Peptide linkers and localization methods for localizing transmembrane proteins in target organelles, and localized fusion proteins

Abstract

Disclosed are a highly rigid peptide linker inserted between a transmembrane protein and an organelle localization signal, a fusion protein comprising the peptide linker, and a localization method comprising a step of inserting the peptide linker.

Description

Technical Field

[0001] The present invention relates to a peptide linker and a localization method for localizing a transmembrane protein in a target cell organelle, as well as a localized fusion protein. Background Art

[0002] Transmembrane proteins are membrane proteins located in cell membranes or organelle membranes. When transmembrane proteins are located in the membrane, the hydrophobic transmembrane region exists in a way that penetrates the lipid bilayer membrane, and the other regions are exposed to the outside of the lipid bilayer membrane. As the main transmembrane proteins, channels and pumps are known, which are transmembrane proteins (transporters) that transport specific substances to the opposite side of the lipid bilayer membrane; cell membrane receptors and membrane-localized enzymes, which are transmembrane proteins that transmit specific inputs from outside the cell as signals inside the cell; etc., which play a variety of functions of biological membranes.

[0003] Failure to biosynthesize proteins essential for the function of an organism is a potentially life-threatening event, and it is extremely difficult to cure the disease when the biosynthesis of proteins with appropriate structures cannot be performed due to genetic diseases, etc. Although gene therapy (gene therapy) is expected to be used to introduce genes into human somatic cells from the outside, there are few examples of actual application.

[0004] Mitochondrial disease, one of the congenital genetic diseases, is a general term for diseases caused by mitochondrial dysfunction, and is a disease that causes various severe symptoms, including decreased muscle strength and developmental delay. In eukaryotic cells, mitochondria are the only organelles other than the nucleus that have organelle-specific DNA called mitochondrial DNA (mtDNA) and can express proteins within the organelle. Mitochondrial proteins expressed by mitochondrial DNA are essential for the function of mitochondria, and therefore, in mitochondrial diseases, abnormalities in protein expression due to mitochondrial DNA have been confirmed.

[0005] The effects of the transport of substances and the transmission of signals by transmembrane proteins on cells are greatly dependent on the position of the transmembrane proteins, and therefore, the control of their positioning has been studied. For example, non-patent document 1 discloses a technique for improving the localization to mitochondria by repeating the mitochondrial localization signal at the N-terminus of subunit 9 of ATP synthase, which is a transmembrane protein. In addition, non-patent document 2 discloses a technique for localizing ATP6, which is a transmembrane protein of five transmembrane types, by adding the mitochondrial localization signal region of the protein SOD2 present in the mitochondria at the 5' end of the mRNA itself and adding the 3' untranslated region of SOD2 at the 3' end, thereby localizing mitochondria.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent document 1: Galanis M. et al., “Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency”, FEBS Letters, 282, 425-430 (1991).

[0009] 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).

[0010] Non-patent document 3: Miyazaki E. 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). Summary of the invention

[0011] Technical problem to be solved by the invention

[0012] The technology for localizing transmembrane proteins in specific organelles can be expected to be applied to medical applications such as gene therapy for congenital genetic diseases and biochemical applications. For example, mitochondria are organelles composed of double membranes, and most mitochondrial proteins are transmembrane proteins localized in the membrane. Therefore, the technology for localizing transmembrane proteins in mitochondria can be expected to be applied to medical applications such as the treatment of mitochondrial diseases by supplementing mitochondrial proteins.

[0013] However, it is known that, unlike the localization of ordinary proteins (e.g., cytoplasmic localization proteins) in target organelles, even if organelle localization signals are added to transmembrane proteins, transmembrane proteins aggregate between molecules or move to the endoplasmic reticulum due to their highly hydrophobic nature, and in most cases cannot be successfully localized in the target organelles.

[0014] Therefore, an object of the present invention is to provide a method for localizing a transmembrane protein in a target cell organelle.

[0015] Technical solutions for solving technical problems

[0016] The inventors of the present invention conducted intensive research and found that when an organelle localization signal is added to the end of a transmembrane protein via a proline-rich peptide linker known as a highly rigid linker or a peptide linker whose two-dimensional structure forms an α-helix (hereinafter collectively referred to as a "rigid linker"), the transmembrane protein is localized in the organelle corresponding to the organelle localization signal, thereby completing the invention of the present application.

[0017] That is, the present invention relates to the following [1] to

[15] .

[0018] [1] A peptide linker, wherein the peptide linker is used to localize a transmembrane protein in a target organelle, the peptide linker is inserted between the transmembrane protein and an organelle localization signal corresponding to the target organelle, the number of proline residues contained in the peptide linker exceeds 40% of the number of all amino acid residues contained in the peptide linker, and the number of amino acid residues constituting the peptide linker is 60 residues or more.

[0019] [2] A peptide linker, wherein the peptide linker is used to localize a transmembrane protein in a target organelle, the peptide linker is inserted between the transmembrane protein and an organelle localization signal corresponding to the target organelle, the peptide linker is composed of a peptide having a two-dimensional structure of an alpha helix, and the number of amino acid residues constituting the peptide linker is greater than 70 residues.

[0020] [3] A fusion protein, wherein the fusion protein comprises: a transmembrane protein, an organelle localization signal for localizing in 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 number of all amino acid residues contained in the peptide linker, and the number of amino acid residues constituting the peptide linker is 60 residues or more.

[0021] [4] A fusion protein, wherein the fusion protein comprises: a transmembrane protein, an organelle localization signal for localizing in a target organelle, and a peptide linker inserted between the transmembrane protein and the organelle localization signal, wherein the peptide linker is composed of a peptide having a two-dimensional structure of an α-helix, and the number of amino acid residues constituting the peptide linker is greater than 70 residues.

[0022] [5] A method for localizing a transmembrane protein in a target organelle, comprising the step of inserting a peptide linker between the transmembrane protein and an organelle localization signal for localizing the protein in 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 residues.

[0023] [6] A method for localizing a transmembrane protein in a target organelle, comprising the step of inserting a peptide linker between the transmembrane protein and an organelle localization signal for localizing the protein in the target organelle, wherein the peptide linker is composed of a peptide having a two-dimensional structure of an α-helix, and the number of amino acid residues constituting the peptide linker is greater than 70 residues.

[0024] [6-1] A nucleic acid comprising a sequence encoding the peptide linker described in [1] or [2].

[0025] [7] A nucleic acid, wherein the nucleic acid encodes the fusion protein described in [3] or [4].

[0026] [8] A vector, wherein the vector expresses the fusion protein described in [3] or [4].

