Polypeptide, fusion protein, method for producing fusion protein, method for localizing protein in exosome, exosome, and method for producing exosome

Abstract

Provided is a polypeptide for localizing a fusion protein of a protein and the polypeptide in an exosome by being added to the C-terminus of the protein, wherein, in the polypeptide, the N-terminal amino acid sequence includes an amino acid sequence represented by formula 1 and the C-terminal amino acid sequence includes an amino acid sequence represented by formula 2.　Formula 1: K-X11-X12-X13-K, wherein X11 and X12 each independently represent any one amino acid residue selected from the group consisting of an L-lysine residue, an L-arginine residue, an L-serine residue, an L-threonine residue, and an aliphatic amino acid residue, and X13 represents an L-lysine residue or an L-arginine residue.　Formula 2: C-X21-X22-X23, wherein X21 and X22 each independently represent an aliphatic amino acid residue, and X23 represents an amino acid residue.

Description

Polypeptide, fusion protein, method for producing fusion protein, method for localizing protein in exosome, exosome, and method for producing exosome

[0001] The present invention relates to a polypeptide, a fusion protein, a method for producing a fusion protein, an exosome, and a method for producing an exosome. This application claims priority based on Japanese Patent Application No. 2023-192277, filed on November 10, 2023, the contents of which are incorporated herein by reference.

[0002] Exosomes are small vesicles (approximately 100 nm in size) produced by cells that play a variety of biological roles. Exosomes contain proteins and / or nucleic acids and are known to mediate intercellular signaling. Due to these characteristics, exosomes are considered promising candidates for use as biomarkers or drug delivery vehicles.

[0003] Patent Document 1 discloses "exosomes comprising a target protein, wherein at least a portion of the target protein is expressed from an exogenous sequence, and the target protein comprises MARCKS, MARCKSL1, BASP1, or a fragment or variant thereof." and "a method for producing engineered exosomes, the method comprising: a. introducing into cells a nucleic acid construct encoding a fusion polypeptide comprising (i) a first sequence encoding MARCKS, MARCKSL1, BASP1, or a fragment or variant thereof, and (ii) a second sequence encoding a cargo peptide; b. maintaining the cells under conditions that allow the cells to express the fusion polypeptide; and c. obtaining the engineered exosomes comprising the fusion polypeptide from the cells."

[0004] Patent Document 2 discloses "isolated extracellular vesicles (EVs) comprising a biologically active molecule linked to a scaffold protein, the scaffold protein comprising an N-terminal domain (ND) and an effector domain (ED), the ND associated with the luminal surface of the EV, the ED associated with the luminal surface of the EV through ionic interactions, and the ED comprising at least two consecutive lysines (Lys) in its sequence."

[0005] Patent Document 3 discloses "extracellular vesicles (EVs) comprising a biologically active molecule (BAM) covalently linked to the EV via an anchoring moiety (AM), wherein the anchoring moiety comprises a formula (I) of [AM]-[linker]n-[BAM] (wherein n is any number)."

[0006] Non-Patent Document 1 discloses that in addition to conventionally known proteins such as CD9 and CD63, PTGFRN (prostaglandin F2 receptor inhibitor) and BASP1 (brain abundant membrane attached signal protein 1) are present in EVs. Non-Patent Document 1 also discloses that fusion proteins of PTGFRN or BASP1 with other proteins are localized in EVs (extracellular vesicles), and that simply adding a polypeptide consisting of the N-terminal 8 to 10 amino acid sequence of BASP1 to the N-terminus of any protein can cause it to be localized in EVs.

[0007] Special Table No. 2021-503300 Publication Special Table No. 2022-513049 Publication Special Publication No. 2022-550248

[0008] Dooley K., et al., "A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties," Molecular Therapy, 2021, Vol. 29, No. 5, pp. 1729-1743

[0009] The technology disclosed in Patent Document 1 requires the addition of a polypeptide consisting of the N-terminal 8-10 amino acid sequence of BASP1 to the N-terminus of a target protein. However, for example, cell membrane proteins require a secretion signal at their N-terminus, making it theoretically impossible to add such a polypeptide.

