Membrane structure and method for producing membrane structure

Abstract

To provide a membrane structure that enables easy and rapid identification of a target protein expressed in a target cell.SOLUTION: Provided is a membrane structure having a PL (Proximity labeling) enzyme on a membrane surface.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a membrane structure and a method for manufacturing a membrane structure. [Background technology]

[0002] Traditionally, viral pandemics have been feared as a threat to humanity, and the COVID-19 (SARS-CoV-2) pandemic has caused significant damage to the entire world since 2020. These pandemics are likely to occur in the future, and understanding the biological characteristics of the causative viruses is essential to reduce the threat as humanity responds to them. In particular, identification of infectious factors such as viral receptors and coreceptors involved in viral entry into host cells is expected to provide important knowledge that will contribute to the development of vaccines and antiviral drugs.

[0003] However, the identification of these infectious agents requires a complex molecular biology experimental process, which is time-consuming and costly. The fact that the SARS-CoV-2 receptor is ACE2 was discovered relatively early on, but this was due to the availability of research results for SARS-CoV-1, which emerged in 2003, and it is not certain that the next pandemic virus will follow a similar course. Given this background, in preparation for the next pandemic, it is considered necessary to develop a system that can easily and rapidly identify viral receptors and infectious factors (hereinafter referred to as "target proteins") expressed on the membrane surface of host cells.

[0004] In addition to identifying virus receptors and infectious agents, autoimmunity caused by cancer cells and immune cells is also feared as a threat to humanity, and in recent years there has been a demand for the early development of medicines that act on these target cells using extracellular vesicles (which includes all vesicles made of lipid membranes, including exosomes. Hereinafter, they may be referred to as "lipid membrane vesicles") It is known that extracellular vesicle drugs exert their effects by binding and invading through "receptors" on target cells (host cells in the case of viruses), just like the above-mentioned viruses. Identification of these "extracellular vesicle receptors" (hereinafter sometimes referred to as "target proteins") also requires a complex molecular biology experimental process, just like virus receptors and infectious factors. Therefore, there is a problem that identifying these target proteins is time-consuming and costly.

[0005] In recent years, therefore, proximity labeling using a PL (proximity labeling) enzyme has attracted attention as a method for identifying the above-mentioned target protein (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Norihiro Kotani et al, J. Biol. Chem. (2022) 298(11) 102500 : 1-17 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of one aspect of the present invention is to provide a membrane structure that enables easy and rapid identification of a target protein expressed in a host cell or a target cell. [Means for solving the problem]

[0008] The means for solving the above problems are as follows. <1> The membrane structure is characterized by having a PL enzyme on the membrane surface. <2> The PL enzyme is at least one of HRP (Horseradish peroxidase), APEX (Ascorbate peroxidase), and BioID (Biotin ligase). <1> 2. The membrane structure according to claim 1 . <3> The membrane structure is at least one of a virus and a lipid membrane vesicle. <1> from <2> 13. The membrane structure according to claim 12, <4> The membrane structure is used in a proximity labeling method. <1> from <3> 13. The membrane structure according to claim 12, <5> A step of introducing a PL enzyme expression vector into a cell to form a PL enzyme-expressing cell that expresses the PL enzyme on the cell membrane; forming a membrane structure having the PL enzyme on a membrane surface from the PL enzyme-expressing cells; The present invention relates to a method for producing a membrane structure, comprising the steps of: <6> The PL enzyme is at least one of HRP (Horseradish peroxidase), APEX (Ascorbate peroxidase), and BioID (Biotin ligase). <5> The present invention relates to a method for producing the membrane structure described above. <7> The cell is a packaging cell or a lipid membrane vesicle-producing cell, The membrane structure is a virus or a lipid membrane vesicle; <5> from <6> 13. A method for producing the membrane structure according to claim 12. Effect of the Invention