[0027] [9] The peptide linker, fusion protein, localization method, nucleic acid or vector as described in any one of [1] to [8], wherein the target organelle is selected from the group consisting of a nucleus, a mitochondria, a chloroplast, an endoplasmic reticulum and a peroxisome.

[0028]

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

[0029]

[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.

[0030]

[12] A drug for treating mitochondrial disease, wherein the target organelle is a mitochondria, and the drug for treating mitochondrial disease comprises the fusion protein described in [3] or [4] or the vector described in [8].

[0031]

[13] A method for treating mitochondrial disease, wherein the target organelle is mitochondria, and the fusion protein described in [3] or [4] or the vector described in [8] is administered to a patient.

[0032]

[14] The fusion protein described in [3] or [4] or the vector described in [8], wherein the target organelle is a mitochondria, and the fusion protein or vector is used for the treatment of mitochondrial disease.

[0033]

[15] Use of the fusion protein described in [3] or [4] or the vector described in [8] in the manufacture of a therapeutic drug for mitochondrial disease, wherein the target organelle is a mitochondria.

[0034] Effects of the Invention

[0035] According to the present invention, a peptide linker and a localization method for localizing a transmembrane protein in a target organelle, and a localized fusion protein can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a diagram schematically showing the structure of the peptide linker, fusion protein, and fusion protein involved in the localization method of the present invention.

[0037] Figure 2 This is a diagram schematically showing a fusion protein in which a flexible linker is inserted between a transmembrane protein and an organelle localization signal. It is presumed that since the peptide linker is flexible, the organelle localization signal and the transmembrane protein undergo intramolecular hydrophobic interactions, thereby preventing localization in the target organelle.

[0038] Figure 3 This is a diagram schematically showing a fusion protein in which a rigid linker is inserted between a transmembrane protein and an organelle localization signal. It is presumed that since the peptide linker is rigid, the organelle localization signal and the transmembrane protein do not undergo intramolecular hydrophobic interaction, thereby being localized in the target organelle.

[0039] Figure 4 This is a diagram showing the mechanism of treating mitochondrial diseases through complementary therapies.

[0040] Figure 5 This is a diagram showing an outline of a nucleic acid corresponding to the translation region of a plasmid used for expressing a protein in a cell in the Examples.

[0041] Figure 6 This is a graph showing the relationship between the number of amino acid residues contained in the proline linker in Example 1 and the correlation coefficient in the colocalization analysis.

[0042] Figure 7The diagram shows a representative fluorescence image of a group in which the number of amino acid residues contained in the proline linker is 0 residues in Example 1.

[0043] Figure 8 This is a diagram showing a representative fluorescent image of a plurality of cells in the group in which the number of amino acid residues contained in the proline linker is 0 residues in Example 1.

[0044] Fig. 9 The diagram shows representative fluorescence images in the groups where the number of amino acid residues contained in the proline linker in Example 1 is 70, 80, 90, 100, 150, and 200 residues.

[0045] Fig.10 This is a graph showing the relationship between the ratio of proline residues in the amino acid residues contained in the proline linker in Example 2 and the correlation coefficient in the colocalization analysis.

[0046] Fig.11 The diagram shows a representative fluorescence image of the group in Example 2 in which the ratio of proline residues among the amino acid residues contained in the proline linker is 30%.

[0047] Fig.12 The figure shows a representative fluorescence image of a plurality of cells in the group in which the ratio of proline residues among the amino acid residues contained in the proline linker in Example 2 is 30%.

[0048] Fig.13 This is a graph showing the relationship between the number of amino acid residues contained in the α-helical linker and the correlation coefficient in the colocalization analysis in Example 3.

[0049] Fig.14 The diagram shows a representative fluorescence image of a group of 300 amino acid residues contained in the α-helical linker in Example 3.

[0050] Fig.15 The diagram shows a representative fluorescence image of a group of 200 amino acid residues contained in the α-helical linker in Example 3. DETAILED DESCRIPTION

[0051] One aspect of the present invention relates to a peptide linker, wherein the peptide linker is used to localize a transmembrane protein in a target organelle, and the peptide linker is inserted between the transmembrane protein and an organelle localization signal corresponding to the target organelle.

[0052] Another aspect of the present invention relates to a fusion protein, wherein the fusion protein comprises: a transmembrane protein, an organelle localization signal, and a peptide linker inserted between the transmembrane protein and the organelle localization signal.

[0053] Another aspect of the present invention relates to a method for localizing a transmembrane protein in an organelle corresponding to an organelle localization signal (hereinafter also referred to as a "localization method"), which includes the step of inserting a peptide linker between the above-mentioned transmembrane protein and the above-mentioned organelle localization signal.

[0054] like Figure 1 As shown, the fusion protein involved in the peptide linker, fusion protein and localization method of the present invention at least comprises: a transmembrane protein, an organelle localization signal, and a peptide linker inserted between the transmembrane protein and the organelle localization signal, and the peptide linker is a rigid linker.

[0055] Although not wishing to be bound by any theory, the mechanism by which the fusion protein involved in the peptide linker, fusion protein, and localization method of the present invention can be localized in the 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 has high hydrophobicity, and therefore, Figure 2 As shown, the hydrophobic regions of the transmembrane protein and the organelle localization signal undergo hydrophobic interactions within the molecule. In this case, the recognition mechanism of the organelle localization signal such as the signal recognition particle (SRP) or the pre-sequence receptor cannot recognize the organelle localization signal, so the fusion protein cannot be localized in the target organelle. In contrast, when the peptide linker inserted between the transmembrane protein and the organelle localization signal is a rigid linker involved in the peptide linker, fusion protein and localization method of the present invention, the rigid linker cannot bend, and the transmembrane protein and the organelle localization signal do not undergo hydrophobic interactions within the molecule. Therefore, as Figure 3 As shown, the organelle localization signal can exist in a free state with the transmembrane protein. Therefore, the recognition mechanism of the organelle localization signal such as the signal recognition particle (SRP) or the pre-sequence receptor can recognize the organelle localization signal, so that the fusion protein can be localized in the target organelle.

[0056] (1) Transmembrane proteins

[0057] Transmembrane proteins are membrane proteins that exist through the lipid bilayer membrane of biological membranes and become the main constituent elements of biological membranes. They exist in the form of pumps, transporters, ion channels, receptors, enzymes or membrane structural proteins, and play the various functions of biological membranes. There are one or more hydrophobic regions (hereinafter, also referred to as "transmembrane domains") in the molecules of transmembrane proteins, which are fixed to biological membranes by hydrophobic interactions, and other regions are exposed to the outside of lipid bilayer membranes. Generally speaking, the hydrophobic region is composed of about 20 hydrophobic amino acids, and there are high hydrophilic amino acids such as charged amino acids at both ends.