[0010] The present invention has been made in view of the above circumstances, and aims to provide a technique capable of localizing any protein into exosomes.

[0011] As a result of extensive research aimed at solving the above problems, the present inventors discovered that adding a polypeptide consisting of a specific amino acid sequence (hereinafter also referred to as an "exosome-localization sequence") to the C-terminus of a protein can cause a fusion protein of the protein and the polypeptide to be localized in exosomes, and thus completed the present invention. That is, the present invention includes the following aspects.

[0012] [1] A polypeptide for localizing a fusion protein with a protein to an exosome by adding the polypeptide to the C-terminus of the protein, the N-terminal amino acid sequence of which comprises the amino acid sequence represented by formula 1 and the C-terminal amino acid sequence of which comprises the amino acid sequence represented by formula 2. １１ -X １２ -X １３ -K Formula 1 where, in Formula 1, X １１ and X １２ are each independently any one amino acid residue selected from the group consisting of an L-lysine residue, an L-arginine residue, an L-serine residue, an L-threonine residue, and an aliphatic amino acid residue; X １３ is an L-lysine residue or an L-arginine residue. ２１ -X ２２ -X ２３ ...Formula 2, where X ２１ and X ２２ are each independently an aliphatic amino acid residue, and X ２３[2] The polypeptide according to [1], wherein the N-terminal amino acid sequence of the polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 1 to 12. [3] In formula 2, X ２１ is L-valine, and X ２２ is L-isoleucine, and X ２３is L-leucine or methionine. [4] The polypeptide according to any one of [1] to [3], consisting of an amino acid sequence represented by any one of SEQ ID NOs: 13 to 27. [5] The polypeptide according to [4], in which at least one cysteine ​​residue is isopropenylated, geranylgeranylated, or farnesylated. [6] A fusion protein comprising a first protein and a polypeptide according to any one of [1] to [5] linked to the C-terminus of the first protein. [7] The fusion protein according to [6], in which the C-terminus of the first protein and the N-terminus of the polypeptide are linked via a spacer. [8] The fusion protein according to [6] or [7], in which the first protein is a transmembrane protein or a fluorescent protein. [9] The fusion protein according to any one of [6] to [8], which is localized to exosomes.

[10] A fusion protein comprising a first protein and a second protein linked to the C-terminus of the second protein, and a polypeptide according to any one of [1] to [5] linked to the C-terminus of the second protein.

[11] The fusion protein according to

[10] , wherein at least one of the C-terminus of the first protein and the N-terminus of the second protein and the C-terminus of the second protein and the N-terminus of the polypeptide are linked via a spacer.

[12] The fusion protein according to

[10] or

[11] , wherein the first protein is a transmembrane protein.

[13] The fusion protein according to any one of

[10] to

[12] , wherein the second protein is a fluorescent protein.

[14] The fusion protein according to any one of

[10] to

[13] , which is localized to exosomes.

[15] A method for producing a fusion protein, comprising the steps of: preparing a construct that is an expression vector incorporating a cDNA of the fusion protein according to any one of [6] to [9]; transfecting the construct into a host cell; expressing the fusion protein in the host cell; and isolating and recovering the fusion protein from the host cell.

[16] A method for producing a fusion protein, comprising the steps of: preparing a construct that is an expression vector incorporating the cDNA of the fusion protein described in any one of

[10] to

[14] ; transfecting the construct into a host cell; expressing the fusion protein in the host cell; and isolating and recovering the fusion protein from the host cell.

[17] A method for localizing a protein in an exosome, comprising the steps of: preparing a construct that is an expression vector incorporating the cDNA of the fusion protein described in any one of [6] to [9]; transfecting the construct into a host cell; and expressing the fusion protein in the host cell.

[18] A method for localizing a protein in an exosome, comprising the steps of: preparing a construct that is an expression vector incorporating the cDNA of the fusion protein described in any one of

[10] to

[14] ; transfecting the construct into a host cell; and expressing the fusion protein in the host cell.