[0009] According to one aspect of the present invention, a membrane structure can be provided that enables simple and rapid identification of a target protein expressed in a host cell or a target cell. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing the structure of a virus as a membrane structure according to one embodiment of the present invention. [Figure 2A] FIG. 1 is a schematic diagram showing an example of a method for labeling a target protein with a PL enzyme using a membrane structure according to one embodiment of the present invention. [Figure 2B] FIG. 1 is a schematic diagram showing an example of a method for labeling a target protein with a PL enzyme using a membrane structure according to one embodiment of the present invention. [Figure 2C] FIG. 1 is a schematic diagram showing an example of a method for labeling a target protein with a PL enzyme using a membrane structure according to one embodiment of the present invention. [Figure 2D] FIG. 1 is a schematic diagram showing an example of a method for labeling a target protein with a PL enzyme using a membrane structure according to one embodiment of the present invention. [Diagram 3] 1 shows the results of Western blotting to confirm whether HRP is expressed in one embodiment of the present invention. [Figure 4] 1 shows the results of production efficiency of DAF-HRP virus in one embodiment of the present invention. [Diagram 5] 1 shows the results of confirming infection of virus-infected cells using a fluorescent microscope in one embodiment of the present invention. [Figure 6] 1 shows the results of electrophoresis and fluorescence detection of labeled proteins after a proximity labeling method (EMARS as one method of proximity labeling methods) in one embodiment of the present invention, and the results of Western blotting using an anti-FITC antibody. [Figure 7] This shows the results of Western blot confirming whether HRP is expressed in EVs (extracellular vesicles) in one embodiment of the present invention, and the results of electrophoresis and fluorescence detection of EMARS products using EV-DAF-HRP. [Figure 8] FIG. 2 is a schematic diagram showing an example of the structure of a vector used in a method for producing a membrane structure in one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] (membrane structure) The membrane structure according to one embodiment of the present invention has a PL (proximity labeling) enzyme on the membrane surface. Due to the presence of the PL enzyme on the membrane surface of the membrane structure, the membrane structure according to one embodiment of the present invention can be used for applications such as proximity labeling method (hereinafter, sometimes referred to as "EMARS") and analysis of proteins targeting cancer cells and immune cells.

[0012] The membrane structure is not particularly limited as long as it has a membrane structure capable of harboring a PL enzyme, and can be appropriately selected depending on the purpose, and examples thereof include viruses, lipid membrane vesicles, etc. When the membrane structure is a virus, it can be used for the application of a proximity labeling method, and when the membrane structure is a lipid membrane vesicle, it can be used for the application of analyzing proteins targeting cancer cells or immune cells.

[0013] <Virus> FIG. 1 is a schematic diagram showing the structure of a virus as a membrane structure according to one embodiment of the present invention. Here, "virus" refers to a particle composed of an envelope and a shell of capsid protein. Furthermore, in one embodiment of the present invention, "virus" includes not only those containing a viral genome (nucleic acid form), but also hollow particles (e.g., hollow particles of VSVG virus, coronavirus, etc.) that are virus-like particles composed of an envelope and capsid protein that do not contain a viral genome. Therefore, VSVG virus and coronavirus refer to either a virus particle containing a viral genome or a hollow particle. Hollow particles include hollow particles such as VSVG virus and coronavirus. In the present invention, the term "viral vector" includes both the above-mentioned viral particles and the viral genome (nucleic acid form) contained in the viral particles; for example, in the case of VSVG virus and coronavirus, recombinant VSVG virus and coronavirus vectors refer to either particles of VSVG virus, coronavirus, etc., or viral genomic DNA present within particles of VSVG virus, coronavirus, etc. A virus according to one embodiment of the present invention has a PL enzyme, and may further have other components such as nucleic acid and capsid protein, as necessary.

[0014] 2A to 2D are schematic diagrams showing an example of a method for labeling a target protein with a PL enzyme. First, as shown in FIG. 2A, on the membrane surface of a cell 100 serving as an analysis sample, there are present a protein 110A to which the PL enzyme binds (hereinafter referred to as "PL enzyme-binding protein 110A"), a target protein 110B that interacts with the PL enzyme-binding protein 110A, and a non-target protein 110C that does not interact with the PL enzyme-binding protein 110A. As shown in FIG. 2B, a membrane structure 130 (virus 130 ) of the present invention expressing a PL enzyme 120 is bound to a PL enzyme-binding protein 110 A on the membrane surface of a cell 100 . Next, as shown in FIG. 2C, a substrate 150 of the PL enzyme labeled with a fluorescent dye 140 is added to the cell 100, whereby the target protein 110B is labeled with the fluorescent dye 140 by the PL enzyme 120 as shown in FIG. 2D. The target protein 110B labeled with the fluorescent dye 140 can be identified by protein analysis.

[0015] The proximity labeling method using a membrane structure (virus) according to one embodiment of the present invention shown in Figures 2A to 2D can label virus receptors physiologically under conditions of infection by actual virus particles, more so than the conventional proximity labeling method using a conjugated protein (recombinant spike protein) of PL enzyme and an antibody against PL enzyme binding protein.

