Peptide linker and localization method for localizing transmembrane proteins to target organelles, and localized fusion protein

Abstract

To provide a method for localizing a transmembrane protein to target organelle.SOLUTION: Disclosed are a highly rigid peptide linker inserted between a transmembrane protein and an organelle transport signal, a fusion protein containing the same, and a localization method that includes inserting a peptide linker.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a peptide linker for localizing a transmembrane protein to a target organelle, a localization method, and a fusion protein to be localized.

Background Art

[0002] Transmembrane proteins are a type of membrane protein localized in cell membranes or organelle membranes. When a transmembrane protein is localized in a membrane, the hydrophobic transmembrane region exists so as to penetrate the lipid bilayer, and the other regions are exposed outside the lipid bilayer. Major transmembrane proteins include channels and pumps, which are transmembrane proteins (transporters) that transport specific substances to the opposite side of the lipid bilayer, cell membrane receptors that are transmembrane proteins transmitting specific extracellular inputs as intracellular signals, and membrane-localized enzymes. These proteins perform diverse functions of biological membranes.

[0003] The inability to perform biosynthesis of proteins essential for biological function is an event that may put life in a serious crisis. In particular, when congenital inability to biosynthesize proteins with appropriate structures occurs due to genetic diseases, radical cure is extremely difficult. As a treatment method for such congenital genetic diseases, although treatment by introducing genes into human somatic cells from the outside (gene therapy) is expected, there are few examples that have been put into practical use.

[0004] Mitochondrial diseases, a type of congenital genetic disorder, are a group of diseases caused by mitochondrial dysfunction and can lead to various serious symptoms, including muscle weakness and developmental delays. In eukaryotic cells, mitochondria are the only organelles other than the nucleus that possess organelle-specific DNA called mitochondrial DNA (mtDNA) and are capable of expressing proteins within the organelle. Mitochondrial proteins expressed by mitochondrial DNA are essential for the functioning of mitochondria, and abnormalities in protein expression by mitochondrial DNA are observed in mitochondrial diseases.

[0005] The effects of transmembrane proteins on cells, such as substance transport and signal transmission, depend heavily on the location where these proteins are localized, and attempts are being made to control their localization. For example, Non-Patent Literature 1 discloses a technique to improve mitochondrial localization by duplicating the mitochondrial localization signal at the N-terminus of subunit 9 of the transmembrane protein ATP synthase. Non-Patent Literature 2 discloses a technique to localize ATP6, a five-transmembrane protein, to mitochondria by adding the mitochondrial localization signal region of the protein SOD2 (whose mRNA itself is present in mitochondria) to the 5' end and the 3' untranslated region of SOD2 to the 3' end. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] GalanisM. et al., "Duplication of leader sequence for protein targeting tomitochondria leads to increased import efficiency", FEBS Letters, 282,425-430 (1991). [Non-Patent Document 2] Kaltimbacher V. et al., "mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein", RNA,12,1408-1417 (2006). [Non-Patent Document 3] MiyazakiE. et al., "Switching the Sorting Mode of Membrane Proteins from Cotranslational Endoplasmic Reticulum Targeting to Posttranslational Mitochondrial Import", Mol. Biol. Cell, 16,1788-1799 (2005). [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The technology to localize transmembrane proteins to specific organelles holds promise for medical and biochemical applications, including gene therapy for congenital genetic diseases. For example, mitochondria are organelles composed of a double membrane, and many mitochondrial proteins are transmembrane proteins localized to the membrane. Therefore, the technology to localize transmembrane proteins to mitochondria holds promise for medical applications in treating mitochondrial diseases through mitochondrial protein supplementation.

[0008] However, unlike the localization of ordinary proteins (e.g., cytoplasmic proteins) to target organelles, it is known that even if an organelle localization signal is applied to a transmembrane protein, due to its high hydrophobicity, transmembrane proteins often aggregate intermolecularly or migrate to the endoplasmic reticulum, and therefore cannot be successfully localized to the target organelle.

[0009] Therefore, an object of the present invention is to provide a method for localizing transmembrane proteins to target organelles. [Means for solving the problem]

[0010] Through diligent research, the inventors discovered that when an organelle localization signal is attached to the end of a transmembrane protein via a proline-rich peptide linker known as a highly rigid linker, or a peptide linker with an α-helix secondary structure (hereinafter collectively referred to as "rigid linker"), the transmembrane protein localizes to the organelle corresponding to the organelle localization signal, thus completing the present invention.

[0011] In other words, the present invention relates to the following [1] to

[15] . [1] A peptide linker used to localize a transmembrane protein to a target organelle, which is inserted between the transmembrane protein and an organelle transition signal corresponding to the target organelle, wherein the number of proline residues contained in the peptide linker exceeds 40% of the total number of amino acid residues contained in the peptide linker, and the number of amino acid residues constituting the peptide linker is 60 or more. [2] A peptide linker used to localize a transmembrane protein to a target organelle, which is inserted between the transmembrane protein and an organelle transition signal corresponding to the target organelle, wherein the peptide linker consists of a peptide whose secondary structure is an α-helix, and the number of amino acid residues constituting the peptide linker is 70 or more. [3] A fusion protein comprising a transmembrane protein, an organelle localization signal for localization to a target organelle, and a peptide linker inserted between the transmembrane protein and the organelle localization signal, wherein the number of proline residues contained in the peptide linker exceeds 40% of the total number of amino acid residues contained in the peptide linker, and the number of amino acid residues constituting the peptide linker is 60 or more. [4] A fusion protein comprising a transmembrane protein, an organelle localization signal for localization to a target organelle, and a peptide linker inserted between the transmembrane protein and the organelle localization signal, wherein the peptide linker consists of a peptide whose secondary structure is an α-helix, and the number of amino acid residues constituting the peptide linker is 70 or more. [5] A method for localizing a transmembrane protein to a target organelle, comprising inserting a peptide linker between the transmembrane protein and an organelle localization signal for localization to the target organelle, wherein the number of proline residues contained in the peptide linker exceeds 40% of the total number of amino acid residues contained in the peptide linker, and the number of amino acid residues constituting the peptide linker is 60 or more. [6] A method for localizing a transmembrane protein to a target organelle, comprising inserting a peptide linker between the transmembrane protein and an organelle localization signal for localization to the target organelle, wherein the peptide linker consists of a peptide whose secondary structure is an α-helix, and the number of amino acid residues constituting the peptide linker is 70 or more. A nucleic acid encoding the fusion protein described in [7][3] or [4]. A vector expressing the fusion protein described in [8][3] or [4]. [9] The target organelle is selected from the group consisting of the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, and peroxisomes, and the peptide linker, fusion protein, localization method, nucleic acid, or vector according to any one of [1] to [8].

