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CN-121991243-A - Fusion protein containing transmembrane protein and tetramer fluorescent protein and application thereof

CN121991243ACN 121991243 ACN121991243 ACN 121991243ACN-121991243-A

Abstract

The present disclosure relates to fusion proteins comprising a transmembrane protein and a tetrameric fluorescent protein and uses thereof. In particular, the present disclosure provides a fusion protein comprising a transmembrane protein and a tetrameric fluorescent protein. The disclosure also provides extracellular vesicles comprising the fusion proteins, and uses of the fusion proteins or compositions or cells comprising the fusion proteins.

Inventors

  • YANG LINGYAN
  • ZENG YONGSHENG

Assignees

  • 广州国家实验室

Dates

Publication Date
20260508
Application Date
20241101

Claims (12)

  1. 1. A fusion protein is characterized by comprising a transmembrane protein and a tetrameric fluorescent protein.
  2. 2. The fusion protein of claim 1, wherein the transmembrane protein is an extracellular vesicle transmembrane protein, preferably comprising one or more of the group consisting of lysosomal associated membrane protein 2 (LAMP 2), LAMP2B, CD, lactadherin C1/C2, CD9, CD81, platelet-derived growth factor receptor (PDGFR), glycosylated Phosphatidylinositol (GPI) ankyrin 、CD20、Claudin18.2、Claudin6、TM4SF、LAPTM4B、Tarp、CD133、GPRC5D、CXCR4、CCR5、CCR8、SSTR2、L1CAM、β-Klotho、CD147、PTGFRN、BASP1、WLS, or a functional variant or fragment thereof, more preferably comprising one or more of CD63, LAMP2B, WLS or a functional variant or fragment thereof, and/or The tetrameric fluorescent protein comprises one or more of ZsGreen、DsRed、DsRed2、DsRed-Express、DsRed-Express2、DsRed-Max、AsRed2、eqFP611、RFP611、zFP538、ZsYellow1、mcCFP、aeBlue、copGFP、AmCyan1、AQ143, or functional variants or fragments thereof, preferably one or more of ZsGreen, dsRed, zsYellow, mcCFP and aeBlue.
  3. 3. The fusion protein of claim 1, wherein the transmembrane protein is linked to the tetrameric fluorescent protein directly or via a linker, Preferably, the linker is selected from (G n S) m 、(G) n 、(EAAAK) n or (XP) n , wherein n and m are each independently selected from integers from 0 to 5, Preferably, the linker is selected from one or more of SEQ ID NO 1-9.
  4. 4. The fusion protein of claim 1, wherein the fusion protein comprises the transmembrane protein and the tetrameric fluorescent protein or the tetrameric fluorescent protein and the transmembrane protein from N-terminus to C-terminus; Preferably, the fusion protein comprises WLS and ZsGreen, CD63 and ZsGreen, and/or LAMP2B and ZsGreen from N-terminus to C-terminus, Preferably, the fusion proteins include WLS and DsRed, CD63 and DsRed, and/or LAMP2B and DsRed from the N-terminus to the C-terminus.
  5. 5. The fusion protein of claim 1, wherein the fusion protein is capable of forming and/or localizing to a vesicle structure.
  6. 6. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 5.
  7. 7. A vector comprising the nucleic acid molecule of claim 6.
  8. 8. A composition comprising the fusion protein of any one of claims 1 to 5.
  9. 9. A cell comprising the fusion protein of any one of claims 1 to 5, the nucleic acid molecule of claim 6, or the vector of claim 7, preferably forming and/or localizing a vesicle structure in the cell.
  10. 10. The cell of claim 9, wherein the cell is an immortalized cell line or a primary cell; preferably, the cells comprise cells derived from a human or non-human mammal; preferably, the non-human mammal is a mouse, rat, hamster, monkey, dog, cow, sheep, pig, rabbit or horse; Preferably, the cell is selected from the group consisting of HEK293 cells, HEK293T cells, HEK293FT, CHO cells, vero cells, COS-7 cells, BHK cells, MDCK cells, human non-small cell lung cancer A549 cells, human liver cancer HepG2 cells, human liver cancer Hep3B cells, hela, human skin fibroblasts BJ, human embryonic lung cells MCR-5, embryonic stem cells, induced pluripotent stem cells, induced totipotent stem cells or mesenchymal stem cells.
  11. 11. An extracellular vesicle comprising the fusion protein of any one of claims 1 to 5, preferably the extracellular vesicle is a microvesicle and/or an exosome, more preferably an exosome.
  12. 12. The fusion protein of any one of claims 1 to 5, the composition of claim 8 or the cell of claim 9 or 10 for use in one or more of: 1) Visualizing subcellular localization of the transmembrane protein; 2) Visualizing the protein subcellular localization of the transmembrane protein to which it interacts; 3) Detecting binding and localization of the transmembrane protein and its interacting proteins; 4) Screening for proteins that interact with the transmembrane protein; 5) Obtaining a cell in which a vesicle structure supporting the fusion protein is formed; 6) Increasing the secretion level of extracellular vesicles; 7) Increasing the level of loading of the fusion protein and/or ligand thereof on extracellular vesicles.

