Modified mitochondria and uses thereof

Abstract

To provide a recombinant protein for effective drug delivery, and to provide modified mitochondria produced using the same.SOLUTION: Provided are modified mitochondria in which a foreign protein is bound to the outer mitochondrial membrane.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a fusion protein capable of modifying mitochondria, mitochondria modified by the fusion protein, and a pharmaceutical composition containing the same as an active ingredient. [Background technology]

[0002] Mitochondria are eukaryotic cellular organelles responsible for the synthesis and regulation of adenosine triphosphate (ATP), the intracellular energy source. Mitochondria are involved in various in vivo metabolic pathways, such as cell signaling, cell differentiation, cell death, and the regulation of the cell cycle and cell proliferation. Mitochondria are organelles with their own genome that play a central role in cellular energy metabolism. Mitochondria produce energy through electron transport and oxidative phosphorylation processes and play an important role in the apoptosis signaling pathway.

[0003] It has been reported that reduced energy production due to mitochondrial dysfunction can lead to various diseases. When the function of the electron transport chain reaction is impaired due to mutations in the mitochondrial genome and proteome, reduced ATP production, excessive production of reactive oxygen species, and impaired calcium regulation can occur. In such cases, changes in mitochondrial membrane permeability can occur, leading to abnormal apoptosis, which can lead to cancer and incurable diseases.

[0004] Mitochondrial dysfunction has been reported to be responsible for a variety of human diseases, including mitochondrial-associated genetic disorders (Wallace DC 1999), diabetes (Maechler P 2001), heart disease (Sorescu D 2002), senile dementias such as Parkinson's disease and Alzheimer's disease (Lin MT 2006), and various cancers (Petros JA, 2005) and cancer metastasis (Ishikawa K, 2008). Furthermore, common features of over 200 different cancers include abnormal apoptosis, increased inflammatory responses, and abnormal metabolic processes. All of these processes are closely related to mitochondrial function, raising interest in the relationship between cancer and mitochondria.

[0005] While normal cells produce 36 ATP per mole of glucose through the electron transport chain process, cancer cells, unlike normal cells, are known to produce 2 ATP per mole of glucose through glycolysis under sufficient oxygen conditions (aerobic glycolysis). Therefore, unlike normal cells, cancer cells are known to use an energy-inefficient glycolytic process to produce amino acids, lipids, nucleic acids, and other nutrients necessary for rapid cell proliferation. Therefore, cancer cells require less oxygen than normal cells and produce more lactic acid.

[0006] Therefore, the abnormal metabolism in cancer cells causes changes in the composition of the cancer microenvironment, inhibits apoptosis due to dysfunctional mitochondria, and increases inflammatory responses, as well as abnormal metabolic responses in cancer cells, play a very important role in cancer growth. Therefore, developing metabolically related anticancer drugs that utilize such characteristics may be an excellent way to eliminate the side effects and economic problems of conventional anticancer drugs.

[0007] It is known that mitochondria can enter cells when mitochondria present in cells are isolated and used to treat cells in vitro, or when mitochondria are introduced into the body. Utilizing this phenomenon, it is possible to treat diseases caused by mitochondrial dysfunction by introducing normal mitochondria isolated from cells into the body, or in particular, by using mitochondria as a carrier to effectively deliver specific proteins into cells, thereby treating the disease, but this has not yet been reported. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide an effective protein delivery system by demonstrating that mitochondria can be used as a means for effectively delivering proteins capable of exhibiting various pharmacological effects into cells. Another object of the present invention is to provide a recombinant protein for effective drug delivery and modified mitochondria produced using the same. Another object of the present invention is to provide a pharmaceutical composition containing the modified mitochondria as an active ingredient. [Means for solving the problem]

[0009] To solve the above problems, modified mitochondria in which a foreign protein is bound to the outer mitochondrial membrane are provided. Furthermore, for preparing the modified mitochondria, a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a desired pharmacological protein is provided. Furthermore, a fusion protein comprising an antibody or a fragment thereof and a mitochondrial outer membrane anchoring peptide is provided. [Effects of the Invention]