[10] The peptide linker, fusion protein, method, nucleic acid, or vector according to any one of [1] to [8], wherein the target organelle is the nucleus or mitochondria.

[11] The peptide linker, fusion protein, method, nucleic acid, or vector according to any one of [1] to [8], wherein the target organelle is a mitochondria.

[12] A therapeutic agent for mitochondrial disease comprising a fusion protein or vector according to any one of [3], [4], or [8], wherein the target organelle is mitochondria.

[13] A method for treating mitochondrial disease, comprising administering to a patient a fusion protein or vector according to any one of [3], [4], or [8], wherein the target organelle is mitochondria.

[14] A fusion protein or vector according to any one of [3], [4], or [8], for use in the treatment of mitochondrial diseases, wherein the target organelle is mitochondria.

[15] Use of a fusion protein or vector according to any one of [3], [4] or [8] for the manufacture of a therapeutic agent for mitochondrial disease, wherein the target organelle is mitochondria. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a peptide linker and a method for localizing a transmembrane protein to a target organelle, as well as a fusion protein that is localized. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows a schematic diagram of the structure of the peptide linker, fusion protein, and fusion protein related to the localization method of the present invention. [Figure 2]Figure 2 is a schematic diagram of a fusion protein in which a flexible linker is inserted between a transmembrane protein and an organelle transit signal. It is inferred that because the peptide linker is flexible, the organelle transit signal and the transmembrane protein undergo intramolecular hydrophobic interaction, and cannot localize to the target organelle. [Figure 3] Figure 3 is a schematic diagram of a fusion protein in which a rigid linker is inserted between a transmembrane protein and an organelle transit signal. It is inferred that because the peptide linker is rigid, the organelle transit signal and the transmembrane protein do not undergo intramolecular hydrophobic interaction, and can localize to the target organelle. [Figure 4] Figure 4 is a diagram showing the mechanism of mitochondrial disease treatment by replacement therapy. [Figure 5] Figure 5 is a diagram outlining the nucleic acid corresponding to the translation region of a plasmid used for expressing a protein in cells in the examples. [Figure 6] Figure 6 is a diagram showing the relationship between the number of amino acid residues contained in a proline linker and the correlation coefficient in colocalization analysis in Example 1. [Figure 7] Figure 7 is a diagram showing representative fluorescence images of a group in which the number of amino acid residues contained in a proline linker is 0 residues in Example 1. [Figure 8] Figure 8 is a diagram showing a representative fluorescence image containing a plurality of cells in a group in which the number of amino acid residues contained in a proline linker is 0 residues in Example 1. [Figure 9] Figure 9 is a diagram showing representative fluorescence images of groups in which the number of amino acid residues contained in a proline linker is 70, 80, 90, 100, 150 and 200 residues in Example 1. [Figure 10] Figure 10 is a diagram showing the relationship between the proportion of proline residues among the amino acid residues contained in a proline linker and the correlation coefficient in colocalization analysis in Example 2. [Figure 11]Figure 11 shows representative fluorescence images from Example 2 in the group where the proportion of proline residues among the amino acid residues contained in the proline linker was 30%. [Figure 12] Figure 12 shows representative fluorescence images of multiple cells in the group in Example 2 where the proportion of proline residues among the amino acid residues contained in the proline linker was 30%. [Figure 13] Figure 13 shows the relationship between the number of amino acid residues contained in the α-helix linker and the correlation coefficient in colocalization analysis in Example 3. [Figure 14] Figure 14 shows representative fluorescence images from Example 3 for the group containing 300 amino acid residues in the α-helix linker. [Figure 15] Figure 15 shows representative fluorescence images from Example 3 for the group containing 200 amino acid residues in the α-helix linker. [Modes for carrying out the invention]

[0014] One aspect of the present invention is a peptide linker used to localize a transmembrane protein to a target organelle, which is inserted between the transmembrane protein and an organelle localization signal corresponding to the target organelle.

[0015] Another aspect of the present invention is a fusion protein comprising a transmembrane protein, an organelle transition signal, and a peptide linker inserted between the transmembrane protein and the organelle transition signal.

[0016] Another aspect of the present invention is a method for localizing a transmembrane protein to an organelle corresponding to the organelle transition signal, which includes inserting a peptide linker between the transmembrane protein and the organelle transition signal (hereinafter also referred to as the "localization method").

[0017] The peptide linker, fusion protein, and localization method of the present invention, as shown in Figure 1, comprises at least a transmembrane protein, an organelle transition signal, and a peptide linker inserted between the transmembrane protein and the organelle transition signal, wherein the peptide linker is a rigid linker.

[0018] The mechanism by which the peptide linker, fusion protein, and localization method of the present invention can localize to a target organelle is presumed to be as follows. When the peptide linker inserted between the transmembrane protein and the organelle localization signal is a linker with low rigidity (hereinafter also referred to as a flexible linker), the transmembrane protein is highly hydrophobic, and as shown in Figure 2, the hydrophobic regions of the transmembrane protein and the organelle localization signal undergo intramolecular hydrophobic interactions. Consequently, the recognition mechanism of the organelle localization signal, such as a signal recognition particle (SRP) or pre-sequence receptor, cannot recognize the organelle localization signal, and therefore the fusion protein cannot localize to the target organelle. In contrast, when the peptide linker inserted between the transmembrane protein and the organelle localization signal is a rigid linker according to the peptide linker, fusion protein, and localization method of the present invention, the rigid linker does not bend, and the transmembrane protein and the organelle localization signal do not interact hydrophobically within the molecule. As a result, as shown in Figure 3, the organelle localization signal can exist freely from the transmembrane protein. Consequently, it is inferred that the recognition mechanism of the organelle localization signal, such as a signal recognition particle (SRP) or pre-sequence receptor, can recognize the organelle localization signal, and the fusion protein can localize to the target organelle.

