9 results about "Cytoplasmic protein" patented technology

The invention belongs to the field of biomedical technology and nano material preparation, and discloses a selenium-based targeted protein degradation nano material as well as a preparation method and application thereof. An antibody of a target protein is connected to the surface of nano-selenium through dopamine hydrochloride, and the universal selenium-based targeted protein degradation nano-material is obtained. The preparation method is simple in step and easy to implement, and the development process of the traditional TPD technology is simplified. In addition, the selenium-based targeted protein degradation nano material can also degrade membrane protein and cytoplasm protein at the same time, and the limitation that the membrane protein or cytoplasm protein can only be selectively degraded in the existing TPD technology is overcome, so that the curative effect of immunotherapy is enhanced. In addition, under X rays, the selenium-based targeted protein degradation nano material has a radiation sensitization effect. Therefore, the selenium-based targeted protein degradation nano material can be used for preparing antitumor drugs or used as an X-ray sensitizer.

The invention discloses an engineered protein and application thereof in mediating non-ubiquitination degradation, and belongs to the technical field of biological medicine. The structural domains CATCH1 and CATCH2 of the MIDN protein and the region between the structural domains CATCH1 and CATCH2 are modified for the first time, the modification mode comprises the steps that the structural domains CATCH1 and CATCH2 and the region between the structural domains CATCH1 and CATCH2 are replaced with antibodies or polypeptides, then structure or sequence optimization is conducted on the basis, and the obtained MIDN engineered protein not only retains the proteasome binding capacity of MIDN, but also has the advantages of being capable of improving the protein quality and the like. According to the present invention, the MIDN-based target protein degradation system has the MIDN-based target protein degradation system, can expand the target range to the membrane protein or the cytoplasm protein, can improve the degradation efficiency of the MIDN-based target protein degradation system, further has the antibody-mediated target protein specificity so as to achieve the pathological / physiological protein distinguishing effect, and does not have the obvious toxicity; therefore, the engineered protein provided by the invention has a good application prospect.

The invention discloses a method for enriching and extracting soluble protein in a tissue sample, which comprises the following steps: adding the tissue sample into a low-salt buffer solution, homogenizing, centrifuging, collecting supernatant precipitate, resuspending the precipitate by using Triton, and obtaining the supernatant to obtain an extracting solution; adding anhydrous acetonitrile to precipitate protein and collecting precipitate; carrying out proteolysis reductive alkylation and quenching reaction treatment on the precipitate to obtain a peptide fragment sample; desalting by using a peptide fragment sample desalting cleaning solution; after washing, adding a desalted eluent for eluting, collecting an eluent containing the peptide fragment, and drying to obtain a desalted peptide fragment; performing reversed-phase fractionation chromatography under an alkaline condition; and finally, LC-MS / MS detection is carried out. Under the condition that a strong denaturing agent is not introduced, secretory protein, cytoplasm protein and membrane protein can be selectively enriched, meanwhile, most of cell nucleus protein and tissue interstitial fibrin are effectively removed, and therefore the detection capacity of follow-up mass spectrometric detection on extremely-low-abundance circulating markers is remarkably improved.

The invention relates to application of a nano-micelle based on functional nucleic acid in targeted protein degradation. Specifically, the invention provides a nucleic acid aptamer-connexon-lipid nanostructure, constructs editable universal platforms respectively utilizing two degradation systems of lysosome and ubiquitin-proteasome, and is respectively used for degradation of membrane protein and cytoplasm protein. A new thought is provided for avoiding the problems of degradation system overload and safety possibly caused by a single degradation path in traditional targeted protein degradation, and technical support is provided for treatment of diseases such as tumors.

Compositions and methods are provided for the cell-mediated targeted killing of diseased cells based on the presence of an intracellular antigen, rather than a surface-bound marker. The targeting cells are modified to express a cytotoxic protein that is delivered into a targeted cell, and after delivery is selectively activated by the presence of a cytoplasmic protein of interest. In one embodiment of the invention, the cytotoxic molecule is a Granzyme B (GrB) polypeptide. In the compositions of the invention, GrB is modified to render its cytotoxic enzymatic functions inactive, until the presence of an intracellular antigen unlocks the GrB molecule to enable enzymatic activities.