[0010] When mitochondria bound to a foreign protein are administered to the human body, the foreign protein can be effectively delivered into cells. Furthermore, the pharmacologically active protein delivered into cells can restore impaired cellular functions. Furthermore, when mitochondria bound to a foreign protein containing a pharmacologically active protein are delivered into cells, the pharmacologically active protein is dissociated from the mitochondria in the cell and is expected to play a useful role. Furthermore, modified mitochondria containing antibody fragments can be effectively delivered to target cells. In particular, when an antibody fragment targeting a protein present on the surface of cancer tissue is bound to the mitochondrial surface, the modified mitochondria can be effectively delivered into cancer cells. Therefore, the introduction of modified mitochondria can not only restore a damaged electron transport system in cells, but also prevent or treat various diseases through the pharmacologically active protein bound to the modified mitochondria. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a method for producing pTA-p53. [Figure 2] FIG. 2 shows a method for constructing the pET15b-UB-p53 vector. [Figure 3] FIG. 3 shows the expression of UB-p53 protein in E. coli. [Figure 4] FIG. 4 shows a method for constructing the pET11C-TOM70-UB-p53 vector. [Figure 5] FIG. 5 shows the expression of TOM70-UB-p53 protein in E. coli. [Figure 6] FIG. 6 shows a method for constructing the pET11C-TOM70-(GGGGS)3-UB-p53 vector. [Figure 7] FIG. 7 shows the expression of TOM70-(GGGGS)3-UB-p53 protein in E. coli. [Figure 8] FIG. 8 shows the method for constructing the pET11C-TOM70-(GGGGS)3-p53 vector. [Figure 9] FIG. 9 shows the expression of TOM70-(GGGGS)3-p53 protein in E. coli. [Figure 10] FIG. 10 shows the method for constructing the pET15b-UB-p53-TOM7 vector. [Figure 11] FIG. 11 shows the expression of UB-p53-TOM7 protein in E. coli. [Figure 12] FIG. 12 shows a method for constructing the pCMV-p53-myc / His vector. [Figure 13] FIG. 13 shows the expression of p53-myc / His protein in transfected CHO. [Figure 14] FIG. 14 shows the results of purifying and then characterizing the TOM70-(GGGGS)3-p53 protein. [Figure 15] FIG. 15 shows purified TOM70-(GGGGS)3-p53 protein. [Figure 16] FIG. 16 shows the results of purifying and then characterizing the TOM70-(GGGGS)3-UB-p53 protein. [Figure 17] FIG. 17 shows purified TOM70-(GGGGS)3-UB-p53 protein. [Figure 18] FIG. 18 shows the results of purifying and then characterizing the UB-p53 protein. [Figure 19] FIG. 19 shows purified UB-p53 protein. [Figure 20] FIG. 20 shows the results of purifying and then characterizing the UB-p53-TOM7 protein. [Figure 21] FIG. 21 shows purified UB-p53-TOM7 protein. [Figure 22] FIG. 22 shows the construction method of the pTA-granzyme B vector. [Figure 23] FIG. 23 shows the construction method of the pET11C-TOM70-(GGGGS)3-UB-Granzyme B vector. [Figure 24]FIG. 24 shows the expression of TOM70-(GGGGS)3-UB-Granzyme B protein in E. coli. [Figure 25] FIG. 25 shows the construction method of the pET15b-UB-Granzyme B-TOM7 vector. [Figure 26] FIG. 26 shows the expression of UB-granzyme B-TOM7 protein in E. coli. [Figure 27] FIG. 27 shows the results of purification of TOM70-(GGGGS)3-UB-Granzyme B protein. [Figure 28] FIG. 28 shows purified UTOM70-(GGGGS)3-UB-Granzyme B protein. [Figure 29] FIG. 29 shows the construction method of the pTA-RKIP vector. [Figure 30] Figure 30 shows a method for constructing the pET11C-TOM70-(GGGGS)3-UB-RKIP vector. [Figure 31] FIG. 31 shows expression of TOM70-(GGGGS)3-UB-RKIP protein in E. coli. [Figure 32] FIG. 32 shows the results of purification of the TOM70-(GGGGS)3-UB-RKIP protein. [Figure 33] FIG. 33 shows purified TOM70-(GGGGS)3-UB-RKIP protein. [Figure 34] FIG. 34 shows the method for constructing the pTA-PTEN vector. [Figure 35] Figure 35 shows the method for constructing the pET11C-TOM70-(GGGGS)3-UB-PTEN vector. [Figure 36] Figure 36 shows expression of TOM70-(GGGGS)3-UB-PTEN protein in E. coli. [Figure 37] Figure 37 shows the results of purification of the TOM70-(GGGGS)3-UB-PTEN protein. [Figure 38] FIG. 38 shows purified TOM70-(GGGGS)3-UB-PTEN protein. [Figure 39] FIG. 39 shows the results of purifying and then characterizing the UB-GFP-TOM7 protein. [Figure 40] FIG. 40 shows purified UB-GFP-TOM7 protein. [Figure 41] Figure 41 shows the results of purifying and then characterizing the TOM70-(GGGGS)3-UB-GFP protein. [Figure 42] FIG. 42 shows purified TOM70-(GGGGS)3-UB-GFP protein. [Figure 43] Figure 43 shows the method for constructing the pET15b-UB-scFvHER2-TOM7 vector. [Figure 44] FIG. 44 shows the expression of UB-scFvHER2-TOM7 protein in E. coli. [Figure 45] Figure 45 shows the method for constructing the pCMV-scFvHER2-TOM7-myc / His vector. [Figure 46] FIG. 46 shows the expression of scFvHER2-TOM7-myc / His protein in transfected CHO. [Figure 47] FIG. 47 shows the results of purification of the UB-ScFvHER2-TOM7 protein. [Figure 48] FIG. 48 shows purified UB-ScFvHER2-TOM7 protein. [Figure 49] Figure 49 shows the method for constructing the pET15b-UB-scFvMEL-TOM7 vector. [Figure 50] Figure 50 shows the expression of UB-scFvMEL-TOM7 protein in E. coli. [Figure 51] Figure 51 shows the method for constructing the pCMV-scFvMEL-TOM7-myc / His vector. [Figure 52] FIG. 52 shows the expression of scFvMEL-TOM7-myc / His protein in transfected CHO. [Figure 53]Figure 53 shows a method for constructing the pCMV-scFvPD-L1-TOM7-myc / His vector. [Figure 54] Figure 54 shows the expression of scFvPD-L1-TOM7-myc / His protein in transfected CHO. [Figure 55] Figure 55 shows the binding of fluorescent proteins to the outer mitochondrial membrane. In this case, mitochondria stained with MitoTracker CMXRos appear red, while TOM70-UB-GFP appears green. The overlapping area of ​​the two appears yellow. In this case, the magnification of Figure 55a is 200x, and the magnification of Figure 55b is 600x. [Figure 56] FIG. 56 shows the results of Western blot analysis confirming that the recombinant proteins TOM70-(GGGGS)3-UB-p53 and UB-p53-TOM7 were bound to the outer mitochondrial membrane. [Figure 57] FIG. 57 shows the results of isolating foreign mitochondria, introducing the mitochondria into cells, and then observing the degree of intracellular introduction depending on the mitochondrial concentration using a fluorescence microscope. [Figure 58] FIG. 58 confirms the effect of normal mitochondria on the proliferation of skin cancer cells. [Figure 59] FIG. 59 is a graph confirming the effect of normal mitochondria on suppressing reactive oxygen species (ROS) production in skin cancer cells. [Figure 60] FIG. 60 confirms the effect of normal mitochondria on drug resistance. [Figure 61] FIG. 61 confirms the effect of intact mitochondria on antioxidant gene expression in cells. [Figure 62] FIG. 62 shows the effect of normal mitochondria on the expression of genes involved in cancer cell metastasis. [Figure 63] FIG. 63 shows a method for confirming the binding of recombinant p53 protein to the outer membrane of foreign mitochondria and the introduction of recombinant p53 protein into cells. [Figure 64]Figure 64 confirms that the recombinant protein p53 was bound to the outer mitochondrial membrane and that p53 was introduced into the cells. In this case, the magnification is 200x. [Figure 65] Figure 65 confirms that the recombinant protein p53 was bound to the outer mitochondrial membrane and that p53 was introduced into the cells. In this case, the magnification is 600x. [Figure 66] FIG. 66 shows a method for confirming the apoptotic ability of p53-bound modified mitochondria introduced into cells using a gastric cancer cell line. [Figure 67a] Figure 67a shows the confirmation of the apoptotic ability of p53-bound modified mitochondria introduced into gastric cancer cells by TUNEL assay, magnification 600x. [Figure 67b] FIG. 67b shows fluorescence measurement confirmation of the apoptotic potential of p53-bound modified mitochondria introduced into gastric cancer cells. [Figure 68] FIG. 68 is a diagram confirming the cancer cell metastasis suppression effect of RKIP-bound modified mitochondria in MDA-MB-231 cells. [Figure 69] FIG. 69 confirms that single chain variable fragment (ScFv) antibodies that target cancer cells were expressed in cells. [Figure 70] Figure 70 shows immunocytochemistry (ICC) experiments confirming that a single-chain variable fragment (ScFv) antibody targeting cancer cells was expressed and bound to mitochondria present in the cells, at 200x magnification. [Figure 71] Figure 71 shows immunocytochemistry (ICC) experiments confirming that a single-chain variable fragment (ScFv) antibody targeting cancer cells was expressed and bound to mitochondria present in the cells, at 600x magnification. [Figure 72] FIG. 72 is a diagram comparing the effects of introducing mitochondria bound to a single-chain variable fragment antibody that targets cancer cells into gastric cancer cell lines. [Figure 73] FIG. 73 shows the schedule of animal experiments using modified mitochondria. [Figure 74] FIG. 74 shows photographs visually observing the growth of tumor tissue. [Figure 75] Figure 75 is a graph confirming changes in mouse weight following administration of mitochondria and modified mitochondria. [Figure 76] Figure 76 confirms tumor size after administration of mitochondria and modified mitochondria. [Figure 77] FIG. 77 is a diagram confirming that modified mitochondria bound by TOM-UB-p53 protein are effective in suppressing the proliferation of A431 cells. [Figure 78] FIG. 78 shows the function of isolated mitochondria confirmed by ATP levels. [Figure 79] FIG. 79 shows the function of isolated mitochondria confirmed by membrane potential. [Figure 80] FIG. 80 shows the extent of damage to isolated mitochondria confirmed by measuring mitochondrial ROS (mROS production). [Figure 81a] FIG. 81a shows the structure of proteins present in the outer mitochondrial membrane and the amino acid sequence of the N-terminal region of TOM70, TOM20 or OM45. [Figure 81b] Figure 81b shows the amino acid sequence of the C-terminal region of TOM5, TOM7, FIs1, VAMP1B, Cytb5, BCL-2 or BCL-X. [Figure 82] FIG. 82 is a diagram confirming whether the desired protein is released depending on the presence or absence of a linker between the outer membrane anchoring peptide and ubiquitin. [Figure 83] Figure 83 confirms that the desired protein bound to the engineered mitochondria is sequestered from the mitochondria in cells. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. One aspect of the present invention provides modified mitochondria in which a foreign protein is bound to the outer mitochondrial membrane.