[0058] Regarding the transmembrane proteins involved in the peptide linker, fusion protein and positioning method of the present invention, as long as it is a transmembrane protein that penetrates the lipid bilayer membrane more than once when positioned on the cell membrane or organelle membrane, for example, it can be a single-transmembrane type protein or a multi-transmembrane type protein. In particular, single-transmembrane type proteins are divided into type I that exposes the N-terminal side in the lumen of the endoplasmic reticulum when inserted into the endoplasmic reticulum membrane during the translation process and type II that exposes the C-terminal side. The single-transmembrane type proteins involved in the peptide linker, fusion protein and positioning method of the present invention can be either type I or type II. In addition, there is no particular limitation on the secondary structure of the transmembrane domain of the transmembrane protein, for example, it can be either an α-helical type or a β-folded type.

[0059] There is no particular limitation on the source of the transmembrane protein involved in the peptide linker, fusion protein and positioning method of the present invention, and the transmembrane protein may be a transmembrane protein existing in nature, or a transmembrane protein obtained by replacing or deleting a part of the amino acid residues within the scope of not damaging the function of the protein existing in nature, or an artificially synthesized transmembrane protein. In addition, when the transmembrane protein is derived from eukaryotic cells, there is no particular limitation on its positioning under physiological conditions, for example, it may be a protein of cell membrane positioning, or a protein of membrane positioning of an organelle such as a cell nucleus, mitochondria, chloroplast, endoplasmic reticulum, Golgi body, secretory granules, secretory vesicles, lysosomes, phagosomes, endosomes, vacuoles or peroxisomes. In addition, the transmembrane protein may be a transmembrane protein obtained by replacing or deleting a part or the whole of the amino acid sequence equivalent to the organelle positioning signal of the transmembrane protein of organelle positioning.

[0060] Examples of the transmembrane proteins involved in the present invention include aquaporins, CLC family ion channels, bestrophin, cysteine-loop (Cys-loop) type ligand-dependent ion channels, ion channel-type glutamate receptors, ion channel-type ATP receptors, potential-dependent potassium ion channels, cyclic nucleotide-dependent channels, calcium-activated potassium ion channels, TRP channels, sodium ion channels, calcium ion channels, CatSper channels, two-pore potassium ion channels, inwardly rectifying potassium ion channels, potential-dependent anion channels (VDAC), ABC transporters, P-type ATPases, V-type ATPases, SLC35 gene family transporters, SLC family transporters, APC superfamily transporters, mitochondrial carriers, MFS-type transporters, Sider oflexin, vesicle-associated membrane protein (VAMP), rhodopsin phosphodiesterase, adhesion-type G protein-coupled receptor, secretin receptor, metabotropic glutamate receptor, Frizzled / Taste2 receptor, EGF receptor-activated kinase, FGF receptor-activated kinase, ephrin receptor-activated kinase, serine / threonine kinase receptor, AXL-activated kinase, insulin receptor, receptor-type guanylate cyclase, PDGF receptor, TNF / NGF receptor, integrin, receptor-type tyrosine phospholipase, plexin, Notch protein, selectin, contactin, NADH-ubiquinone oxidoreductase, cytochrome c oxidase, flavin-containing monooxygenase (Flavin containing monooxygenases), non-specific monooxygenases, cytochrome P450, acyltransferases, glycosyltransferases, phosphotransferases, sulfotransferases, non-receptor tyrosine phosphatases, phosphodiester hydrolases, membrane-localized peptidases, nucleoside diphosphatases, adenylate cyclases, SAM complex-forming proteins, TOM complex-forming proteins, TIM complex-forming proteins, mitochondrial fusion proteins (Mitofusin) and ERMES complex-forming proteins, etc.

[0061] (2) Organelle localization signals

[0062] In this specification, an organelle localization signal refers to a peptide having the property of localizing a protein to an organelle (target organelle) corresponding to the organelle localization signal by addition to the protein. Generally speaking, proteins existing in a free state in cells, such as cytoplasmic localization proteins, are easily localized to the target organelle by adding an organelle localization signal, whereas proteins showing strong localization to a specific organelle under physiological conditions are difficult to localize by an organelle localization signal.

[0063] The organelle localization signal involved in the peptide linker, fusion protein and localization method of the present invention is not particularly limited as long as it is an organelle localization signal that can localize the protein to the target organelle by adding it to the protein. It can be an organelle localization signal existing in nature, or an organelle localization signal obtained by replacing or deleting a part of amino acid residues within the range that does not damage the function of the organelle localization signal existing in nature, or an artificially synthesized organelle localization signal. When the organelle localization signal is an organelle localization signal existing in nature, the organelle localization signal can be the full length of the protein localized in the target organelle, or it can be a part of the protein localized in the target organelle, but from the viewpoint of avoiding inhibition of the function of the transmembrane protein, it is preferably a part of the protein localized in the target organelle.

[0064] The amino acid sequence of a protein localized in a target organelle can be obtained from a known protein database such as the protein database of the National Center for Biotechnology Information (NCBI) of the United States.

[0065] As target organelles, for example, nuclei, mitochondria, chloroplasts, endoplasmic reticulum, Golgi bodies, secretory granules, secretory vesicles, lysosomes, phagosomes, endosomes, vacuoles, peroxisomes, etc. From the viewpoint of controlling the dynamics of proteins in cells or energy production, nuclei, mitochondria, chloroplasts, endoplasmic reticulum, or peroxisomes are preferred, and from the same viewpoint, nuclei or mitochondria are more preferred, and from the viewpoint of controlling the functions of cells, mitochondria are particularly preferred.

[0066] When the target organelle is mitochondria, generally speaking, the organelle localization signal is added to the N-terminus. As a mitochondrial localization signal (Mitochondria targeting signal: also recorded as MTS.), a peptide described in a known document (e.g., Gunnar von HEIJNE et al., "European Journal of Biochemistry", 1989, Vol. 180, p535-545) can be used. From the viewpoint of improving the localization to mitochondria, the number of amino acid residues constituting the mitochondrial localization signal is preferably 15 residues or more and 300 residues or less, more preferably 16 residues or more and 250 residues or less, further preferably 17 residues or more and 200 residues or less, and further preferably 18 residues or more and 160 residues or less. In addition, from the viewpoint of improving the localization to mitochondria by interaction with the Tom complex, the number of leucine residues contained in the mitochondrial localization signal can be more than 10% of the number of all amino acid residues contained in the organelle localization signal. From the same viewpoint, the number of arginine residues contained in the mitochondrial localization signal may be 10% or more of the number of all amino acid residues contained in the organelle localization signal. As an example of a mitochondrial localization signal, for example, a peptide having an amino acid sequence shown in the following sequence numbers 1 to 3 and added to the N-terminus of a protein can be cited.