[19] A method for producing exosomes, comprising the steps of preparing a construct that is an expression vector incorporating the cDNA of the fusion protein according to any one of [6] to [9], transfecting host cells with the construct, expressing the fusion protein in the host cells, and isolating and recovering exosomes from the host cells.

[20] A method for producing exosomes, comprising the steps of preparing a construct that is an expression vector incorporating the cDNA of the fusion protein according to any one of

[10] to

[14] , transfecting host cells with the construct, expressing the fusion protein in the host cells, and isolating the fusion protein from the host cells.

[0013] The present invention is free from the limitations of the technique disclosed in Non-Patent Document 1 and can localize any protein in exosomes.

[0014] Fig. 1 is a graph showing the measurement results of Reference Example 1, Examples 1 and 2, and Comparative Examples 1 to 4. Fig. 2 is a graph showing the measurement results of Reference Example 1 and Examples 1 and 3 to 5. Fig. 3 is a graph showing the measurement results of Comparative Examples 5 and 6 and Example 6. Fig. 4 is a graph showing the measurement results of Comparative Example 7 and Example 7.

[0015] Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described below, and various modifications are possible without departing from the gist of the present invention. In the present invention, when a numerical range is expressed using "to", the numerical values ​​on both sides of "to" are included in the numerical range. In the present invention, the names and symbols of amino acids are in accordance with IUPAC-IUB Joint Commission on Biochemical Nomenclature. Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem. 138: 9-37 (1984). In the present invention, the optical isomerism of amino acids is preferably the L-form, unless otherwise specified.

[0016] [Polypeptide] The polypeptide of this embodiment is a polypeptide whose N-terminal amino acid sequence comprises the amino acid sequence shown in Formula 1 and whose C-terminal amino acid sequence comprises the amino acid sequence shown in Formula 2, and which is added to the C-terminus of a protein to localize a fusion protein with the protein in an exosome. １１ -X １２ -X １３ -K Formula 1 where, in Formula 1, X １１ and X １２ are each independently any one amino acid residue selected from the group consisting of an L-lysine residue, an L-arginine residue, an L-serine residue, an L-threonine residue, and an aliphatic amino acid residue; X １３ is an L-lysine residue or an L-arginine residue. ２１ -X ２２ -X ２３ ...Formula 2, where X ２１ and X ２２are each independently an aliphatic amino acid residue, and X ２３ is any amino acid residue.

[0017] The aliphatic amino acid residue is not particularly limited as long as it is an amino acid residue having an aliphatic hydrocarbon group, but is preferably any one selected from the group consisting of L-alanine residue, L-valine residue, L-leucine residue, and L-isoleucine residue, and more preferably L-leucine residue or L-isoleucine residue. Some of the hydrogen atoms in the aliphatic hydrocarbon group in the side chain of the aliphatic amino acid residue may be substituted with halogen atoms such as chlorine atoms or fluorine atoms.

[0018] The X ２１ and X ２２ is not particularly limited as long as it is an amino acid residue having an aliphatic hydrocarbon group, but is preferably any one selected from the group consisting of L-alanine residue, L-valine residue, L-leucine residue, and L-isoleucine residue, and more preferably an L-valine residue or an L-isoleucine residue having a branched aliphatic hydrocarbon group. A portion of the hydrogen atoms of the aliphatic hydrocarbon group may be substituted with halogen atoms such as chlorine atoms or fluorine atoms.

[0019] The X ２３ is preferably an amino acid residue having a hydrophobic functional group in the side chain, more preferably any one selected from the group consisting of L-alanine residue, L-valine residue, L-leucine residue, L-isoleucine residue, and L-methionine residue, and even more preferably any one selected from the group consisting of L-alanine residue, L-leucine residue, and L-methionine residue. Some of the hydrogen atoms directly bonded to the carbon atoms of the hydrophobic functional group may be substituted with halogen atoms such as chlorine atoms or fluorine atoms.