[0013] Mitochondria can be obtained from mammals, and can be obtained from humans. In particular, mitochondria can be isolated from cells or tissues. For example, mitochondria can be obtained from somatic cells, germ cells, or stem cells. Furthermore, mitochondria can be normal mitochondria obtained from cells with normal mitochondrial biological activity. Furthermore, mitochondria can be cultured in vitro.

[0014] Furthermore, mitochondria can be obtained from autologous, allogeneic, or xenogeneic subjects. Specifically, autologous mitochondria refers to mitochondria obtained from a subject's own tissues or cells. Furthermore, allogeneic mitochondria refers to mitochondria obtained from a subject of the same species as the subject but with a different allelic genotype. Furthermore, xenogeneic mitochondria refers to mitochondria obtained from a subject of a different species than the subject.

[0015] In particular, the somatic cells may be muscle cells, hepatocytes, nerve cells, fibroblasts, epithelial cells, adipocytes, bone cells, leukocytes, lymphocytes, platelets, or mucosal cells. Furthermore, the germ cells are cells that undergo meiosis and mitosis, and may be sperm or eggs. Furthermore, the stem cells may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, corneal epithelial stem cells, and tissue-derived stem cells. In this case, the mesenchymal stem cells may be any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0016] On the other hand, when mitochondria are isolated from specific cells, they can be isolated by various known methods, for example, by using a specific buffer solution or by utilizing a potential difference and a magnetic field.

[0017] As used herein, the term "foreign protein" refers to a protein, including a desired protein, that can function inside and outside a cell. In this case, the foreign protein may be a protein not present in mitochondria and may be a recombinant protein. In particular, the foreign protein may include a mitochondrial anchoring peptide and a desired protein. Furthermore, the foreign protein may be a recombinant fusion protein containing a mitochondrial anchoring peptide and a desired protein. In this case, the foreign protein may include a mitochondrial anchoring peptide. Preferably, the mitochondrial anchoring peptide may be a peptide that can be located on the outer mitochondrial membrane. Thus, the foreign protein can be bound to the outer mitochondrial membrane by the mitochondrial anchoring peptide. The mitochondrial anchoring peptide may be a peptide containing the N-terminal or C-terminal region of a protein present in a mitochondrial membrane protein, and the N-terminal or C-terminal region of a protein present in the outer mitochondrial protein membrane may be located on the outer mitochondrial membrane. In this case, the anchoring peptide may further include a mitochondrial signal sequence.