[0067] (a)MLSLRQSIRFFKPATRTLCSSRYL(sequence number 1)

[0068] (b)MRAPSARALLLIPRRGPAVRAWAPAVSSRIWLASEWTPLVR AWTSLIHKPGSGLRFPAPLSGLPGGVGQWATSSGARRCWVLAGPR AAHPLFARLQGAAATGVRDLGNDSQRRPAATGRSEVWKLLGLVRPERGRLSAAV (serial number 2)

[0069] (c)MALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPE PNNPHADRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASP LSTSSSSSRPADKAQLTWVDKWIPEAARPY (Serial Number 3)

[0070] When the target organelle is the nucleus, generally speaking, the organelle localization signal can also be added to a position other than the end of the peptide, so the nuclear localization signal (Nuclear localization signal: also referred to as NLS.) can be added to the N-terminus or the C-terminus, and further, the peptide chain can be added to the N-terminus side of the nuclear localization signal added to the N-terminus or to the C-terminus side of the nuclear localization signal added to the C-terminus. As the nuclear localization signal, the peptides described in the known literature (e.g., Shunichi Kosugi et al., Journal of Biochemistry, 2009, Vol. 284, p478-485 and Allison Lange et al., Journal of Biochemistry, 2007, Vol. 282, p5101-5105) can be used. There is a single segment type in 1 region and a double segment type in 2 regions in the cell nuclear localization signal in which the signal sequence is located in the peptide, and the cell nuclear localization signal involved in the peptide connexon of the present invention, fusion protein and localization method can be either a single segment type or a double segment type. The number of amino acid residues constituting the cell nuclear localization signal is, for example, below 20 residues, can be below 18 residues, or can be below 16 residues. In addition, from the viewpoint of improving the cell nuclear localization, the ratio occupied by the total number of lysine residues and arginine residues in the number of amino acid residues constituting the cell nuclear localization signal can be more than 20%. As an example of the cell nuclear localization signal, a peptide with the amino acid sequence shown in the following sequence number 4 can be enumerated.

[0071] (a)PKKKRRV (sequence number 4)

[0072] When the target organelle is a peroxisome, the peroxisome targeting signal (PTS) as an organelle targeting signal is divided into PTS1 added to the C-terminal side and PTS2 added to the N-terminal side. The PTS involved in the peptide linker, fusion protein and positioning method of the present invention can be either PTS1 or PTS2. As PTS1, for example, the peptides shown below can be exemplified, and as PTS2, for example, the peptides shown below can be exemplified. In addition, if there is no special record below, X in the recorded amino acid sequence represents an arbitrary amino acid.

[0073] (a)SKL

[0074] (b)RLXXXXXHL

[0075] When the target organelle is a chloroplast, generally speaking, the organelle localization signal is added to the N-terminus. As a chloroplast localization signal, a peptide described in a known document (e.g., Gunnar von HEIJNE, "European Journal of Biochemistry" (EUROPEAN JOURNAL OF BIOCHEMISTRY), 1989, vol. 180, p535-545) can be used. In addition, a chloroplast localization signal peptide (transit peptide) of the small subunit of Rubisco (rubisco, ribulose bisphosphate carboxylase / oxygenase) of any plant species can also be used. From the viewpoint of improving the localization to the chloroplast, the number of amino acid residues constituting the chloroplast localization signal is preferably 30 or more and 100 or less.

[0076] (3) Peptide linker

[0077] The peptide linker involved in the peptide linker, fusion protein and positioning method of the present invention is a peptide linker composed of α amino acids (a linker formed by peptide-bonded amino acids), a proline-rich peptide linker known as a linker with high rigidity (hereinafter, also referred to as a proline linker) or a peptide linker whose two-dimensional structure forms an α helix (hereinafter, also referred to as an α helix linker). In addition, in the present specification, α amino acids include proline.

[0078] When the peptide linker, fusion protein and positioning method of the present invention involve a proline linker, from the perspective of improving 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 can be 30% or more of the total number of amino acid residues contained in the peptide linker, preferably 40% or more, more preferably more than 40%, and more preferably 50% or more. In addition, from the same point of view, with respect to amino acid residues other than proline residues contained in the peptide linker, the average number of proline residues bonded to each amino acid residue peptide is preferably 0.8 or more, more preferably 1.0 or more, more preferably 1.2 or more, more preferably 1.5 or more, and more preferably 1.8 or more. In addition, from the same point of view, the number of amino acid residues constituting the peptide linker can be 60 residues or more, preferably more than 60 residues, more preferably 70 residues or more, and, from the perspective of improving expression efficiency, for example, 600 residues or less, 500 residues or less, 400 residues or less, or 300 residues or less.

[0079] The α-helical linker forms a helical structure, i.e., an α-helix, which is a two-dimensional structure with 3.6 residues around it, thereby becoming a rigid linker that is difficult to bend. That is, when the peptide linker, fusion protein, and positioning method of the present invention involve an α-helical linker, the amino acid sequence constituting the peptide linker can be an amino acid sequence that can form an α-helix, without any particular limitation, and can be a naturally occurring L-type amino acid, or can include non-natural amino acids and D-type amino acids. When the peptide linker, fusion protein, and positioning method of the present invention involve an α-helical linker, from the perspective of improving 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 can be 70 residues or more, preferably 80 residues or more, more preferably 90 residues or more, and, from the perspective of improving expression efficiency, for example, 600 residues or less, 500 residues or less, 400 residues or less, or 300 residues or less. Examples of the α-helical linker include peptide linkers having the following SEQ ID NOs: 5 to 10 as repeating units.

[0080] (a) EAAAK (sequence number 5)

[0081] (b) EAAAR (sequence number 6)

[0082] (c) EAAAQ (sequence number 7)

[0083] (d) DAAAK (sequence number 8)

[0084] (e)DAAAR (serial number 9)

[0085] (f) DAAAQ (serial number 10)

[0086] (4) Fusion protein

[0087] The fusion protein involved in the peptide linker, fusion protein and localization method of the present invention comprises at least a transmembrane protein, an organelle localization signal, and a peptide linker inserted between the transmembrane protein and the organelle localization signal. The fusion protein may be a simple protein composed only of amino acids or a complex protein containing constituent elements other than amino acids, and is usually a simple protein from the perspective of ease of acquisition.