[0019] (1) Transmembrane proteins Transmembrane proteins are membrane proteins that exist across the lipid bilayer of biological membranes and are major components of biological membranes. They exist as pumps, transporters, ion channels, receptors, enzymes, or membrane structural proteins, performing diverse functions of biological membranes. Within the molecule of a transmembrane protein, there is one or more hydrophobic regions (hereinafter also called "transmembrane domains") which are fixed to the biological membrane by hydrophobic interactions, while the remaining regions are exposed on the outside of the lipid bilayer. Generally, the hydrophobic region consists of about 20 highly hydrophobic amino acids, with highly hydrophilic amino acids, such as charged amino acids, at both ends.

[0020] The peptide linker, fusion protein, and transmembrane protein according to the present invention only need to penetrate the lipid bilayer once or more when localized to the cell membrane or organelle membrane. For example, it may be a single-pass transmembrane protein or a multi-pass transmembrane protein. In particular, single-pass transmembrane proteins are classified into type I, where the N-terminus is exposed to the lumen of the endoplasmic reticulum when inserted into the endoplasmic reticulum membrane during translation, and type II, where the C-terminus is exposed. The single-pass transmembrane protein according to the peptide linker, fusion protein, and localization method of the present invention may be type I or type II. Furthermore, the secondary structure of the transmembrane domain of the transmembrane protein is not particularly limited and may be, for example, an α-helix type or a β-sheet type.

[0021] The origin of the peptide linker, fusion protein, and transmembrane protein related to the localization method of the present invention is not particularly limited. It may be a naturally occurring protein, or a naturally occurring protein in which some amino acid residues have been substituted or deleted without impairing its function, or it may be artificially created. Furthermore, if the transmembrane protein is derived from a eukaryotic cell, its localization under physiological conditions is not particularly limited. For example, it may be a cell membrane-localized protein, or a membrane-localized protein of an organelle such as the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, secretory granules, secretory vesicles, lysosomes, phagosomes, endosomes, vacuoles, or peroxisomes. In addition, the transmembrane protein may be an organelle-localized transmembrane protein in which part or all of the amino acid sequence corresponding to the organelle transition signal has been substituted or deleted.

[0022] Examples of transmembrane proteins according to the present invention include aquaporins, CLC family ion channels, bethrophine, Cys loop ligand-gated ion channels, ion channel glutamate receptors, ion channel ATP receptors, voltage-gated potassium channels, cyclic nucleotide-gated channels, calcium-activated potassium channels, TRP channels, sodium channels, calcium channels, CatSper channels, two-porous potassium channels, inwardly rectifying potassium channels, voltage-gated anion channels (VDACs), ABC transporters, P-type ATPases, V-type ATPases, SLC35 gene family transporters, SLC family transporters, APC superfamily transporters, mitochondrial carriers, MFS-type transporters, sideroflexins, vesicle-associated membrane proteins (VAMPs), rhodopsin phosphodiesterases, adhesion G protein-coupled receptors, secretin receptors, and metabotropic glutamate receptors. Examples include Frizzled / Taste2 receptors, EGF receptor-activated kinases, FGF receptor-activated kinases, ephrin receptor-activated kinases, serine / threonine kinase receptors, AXL-activated kinases, insulin receptors, receptor-type guanylate cyclases, PDGF receptors, TNF / NGF receptors, integrins, receptor-type tyrosine phosphatases, plexin, Notch, selectin, contactin, NADH-ubiquinone oxidoreductases, cytochrome c oxidases, flavin-containing monooxygenases, nonspecific monooxygenases, cytochrome P450, acyltransferases, glycosyltransferases, phosphatetransferases, sulfotransferases, non-receptor-type tyrosine phosphorylation enzymes, phosphate diester hydrolases, membrane-localized peptidases, nucleoside diphosphatases, adenylyl cyclases, SAM complex constituent proteins, TOM complex constituent proteins, TIM complex constituent proteins, mitofucin, and ERMES complex constituent proteins.

[0023] (2) Organelle transition signals In this specification, an organelle localization signal refers to a peptide that, when attached to a protein, causes that protein to localize to the organelle corresponding to the organelle localization signal (target organelle). Generally, proteins that exist in a free state in cells, such as cytoplasmic localized proteins, are easily localized to target organelles by the attachment of organelle localization signals, whereas proteins that show strong localization to specific organelles in their physiological state are not easily localized by organelle localization signals.

[0024] The organelle localization signal according to the peptide linker, fusion protein, and localization method of the present invention only needs to be one that has been confirmed to localize any protein to a target organelle when attached to that protein. It may be one that exists in nature, one in which some amino acid residues have been substituted or deleted without impairing the function of a naturally occurring organelle localization signal, or one that is artificially created. If the organelle localization signal exists in nature, it may be the full length or a part of the protein localized to the target organelle, but from the viewpoint of avoiding inhibition of the function of transmembrane proteins, it is preferably a part of the protein localized to the target organelle.

[0025] The amino acid sequences of proteins localized to target organelles can be obtained from known protein databases, such as the National Center for Biotechnology Information (NCBI) protein database.

[0026] Examples of target organelles include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, secretory granules, secretory vesicles, lysosomes, phagosomes, endosomes, vacuoles, or peroxisomes. From the viewpoint of controlling protein dynamics or energy production in cells, the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, or peroxisomes are preferred, from the same viewpoint, the nucleus or mitochondria are more preferred, and from the viewpoint of controlling cellular function, mitochondria are particularly preferred.

[0027] When the target organelle is mitochondria, the organelle targeting signal is generally conjugated at the N-terminus. As the mitochondrial targeting signal (MTS), peptides described in publicly available literature (e.g., Gunnar von HEIJNE et al., "EUROPEAN JOURNAL OF BIOCHEMISTRY", 1989, Vol. 180, pp. 535-545) can be used. From the viewpoint of enhancing localization to mitochondria, the number of amino acid residues constituting the mitochondrial targeting signal is preferably 15 to 300 residues, more preferably 16 to 250 residues, even more preferably 17 to 200 residues, and even more preferably 18 to 160 residues. Furthermore, from the viewpoint of enhancing localization to mitochondria through interaction with the Tom complex, the number of leucine residues included in the mitochondrial targeting signal may be 10% or more of the total number of amino acid residues included in the organelle targeting signal. From a similar perspective, the number of arginine residues included in the mitochondrial localization signal may be 10% or more of the total number of amino acid residues included in the organelle localization signal. An example of a mitochondrial localization signal is a peptide having the amino acid sequence shown in SEQ ID NOs: 1-3 below, which is attached to the N-terminus of a protein. (a)MLSLRQSIRFFKPATRTLCSSRYL(Sequence ID 1) (b)MRAPSARALLLIPRRGPAVRAWAPAVSSRIWLASEWTPLVRAWTSLIHKPGSGLRFPAPLSGLPGGVGQWATSSGARRCWVLAGPRAAHPLFARLQGAAATGVRDLGNDSQRRPAATGRSEVWKLLGLVRPERGRLSAAV (Sequence ID 2) (c) MALLRAAVSELRRRGRGALTPLPALSSLLSSLSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPY (SEQ ID NO: 3)