[0018] Examples of proteins present in mitochondrial membrane proteins may be any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. In particular, when the mitochondrial anchoring peptide is derived from any one selected from the group consisting of TOM20, TOM70, and OM45, it may include the N-terminal region of TOM20, TOM70, and OM45. Examples of mitochondrial anchoring peptides may be yeast-derived TOM70 of SEQ ID NO: 75 or human-derived TOM70 of SEQ ID NO: 76. Other examples may be yeast-derived TOM20 of SEQ ID NO: 77 or human-derived TOM20 of SEQ ID NO: 78. Another example may be yeast-derived OM45 of SEQ ID NO: 79.

[0019] Furthermore, when the mitochondrial anchoring peptide is derived from any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B, it may comprise the C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. An example of a mitochondrial anchoring peptide may be yeast-derived TOM5 of SEQ ID NO: 80 or human-derived TOM5 of SEQ ID NO: 81. Another example may be yeast-derived TOM7 of SEQ ID NO: 82 or human-derived TOM7 of SEQ ID NO: 83. Another example may be yeast-derived TOM22 of SEQ ID NO: 84 or human-derived TOM22 of SEQ ID NO: 85. Another example may be yeast-derived Fis1 of SEQ ID NO: 86 or human-derived Fis1 of SEQ ID NO: 87. Another example may be human-derived Bcl-2α of SEQ ID NO: 88. Another example may be yeast-derived VAMP1 of SEQ ID NO:89 or human-derived VAMP1 of SEQ ID NO:90.

[0020] In this case, the desired protein contained in the foreign protein and capable of functioning inside and outside the cell may be any one selected from the group consisting of active proteins that exhibit activity in cells, proteins present in cells, and proteins that have the ability to bind to ligands or receptors present in the cell membrane.

[0021] Examples of active proteins or proteins present in cells can be any one selected from the group consisting of p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun / Fos), EGR-1, retinoblastoma (RB), phosphatase and tensin homolog (PTEN), E-cadherin, neurofibromin-2 (NF-2), poly[ADP-ribose] synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis coli (APC), tumor necrosis factor receptor-associated factors (TRAFs), RAF kinase inhibitory protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, Oct3 / 4, Sox2, Klf4, and c-Myc. When the desired protein is selected from the above group, the desired protein can be linked to an anchoring peptide comprising the N-terminal region of TOM20, TOM70, or OM45.

[0022] Such fusion proteins may be linked in the following order: N-terminus—anchoring peptide containing the N-terminal region of TOM20, TOM70, or OM45—desired protein—C-terminus.

[0023] Furthermore, the foreign protein may further contain an amino acid sequence recognized by a protease in eukaryotic cells, or ubiquitin or a fragment thereof, between the mitochondrial anchoring peptide and the desired protein. A protease in eukaryotic cells refers to an enzyme that degrades proteins present in eukaryotic cells. In this case, since the foreign protein contains an amino acid sequence recognized by the protease, the foreign protein bound to the outer mitochondrial membrane can be degraded into the anchoring peptide and the desired protein in the cell.

[0024] In this case, the ubiquitin fragment may contain the C-terminal Gly-Gly of the amino acid sequence of SEQ ID NO: 71, or may contain 3 to 75 consecutive amino acids from the C-terminus. Furthermore, the foreign protein may further contain a linker between the desired protein and ubiquitin or a fragment thereof. In this case, the linker may consist of, but is not limited to, 1 to 150 amino acids, 10 to 100 amino acids, or 20 to 50 amino acids. The linker may consist of an amino acid appropriately selected from 20 amino acids, preferably glycine and / or serine. For example, the linker may consist of 5 to 50 amino acids consisting of glycine and serine. An example of a linker is (G4S)n, where n is an integer from 1 to 10, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0025] Furthermore, the protein capable of binding to a ligand or receptor present on the cell membrane may be a ligand or receptor present on the surface of tumor cells. In this case, the ligand or receptor present on the surface of tumor cells may be CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), membrane-bound mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosine-linked protein 1 (tyrp1), tyrosine-linked protein 1 (tyrp2), Brachyury, mesothelin, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms' tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, or UNC5H2. It may be, but is not limited to, any one selected from the group consisting of LILRB2, CEACAM, nectin-3, and combinations thereof.

[0026] Furthermore, the protein capable of binding to a ligand or receptor present on the cell membrane may be an antibody or a fragment thereof that binds to any one selected from the above group. In particular, the antibody fragment refers to a fragment having the same complementarity-determining region (CDR) as the antibody. This may be, in particular, Fab, scFv, F(ab')2, or a combination thereof.

[0027] In this case, the desired protein may be linked to an anchoring peptide comprising the C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B, and the foreign protein may be linked in the following order: an anchoring peptide comprising the C-terminal region of any one selected from the group consisting of N-terminus-desired protein-TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B; and a C-terminus.

[0028] Furthermore, the foreign protein may further comprise a linker between the desired protein and the C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. In this case, the linker is as described above. In this case, the desired protein, such as an active protein, a protein present in a cell, or a protein capable of binding to a ligand or receptor present in a cell membrane, is as described above.

[0029] In one form of the desired protein, an antibody or fragment thereof that targets a specific cell may be linked to an anchoring peptide comprising the C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. Modified mitochondria to which such a desired protein is bound can be easily introduced into a specific target, thereby enabling the mitochondria to be effectively introduced into specific cells.

[0030] One embodiment of the modified mitochondria may be a form in which one or more desired proteins are bound. In particular, the modified mitochondria may be a form in which a desired protein including p53 and a desired protein including an anti-HER-2 antibody or a fragment thereof are bound. Such modified mitochondria can effectively deliver mitochondria to HER-2-expressing cancer cells. Furthermore, cancer cells can be effectively killed by p53 bound to the modified mitochondria.