[0020] The polypeptide of this embodiment preferably comprises the amino acid sequences shown in SEQ ID NOs: 1 to 12. In the amino acid sequences below, the portions corresponding to the amino acid sequence shown in Formula 1 are underlined. SEQ ID NO: 1: MGGKLSKK SEQ ID NO: 2: PKKKRKV SEQ ID NO: 3: PKTKRKV SEQ ID NO: 4: PKTKKKV SEQ ID NO: 5: KKTKRKV SEQ ID NO: 6: KKTKKKV SEQ ID NO: 7: PKTKRKV SEQ ID NO: 8: PKTKKKV SEQ ID NO: 9: KKTKRKV SEQ ID NO: 10: KKTKKKV SEQ ID NO: 11: KKKKK SEQ ID NO: 12: KKRKK

[0021] The polypeptide of this embodiment preferably has an N-terminal amino acid sequence comprising an amino acid sequence represented by any one of SEQ ID NOS: 1 to 10. Here, "the N-terminal amino acid sequence of the polypeptide" refers to an amino acid sequence located N-terminally of the amino acid sequence represented by Formula 2 in the molecular chain of the polypeptide. As exemplified below by specific full-length polypeptide sequences, a spacer consisting of any amino acid sequence may or may not be present between the amino acid sequence represented by Formula 1 and the amino acid sequence represented by Formula 2 in the molecular chain of the polypeptide. The number of amino acid residues constituting the spacer may be, for example, 0 to 20, preferably 1 to 15, more preferably 1 to 10, and even more preferably 1 to 5. The spacer sequence preferably comprises one or more residues selected from L-valine residues, L-lysine residues, and L-cysteine ​​residues.

[0022] The polypeptide of this embodiment preferably consists of an amino acid sequence shown in any one of SEQ ID NOs: 13 to 27. In the amino acid sequences below, the portion corresponding to the amino acid sequence shown in Formula 2 is underlined. SEQ ID NO: 13: MGGKLSKKKKCCVIL SEQ ID NO: 14: MGGKLSKKKKCCVIL SEQ ID NO: 15: MGGKLSKKKKCCVIL SEQ ID NO: 16: MGGKLSKKKKKYMGGKLSKKKKCCVIL SEQ ID NO: 17: PKKKRKVCCVIL SEQ ID NO: 18: PKTKRKVCCVIL SEQ ID NO: 19: PKTKKKVCCVIL SEQ ID NO: 20: KKTKRKVCCVIL SEQ ID NO: 21: KKTKKKVCCVIL SEQ ID NO: 22: PKTKRKVCCVIL SEQ ID NO: 23: PKTKKKVCCVIL SEQ ID NO: 24: KKTKRKVCCVIL SEQ ID NO: 25: KKTKKKVCCVIL SEQ ID NO: 26: KKKKKCCVIL SEQ ID NO: 27: KKRKKCCVIL

[0023] At least one of the C-terminal L-cysteine ​​residues of a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs: 13 to 27 may be isopropenylated, geranylgeranylated, or farnesylated. The polypeptide of this embodiment is lipid-modified by introducing an isopropenyl group, geranylgeranyl group, or farnesyl group into the C-terminal L-cysteine ​​residue, which makes it easier for these lipids to penetrate the phospholipid bilayer membrane of exosomes, thereby facilitating localization in the exosome membrane. Because the L-cysteine ​​residue has a thiol group (—SH) in its side chain, it can easily be bound to an isopropenyl group, geranylgeranyl group, or farnesyl group via a thioether bond.

[0024] [Fusion Protein and Method for Producing Fusion Protein] <First Aspect> One aspect of the fusion protein of this embodiment is a fusion protein in which the above-described polypeptide is linked to the C-terminus of a first protein.

[0025] The first protein is preferably a target protein, i.e., a protein to be localized in exosomes.