[0028] When the target organelle is the nucleus, organelle localization signals can generally be conjugated at locations other than the terminal. Therefore, the nuclear localization signal (also known as NLS) may be conjugated at the N-terminus, at the C-terminus, or a peptide chain may be conjugated further N-terminally to a nuclear localization signal conjugated at the N-terminus, or further C-terminally to a nuclear localization signal conjugated at the C-terminus. As nuclear localization signals, peptides described in publicly available literature (for example, Shunichi Kosugi et al., "JOURNAL OF BIOLOGICAL CHEMISTRY", 2009, Vol. 284, pp. 478-485 and Allison Lange et al., "JOURNAL OF BIOLOGICAL CHEMISTRY", 2007, Vol. 282, pp. 5101-5105) can be used. Nuclear localization signals exist in two forms: monosegmental, where the signal sequence is region 1 in the peptide, and bisegmental, where the signal sequence is region 2. The nuclear localization signal according to the peptide linker, fusion protein, and localization method of the present invention may be monosegmental or bisegmental. The number of amino acid residues constituting the nuclear localization signal may be, for example, 20 residues or less, 18 residues or less, or 16 residues or less. Furthermore, from the viewpoint of enhancing nuclear localization, the ratio of the total number of lysine residues and arginine residues to the total number of amino acid residues constituting the nuclear localization signal may be 20% or more. An example of a nuclear localization signal is a peptide having the amino acid sequence shown in SEQ ID NO: 4 below. (a)PKKKRRV (Sequence ID 4)

[0029] When the target organelle is a peroxisome, the peroxisomal targeting signals (PTS), which are organelle targeting signals, are classified into PTS1, which is attached to the C-terminus, and PTS2, which is attached to the N-terminus. However, the PTS related to the peptide linker, fusion protein, and localization method of the present invention may be either PTS1 or PTS2. Examples of PTS1 include the peptides shown below, and examples of PTS2 include the peptides shown below. In the following, unless otherwise specified, X in the amino acid sequences represents any amino acid. (a) SKL (b)RLXXXXXHL

[0030] When the target organelle is a chloroplast, the organelle translocation signal is generally conjugated at the N-terminus. As a chloroplast translocation signal, peptides described in public literature (e.g., Gunnar von HEIJNE et al., "EUROPEAN JOURNAL OF BIOCHEMISTRY", 1989, Vol. 180, pp. 535-545) can be used, or the chloroplast translocation signal peptide (transit peptide) of the small subunit of rubisco (ribulose bisphosphate carboxylase / oxygenase) from any plant species can be used. From the viewpoint of enhancing localization to chloroplasts, the number of amino acid residues constituting the chloroplast translocation signal is preferably between 30 and 100.

[0031] (3) Peptide linker The peptide linker according to the peptide linker, fusion protein, and localization method of the present invention is a peptide linker consisting of α-amino acids (a linker formed by peptide-bonded amino acids), and is a proline-rich peptide linker (hereinafter also referred to as a proline linker) or a peptide linker having an α-helix secondary structure (hereinafter also referred to as an α-helix linker), which is known to be a highly rigid linker. In this specification, α-amino acids include proline.

[0032] When the peptide linker according to the peptide linker, fusion protein, and localization method of the present invention is a proline linker, from the viewpoint of enhancing the localization of the fusion protein to the target organelle by inhibiting intramolecular hydrophobic interactions, the number of proline residues contained in the peptide linker may be 30% or more of the total number of amino acid residues contained in the peptide linker, preferably 40% or more, more preferably exceeding 40%, and even more preferably 50% or more. Also from a similar viewpoint, with respect to amino acid residues other than proline residues contained in the peptide linker, the average number of proline residues peptide-bonded to each amino acid residue is preferably 0.8 or more, more preferably 1.0 or more, even more preferably 1.2 or more, even more preferably 1.5 or more, and even more preferably 1.8 or more. Also from a similar viewpoint, the number of amino acid residues constituting the peptide linker may be 60 or more, preferably exceeding 60, more preferably 70 or more, and from the viewpoint of increasing expression efficiency, for example, it may be 600 or less, 500 or less, 400 or less, or 300 or less.

[0033] The α-helix linker forms an α-helix, a helical structure consisting of 3.6 residues in one turn, as a secondary structure, resulting in a rigid linker that is difficult to bend. That is, when the peptide linker according to the peptide linker, fusion protein, and localization method of the present invention is an α-helix linker, the amino acid sequence constituting the peptide linker is not particularly limited as long as it can form an α-helix, and may include unnatural amino acids and D-amino acids in addition to naturally occurring L-amino acids. When the peptide linker according to the peptide linker, fusion protein, and localization method of the present invention is an α-helix linker, from the viewpoint of enhancing the localization of the fusion protein to the target organelle by inhibiting intramolecular hydrophobic interactions, the number of amino acid residues constituting the peptide linker should be 70 residues or more, preferably 80 residues or more, and more preferably 90 residues or more. Furthermore, from the viewpoint of increasing expression efficiency, it may be, for example, 600 residues or less, 500 residues or less, 400 residues or less, or 300 residues or less. Examples of α-helix linkers include peptide linkers using the following sequence numbers 5 to 10 as repeating units. (a) EAAAK (Sequence ID 5) (b) EAAAR (Sequence ID 6) (c)EAAAQ (Sequence ID 7) (d) DAAAK (Sequence No. 8) (e) DAAAR (Sequence ID 9) (f)DAAAQ(Sequence ID 10)

[0034] (4) Fusion protein The peptide linker, fusion protein, and localization method of the present invention comprise at least a transmembrane protein, an organelle transition signal, and a peptide linker inserted between the transmembrane protein and the organelle transition signal. The fusion protein may be a simple protein consisting only of amino acids, or a complex protein containing components other than amino acids, but from the viewpoint of ease of acquisition, it is usually a simple protein.