Description

Fusion protein containing transmembrane protein and tetramer fluorescent protein and application thereof Technical Field The disclosure belongs to the technical field of molecular biology, and in particular relates to a fusion protein containing extracellular vesicle transmembrane protein and tetramer fluorescent protein and application thereof. Background Exosomes are a class of nanoscale extracellular vesicles released by living cells, with a typical bilayer membrane structure. Exosomes can carry a variety of biologically active substances of parent cell origin, including proteins, nucleic acids, lipids, and the like, and by acting on recipient cells in close or remote proximity, play an important role in cell-to-cell mass transport and information transfer. In recent years, exosomes have been widely used as drug delivery vehicles, since they can carry endogenous or exogenous therapeutic drugs. However, normal cultured mammalian cells have limited levels of exosomes released, which are difficult to use in industrial production and clinical therapy, and present a significant challenge in the use of exosomes as drug delivery vehicles. To better enhance the level of exosomes produced by cells, researchers have explored a variety of physical methods such as mechanical loading, geometric, acoustic, electrical stimulation or cellular nanopore chips, etc., as well as non-physical methods including molecular interference (e.g., tumor necrosis factor and interferon, etc.), environmental factors (e.g., hypoxia, heat shock, pH adjustment and glucose concentration adjustment), and external inducers, role in exosome production. In addition, the cell specific genes can be regulated and controlled by utilizing the genetic engineering technology, so that the capability of exosome production is increased, for example, genes such as an exosome cell membrane protein CD63, STEAP3, syndecan-4 and the like are over-expressed. The human WNT transporter Wntless (WLS) is an eight-pass transmembrane G protein coupled protein receptor (GPCR) protein that possesses two domains, a transmembrane domain and a secondary domain resembling an ancient fatty acid regulator, with WNT fatty acid tails capable of being inserted into conserved cavities of WLS transmembrane domains. WNT protein is a secreted glycoprotein, which is widely involved in the regulation of developmental processes of the body by activating WNT signal pathways to regulate cell behaviors and functions, and is associated with the occurrence of various diseases such as tumors, bones, muscles, nerves, skin, viscera, and the like. Therefore, the WNT protein has important basic research and clinical application value in future regenerative medicine. WNT proteins undergo lipidation modifications on the internal network after translation in mammalian cells, resulting in increased hydrophobicity. While lipidated modified WNT proteins can bind to intracellular WLS, WNT proteins can only be transported to the cell membrane and secreted into the extracellular space with the help of WLS. We have previously found that the use of WLS protein increases the efficiency of WNT protein loading and allows WNT protein to be loaded on the outer surface of extracellular vesicle membrane, enhancing WNT protein signal modulating activity. In addition, WLS is currently widely believed to be able to load all 19 human WNT proteins, but lacks direct evidence. In recent years, scientists only determine the interaction relation between WNT3A, WNT, A, WNT A and WLS through a refrigeration electron microscope technology, but the difficulty is high, and the requirements on an instrument platform are high. Disclosure of Invention The present disclosure unexpectedly found that fusion proteins formed by linking transmembrane proteins with tetrameric fluorescent proteins are capable of being specifically localized in intracellular vesicle structures and capable of being secreted to extracellular load on extracellular vesicles, while increasing the secretion of extracellular vesicles by cells. The use of the fusion protein of WLS can also detect and study interactions between WLS and WNT by visualizing subcellular localization of both. According to one aspect of the present disclosure, there is provided a fusion protein comprising a transmembrane protein, and a tetrameric fluorescent protein. In some embodiments, the transmembrane protein may comprise any known transmembrane protein on a cell membrane or organelle membrane. In some embodiments, the transmembrane protein may comprise a transmembrane protein on an extracellular vesicle. In some embodiments, the transmembrane proteins may include, but are not limited to, one or more of lysosomal associated membrane protein 2 (LAMP 2), LAMP2B, CD63, lactadherin C1/C2, CD9, CD81, platelet Derived Growth Factor Receptor (PDGFR), glycosylated Phosphatidylinositol (GPI) ankyrin 、CD20、Claudin18.2、Claudin6、TM4SF、LAPTM4B、Tarp、CD133、GPRC5D、CXCR4、CCR5、CCR8、SSTR2、L1CAM、β-K