[0026] The first protein is preferably a transmembrane protein or a fluorescent protein. Specific examples of the transmembrane protein include growth factor receptors such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptors (VEGFR1, VEGFR2), GPCRs such as GLP1 receptor (GLP1R), adrenergic receptor (βAR), fatty acid receptors (e.g., GPR40), and human chemokine receptors (e.g., CCR8), intercellular adhesion proteins such as connexin 26 and connexin 43, and cell adhesion proteins such as integrins.

[0027] Specific examples of the fluorescent protein include blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2 and HyPer; TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsY yellow fluorescent proteins such as DsRed-benzylidene fluorescein (DsRed) and mBanana; orange fluorescent proteins such as KusabiraOrange and mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, and mStrawberry; and near-infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed, mRasberry, and mPlum.

[0028] In the fusion protein according to the first aspect described above, the C-terminus of the first protein and the N-terminus of the polypeptide may be linked via a spacer, or they may be linked directly without a spacer. The spacer is not particularly limited, but is preferably a spacer consisting of a polypeptide chain. When the spacer consists of a polypeptide chain, it is preferable because it can be produced together with the fusion protein intracellularly. When a spacer is interposed between the N-terminal L-lysine residue of the amino acid sequence of the polypeptide represented by Formula 1 and the C-terminal amino acid of the first protein, the number of amino acid residues constituting the spacer can be, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and even more preferably 1 to 5. The spacer sequence preferably contains one or more residues selected from L-methionine residues, glycine residues, L-proline residues, and L-lysine residues.

[0029] The fusion protein according to the first aspect described above can be localized in exosomes.

[0030] <Second Aspect> Another aspect of the fusion protein of the present embodiment is a fusion protein obtained by linking a second protein to the C-terminus of a first protein and linking the above-mentioned polypeptide to the C-terminus of the second protein.

[0031] The first protein is preferably a target protein, i.e., a protein to be localized in exosomes.

[0032] The first protein is preferably a transmembrane protein. Specific examples of the transmembrane protein include growth factor receptors such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptors (VEGFR1, VEGFR2), GPCRs such as GLP1 receptor (GLP1R), adrenergic receptor (βAR), fatty acid receptor (GPR40) (e.g., GPR40), and human chemokine receptor (e.g., CCR8), intercellular adhesion proteins such as connexin 26 and connexin 43, and cell adhesion proteins such as integrin.

[0033] The second protein is preferably a fluorescent protein. Specific examples of the fluorescent protein include blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, and HyPer; TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, and ZsY. yellow fluorescent proteins such as DsRed-benzylidene fluorescein (DsRed) and mBanana; orange fluorescent proteins such as KusabiraOrange and mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, and mStrawberry; and near-infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed, mRasberry, and mPlum.

[0034] In the fusion protein according to the second aspect described above, at least one of the C-terminus of the first protein and the N-terminus of the second protein and the C-terminus of the second protein and the N-terminus of the polypeptide may be linked via a spacer. The spacer is not particularly limited, but is preferably a spacer consisting of a polypeptide chain. When the spacer consists of a polypeptide chain, it is preferable because it can be produced together with the fusion protein in cells. Examples of the spacer between the C-terminus of the second protein and the N-terminus of the polypeptide include the same ones as those exemplified in the first aspect.

[0035] The fusion protein according to the second aspect can be localized in exosomes.

[0036] <Method for Producing Fusion Protein> The fusion protein according to the first or second aspect described above can be produced by a method for producing a fusion protein comprising the following steps (1) to (4): (1) preparing a construct, which is an expression vector incorporating the cDNA of the fusion protein according to the first or second aspect described above; (2) transfecting the construct into a host cell; (3) expressing the fusion protein in the host cell; and (4) isolating and recovering the fusion protein from the host cell. These steps can be carried out by standard methods.

[0037] [Method for localizing proteins in exosomes] The fusion proteins according to the first and second aspects described above can be localized in exosomes by a method comprising the following steps (1) to (3): (1) preparing a construct that is an expression vector incorporating the cDNA of the fusion protein according to the first or second aspect described above; (2) transfecting the construct into a host cell; and (3) expressing the fusion protein in the host cell. These steps can be carried out by standard methods.