[0031] Desired proteins, including one or more active proteins, can be constructed and associated with mitochondria depending on the purpose of the modified mitochondria. Additionally, desired proteins targeted to cells can be constructed in a variety of ways depending on the target cell.

[0032] In another aspect of the present invention, there is provided a pharmaceutical composition comprising the modified mitochondria as an active ingredient. In this case, the pharmaceutical composition may be used for the prevention or treatment of cancer. In this case, the cancer may be any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0033] In particular, when an activated protein such as p53 that kills tumor cells or a protein that suppresses growth is bound to mitochondria, modified mitochondria with bound p53 can be used as an anti-cancer drug. Furthermore, when a protein such as RKIP that can suppress metastasis of cancer cells is bound to mitochondria, modified mitochondria with bound RKIP can be used as a tumor metastasis inhibitor. When any one selected from the group consisting of granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun / Fos), EGR-1, retinoblastoma (RB), phosphatase and tensin homolog (PTEN), E-cadherin, neurofibromin-2 (NF-2), poly[ADP-ribose] synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis coli (APC), tumor necrosis factor receptor-associated factor (TRAF), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, and combinations thereof, which are proteins that suppress the proliferation of cancer cells, or that control phosphorylation reactions in cancer cells, or that suppress the metastasis of cancer cells, is bound to mitochondria, the modified mitochondria to which the active protein is bound can be used as an anticancer agent.

[0034] Furthermore, in the pharmaceutical composition, mitochondria may be contained at a concentration of, but not limited to, 0.1 μg / ml to 500 μg / ml, 0.2 μg / ml to 450 μg / ml, or 0.5 μg / ml to 400 μg / ml. Setting the mitochondrial content within the above ranges may facilitate adjustment of the mitochondrial dosage during administration, thereby improving the degree of improvement in patient disease symptoms. In this case, the mitochondrial dosage may be determined by quantifying mitochondria by quantifying membrane proteins of isolated mitochondria. In particular, isolated mitochondria may be quantified by the Bradford protein assay (James D. McCully, J Vis Exp. 2014; (91): 51682).

[0035] Furthermore, the active protein bound to mitochondria in the pharmaceutical composition may be contained at a concentration of, but not limited to, 0.1 μg / ml to 500 μg / ml, 0.2 μg / ml to 450 μg / ml, or 0.5 μg / ml to 400 μg / ml. When the active protein content is within the above range, it can be easy to adjust the active protein dosage during administration, and the degree of improvement in the patient's disease symptoms can be improved.

[0036] Furthermore, in the pharmaceutical composition, the targeting protein capable of delivering mitochondria to specific cells may be contained at a concentration of, but not limited to, 0.1 μg / ml to 500 μg / ml, 0.2 μg / ml to 450 μg / ml, or 0.5 μg / ml to 400 μg / ml. When the targeting protein content is within the above range, it can be easily adjusted for the targeting protein dose during administration, and the degree of improvement in patient disease symptoms can be improved.

[0037] In particular, the pharmaceutical composition of the present invention may be administered at a mitochondrial dose of 0.01 to 5 mg / kg, 0.1 to 4 mg / kg, or 0.25 to 2.5 mg / kg per administration based on the body weight of the individual receiving the administration, but is not limited thereto. That is, in terms of cellular activity, it is most preferable to administer the pharmaceutical composition so that the amount of modified mitochondria based on the body weight of the individual bearing cancer tissue falls within the above range. Furthermore, the pharmaceutical composition may be administered 1 to 10 times, 3 to 8 times, or 5 to 6 times, preferably 5 times. In this case, the administration interval may be 1 to 7 days, or 2 to 5 days, preferably 3 days.

[0038] Furthermore, the pharmaceutical composition of the present invention can be administered to humans or other mammals that may be affected by cancer or that already have cancer. Furthermore, the pharmaceutical composition can be an injectable formulation that can be administered intravenously or a locally administered injectable formulation, preferably an injectable formulation.

[0039] Therefore, to ensure stability of the injectable formulation during distribution, the pharmaceutical composition of the present invention may be prepared as an injectable formulation with high physical or chemical stability by adjusting the pH of the composition with a buffer solution that can be used in the injectable formulation, such as an aqueous acid or phosphate.

[0040] In particular, the pharmaceutical composition of the present invention may contain water for injection, which is distilled water prepared for dissolving an injectable solid preparation or for diluting a water-soluble injectable preparation, and may be glucose injection, xylitol injection, D-mannitol injection, fructose injection, saline, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactate-Ringer's solution, phosphate buffer of pH 3.5 to 7.5, sodium dihydrogen phosphate-citrate buffer, etc.

[0041] Furthermore, the pharmaceutical composition of the present invention may contain a stabilizer or solubilizer. For example, the stabilizer may be sodium pyrosulfite or ethylenediaminetetraacetic acid, and the solubilizer may be hydrochloric acid, acetic acid, sodium hydroxide, sodium bicarbonate, sodium carbonate, or potassium hydroxide.

[0042] Furthermore, the present invention provides a method for preventing or treating cancer, which comprises administering the pharmaceutical composition to an individual, wherein the individual may be a mammal, preferably a human.

[0043] One aspect of the present invention provides a method for preparing modified mitochondria, comprising mixing isolated mitochondria with a desired protein, including an active protein, and / or a desired protein, including a target-targeting protein.

[0044] In this case, the desired protein and mitochondria can be mixed at an appropriate ratio. For example, the desired protein:mitochondria ratio can be 1:100 to 100:1 by weight. In particular, the ratio can be 1:10, 1:5, 1:4, 1:3, 1:2, or 1:1. Furthermore, the ratio can be 10:1, 5:1, 4:1, 3:1, or 2:1.