[0035] The peptide linker, fusion protein, and localization method of the present invention may have an additional linker inserted between the peptide linker and the transmembrane protein and / or between the peptide linker and the organelle localization signal. The structure of the inserted linker is not particularly limited, but from the viewpoint of ease of acquisition, the linker may be a linker formed from peptide-bonded amino acids. Furthermore, from the viewpoint of ensuring the rigidity of the peptide chain between the fusion protein and the organelle localization signal and inhibiting intramolecular hydrophobic interactions to enhance the localization of the fusion protein to the target organelle, the length of the linker may be, for example, 200 nm or less, 150 nm or less, 100 nm or less, 50 nm or less, or 25 nm or less. If the linker is formed by amino acids linked by peptide bonds, from a similar viewpoint, the number of amino acid residues constituting the linker may be, for example, 400 residues or less, 300 residues or less, 200 residues or less, 100 residues or less, 50 residues or less, or, for example, 2.0 times or less the number of amino acid residues constituting the peptide linker, 1.5 times or less, 1.0 times or less, or 0.5 times or less.

[0036] The peptide linker, fusion protein, and localization method of the present invention may further contain an optional peptide domain at the peptide terminus of the fusion protein that is not bound to an organelle transition signal. The peptide domain may include, for example, a fluorescent protein domain such as GFP, CFP, and RFP, and a functional domain such as an affinity tag domain such as a His tag, FLAG tag, and GST tag.

[0037] (5) Nucleic acids, vectors, and methods for obtaining them Another aspect of the present invention is a nucleic acid encoding a fusion protein according to one aspect of the present invention, or a vector expressing a fusion protein according to one aspect of the present invention.

[0038] The nucleic acid according to one embodiment of the present invention is not particularly limited as long as it contains a base sequence corresponding to the amino acid sequence of a fusion protein according to one aspect of the present invention or a base sequence having 90% or more sequence identity with said base sequence, and may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a complex of DNA and RNA, and may be single-stranded nucleic acid or double-stranded nucleic acid, and may be genomic DNA or cDNA (complementary DNA).

[0039] The vector according to one embodiment of the present invention is not particularly limited as long as it can express the fusion protein according to one aspect of the present invention in cells, and may be, for example, a transient vector or a stable expression vector, or a plasmid containing at least a promoter sequence and a nucleotide sequence corresponding to the amino acid sequence of the fusion protein, or a viral vector such as an adeno-associated virus, adenovirus, retrovirus, or lentivirus into which a nucleotide sequence corresponding to the amino acid sequence of the fusion protein has been incorporated. The vector into which the nucleic acid is incorporated may contain various sequences such as restriction enzyme sites, regulatory sequences for the expression of the inserted gene, antibiotic resistance genes, sequences for selecting transformants, etc. The vector according to one embodiment of the present invention can be easily obtained by methods commonly used by those skilled in the art, for example, by introducing the nucleic acid according to one embodiment of the present invention as an insert into any vector by ligation and cloning. As the above-mentioned vector into which the nucleic acid is incorporated, commercially available products such as pEBMulti-Hygro vector (Fujifilm Wako Pure Chemical Corporation) can be used.

[0040] (6) Method for implementing localization One aspect of the present invention can be carried out, for example, by introducing a vector expressing a fusion protein according to one aspect of the present invention into cells. Introduction into cells can be easily carried out by methods commonly used by those skilled in the art. For example, if the vector is a viral vector, it can be introduced into cells by adding the viral vector to the cell culture environment or to an animal. Furthermore, if the vector is a plasmid or a fusion protein, it can be introduced into cells by lipofection, electroporation, microinjection, sonoporation, or magnetofection, etc.

[0041] (7) Method for obtaining fusion proteins A fusion protein according to one aspect of the present invention can be obtained, for example, by isolating and purifying it from host cells expressing the fusion protein in a manner similar to the localization method according to one aspect of the present invention. The host cell is not particularly limited and may be a prokaryotic cell such as Escherichia coli or Bacillus subtilis, or a eukaryotic cell such as yeast or animal cell. The animal cell may be, for example, a mammalian cell, and more specifically, a mouse cell or a human cell. Furthermore, the method for isolating the fusion protein from the host cell is not particularly limited and can be carried out by a method commonly used by those skilled in the art. For example, after solubilizing the membrane of the host cell, it can be isolated and purified by methods such as dialysis, salting out, filtration, fractional precipitation, affinity chromatography, ion exchange chromatography, gel electrophoresis, isoelectric focusing, hydrophobic chromatography, gel filtration column chromatography, or reverse-phase chromatography. These methods may be used individually or in combination.

[0042] (8) Treatment of mitochondrial diseases Mitochondrial diseases, a type of congenital genetic disorder, are a group of diseases caused by mitochondrial dysfunction, resulting in a variety of serious symptoms, including muscle weakness and developmental delays. Mitochondria are the only organelles other than the nucleus that possess organelle-specific DNA called mitochondrial DNA (mtDNA), and are capable of expressing proteins within the organelle. Mitochondrial proteins expressed by mitochondrial DNA are essential for mitochondrial function, and in mitochondrial diseases, abnormalities in protein expression by mitochondrial DNA are observed. In other words, in mitochondrial diseases, a deficiency or dysfunction of proteins expressed by mitochondrial DNA leads to mitochondrial dysfunction itself, causing a variety of serious symptoms.

[0043] One possible treatment method for mitochondrial diseases is to replenish them by expressing proteins expressed by mitochondrial DNA from nuclear DNA and transporting them to the mitochondria, as shown in Figure 4 (hereinafter also referred to as "replacement therapy"). However, mitochondria are organelles with lipid bilayer membranes, an inner membrane and an outer membrane, and many mitochondrial proteins essential for life functions are transmembrane proteins. Conventional techniques have made it difficult to localize transmembrane proteins to mitochondria, making replacement therapy difficult. On the other hand, the fusion protein or vector according to one aspect of the present invention can localize transmembrane proteins to target organelles in cells, and is therefore considered usable for replacement therapy of mitochondrial diseases.