[0038] [Exosomes and Method for Producing Exosomes] The exosomes of this embodiment are exosomes formed by localizing the fusion protein described above. The exosomes of this embodiment can be produced by a production method including the following steps (1) to (4): (1) preparing a construct, which is an expression vector incorporating the cDNA of the fusion protein described above; (2) transfecting the construct into host cells; (3) expressing the fusion protein in the host cells; and (4) isolating and recovering exosomes from the host cells. Steps (1) to (3) can be performed by standard methods.

[0039] Step (4) is preferably carried out by the zinc precipitation method. The zinc precipitation method utilizes the binding of zinc to phospholipids on the surface of exosomes. Phosphate and zinc generally bind tightly. Zinc phosphate forms an insoluble precipitate. It is also known that DNA and zinc ions can also form precipitates.

[0040] Specific examples of zinc precipitation methods include the following: The cell culture supernatant is centrifuged at 1000 × g for 3 minutes, 2000 × g for 30 minutes, and 10,000 × g for 30 minutes to remove cell fragments (standard method). ２ Add the solution and ２ The final concentration of is adjusted to 8-10 mM, and the mixture is immediately centrifuged (at 30,000 x g for 1-3 hours). After centrifugation, the supernatant is discarded, and Tris-EDTA buffer (10 mM Tris (pH 8.0), 3-10 mM EDTA) is added to the precipitate to suspend it.

[0041] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples described below.

[0042] Proteins are localized to exosomes by adding a polypeptide sequence consisting of an exosome-localization sequence to the C-terminus of the protein to be localized to exosomes. At this time, as a control, the MGGKLSKKKK sequence (10 residues from the N-terminus of BASP1, hereinafter referred to as "BASP") was added to the N-terminus of mCherry. １０ This sequence is already known as a localization sequence for exosomes. However, it is essential to add it to the N-terminus, as the second glycine is myristoylated (mutating this to Ala is known to eliminate the activity).

[0043] <Fusion Proteins> Expression vectors for the following fusion proteins were prepared by standard methods. Reference Example 1 (positive control): A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28 was added to the N-terminus of mCherry. SEQ ID NO: 28: MGGKLSKKKK Reference Example 2 (negative control): No expression vector Example 1: A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15 was added to the C-terminus of mCherry. SEQ ID NO: 15: MGGKLSKKKKCCVIL Example 2: A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 was added to the C-terminus of mCherry. SEQ ID NO: 16: MGGKLSKKKKKYMGGKLSKKKKCCVIL Example 3: A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 21 was added to the C-terminus of mCherry. SEQ ID NO:21: KKTKKKVCCVIL Example 4 A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:26 has been added to the C-terminus of mCherry. SEQ ID NO:26: KKKKKCCVIL Example 5 A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:27 has been added to the C-terminus of mCherry. SEQ ID NO:27: KKRKKCCVIL Comparative Example 1 A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:29 has been added to the C-terminus of mCherry. SEQ ID NO:29: CCLVKSIRSGYEVMCCVIL Comparative Example 2 A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:30 has been added to the C-terminus of mCherry. SEQ ID NO:30: CCAIRRNREMVCCVIL Comparative Example 3 A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:31 has been added to the C-terminus of mCherry. SEQ ID NO: 31: CCGIRNSSVYCCVIL Comparative Example 4 A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32 was added to the C-terminus of mCherry. SEQ ID NO: 32: CCKKEVQETRRERRRLMSMEMDCCVIL