[0088] The fusion protein involved in the peptide linker, fusion protein and positioning method of the present invention can be further inserted with a linker between the peptide linker and the transmembrane protein and / or between the peptide linker and the organelle localization signal. There is no particular limitation on the structure of the inserted linker. From the perspective of the ease of acquisition, the linker can be a linker formed by peptide-bonded amino acids. In addition, from the perspective of ensuring the rigidity of the peptide chain between the fusion protein and the organelle localization signal and improving the localization of the fusion protein to the target organelle by suppressing intramolecular hydrophobic interactions, the length of the linker can be, for example, less than 200nm, less than 150nm, less than 100nm, less than 50nm, or less than 25nm. When the linker is a linker formed by amino acids bonded by a peptide, from the same point of view, the number of amino acid residues constituting the linker can be, for example, less than 400 residues, less than 300 residues, less than 200 residues, less than 100 residues, less than 50 residues, and, for example, can be less than 2.0 times, less than 1.5 times, less than 1.0 times, or less than 0.5 times the number of amino acid residues constituting the peptide linker.

[0089] The fusion protein involved in the peptide linker, fusion protein and localization method of the present invention may further include any peptide domain at the end of the peptide end of the fusion protein that is not bound to the organelle localization signal. The peptide domain may include functional domains such as fluorescent protein domains such as GFP, CFP and RFP, and affinity tag domains such as His tags, FLAG tags and GST tags.

[0090] (5) Nucleic acids, vectors, and methods for obtaining them

[0091] Another aspect of the present invention is a nucleic acid encoding the fusion protein involved in one aspect of the present invention or a vector expressing the fusion protein involved in one aspect of the present invention.

[0092] The nucleic acid involved in 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 the fusion protein involved in one aspect of the present invention or a base sequence having a sequence identity of more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with the base sequence. It can be any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a complex of DNA and RNA, and can also be a single-stranded nucleic acid or a double-stranded nucleic acid, and can also be genomic DNA or cDNA (complementary DNA). The nucleic acid involved in a preferred embodiment of the present invention is a nucleic acid containing a base sequence corresponding to the amino acid sequence of the fusion protein involved in one aspect of the present invention or a base sequence having a sequence identity of more than 90% with the base sequence.

[0093] For the vector involved in one embodiment of the present invention, as long as it can express the fusion protein involved in one aspect of the present invention in cells, there is no particular limitation. For example, it can be a transient expression vector or a stable expression vector, and it can be a plasmid containing at least a promoter sequence and a base sequence corresponding to the amino acid sequence of the fusion protein, or a vector into which a base sequence corresponding to the amino acid sequence of the fusion protein is introduced into a viral vector such as an adeno-associated virus, an adenovirus, a retrovirus, or a lentivirus. The vector into which the nucleic acid is introduced can contain various sequences such as restriction enzyme sites, regulatory sequences for expressing the inserted gene, antibiotic resistance genes, and sequences for identifying transformants. The vector involved in one embodiment of the present invention can be easily obtained by a method commonly performed by a person skilled in the art, for example, it can be obtained by introducing the nucleic acid involved in one embodiment of the present invention as an insert into any vector using a ligation reaction and cloning. As a vector into which the above-mentioned nucleic acid is introduced, for example, a commercially available product such as pEBMulti-Hygro vector (Fujifilm Wako Pure Chemical Industries, Ltd.) can be used.

[0094] (6) Implementation method of positioning method

[0095] The localization method involved in one aspect of the present invention can be implemented, for example, by introducing the fusion protein involved in one aspect of the present invention or a vector expressing the protein into a cell. The introduction into the cell can be easily performed by a method commonly performed by those skilled in the art, for example, when the vector is a viral vector, the vector is introduced into the cell by adding the viral vector to the culture environment of the cell or an animal individual. In addition, for example, when the vector is a plasmid or when the fusion protein itself is introduced into the cell, it can be introduced into the cell by liposome transfection, electroporation, microinjection, sonoporation or magnetofection, etc.

[0096] (7) Method for obtaining fusion protein

[0097] The fusion protein involved in one aspect of the present invention can be obtained by, for example, separation and purification from a host cell, wherein the fusion protein is expressed in the host cell by the same method as the localization method involved in one aspect of the present invention. There is no particular limitation on the host cell, and it can be a prokaryotic cell of Escherichia coli, Bacillus subtilis, etc., or a eukaryotic cell of yeast, animal cells, etc. Regarding animal cells, for example, it can be a mammalian cell, more specifically, a mouse cell or a human cell. In addition, there is no particular limitation on the method for separating the fusion protein from the host cell, and it can be carried out by the method commonly performed by those skilled in the art, for example, it can be separated and purified by dialysis, salting out, filtration, precipitation, affinity chromatography, ion exchange chromatography, gel electrophoresis, isoelectric point electrophoresis, hydrophobic chromatography, gel filtration column chromatography or countercurrent chromatography, etc. after the membrane of the host cell is dissolved. For these methods, it can be used alone or in combination with a variety of methods.

[0098] (8) Treatment of mitochondrial disease

[0099] Mitochondrial disease, one of the congenital genetic diseases, is a general term for diseases caused by mitochondrial dysfunction. It is a disease that causes various severe symptoms, including decreased muscle strength and developmental delay. Mitochondria are the only organelles other than the nucleus that have organelle-specific DNA called mitochondrial DNA (mtDNA) and can express proteins in the organelles. Mitochondrial proteins expressed by mitochondrial DNA are essential for the function of mitochondria. Therefore, in mitochondrial diseases, abnormal protein expression caused by mitochondrial DNA is confirmed. That is, in mitochondrial diseases, due to the defect or dysfunction of the protein expressed by mitochondrial DNA, the mitochondria themselves become dysfunctional, causing various severe symptoms.

[0100] As one of the treatments for mitochondrial diseases, Figure 4 As shown, the following scheme can be considered: the protein expressed by mitochondrial DNA is expressed by the DNA of the cell nucleus and transported to the mitochondria, thereby supplementing the protein (hereinafter also referred to as "supplementation therapy"). However, mitochondria are small cell organs with lipid bilayer membranes, i.e., inner and outer membranes, and most of the mitochondrial proteins necessary for life functions are transmembrane proteins. Therefore, it is difficult to localize transmembrane proteins in mitochondria in the prior art, so it is difficult to use supplementation therapy for treatment. On the other hand, the fusion protein or vector involved in one aspect of the present invention can localize the transmembrane protein in the target organelle in the cell, and therefore, it is believed that it can be applied to supplementation therapy for mitochondrial diseases.