[0044] In other words, another aspect of the present invention is a therapeutic agent for mitochondrial disease comprising a fusion protein or vector relating to one aspect of the present invention, wherein the target organelle is mitochondria. The transmembrane protein relating to the fusion protein or vector in this embodiment is a transmembrane protein that is deficient or dysfunctional in mitochondrial disease, or a transmembrane protein capable of substituting the function of said transmembrane protein. The organelle localization signal relating to the fusion protein or vector in this embodiment is a mitochondrial localization signal.

[0045] The fusion protein or vector contained in the mitochondrial disease therapeutic agent according to one embodiment of the present invention may be encapsulated in a drug delivery carrier such as a liposome, microsphere, nanosphere, polymer micelle, or exosome. Furthermore, the method of administering the mitochondrial disease therapeutic agent according to one embodiment of the present invention is not particularly limited, and can be administered by methods such as oral administration, inhalation, intravenous administration, intramuscular administration, subcutaneous administration, or transdermal administration.

[0046] The present invention will be described in detail below with reference to examples, etc., but the present invention is not limited to these. [Examples]

[0047] [Experimental Method 1: Fusion protein and the vector used for its expression] Figure 5 shows a schematic of the nucleic acids corresponding to the translational region of the plasmid used to express the fusion protein in cells in the example. In Figure 5, the left is the 5' end. The plasmid containing the above translational region was prepared as follows: The nucleic acids encoding COX8N25 and the spacer were obtained by having primers containing these sequences synthesized by Hokkaido System Science Co., Ltd. The nucleic acid encoding the peptide linker was obtained by having it synthesized by Eurofins Genomics Co., Ltd. The nucleic acid encoding CFP was purchased from ATUM (FPB-47-609). The nucleic acid encoding the transmembrane protein was obtained from mouse cDNA (Quick Clone cDNA, 637304, Takara Bio Inc.). These nucleic acids were PCR-cloned using KOD One PCR Master Mix -Blue- (KMM-201, Toyobo Co., Ltd.), and a construct for protein expression was prepared by inserting them into the pEX-A2J2 vector purchased from Eurofins Genomics Co., Ltd. using In-Fusion Snap Assembly Master Mix (Z8947N, Takara Bio Inc.). The construct was introduced into Toyobo Co., Ltd.'s competent cells (Competent Quick DH5a, DNA-913F) and amplified, and the plasmid was obtained by purification using Takara Bio Inc.'s plasmid purification kit (NucleoSpin Plasmid, U0588A). The pEX-A2J2 vector incorporates a portion of the Hipp 11 region (1615 bp) of the Chinese hamster, and is a vector that allows for knock-in by homologous recombination if the Cas9 protein is expressed simultaneously (in the examples described herein, transient expression was sufficient, so Cas9 was not expressed).The proteins expressed in cells using this plasmid consist, in order from the N-terminus, of a mitochondrial localization signal (SEQ ID NO: 1, COX8N25), a peptide of 6 extra residues in cloning (glycine-serine-alanine-glycine-serine-alanine), a peptide linker, a transmembrane protein mABCB10d132 (SEQ ID NO: 11), a spacer (SEQ ID NO: 12), a fluorescent protein CFP (SEQ ID NO: 13), and a histidine tag (His tag, 6 histidine residues). mABCB10d132 is a protein in which the N-terminal 132 amino acids of the mitochondrial inner membrane localized protein mouseABCB10 are deleted. By deleting the N-terminal 132 amino acids corresponding to the mitochondrial localization signal, it loses mitochondrial localization and localizes to the endoplasmic reticulum (Non-Patent Literature 3). Furthermore, it has been reported that even if the MTS of other types of mitochondrial localized proteins is added in place of the deleted N-terminal 132 amino acids, localization to mitochondria is not possible (Non-Patent Literature 3).

[0048] [Experimental Method 2: Preparation of cells expressing the fusion protein] The method for preparing the cells expressing the fusion protein used in the examples is as follows:

[0049] First, regarding the cells, CHO-K1 cells (distributed from the JCRB Cell Bank; cell registration number: JCRB9018; hereinafter simply referred to as cells) cultured in Ham's F-12 Nutrient Mix (Thermo Fisher Scientific, product number 11765054) medium (hereinafter simply referred to as medium) containing 10% final concentration fetal bovine serum were placed in 3 welldishes (AGC Technoglass Co., Ltd., product number 3970-103) in 0.7 × 10⁶ units. 4 Seeds were sown to achieve a cell / well ratio.

[0050] Next, regarding the solution to be used for transformation, 20 μL of Opti-MEM (Thermo Fisher Scientific, product number 31985062), 200 ng of plasmid prepared according to experimental method 1, 0.2 μL of Plus reagent, and 0.8 μL of Lipofectamine LTX (Thermo Fisher Scientific, product number 15338030) were mixed and allowed to stand at room temperature for 25 minutes to prepare the solution (transformation solution).

[0051] Finally, regarding the preparation of cells expressing the fusion protein, seeded cells were cultured in culture medium at 37°C under a 5% CO2 atmosphere for 1 day. Then, 10 μL of the transformation solution was added per well to the culture medium, and the cells were allowed to stand for 4-5 hours at 37°C under a 5% CO2 atmosphere. After that, the culture medium was replaced with a medium that did not contain the transformation solution, and the cells were cultured for another day at 37°C under a 5% CO2 atmosphere.

[0052] [Experimental Method 3: Fluorescent staining of mitochondria] Cells expressing the fusion protein prepared in Experimental Method 2 were cultured for 1 hour at 37°C under a 5% CO2 atmosphere in a medium containing 50 nM Mitotracker Deep Red (Thermo Fisher Scientific, product number M22426, hereinafter also referred to as MTDR) at a final concentration of 50 nM, 24 hours after the addition of the transformation solution, to fluorescently stain the mitochondria.

[0053] [Experimental Method 4: Fluorescence Microscopy] The cells obtained in Experimental Method 3 were observed under a fluorescence microscope according to the following protocol. 1 to 6 hours after mitochondrial fluorescence staining, the cells were observed under a fluorescence microscope using an Olympus IX83 motorized inverted microscope equipped with a 100x objective lens UplanSApo 100x and a Hamamatsu Photonics ORCA-flash4.0V3 digital CMOS camera (C13440-20CU), and fluorescence images were acquired. For fluorescence observation of MTDR (mitochondrial cells), the X3-FGWXL (excitation wavelength 530-550 nm, fluorescence wavelength 570 nm or higher) was used, and for fluorescence observation of CFP (fusion protein), the IX3-FCFPXL (excitation wavelength 425-445 nm, fluorescence wavelength 460-510 nm) was used.