[0044] Experimental Method: HEK293T (40,000 cells) were transfected with 100 ng of each expression vector using PEImax (Polysciences) and seeded onto a 96-well plate. After three days, the supernatant was collected, and exosomes were recovered using zinc precipitation. Fluorescence was measured. The zinc precipitation method utilizes the binding of zinc to phospholipids on the exosome surface. Phosphate and zinc generally bind tightly. Zinc phosphate forms an insoluble precipitate. It is also known that DNA and zinc ions can also form precipitates. The specific method for the zinc precipitation method is as follows: The cell culture supernatant was centrifuged sequentially at 1000 x g for 3 minutes, 2000 x g for 30 minutes, and 10,000 x g for 30 minutes to remove cell fragments. After centrifugation, 400 mM zinc(II) chloride aqueous solution was added to a final concentration of 8-10 mM, and the mixture was immediately centrifuged (30,000 x g for 1-3 hours). After centrifugation, the supernatant was discarded, and Tris-EDTA buffer (10 mM Tris (pH 8.0), 3-10 mM EDTA) was added to the precipitate to suspend it. The fluorescence of the resulting suspension was measured to determine the fluorescence value of the exosomes.

[0045] The measurement results for Reference Example 1, Examples 1 and 2, and Comparative Examples 1 to 4 are shown in Figure 1. The measurement results for Reference Example 1 and Examples 1, 3 to 5 are shown in Figure 2. All fusion proteins in which a polypeptide was added to mCherry were localized to exosomes. Figure 1 shows that Examples 1 and 2 localized to exosomes to a greater extent than Reference Example 1 (positive control), which is already known to localize to exosomes. Figure 2 shows that Examples 3 to 5 also localized to exosomes to a greater extent than Reference Example 1 (positive control), which is already known to localize to exosomes.

[0046] <Discussion> The sequence that showed significant localization this time contains the amino acid sequence CCVIL, which is lipidated, at the C-terminus. In terms of the mechanism of action, it is thought that similar results would be obtained if CVIM or CVIL were used instead of CCVIL.

[0047] [List of amino acid sequences]

[0048]

[0049]

[0050] Expression vectors for the following fusion proteins were prepared by standard methods. Comparative Example 5: A fusion protein in which the red fluorescent protein mCherry is fused to the C-terminus of the human chemokine receptor CCR8, a type of GPCR. No additional polypeptide is added to the C-terminus of the fusion protein. Comparative Example 6: A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 33 is added to the C-terminus of the fusion protein of Comparative Example 5. SEQ ID NO: 33: AAAAACCVIL. Example 6: A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26 is added to the C-terminus of the fusion protein of Comparative Example 5. SEQ ID NO: 26: KKKKKCCVIL

[0051] Experimental Method: HEK293T (40,000 cells) were transfected with 100 ng of each expression vector using PEImax and seeded onto a 96-well plate. After 7 days, the culture supernatant was collected, and the exosome fraction was recovered using the zinc precipitation method described above, and red fluorescence was measured.

[0052] The measurement results of Comparative Examples 5 to 6 and Example 6 are shown in Figure 3. It was found that the amount of the fusion protein containing a GPCR transferred to exosomes increased by adding the polypeptide shown in SEQ ID NO: 26 to the C-terminus.

[0053] The following expression vectors for fusion proteins were prepared by common methods. Comparative Example 7: A fusion protein in which the red fluorescent protein mCherry is fused to the C-terminus of connexin 26 (Cx26), a component protein of gap junction. No additional polypeptide is added to the C-terminus of the fusion protein. Example 7: A fusion protein in which a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26 is added to the C-terminus of the fusion protein of Comparative Example 7. SEQ ID NO: 26: KKKKKCCVIL

[0054] Experimental Method: HEK293T (40,000 cells) were transfected with 100 ng of each expression vector using PEImax and seeded onto a 96-well plate. After 4 days, the culture supernatant was collected, and the exosome fraction was recovered using the zinc precipitation method described above, and red fluorescence was measured.

[0055] The measurement results of Comparative Example 7 and Example 7 are shown in Figure 4. It was found that the amount of the fusion protein containing Cx26 transferred to exosomes was increased by adding the polypeptide shown in SEQ ID NO: 26 to the C-terminus.

[0056] Membrane proteins such as GPCRs (G protein-coupled receptors) can be targeted by drugs, but producing antibodies against them is known to be extremely difficult. This technology is expected to enable stable antibody production by localizing these membrane proteins or cytoplasmic proteins into exosomes. It is also expected that this technology can be applied to vaccine development.

