), so that the protein can be enriched by streptavidin-beads for mass spectrum identification.

The present invention relates to a nanobody-proximity labeling enzyme fusion protein and the use thereof. Specifically, provided is a fusion protein used for proximity labeling; the fusion protein comprises (a) a nanobody and (b) a proximity labeling enzyme which are operably linked; optionally, there is a peptide linker between the nanobody and the proximity labeling enzyme. The nanobody-proximity labeling enzyme fusion protein is obtained by means of in vitro expression and purification; and said fusion protein uses an antibody to target a target protein, and therefore is not dependent on an overexpression system and is compatible with various types of cells, tissue and clinically immobilized specimens. The fusion protein can target to proteins having post-translational modifications and various organelles, such as membraneless organelles, and can reach a 100% accuracy of localization.