[0016] The PL enzyme is an enzyme used in a proximity labeling method, and examples thereof include HRP (Horseradish peroxidase), APEX (Ascorbate peroxidases), and BioID (Biotin ligase), etc. When the membrane structure has the PL enzyme, it becomes possible to easily and quickly identify a target protein expressed on a target cell.

[0017] The other components are not particularly limited and can be appropriately selected depending on the purpose, and examples of the other components include nucleic acids, envelope proteins, and capsid proteins.

[0018] The nucleic acid means a polymeric organic compound in which nitrogen-containing bases derived from purine or pyrimidine, sugars, and phosphates are regularly bonded, and includes nucleic acid fragments, and analogs of these nucleic acids or their fragments. The nucleic acid is not particularly limited and can be appropriately selected depending on the purpose. Examples of the nucleic acid include DNA, RNA, and cDNA.

[0019] The nucleic acid or nucleic acid fragment may be a natural product obtained from an organism or a processed product thereof, or may be produced using recombinant gene technology or a chemically synthesized artificially synthesized nucleic acid. These may be used alone or in combination of two or more. By using an artificially synthesized nucleic acid, impurities are reduced and it is possible to reduce the molecular weight, thereby improving the initial reaction efficiency. The artificially synthesized nucleic acid refers to a nucleic acid that is artificially synthesized from a nucleic acid that consists of the same components (bases, deoxyribose, phosphate) as naturally occurring DNA or RNA. The artificially synthesized nucleic acid is not limited to a nucleic acid having a base sequence that codes for a protein, but includes a nucleic acid having any base sequence.

[0020] Examples of analogues of nucleic acids or nucleic acid fragments include nucleic acids or nucleic acid fragments bound to non-nucleic acid components, nucleic acids or nucleic acid fragments labeled with labeling agents such as fluorescent dyes or isotopes (e.g., primers or probes labeled with fluorescent dyes or radioisotopes), and artificial nucleic acids in which the chemical structure of some of the nucleotides constituting the nucleic acid or nucleic acid fragment has been changed (e.g., PNA, BNA, LNA, etc.).

[0021] The form of the nucleic acid is not particularly limited and can be appropriately selected depending on the purpose. Examples include double-stranded nucleic acid, single-stranded nucleic acid, partially double-stranded or single-stranded nucleic acid, and the like. Circular or linear plasmids can also be used. The nucleic acid may also be modified or mutated.

[0022] <Extracellular vesicles (lipid membrane vesicles)> The extracellular vesicles (lipid membrane vesicles) are not particularly limited as long as they contain the PL enzyme and can be appropriately selected depending on the purpose. Examples of the extracellular vesicles include liposomes. The liposome refers to a bilayer membrane having a closed space and composed of lipids such as phospholipids and cholesterol. The phospholipid membrane constituting the liposome has a structure in which the phospholipid, which is an amphipathic surfactant, forms an interface with the polar group facing the aqueous phase side and the hydrophobic group facing the opposite side of the interface.

[0023] The lipids such as phospholipids and cholesterol in the liposomes are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected from known lipids such as phospholipids and cholesterol used in liposomes. The lipids such as phospholipids and cholesterol may be used alone or in combination of two or more.

[0024] The method for producing the liposomes is not particularly limited, and any known method can be appropriately selected.

[0025] (Membrane structure manufacturing method) One embodiment of the present invention relates to a method for producing a membrane structure, which includes the steps of introducing a PL enzyme expression vector into a cell to form a PL enzyme-expressing cell that expresses PL enzyme on the cell membrane, and forming a membrane structure having PL enzyme on its membrane surface from the PL enzyme-expressing cell.

[0026] By cell is meant the structural and functional unit that contains amplifiable reagents (eg, nucleic acids) and that forms an organism. The cells are not particularly limited and can be appropriately selected depending on the purpose, and any cells can be used, regardless of whether they are eukaryotic cells, prokaryotic cells, multicellular organism cells, or unicellular organism cells. These may be used alone or in combination of two or more types.

[0027] The eukaryotic cells are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include animal cells, insect cells, plant cells, fungi, algae, protozoa, etc. These may be used alone or in combination of two or more types. Among these, animal cells are preferred.