The application provides a multi-omics method for simultaneously detecting surface proteins, cytoplasmic proteins and nuclear proteins of single cells and a device for single-cell multi-omics detection. The microfluidic unit of the device comprises a plurality of inlets in communication with a microfluidic channel, which are used to supply cells and a plurality of markers to the microfluidic channel to form microdroplets containing single cells, the plurality of inlets comprise a first antibody inlet, the first antibody can bind to the surface proteins of the cells and at least one selected from cytoplasmic proteins and nuclear proteins, and the first antibody is connected with a first DNA sequence; a separation unit is connected with the microfluidic unit, which is used to separate target substances based on the markers in the microdroplets and establish a text library; an analysis and sequencing unit is connected with the microfluidic unit, which is used to perform single-cell transcriptome sequencing and protein information analysis of the cells based on the text library. The device can be used for single-cell multi-omics detection and can simultaneously detect surface proteins, cytoplasmic proteins and nuclear protein information.

Provided are a nanocomposite for targeted degradation of a pathogenic protein, a preparation method therefor, and use thereof, which pertain to the technical field of nanobiological drugs. The nanocomposite for targeted degradation of the pathogenic protein is provided, which comprises a nanocarrier and a protein-targeting binding peptide grafted on the nanocarrier. The nanocarrier is a nanoassembly of maleimide-polyethylene glycol-polylactic acid and cationic lipids; on the other hand, the use of the nanocomposite in the preparation of drugs including an anti-tumor nanodrug and a Huntington's disease inhibiting drug is provided. The nanocomposite can simulate a key receptor protein in a selective autophagy pathway, so that the pathogenic protein to be degraded can be brought into an autophagosome to be degraded by means of an autophagy pathway, thereby effectively solving the problem that PROTACs cannot degrade large-molecular-weight protein aggregates and LYTACs cannot degrade cytoplasmic proteins.

EVs have been recognized as vectors for drug delivery. In particular, loading EVs with targeting and therapeutic agents offers intriguing opportunities for converting EVs into biomimetic selective delivery systems. Indeed, EVs constitute physiological carriers that are potentially less immunogenic than artificial delivery vehicles. Here, we developed a novel method for on-demand controlled cargo loading into EVs. These EVs are equipped with nonviral fusogenic factors, as needed, thus facilitating the delivery of EV-cargo to acceptor cells. To sensitively measure this process, we tracked the fate of luciferase-tagged cargo. Cargo loading was enabled by a drug-reversible, inducible dimerization system. Briefly, donor cells were transfected with plasmids encoding FKBP-tagged CD63, a classical membrane EV marker, and FRB-Nanoluciferase (NLuc), a normally cytoplasmic protein. Upon addition of a dimerizer, FRB-Nluc interacts with FKBP-CD63 and is recruited to secreted EVs, which promotes delivery to acceptor cells. This phenomenon can be further enhanced if EVs are equipped with syncytin 1, a mammalian fusogenic protein that induces fusion between the EV membrane and the plasma membrane of acceptor cells. Using this novel method, we further demonstrated that the catalytic domain of diphtheria toxin (DTA), which is involved in protein synthesis inhibition and ultimately cell death, can be delivered to acceptor cells via functionalized EVs. This resulted in protein synthesis inhibition and death of the acceptor cells. This novel method and its resulting applications are expected to open new doors in precision medicine, especially when EVs are equipped with antibodies raised against cell-specific antigens.

The invention discloses a low-abundance cell nucleus enrichment method which comprises the following steps: (1) splitting a tissue sample to obtain a cell nucleus with extracellular cytoplasm retained; and (2) marking and sorting the cell nucleuses obtained in the step (1) to obtain enriched low-abundance cell nucleuses. According to the method, a mild dissociation technology is used, so that more cytoplasm components can be reserved in cell nucleuses, and then the cell nucleuses are used for marking cytoplasm proteins and are combined with flow sorting, so that directional enrichment of the cell nucleuses with low abundance and specific functional states can be effectively realized. Through the whole process optimization, the method can ensure the completeness of the cell nucleus to the maximum extent, takes downstream sequencing application into consideration, and can obtain high-quality mononuclear transcriptome sequencing data by combining with the screening of cytoplasm sequencing data. Moreover, according to the method, directional enrichment is realized through the cytoplasmic protein, the use cost can be effectively reduced, and the method is suitable for large-scale clinical sample data acquisition and has higher flux compared with a single space transcriptome slice.