[0045] In another aspect of the present invention, there is provided a method for preparing modified mitochondria from cells transformed by introducing a polynucleotide encoding the desired protein into a eukaryotic cell. In particular, there is provided a method for preparing the fusion protein, comprising the steps of introducing the polynucleotide into a prokaryotic cell or a eukaryotic cell lacking a ubiquitinase or protease, and obtaining the fusion protein. This preparation method is suitable when the desired protein does not contain an amino acid sequence recognized by a protease in the eukaryotic cell, or ubiquitin or a fragment thereof.

[0046] In another aspect of the present invention, the desired protein can be prepared using prokaryotic cells or prokaryotic cell extracts. Additionally, methods are provided for preparing modified mitochondria using eukaryotic cells or eukaryotic cell extracts that lack ubiquitinases or protease enzymes.

[0047] Another aspect of the present invention provides the use of mitochondria as a means for delivering foreign proteins. In particular, modified mitochondria can be used as a means for intracellular and extracellular delivery of foreign proteins, including desired proteins capable of functioning inside and outside of cells. Mitochondria can be effectively introduced into cells, and in this case, foreign proteins desired to be delivered to cells can be effectively delivered to cells. In this case, mitochondria can be used as an effective protein delivery system. The desired proteins are as described above.

[0048] Another aspect of the present invention provides a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a desired protein, wherein the desired protein is as described above.

[0049] As used herein, the term "mitochondrial outer membrane anchoring peptide" may be the N-terminus or C-terminus of a protein present in the outer membrane of mitochondria. The mitochondrial outer membrane anchoring peptide may have an amino acid sequence that is specifically present in the outer membrane of mitochondria. In this case, the mitochondrial outer membrane anchoring peptide enables the fusion protein disclosed in the present invention to be bound to the outer membrane of mitochondria. In this case, the mitochondrial outer membrane anchoring peptide may be used interchangeably with the mitochondrial outer membrane targeting peptide.

[0050] Furthermore, the mitochondrial outer membrane anchoring peptide prevents the fusion protein disclosed in the present invention from entering the mitochondria. The TOM (translocase of outer membrane) complex present in the mitochondrial outer membrane may have a mitochondrial targeting sequence and one outer membrane anchoring domain at the amino terminus, with the majority of the carboxy terminus exposed to the cytoplasm (Figure 81a). The TOM (translocase of outer membrane) complex present in the mitochondrial outer membrane may have a mitochondrial targeting sequence and one outer membrane anchoring domain at the carboxyl terminus, with the majority of the amino terminus exposed to the cytoplasm (Figure 81b). Furthermore, the protein present in the mitochondrial outer membrane may be selected from proteins present in mitochondria present in eukaryotic cells. For example, it may be selected from proteins present in the mitochondrial outer membrane present in yeast, animal cells, or human cells.

[0051] In this case, the protein present in the outer mitochondrial membrane may be any one protein selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B, or fragments thereof. In this case, the mitochondrial outer membrane anchoring peptide may be a fragment of any one protein selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. In this case, the outer membrane anchoring peptide may be a C-terminal or N-terminal polypeptide of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B present in the outer mitochondrial membrane.

[0052] In particular, when the mitochondrial outer membrane anchoring peptide is fused to the N-terminus of a desired protein, the mitochondrial outer membrane anchoring peptide may comprise the terminal sequence of a protein selected from the group consisting of TOM20, TOM70, and OM45. Preferably, it may be the N-terminal sequence of a protein selected from the group consisting of TOM20, TOM70, and OM45. Embodiments of the mitochondrial outer membrane anchoring peptide are as described above.

[0053] Furthermore, when the mitochondrial outer membrane anchoring peptide is fused to the C-terminus of a desired protein, the outer membrane targeting protein may comprise the terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X, and VAMP1B. Preferably, it may be the C-terminus of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X, and VAMP1B. Embodiments of the mitochondrial outer membrane anchoring peptide are as described above.

[0054] As used herein, the term "active protein" may refer to a protein that exhibits physiological activity. An embodiment of such an active protein may be a protein with reduced function or an altered protein present in damaged cancer cells. An embodiment of an active protein may be a protein that enhances cellular activity. Examples of such active proteins are described above.

[0055] The fusion protein can be a protein in which a mitochondrial outer membrane targeting protein and a desired protein are linked from the N-terminus to the C-terminus.In this case, it can further contain ubiquitin or a fragment thereof having a ubiquitin protease specific cleavage site (glycine-glycine) between the mitochondrial outer membrane targeting protein and the desired protein.In this case, it can further contain a linker containing hydrophilic and polar amino acids, serine, glycine and threonine, between the mitochondrial outer membrane targeting protein and the ubiquitin protein to promote cleavage by ubiquitin protease.

[0056] As used herein, the term "ubiquitin" refers to a protein involved in the proteolytic process, also referred to as UB. Examples of ubiquitin may be ubiquitin present in the human body or ubiquitin present in yeast. Ubiquitin present in the human body consists of 76 amino acids. In this case, ubiquitin may be used in its mature form. As used herein, the term "mature form" may refer to a protein in a form in which the signal peptide has been removed.

[0057] Furthermore, enzymes called ubiquitin proteases or UBPs (ubiquitin-specific proteases) exist naturally in eukaryotic cells and can induce the spontaneous degradation of desired proteins in cells by cleaving the C-terminal amino acid glycine-glycine moiety of ubiquitin.

[0058] In this case, the ubiquitin fragment may contain the C-terminal Gly-Gly amino acids of ubiquitin, or may contain 3 to 75 consecutive amino acids from the C-terminus. In particular, examples of the ubiquitin fragment may be Arg-Gly-Gly, Leu-Arg-Gly-Gly, Arg-Leu-Arg-Gly-Gly, or Leu-Arg-Leu-Arg-Gly-Gly. Furthermore, the ubiquitin fragment may have the amino acid sequence of SEQ ID NO: 71.