[0028] Adherent cells may be primary cells taken directly from tissues or organs, or primary cells taken directly from tissues or organs that have been passaged for several generations. They can be appropriately selected depending on the purpose, and examples of such cells include differentiated cells and undifferentiated cells.

[0029] The differentiated cells are not particularly limited and can be appropriately selected depending on the purpose, and examples include hepatocytes, which are parenchymal cells of the liver; stellate cells; Kupffer cells; vascular endothelial cells; endothelial cells such as meatal endothelial cells and corneal endothelial cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; tracheal epithelial cells; digestive tract epithelial cells; cervical epithelial cells; epithelial cells such as corneal epithelial cells; mammary cells; pericytes; muscle cells such as smooth muscle cells and cardiac muscle cells; kidney cells; pancreatic islet cells of Langerhans; nerve cells such as peripheral nerve cells and optic nerve cells; chondrocytes; bone cells, etc.

[0030] The undifferentiated cells are not particularly limited and can be appropriately selected depending on the purpose. Examples of undifferentiated cells include pluripotent stem cells such as embryonic stem cells and mesenchymal stem cells having multi-differentiation ability; unipotent stem cells such as vascular endothelial progenitor cells having a unidifferentiation ability; iPS cells, etc.

[0031] The fungus is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include molds and yeasts. These may be used alone or in combination of two or more. Among these, yeasts are preferred because they can regulate the cell cycle and can be used in haploid form. The cell cycle refers to the process by which cells divide when they multiply, and the cells (daughter cells) produced by cell division become cells (mother cells) that undergo cell division again to produce new daughter cells.

[0032] The PL enzyme expression vector is not particularly limited as long as it can express the PL enzyme when introduced into cells, and can be appropriately selected depending on the purpose. For example, a vector containing the base sequence shown in SEQ ID NO: 1 in the sequence listing can be used. EXAMPLES

[0033] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples in any way.

[0034] Example 1 HEK293T cells were used as packaging cells, and transfection was performed using each of the lentiviral vectors pLenti-GFP, pLenti-HRP-DAFGPI (see the upper part of Figure 8 (HRP-DAFGPI): the base sequence from DAFS to DAFGPI in HRP-DAFGPI is sequence number 1, and the amino acid sequence is sequence number 3), or pLenti-HRP-TH1GPI (see the upper part of Figure 8 (HRP-THY1GPI): the sequence from THY1S to THY1GPI in HRP-THY1GPI is sequence number 2, and the amino acid sequence is sequence number 4), mixed with the psPAX2 vector (lentiviral packaging vector) and the pMD2.G vector (VSVG spike protein expression vector), using TransIT2020 reagent (Takara). Each lentivirus secreted from the transformed cells was separately treated again in new 293T cells.Three packaging cell types were stably obtained from this procedure: control packaging cells (hereinafter sometimes referred to as "293"), DAF-HRP packaging cells (hereinafter sometimes referred to as "DAF"), and THY1-HRP packaging cells (hereinafter sometimes referred to as "THY1"). (Both DAF and THY1 are GPI-linked proteins, but their expression sites are different, so we decided to use the two GPI sequences and adopt the one that was more convenient.) The lysates of each packaging cell prepared and the VSVG virus prepared from each packaging were collected and concentrated, and then electrophoresed by SDS-PAGE, after which the expression of HRP was confirmed by Western blotting (packaging cell lysate: packaging cells; VSVG virus: VSVG virus). The results are shown in Figure 3. From the results in Figure 3, THY1 and DAF expressed HRP. There was no significant difference in HRP expression in the virus.

[0035] Regarding the efficiency of DAF-HRP virus production, Lenti-X TM GoStix TM The results are shown in Figure 4. 20 μL of the virus culture medium from 293 packaging cells (producing native VSVG virus) and DAF-HRP packaging cells was transferred to GoStix TM After adding the antibody, add 80 μL of chase buffer (included in the kit) and eluate the reaction band with GoStix. TM The amount of virus was quantified by reading it with the app (Android version). Both native VSVG and DAF-HRP-VSVG viruses were produced under the same conditions, and the results showed that the amount of DAF-HRP-VSVG virus produced was about half that of the native VSVG virus.