[0054] [Experimental method 5: Colocalization analysis] The fluorescence images of MTDR and CFP obtained in Experimental Method 4 were subjected to colocalization analysis according to the following protocol. For the fluorescence images, a median filter with a window size of 35 pixels (pixel resolution of 130 nm / pixel) was used to remove high-frequency components including noise, and this image was subtracted from the original fluorescence image to create an analytical image from which background light had been removed. For the two analytical images obtained from the fluorescence images of MTDR and CFP, the Pearson Correlation Coefficient (hereinafter simply referred to as "correlation coefficient") of the luminance values ​​was calculated according to the method described in the publicly available literature (Costes SV et al., Biophys.J.86,3993-4003(2004)).

[0055] Significant differences in correlation coefficients between different groups were tested using a two-tailed test (Mann-Whitney U test) with a significance level of 1%.

[0056] [Example 1: Relationship between the number of amino acid residues in proline linker and its localization effect] We investigated the relationship between the number of amino acid residues contained in a proline linker and its localization effect when the peptide linker is a proline linker.

[0057] Cells expressing fusion proteins in which the number of amino acid residues in the proline linker was 0, 50, 60, 70, 80, 90, 100, 150, or 200, and in which 50% of the amino acid residues in the proline linker were proline residues, were observed under a fluorescence microscope and colocalization analysis was performed. As an example, the amino acid sequence of the expressed fusion protein in the case where the number of amino acid residues in the proline linker was 100 is shown in SEQ ID NO: 14, and the base sequence of the nucleic acid corresponding to the translational region in the plasmid used to express the fusion protein is shown in SEQ ID NO: 15. When the number of amino acid residues in the proline linker was less than 100, instead of the 100-residue proline linker region (SEQ ID NO: 16) in the amino acid sequence of the fusion protein shown in SEQ ID NO: 14, a number of amino acid residues corresponding to the number of amino acid residues in each group's linker were used as the proline linker region from the C-terminal side of SEQ ID NO: 16. When the number of amino acid residues in the proline linker is 150, instead of the 100-residue proline linker region (SEQ ID NO: 16) in the amino acid sequence of the fusion protein shown in SEQ ID NO: 14, an amino acid sequence having the 50 residues from the N-terminus of SEQ ID NO: 16 in addition to the amino acid sequence shown in SEQ ID NO: 16 was used as the proline linker region. When the number of amino acid residues in the proline linker is 200, instead of the 100-residue proline linker region (SEQ ID NO: 16) in the amino acid sequence of the fusion protein shown in SEQ ID NO: 14, an amino acid sequence having the amino acid sequence shown in SEQ ID NO: 16 in addition to the amino acid sequence shown in SEQ ID NO: 16 in addition to the amino acid sequence shown in SEQ ID NO: 16 was used as the proline linker region. Furthermore, a fusion protein with 0 amino acid residues in the proline linker is a fusion protein that does not have a peptide linker region, and the plasmid used to express the fusion protein is also a plasmid that does not have a peptide linker encoding region.

[0058] Figure 6 shows the correlation coefficients in colocalization analysis according to the number of amino acid residues contained in the proline linker. In Figure 6, the gray bar graph represents the mean, the black error bars represent the standard deviation, and the asterisk (*) indicates a significant difference in the correlation coefficient between the group with 0 amino acid residues and the group with 0 amino acid residues. Furthermore, representative fluorescence images for the group with 0 amino acid residues are shown in Figures 7 and 8, and representative fluorescence images for the groups with 70, 80, 90, 100, 150, and 200 amino acid residues are shown in Figure 9. In Figures 7-9, the numbers written next to the number of amino acid residues or near the outside of the white line indicate the correlation coefficient for the region enclosed by the white line for each image.

[0059] As shown in Figure 6, an increase in the mean correlation coefficient was observed in fusion proteins containing 60 or more amino acid residues in the proline linker, and the difference in correlation coefficient between fusion proteins with 70 or more amino acid residues and fusion proteins with 0 amino acid residues was significant. Furthermore, as shown in Figures 7 and 8, fusion proteins with 0 amino acid residues were strongly suggested to be distributed in a mesh-like pattern throughout the cell and to migrate to the endoplasmic reticulum, whereas, as shown in Figure 9, fusion proteins with 70, 80, 90, 100, 150, and 200 amino acid residues gave fluorescence images very similar to MTDR, strongly suggesting that they localize to mitochondria.

[0060] [Example 2: Relationship between the proportion of proline residues in proline linkers and their localization effect] We investigated the relationship between the proportion of proline residues in a proline linker and its localization effect when the peptide linker is a proline linker.

[0061] Cells expressing a fusion protein containing 100 amino acid residues in the proline linker, with 30, 40, or 50% of the amino acid residues being proline residues, were observed under a fluorescence microscope and colocalization analysis was performed according to experimental methods 1-5. The amino acid sequence of the expressed fusion protein when 30% of the amino acid residues in the proline linker are proline residues is shown in SEQ ID NO: 17, and the nucleotide sequence of the nucleic acid corresponding to the translation region in the plasmid used to express the fusion protein is shown in SEQ ID NO: 18. The amino acid sequence of the expressed fusion protein when 40% of the amino acid residues in the proline linker are proline residues is shown in SEQ ID NO: 19, and the nucleotide sequence of the nucleic acid corresponding to the translation region in the plasmid used to express the fusion protein is shown in SEQ ID NO: 20. When 50% of the amino acid residues in the proline linker are proline residues, the amino acid sequence of the expressed fusion protein is identical to the amino acid sequence shown in SEQ ID NO: 14, and the nucleotide sequence of the nucleic acid corresponding to the translation region in the plasmid used to express the fusion protein is identical to the nucleotide sequence shown in SEQ ID NO: 15.

[0062] Figure 10 shows the correlation coefficients in colocalization analysis according to the proportion of proline residues in the amino acid residues contained in the proline linker. In Figure 10, the gray bar graph shows the mean, the black error bars show the standard deviation, and a sharp (#) indicates a significant difference in the correlation coefficient between the group where 50% of the amino acid residues are proline residues and the other group. Also, representative fluorescence images of the group where 30% of the amino acid residues are proline residues are shown in Figures 11 and 12. In Figures 11 and 12, the numbers written near the outside of the white line indicate the correlation coefficient for the region enclosed by the white line for each image.