[0059] A fusion protein comprising a mitochondrial outer membrane targeting protein and a desired protein may also be referred to as a fusion protein that modifies mitochondrial activity. Such a fusion protein may have any one of the following structures: <Structural formula 1> N-terminus - mitochondrial outer membrane anchoring peptide - desired protein - C-terminus <Structural formula 2> N-terminus - mitochondrial outer membrane anchoring peptide - ubiquitin or its fragment - desired protein - C-terminus <Structural formula 3> N-terminus - mitochondrial outer membrane anchoring peptide - linker 1 - ubiquitin or its fragment - desired protein - C-terminus <Structural formula 4> N-terminus - mitochondrial outer membrane anchoring peptide - ubiquitin or its fragment - linker 2 - desired protein - C-terminus <Structural formula 5> N-terminus - mitochondrial outer membrane anchoring peptide - linker 1 - ubiquitin or its fragment - linker 2 - desired protein - C-terminus

[0060] In the above structural formulas 1 to 5, the outer membrane anchoring peptide may be a terminal sequence of a protein selected from the group consisting of TOM20, TOM70, and OM45, and the desired protein may be any one selected from the group consisting of p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun / Fos), EGR-1, retinoblastoma (RB), phosphatase and tensin homolog (PTEN), E-cadherin, neurofibromin-2 (NF-2), poly[ADP-ribose] synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis coli (APC), tumor necrosis factor receptor-associated factor (TRAF), RAF kinase inhibitor protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, and DKK-3PD1.

[0061] In this case, linkers 1 and 2 can each be a polypeptide consisting of 1 to 100, 1 to 80, 1 to 50, or 1 to 30 amino acids, preferably a polypeptide consisting of 1 to 30 amino acids consisting of serine, glycine, or threonine alone, or a combination thereof. Furthermore, linkers 1 and 2 can each be a polypeptide consisting of 5 to 15 amino acids, preferably a polypeptide consisting of 5 to 15 amino acids consisting of serine, glycine, or threonine alone, or a combination thereof. An example of a linker is (GGGGS)3 (SEQ ID NO: 70).

[0062] <Structural formula 6> N-terminus - desired protein - mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 7> N-terminus - desired protein - ubiquitin or its fragment - mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 8> N-terminus - desired protein - linker 1 - ubiquitin or its fragment - mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 9> N-terminus - desired protein - ubiquitin or its fragment - linker 2 - mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 10> N-terminus - desired protein - linker 1 - ubiquitin or its fragment - linker 2 - mitochondrial outer membrane targeting peptide - C-terminus

[0063] In the above structural formulas 6 to 10, the outer membrane anchoring tether peptide can be a terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X, and VAPM1B, and the desired protein can be p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun / Fos), EGR-1, retinoblastoma (RB), phosphatase and tensin homolog (PTEN), E-cadherin, , neurofibromin-2 (NF-2), poly[ADP-ribose] synthetase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis coli (APC), tumor necrosis factor receptor-associated factors (TRAFs), RAF kinase inhibitory protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, Oct3 / 4, Sox2, Klf4, and c-Myc. In this case, linker 1 or 2 is as described above.

[0064] One aspect of the present invention provides a polynucleotide encoding a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a desired protein.

[0065] Furthermore, one aspect of the present invention provides a vector into which a polynucleotide encoding a fusion protein containing a desired protein has been introduced.

[0066] Furthermore, one aspect of the present invention provides a host cell into which a vector has been introduced, the vector carrying a polynucleotide encoding a fusion protein containing a desired protein.

[0067] One aspect of the present invention provides a fusion protein comprising a targeting protein and a mitochondrial outer membrane targeting protein.

[0068] In this case, the targeting protein and the mitochondrial outer membrane anchoring peptide can be linked from the N-terminus to the C-terminus, and the mitochondrial outer membrane anchoring peptide can be any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

[0069] As used herein, the term "target" refers to a location to which modified mitochondria are delivered. An example of a target may be a cancer cell. In particular, an example of a target may be a biomarker present on the surface of a cancer cell. In particular, a target may be a tumor-associated antigen (TAA). In this case, the tumor-associated antigen may be any one selected from the group consisting of CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), membrane-bound mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosine-linked protein 1 (tyrp1), tyrosine-linked protein 1 (tyrp2), brachyury, mesothelin, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms' tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, nectin-3, and combinations thereof.

[0070] As used herein, the term "targeting protein" may refer to a protein sequence capable of binding to the target. In this case, one embodiment of the targeting protein may be a protein that binds to a biomarker present on the surface of cancer cells. In this case, examples of biomarkers present on the surface of cancer cells may be, but are not limited to, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, or nectin-3. In this case, the targeting protein may be included in the foreign protein.

[0071] An example of a targeting protein may be an antibody or a fragment thereof. In particular, it may be an antibody or a fragment thereof that specifically binds to a tumor-associated antigen. Furthermore, the antibody fragment may be any one selected from the group consisting of Fab, Fab', scFv, and F(ab)2.

[0072] An example of a targeting protein may be scFvHER, which can bind to epidermal growth factor receptor. Another example may be scFvMEL, which can target melanoma. Another example may be scFvPD-L1, which can bind to PD-L1 overexpressed on the surface of cancer cells. Another example may be PD-1, which can bind to PDL-1 overexpressed on the surface of cancer cells.