[0036] Next, the same amount of the virus produced from each packaging cell was used as a virus culture medium to infect 293T cells. Cells infected with each virus expressed GFP and emitted fluorescence, so they were observed under a fluorescent microscope. The results are shown in Figure 5. The infection efficiency of each VSVG virus was in the order of 293>THY1-DAF>DAF-HRP (left panel of Figure 5). In addition, a virus vector for SARS-CoV-2 virus production (System Biosiences) was introduced into each packaging cell using TransIT2020 reagent (Takara), and SARS-CoV-2 virus expressing HRP was produced in the same manner as VSVG. When an infection experiment was performed using 293T-ACE2 cells, the host cells, as in the case of VSVG, infection was observed with almost the same infection efficiency (see the right panel of Figure 5).

[0037] VSVG and SARS-CoV-2 viruses (6 types in total) were produced using 293, DAF-HRP, and THY1-HRP packaging cells prepared by the method described above. Lenti-X Lentivirus Concentration Reagent (Takara) was added to each virus culture solution to concentrate each virus. The concentrated virus was treated with each host cell and incubated at room temperature for 10 minutes, after which unbound virus was washed with PBS and removed. With the virus bound to each host cell, it was treated with EMARS reagent (PBS solution containing 0.1 mM fluorescein-tyramide) and incubated at room temperature for 20 minutes. The host cells after the EMARS reaction were collected, and candidate molecules such as fluorescein (FITC)-labeled virus receptors were concentrated by immunoprecipitation (IP) and electrophoresed by SDS-PAGE. When fluorescein (FITC) was detected directly on the electrophoretic gel using a fluorescent imaging device (Bio-Rad: Chemidoc), a labeled band was detected in the DAF-HRP virus. A slight labeled band was detected in the THY1-HRP virus. The results are shown in the left diagram of Figure 6. After detection, the gel was blotted, and each labeled molecule was detected by Western blotting using an anti-FITC antibody, resulting in the detection of many bands (see the right diagram of Figure 6).

[0038] In the cells (DAF-HRP cells) in which DAF-HRP was expressed in 293T cells by the method described above, no virus was produced by introducing a viral vector, and the culture medium of the DAF-HRP cells was simply collected after culturing them for 3 days. The extracellular vesicles (EVs) secreted into the culture medium can be collected and concentrated in the same way as viruses using Lenti-X Lentivirus Concentration Reagent (Takara), and HRP-expressing EVs were collected and concentrated. Western blot was used to confirm whether HRP was expressed in these EVs. The results are shown in Figure 7. From the results in Figure 7, it was found that HRP was expressed in the extracellular vesicles secreted from the DAF-HRP cells (see the left diagram in Figure 7). In addition, EMARS (proximity labeling) was performed using these HRP-expressing EVs, as was performed in the virus experiment described above, to label the EV receptor with fluorescein, and they were concentrated by immunoprecipitation (IP). These samples were electrophoresed on SDS-PAGE, and the electrophoretic gel was subjected to a fluorescence imaging system (Bio-Rad: Chemidoc) to detect fluorescein (FITC)-labeled molecules. No labeled molecules were detected in EVs secreted from 293T cells (EVs that do not express HRP), but multiple labeled molecules were detected in EVs secreted from DAF-HRP cells. These experiments demonstrated that the problems of the present invention can be solved by using the virus of the present invention. [Explanation of symbols]

[0039] 100 cells 110A PL enzyme binding protein 110B Target Protein 110C Non-target proteins 120 PL Enzyme 130 Membrane structures, viruses 140 Fluorescent Dyes 150 Substrate

Claims

1. A membrane structure having a PL enzyme on a membrane surface.

2. 2. The membrane structure according to claim 1, wherein the PL enzyme is at least one of HRP (horseradish peroxidase), APEX (ascorbate peroxidase), and BioID (biotin ligase).

3. The membrane structure according to claim 1 , wherein the membrane structure is at least one of a virus and a lipid membrane vesicle.

4. The membrane structure according to claim 1 , which is used in a proximity labeling method.

5. A step of introducing a PL enzyme expression vector into a cell to form a PL enzyme-expressing cell that expresses the PL enzyme on the cell membrane; forming a membrane structure having the PL enzyme on its membrane surface from the PL enzyme-expressing cells; A method for producing a membrane structure, comprising the steps of:

6. 6. The method for producing a membrane structure according to claim 5, wherein the PL enzyme is at least one of HRP (horseradish peroxidase), APEX (ascorbate peroxidase), and BioID (biotin ligase).

7. The cell is a packaging cell or a lipid membrane vesicle-producing cell, The method for producing a membrane structure according to claim 5 , wherein the membrane structure is a virus or a lipid membrane vesicle-producing cell.

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