Claims

1. A polypeptide for localizing a fusion protein with a protein to an exosome by adding the polypeptide to the C-terminus of the protein, the N-terminal amino acid sequence of which comprises the amino acid sequence represented by formula 1 and the C-terminal amino acid sequence of which comprises the amino acid sequence represented by formula 2. １１ -X １２ -X １３ -K Formula 1 Where, in Formula 1, X １１ and X １２ are each independently any one amino acid residue selected from the group consisting of L-lysine residue, L-arginine residue, L-serine residue, L-threonine residue, and aliphatic amino acid residues; X １３ is an L-lysine or L-arginine residue. ２１ -X ２２ -X ２３ ...Formula 2, where X ２１ and X ２２ are each independently an aliphatic amino acid residue; X ２３ is any amino acid residue.

2. The polypeptide according to claim 1, wherein the N-terminal amino acid sequence of the polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 1 to 12.

3. In formula 2, X ２１ is L-valine, and X ２２ is L-isoleucine, and X ２３ The polypeptide according to claim 2, wherein is L-leucine or methionine.

4. The polypeptide according to claim 1, which consists of an amino acid sequence shown in any one of SEQ ID NOs: 13 to 27.

5. The polypeptide of claim 4, wherein at least one cysteine ​​residue is isopropenylated, geranylgeranylated, or farnesylated.

6. A fusion protein comprising a polypeptide according to any one of claims 1 to 5 linked to the C-terminus of a first protein.

7. The fusion protein of claim 6, wherein the C-terminus of the first protein and the N-terminus of the polypeptide are linked via a spacer.

8. The fusion protein of claim 6, wherein the first protein is a transmembrane protein or a fluorescent protein.

9. A fusion protein comprising a first protein, a second protein linked to the C-terminus thereof, and a polypeptide according to any one of claims 1 to 5 linked to the C-terminus of said second protein.

10. The fusion protein described in claim 9, wherein at least one of the C-terminus of the first protein and the N-terminus of the second protein and the C-terminus of the second protein and the N-terminus of the polypeptide are linked via a spacer.

11. The fusion protein of claim 9, wherein the first protein is a transmembrane protein.

12. The fusion protein of claim 9, wherein the second protein is a fluorescent protein.

13. A method for producing a fusion protein, comprising the steps of: preparing a construct which is an expression vector incorporating the cDNA of the fusion protein described in claim 6; transfecting the construct into a host cell; expressing the fusion protein in the host cell; and isolating and recovering the fusion protein from the host cell.

14. A method for producing a fusion protein, comprising the steps of: preparing a construct which is an expression vector incorporating the cDNA of the fusion protein described in claim 9; transfecting the construct into a host cell; expressing the fusion protein in the host cell; and isolating and recovering the fusion protein from the host cell.

15. An exosome in which the fusion protein according to claim 6 is localized.

16. An exosome comprising the fusion protein according to claim 9 localized therein.

17. A method for localizing a protein in an exosome, comprising the steps of: preparing a construct which is an expression vector incorporating the cDNA of the fusion protein described in claim 6; transfecting the construct into a host cell; and expressing the fusion protein in the host cell.

18. A method for localizing a protein in an exosome, comprising the steps of: preparing a construct which is an expression vector incorporating the cDNA of the fusion protein described in claim 9; transfecting the construct into a host cell; and expressing the fusion protein in the host cell.

19. A method for producing exosomes, comprising the steps of: preparing a construct which is an expression vector incorporating the cDNA of the fusion protein described in claim 6; transfecting the construct into a host cell; expressing the fusion protein in the host cell; and isolating and recovering exosomes from the host cell.

20. A method for producing exosomes, comprising the steps of: preparing a construct which is an expression vector incorporating the cDNA of the fusion protein described in claim 9; transfecting the construct into a host cell; expressing the fusion protein in the host cell; and isolating the fusion protein from the host cell.