[0101] That is, another aspect of the present invention is a mitochondrial disease therapeutic drug, which comprises the fusion protein or vector involved in one aspect of the present invention, and the target organelle is the mitochondria. The transmembrane protein involved in the fusion protein or vector in this embodiment is a transmembrane protein that is defective or dysfunctional in mitochondrial disease, or a transmembrane protein that can replace the function of the transmembrane protein. The organelle localization signal involved in the fusion protein or vector in this embodiment is a mitochondrial localization signal.

[0102] The fusion protein or vector contained in the mitochondrial disease therapeutic drug involved in one embodiment of the present invention can be encapsulated in a drug delivery carrier such as liposomes, microspheres, nanospheres, polymer micelles or exosomes. In addition, there is no particular limitation on the administration method of the mitochondrial disease therapeutic drug involved in one embodiment of the present invention, and for example, it can be administered by oral administration, inhalation, intravenous administration, intramuscular administration, subcutaneous administration or transdermal administration.

[0103] (9) Nucleic acid encoding peptide linker

[0104] Another aspect of the present disclosure is a nucleic acid comprising a sequence encoding a peptide connector involved in one aspect of the present invention. The nucleic acid involved in 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 the peptide connector involved in one aspect of the present invention or a base sequence having a sequence identity of more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%. It can be any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a complex of DNA and RNA, a single-stranded nucleic acid or a double-stranded nucleic acid, and can also be genomic DNA or cDNA (complementary DNA). The nucleic acid involved in a preferred embodiment of the present invention is a nucleic acid containing a base sequence corresponding to the amino acid sequence of the peptide connector involved in one aspect of the present invention or a base sequence having a sequence identity of more than 90% with the base sequence. The nucleic acid involved in one embodiment of the present invention can be a base sequence corresponding to the amino acid sequence of the peptide linker involved in one aspect of the present invention, or a base sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the base sequence, which accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95% or 100% of the base sequence of the nucleic acid.

[0105] Hereinafter, the present invention will be described in detail with reference to Examples and the like, but the present invention is not limited thereto.

[0106] Example

[0107] [Experimental method 1: Fusion protein and its expression vector]

[0108] exist Figure 5 The following is a summary of the nucleic acid corresponding to the translation region of the plasmid used to express the fusion protein in the cell in the example. Figure 5In the sequence, the left side is the 5' end. The plasmid containing the above translation region was prepared as follows. Regarding the nucleic acid encoding COX8N25 and the spacer sequence, the primers containing these sequences were commissioned to Hokkaido System Science Co., Ltd. for synthesis and obtained. Regarding the nucleic acid encoding the peptide linker, it was commissioned to Eurofins Genomics Co., Ltd. for synthesis and obtained. Regarding the nucleic acid encoding CFP, it was purchased from ATUM (FPB-47-609). Regarding the nucleic acid encoding the transmembrane protein, it was obtained from mouse cDNA (Quick Clone cDNA, 637304, Takara BIO Co., Ltd.). These nucleic acids were PCR cloned using KODOne PCR Master Mix-Blue-(KMM-201, Toyobo Co., Ltd.), and inserted into the pEX-A2J2 vector purchased from Eurofins Genomics Co., Ltd. using In-FusionSnap Assembly Master Mix (Z8947N, Takara BIO Co., Ltd.), thereby preparing a construct for expressing protein. The construct was introduced into competent cells (Competent Quick DH5a, DNA-913F) of Toyobo Co., Ltd. for amplification, and purified using a plasmid purification kit (NucleoSpin Plasmid, U0588A) of TakaraBIO Co., Ltd. to obtain a plasmid. In addition, a portion of the Hipp 11 region (1615 bp) of the Chinese Hamster was introduced into the pEX-A2J2 vector, and when the Cas9 protein was expressed at the same time, it became a vector capable of knocking in by homologous recombination (in the examples described in this specification, transient expression is sufficient, so Cas9 is not expressed). The protein expressed in cells using this plasmid includes, from the N-terminus, a mitochondrial localization signal (SEQ ID NO. 1, COX8N25), a peptide of 6 residues remaining in the clone (Glycine-Serine-Alanine-Glycine-Serine-Alanine), a peptide linker, a transmembrane protein mABCB10d132 (SEQ ID NO. 11), a spacer sequence (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 132 amino acids on the N-terminal side of the mitochondrial inner membrane localization protein mouseABCB10 are deleted. By deleting the 132 amino acids on the N-terminal side corresponding to the mitochondrial localization signal, the mitochondrial localization is lost and localized in the endoplasmic reticulum (Non-Patent Document 3). In addition, it has been reported that by adding the MTS of other types of mitochondrial localization proteins instead of the deleted 132 amino acids on the N-terminal side, localization in the mitochondria is also impossible (Non-Patent Document 3).

[0109] [Experimental method 2: Preparation of cells expressing fusion protein]

[0110] The method for preparing the cells expressing the fusion protein used in the examples is as follows.

[0111] First, regarding cells, CHO-K1 cells (sold by JCRB Cell Bank. Cell accession number: JCRB9018, hereinafter also referred to as “cells”) cultured in Ham's F-12 Nutrition Mix (manufactured by Thermo Fisher Scientific, product number 11765054) medium (hereinafter also referred to as “medium”) containing fetal bovine serum at a final concentration of 10% were cultured to a concentration of 0.7×10 4 The cells were seeded in a 3-well plate (AGC Techno Glass Co., Ltd., product number 3970-103) at 10 cells / well.

[0112] Next, for the transformation solution, 20 μL of Opti-MEM (manufactured by Thermo Fisher Scientific, product number 31985062), 200 ng of the plasmid prepared according to Experimental Method 1, 0.2 μL of Plus reagent, and 0.8 μL of Lipofectamine LTX (manufactured by Thermo Fisher Scientific, product number 15338030) were mixed and allowed to stand at room temperature for 25 minutes to prepare a solution (transformation solution).

[0113] Finally, regarding the preparation process of cells expressing fusion proteins, the seeded cells were cultured in a culture medium at 37°C, 5% CO2 for 1 day, and then 10 μL of the transformation solution was added to each well of the culture medium for culturing cells, and the cells were allowed to stand at 37°C, 5% CO2 for 4 to 5 hours. Thereafter, the culture medium was replaced with a culture medium without the transformation solution, and the cells were further cultured at 37°C, 5% CO2 for 1 day.