[0063] As shown in Figure 10, the correlation coefficient was low for fusion proteins in which the proportion of proline residues in the amino acid residues contained in the proline linker was 30% and 40%, and the difference in the correlation coefficient was significant compared to the fusion protein in which the proportion of proline residues was 50%. Furthermore, as shown in Figures 11 and 12, it was strongly suggested that the fusion protein in which the proportion of proline residues in the amino acid residues contained in the proline linker was distributed in a mesh-like manner throughout the cell and migrated to the endoplasmic reticulum, similar to the fusion protein that does not have a proline linker region. A similar trend was observed for the fusion protein in which the proportion of proline residues was 40%.

[0064] [Example 3: Relationship between the number of amino acid residues in α-helix linkers and their localization effect] We investigated the relationship between the number of amino acid residues contained in an α-helix linker and its localization effect when the peptide linker is an α-helix linker.

[0065] Cells expressing fusion proteins with α-helix linkers containing 0, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 600 amino acid residues were observed under a fluorescence microscope and colocalization analysis was performed according to experimental methods 1-5. The amino acid sequence of the expressed fusion protein when the α-helix linker contains 50 amino acid residues is shown in SEQ ID NO: 21, and the nucleotide sequence of the nucleic acid corresponding to the translation region in the plasmid used to express the fusion protein is shown in SEQ ID NO: 22. The amino acid sequence of the expressed fusion protein when the α-helix linker contains 60 amino acid residues is shown in SEQ ID NO: 23, and the nucleotide sequence of the nucleic acid corresponding to the translation region in the plasmid used to express the fusion protein is shown in SEQ ID NO: 24. Sequence ID 25 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 70 amino acid residues, and Sequence ID 26 shows the nucleic acid sequence corresponding to the translation region in the plasmid used to express the fusion protein. Sequence ID 27 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 80 amino acid residues, and Sequence ID 28 shows the nucleic acid sequence corresponding to the translation region in the plasmid used to express the fusion protein. Sequence ID 29 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 90 amino acid residues, and Sequence ID 30 shows the nucleic acid sequence corresponding to the translation region in the plasmid used to express the fusion protein. Sequence ID 31 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 100 amino acid residues, and Sequence ID 32 shows the nucleic acid sequence corresponding to the translation region in the plasmid used to express the fusion protein. Sequence ID 33 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 200 amino acid residues, and Sequence ID 34 shows the base sequence of the nucleic acid corresponding to the translational region contained in the plasmid used to express the fusion protein.Sequence ID 35 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 300 amino acid residues, and Sequence ID 36 shows the nucleic acid sequence corresponding to the translation region in the plasmid used to express the fusion protein. Sequence ID 37 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 400 amino acid residues, and Sequence ID 38 shows the nucleic acid sequence corresponding to the translation region in the plasmid used to express the fusion protein. Sequence ID 39 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 500 amino acid residues, and Sequence ID 40 shows the nucleic acid sequence corresponding to the translation region in the plasmid used to express the fusion protein. Sequence ID 41 shows the amino acid sequence of the expressed fusion protein when the α-helix linker contains 600 amino acid residues, and Sequence ID 42 shows the nucleic acid sequence corresponding to the translation region in the plasmid used to express the fusion protein. Furthermore, the fusion protein in which the number of amino acid residues in the α-helix linker is 0, and the plasmid used to express the fusion protein are the same as the fusion protein in which the number of amino acid residues in the linker is 0, and the plasmid used to express the fusion protein, as used in Example 1.

[0066] Figure 13 shows the correlation coefficients in colocalization analysis according to the number of amino acid residues contained in the α-helix linker. In Figure 13, the gray bar graph represents the mean, the black error bars represent the standard deviation, and the asterisk (*) indicates a significant difference in the correlation coefficient between the group with 0 amino acid residues and the group with 300 amino acid residues. In addition, representative fluorescence images for the group with 300 amino acid residues are shown in Figure 14, and representative fluorescence images for the group with 200 amino acid residues are shown in Figure 15. In Figures 14 and 15, the numbers next to the number of amino acid residues indicate the correlation coefficient for the region enclosed by the white line for each image.

[0067] As shown in Figure 13, an increase in the mean correlation coefficient was observed in fusion proteins with 70 or more amino acid residues in the α-helix linker, and the difference in correlation coefficient between fusion proteins with 90 or more amino acid residues and fusion proteins with 0 amino acid residues was significant. Furthermore, as shown in Figures 14 and 15, fusion proteins with 200 and 300 amino acid residues gave fluorescence images very similar to MTDR, strongly suggesting localization to mitochondria.

Claims

1. A fusion protein comprising a transmembrane protein, an organelle localization signal for localization to a target organelle, and a peptide linker inserted between the transmembrane protein and the organelle localization signal, wherein the number of proline residues contained in the peptide linker exceeds 40% of the total number of amino acid residues contained in the peptide linker, and the number of amino acid residues constituting the peptide linker is 60 or more.

2. A fusion protein comprising a transmembrane protein, an organelle localization signal for localization to a target organelle, and a peptide linker inserted between the transmembrane protein and the organelle localization signal, wherein the peptide linker consists of a peptide whose secondary structure is an α-helix, and the number of amino acid residues constituting the peptide linker is 70 or more.

3. A method for localizing a transmembrane protein to a target organelle, comprising inserting a peptide linker between the transmembrane protein and an organelle localization signal for localization to the target organelle, wherein the number of proline residues contained in the peptide linker exceeds 40% of the total number of amino acid residues contained in the peptide linker, and the number of amino acid residues constituting the peptide linker is 60 or more.

4. A method for localizing a transmembrane protein to a target organelle, comprising inserting a peptide linker between the transmembrane protein and an organelle localization signal for localization to the target organelle, wherein the peptide linker consists of a peptide whose secondary structure is an α-helix, and the number of amino acid residues constituting the peptide linker is 70 or more.

5. A nucleic acid encoding the fusion protein described in claim 1 or 2.

6. A vector for expressing the fusion protein described in claim 1 or 2.

7. A therapeutic agent for mitochondrial disease comprising the fusion protein described in claim 1 or 2, or a vector expressing the fusion protein described in claim 1 or 2, wherein the target organelle is a mitochondria.

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