[0114] [Experimental method 3: Fluorescence staining of mitochondria]

[0115] 24 hours after adding the transformation solution, the cells expressing the fusion protein prepared in Experimental Method 2 were cultured in a culture medium containing Mitotracker Deep Red (manufactured by Thermo Fisher Scientific, product number M22426, hereinafter also referred to as MTDR) dissolved at a final concentration of 50 nM at 37°C and 5% CO2 for 1 hour to perform fluorescent staining of the mitochondria.

[0116] [Experimental method 4: fluorescence microscopy]

[0117] The cells obtained in Experimental Method 3 were observed by fluorescence microscopy according to the following operating procedures. One to six hours after the fluorescent staining of mitochondria, the cells were observed by fluorescence microscopy using an electric inverted microscope IX83 (manufactured by Olympus) equipped with a 100x objective lens UplanSApo 100x (manufactured by Olympus) and a digital CMOS camera ORCA-flash4,0V3 (manufactured by Hamamatsu Photonics, C13440-20CU), and a fluorescent image was obtained. As a fluorescent reflector unit, X3-FGWXL (excitation wavelength 530 to 550 nm, fluorescence wavelength 570 nm or more) was used for the fluorescence observation of MTDR (mitochondria), and IX3-FCFPXL (excitation wavelength 425 to 445 nm, fluorescence wavelength 460 to 510 nm) was used for the fluorescence observation of CFP (fusion protein).

[0118] [Experimental method 5: colocalization analysis]

[0119] The fluorescence images of MTDR and CFP obtained in Experimental Method 4 were subjected to colocalization analysis according to the following operating procedures. For the fluorescence image, an image with high-frequency components including noise removed was produced by a median filter with a window size of 35 pixels (pixel resolution 130 nm / pixel), and this was subtracted from the original fluorescence image to produce an analysis image with background light removed from the fluorescence image. For the two analysis images obtained from the fluorescence images of MTDR and CFP, the correlation coefficient of the brightness values ​​- the Pearson correlation coefficient (hereinafter also simply referred to as the "correlation coefficient") was calculated according to the method described in the known literature (Costes SV et al., Biophys. J. 86, 3993-4003 (2004)).

[0120] The significance test of the difference in correlation coefficients between different groups was performed by a two-sided test (Mann-Whitney U test) at a significance level of 1%.

[0121] [Example 1: Relationship between the number of amino acid residues contained in the proline linker and the positioning effect]

[0122] When the peptide linker is a proline linker, the relationship between the number of amino acid residues contained in the proline linker and the positioning effect was studied.

[0123] According to experimental methods 1 to 5, cells expressing fusion proteins (in which the number of amino acid residues contained in the proline linker is 0, 50, 60, 70, 80, 90, 100, 150 or 200 residues, and 50% of the amino acid residues contained in the proline linker are proline residues) were observed by fluorescence microscopy and colocalization analysis was performed. As an example, the amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the proline linker is 100 residues is shown in sequence number 14, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid used to express the fusion protein is shown in sequence number 15. When the number of amino acid residues contained in the proline linker is less than 100 residues, the 100 residues (SEQ ID NO. 16) of the proline linker region contained in the amino acid sequence of the fusion protein shown in SEQ ID NO. 14 are replaced with the amino acid residues of the number corresponding to the number of amino acid residues contained in the linker of each group from the C-terminal side of SEQ ID NO. 16 as the proline linker region. When the number of amino acid residues contained in the proline linker is 150 residues, the 100 residues (SEQ ID NO. 16) of the proline linker region contained in the amino acid sequence of the fusion protein shown in SEQ ID NO. 14 are replaced with an amino acid sequence having 50 residues on the N-terminal side of the amino acid sequence shown in SEQ ID NO. 16 as the proline linker region. When the number of amino acid residues contained in the proline linker is 200 residues, the proline linker region 100 residues (SEQ ID NO. 16) contained in the amino acid sequence of the fusion protein shown in SEQ ID NO. 14 is replaced with an amino acid sequence further having the amino acid sequence shown in SEQ ID NO. 16 on the N-terminal side of the amino acid sequence shown in SEQ ID NO. 16 as the proline linker region. In addition, the fusion protein in which the number of amino acid residues contained in the proline linker is 0 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 region encoding a peptide linker.

[0124] Figure 6 represents the correlation coefficient in the colocalization analysis corresponding to the number of amino acid residues contained in the proline linker. Figure 6 In the figure, the gray bar graph represents the mean value, the black error bar represents the standard deviation, and the asterisk (*) indicates that the correlation coefficient is significantly different from the group with 0 amino acid residues. Figure 7 and Figure 8 The representative fluorescence images in the group with 0 residues are shown in Fig. 9 Representative fluorescence images of groups of 70, 80, 90, 100, 150, and 200 residues are shown in FIG. Figures 7 to 9In FIG. 1 , the numerical value written outside the horizontal line or the white line indicating the number of amino acid residues represents the correlation coefficient in the region surrounded by the white line in each image.

[0125] like Figure 6 As shown in the figure, in the fusion proteins with more than 60 amino acid residues in the proline linker, the average value of the correlation coefficient increases; in the fusion proteins with more than 70 amino acid residues, the difference in the correlation coefficient between the fusion proteins with 0 amino acid residues is significant. Figure 7 and Figure 8 As shown, the fusion protein with 0 amino acid residues was distributed in a sieve-like manner throughout the cell, strongly suggesting that it was transferred to the endoplasmic reticulum. In contrast, Fig. 9 As shown, the fusion proteins with amino acid residues of 70, 80, 90, 100, 150 and 200 residues obtained fluorescence images highly similar to MTDR, strongly suggesting that it is localized in mitochondria.

[0126] [Example 2: Relationship between the ratio of proline residues contained in the proline linker and the positioning effect]

[0127] When the peptide linker is a proline linker, the relationship between the ratio of proline residues contained in the proline linker and the positioning effect was studied.

[0128] According to experimental methods 1 to 5, cells expressing fusion proteins (in which the number of amino acid residues contained in the proline linker is 100 residues, and 30%, 40% or 50% of the amino acid residues are proline residues) were observed by fluorescence microscopy and co-localization analysis was performed. The amino acid sequence of the fusion protein expressed when 30% of the amino acid residues contained in the proline linker are proline residues is shown in sequence number 17, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid used to express the fusion protein is shown in sequence number 18. The amino acid sequence of the fusion protein expressed when 40% of the amino acid residues contained in the proline linker are proline residues is shown in sequence number 19, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid used to express the fusion protein is shown in sequence number 20. When 50% of the amino acid residues contained in the proline linker are proline residues, the amino acid sequence of the expressed fusion protein is the same as the amino acid sequence shown in sequence number 14, and the base sequence of the nucleic acid equivalent to the translation region contained in the plasmid used to express the fusion protein is the same as the base sequence shown in sequence number 15.

[0129] Fig.10 In represents the correlation coefficient in the colocalization analysis corresponding to the ratio of proline residues in the amino acid residues contained in the proline linker. Fig.10In the figure, the gray bar graph represents the mean value, the black error bar represents the standard deviation, and the pound sign (#) indicates that there is a significant difference in the correlation coefficient between the groups in which 50% of the amino acid residues are proline residues. Fig.11 and Fig.12 Representative fluorescence images of a group in which 30% of the amino acid residues are proline residues. Fig.11 and Fig.12 In FIG. 1 , the numerical values ​​written near the outside of the white lines represent the correlation coefficients in the regions surrounded by the white lines in each image.

[0130] like Fig.10 As shown in FIG. 1 , the correlation coefficients of the fusion proteins in which the ratio of proline residues in the amino acid residues contained in the proline linker is 30% and 40% are small, and the difference in the correlation coefficient between the fusion protein in which the ratio of proline residues is 50% is significant. Fig.11 and Fig.12 As shown, for the fusion protein in which the ratio of proline residues in the amino acid residues contained in the proline linker is 30%, the fusion protein has a sieve-like distribution in the whole cell, similar to the fusion protein without the proline linker region, strongly suggesting that it is transferred to the endoplasmic reticulum. The fusion protein in which the ratio of proline residues is 40% also shows the same trend.

[0131] [Example 3: Relationship between the number of amino acid residues contained in the α-helical linker and the positioning effect]

[0132] When the peptide linker is an α-helical linker, the relationship between the number of amino acid residues contained in the α-helical linker and the positioning effect was studied.

[0133] According to experimental methods 1 to 5, cells expressing fusion proteins (in which the number of amino acid residues contained in the α-helical linker is 0, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 600 residues, respectively) were observed by fluorescence microscopy and colocalization analysis was performed. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 50 residues is shown in sequence number 21, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid used to express the fusion protein is shown in sequence number 22. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 60 residues is shown in sequence number 23, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid used to express the fusion protein is shown in sequence number 24. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 70 residues is shown in SEQ ID NO: 25, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 26. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 80 residues is shown in SEQ ID NO: 27, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 28. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 90 residues is shown in SEQ ID NO: 29, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 30. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 100 residues is shown in SEQ ID NO: 31, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 32. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 200 residues is shown in SEQ ID NO: 33, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 34. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 300 residues is shown in SEQ ID NO: 35, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 36. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 400 residues is shown in SEQ ID NO: 37, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 38. The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 500 residues is shown in SEQ ID NO: 39, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 40.The amino acid sequence of the fusion protein expressed when the number of amino acid residues contained in the α-helical linker is 600 residues is shown in SEQ ID NO: 41, and the base sequence of the nucleic acid corresponding to the translation region contained in the plasmid for expressing the fusion protein is shown in SEQ ID NO: 42. In addition, the fusion protein in which the number of amino acid residues contained in the α-helical linker is 0 and the plasmid for expressing the fusion protein are the same as the fusion protein in which the number of amino acid residues contained in the linker is 0 and the plasmid for expressing the fusion protein used in Example 1.

[0134] Fig.13 represents the correlation coefficient in the colocalization analysis corresponding to the number of amino acid residues contained in the α-helical linker. Fig.13 In the figure, the gray bar graph represents the mean value, the black error bar represents the standard deviation, and the asterisk (*) indicates that the correlation coefficient is significantly different from the group with 0 amino acid residues. Fig.14 The number of amino acid residues is shown in the representative fluorescence images of the group of 300 residues. Fig.15 The number of amino acid residues is shown in the representative fluorescence images of the group of 200 residues. Fig.14 and Fig.15 In FIG. 1 , the numerical value written on the horizontal line of the number of amino acid residues represents the correlation coefficient in the region surrounded by the white line in each image.

[0135] like Fig.13 As shown in the figure, in the fusion proteins with 70 or more amino acid residues in the α-helical linker, the average value of the correlation coefficient increases; in the fusion proteins with 90 or more amino acid residues, the difference in the correlation coefficient between the fusion proteins with 0 amino acid residues is significant. Fig.14 and 15 As shown, the fusion proteins with 200 and 300 amino acid residues obtained fluorescence images highly similar to MTDR, strongly suggesting that it is localized in mitochondria.

Claims

1. A peptide linker, wherein This peptide linker is used to localize transmembrane proteins in target organelles. The peptide linker is inserted between the transmembrane protein and the organelle localization signal corresponding to the target organelle. The number of proline residues contained in the peptide linker exceeds 40% of the number of all amino acid residues contained in the peptide linker, and the number of amino acid residues constituting the peptide linker is 60 residues or more.

2. A peptide linker, wherein This peptide linker is used to localize transmembrane proteins in target organelles. The peptide linker is inserted between the transmembrane protein and the organelle localization signal corresponding to the target organelle. The peptide linker is composed of a peptide whose two-dimensional structure is an α-helix, and the number of amino acid residues constituting the peptide linker is 70 residues or more.

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

4. A fusion protein, wherein: The fusion protein comprises: a transmembrane protein, an organelle localization signal for localization in a target organelle, and a peptide linker inserted between the transmembrane protein and the organelle localization signal. The peptide linker is composed of a peptide whose two-dimensional structure is an α-helix, and the number of amino acid residues constituting the peptide linker is 70 residues or more.

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

6. A method for localizing a transmembrane protein in a target organelle, wherein: comprising the step of inserting a peptide linker between the transmembrane protein and an organelle localization signal for localization to the target organelle, The peptide linker is composed of a peptide whose two-dimensional structure is an α-helix, and the number of amino acid residues constituting the peptide linker is 70 residues or more.

7. A nucleic acid, wherein The nucleic acid encodes the fusion protein according to claim 3 or 4.

8. A vector, wherein The vector expresses the fusion protein according to claim 3 or 4.

9. A drug for treating mitochondrial disease, wherein: Comprising: the fusion protein according to claim 3 or 4, or a vector expressing the fusion protein according to claim 3 or 4, The target organelle is mitochondria.

10. A nucleic acid, wherein The nucleic acid comprises a sequence encoding the peptide linker according to claim 1 or 2.

Images