[0073] One aspect of the present invention can further comprise ubiquitin or a fragment thereof between the targeting protein and the mitochondrial outer membrane targeting protein. The fusion protein comprising the mitochondrial targeting protein and a desired protein can be referred to as a fusion protein that modifies mitochondrial activity. Such a fusion protein can have any one of the following structures: <Structural formula 11> N-terminus - Targeted targeting protein - Mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 12> N-terminus - targeting protein - ubiquitin or its fragment - mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 13> N-terminus - target targeting protein - linker 1 - ubiquitin or its fragment - mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 14> N-terminus - targeting protein - ubiquitin or its fragment - linker 2 - mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 15> N-terminus - targeting protein - linker 1 - ubiquitin or its fragment - linker 2 - mitochondrial outer membrane anchoring peptide - C-terminus

[0074] In the above structural formulas 11 to 15, the outer membrane anchoring peptide can be a terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X, and VAPM1B, and the targeting protein can be a tumor-associated antigen, CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), membrane-bound mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate cancer cell line (Proc), or prostate cancer cell line (Proc). The targeting protein may be any one selected from the group consisting of prostate-specific antigen (PSA), survivin, tyrosine-linked protein 1 (tyrp1), tyrosine-linked protein 2 (tyrp2), brachyury, mesothelin, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms' tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, nectin-3, and combinations thereof. Furthermore, the targeting protein may be an antibody or a fragment thereof that specifically binds to a tumor-associated antigen. In this case, the linker 1 or 2 and the amino acid sequence recognized by the protease are as described above.

[0075] <Structural formula 16> N-terminus - mitochondrial outer membrane anchoring peptide - targeting protein - C-terminus <Structural formula 17> N-terminus - mitochondrial outer membrane anchoring peptide - ubiquitin or its fragment - targeting protein - C-terminus <Structural formula 18> N-terminus - mitochondrial outer membrane anchoring peptide - linker 1 - ubiquitin or its fragment - targeting protein - C-terminus <Structural formula 19> N-terminus - mitochondrial outer membrane anchoring peptide - ubiquitin or its fragment - linker 2 - target targeting protein - C-terminus <Structural formula 20> N-terminus - mitochondrial outer membrane anchoring peptide - linker 1 - ubiquitin or its fragment - linker 2 - targeting protein - C-terminus

[0076] In the above structural formulas 16 to 20, the outer membrane anchoring peptide can be any one selected from the group consisting of TOM20, TOM70, and OM45. Furthermore, the targeting protein, ubiquitin or a fragment thereof, and linker 1 or 2 are as described above.

[0077] One aspect of the invention provides a polynucleotide encoding a fusion protein comprising a targeting protein.

[0078] Furthermore, one aspect of the present invention provides a vector into which a polynucleotide encoding a fusion protein containing a target-targeting protein has been introduced.

[0079] Furthermore, one aspect of the present invention provides a host cell containing a vector incorporating a polynucleotide encoding a fusion protein containing a targeting protein. The host cell may be a prokaryotic or eukaryotic cell. In this case, the eukaryotic cell may preferably be a strain deleted for ubiquitin-degrading enzymes.

[0080] Furthermore, one aspect of the present invention provides a method for preparing modified mitochondria from cells transformed by introducing into a eukaryotic cell a polynucleotide encoding the fusion protein. [Example]

[0081] To aid in understanding the present invention, preferred embodiments are shown below. However, the following examples are provided merely to facilitate understanding of the present invention, and the present invention is not limited to the following examples. I. Preparation of a fusion protein containing a mitochondrial outer membrane anchoring peptide, a linker, ubiquitin, and a desired protein Example 1. Preparation of a fusion protein containing p53 Example 1.1. Amplification of the p53 gene To express human p53 as a recombinant protein, total RNA was extracted from human epithelial cells and cDNA was synthesized from the RNA. Specifically, human dermal fibroblasts (1 × 10 cells) were cultured in 10% serum medium under conditions of 5% carbon dioxide and 37°C. 6 The culture medium was then removed, the cells were washed twice with PBS buffer, and 0.5 ml of RNA extraction reagent (Trizol Reagent, Thermo Fisher Scientific) was added directly. The mixture containing the RNA extraction reagent was left at ambient temperature for 10 minutes, after which 0.1 ml of chloroform was added, the cells were stirred for 15 seconds, and then centrifuged at approximately 12,000 × g for 10 minutes. The separated supernatant was then removed, an equal volume of isopropyl alcohol was added, and the cells were centrifuged again at 12,000 × g for 10 minutes. The liquid was then removed, the cells were washed once with 75% ethanol, and the RNA was dried at ambient temperature.

[0082] Approximately 50 μl of RNAse-free purified distilled water was added, and the quantity and purity of RNA were measured using a spectrophotometer. To synthesize cDNA, 2 μg of purified total RNA was subjected to a ligation reaction with oligo(dT) at 70°C for 5 minutes. Then, 10× reverse transcription buffer, 10 mM dNTPs, RNAse inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea) were added, and the cDNA synthesis reaction was carried out at 42°C for 60 minutes. The reverse transcriptase was then inactivated by heating at 72°C for 5 minutes. RNase H was then added to remove single-stranded RNA, which was then used as a template for polymerase chain reaction of the p53 gene.

[0083] To obtain the p53 gene from human skin fibroblasts without the signal peptide sequence, a primer encoding the amino-terminal glutamic acid (T2p53) and a primer encoding the carboxyl-terminus (Xp53) were synthesized and PCR was performed using the cDNA prepared as described above as a template. The sequences of these primers are listed in Table 1.

[0084] [Table 1]

[0085] 0.2 pmol of T2p53 primer and 0.2 pmol of Xp53 primer were mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The amplification reaction was then performed in a polymerase chain reaction (PCR) machine for 40 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. After the reaction, the amplified DNA fragment of approximately 1.2 kbp was isolated by electrophoresis on a 1% agarose gel and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. Sequencing of the resulting DNA confirmed that a cDNA encoding human p53 protein had been obtained. The resulting p53 gene, designated pTA-p53, has the same nucleotide sequence as SEQ ID NO: 3 (Figure 1).

[0086] Example 1.2. Construction of an E. coli expression vector for p53 Example 1.2.1. Construction of plasmid pET15b-UB-p53 To prepare ubiquitin-fused p53 protein, the following expression vector was constructed. To obtain the ubiquitin gene, NdeUB and T2UB primers were constructed. The sequences of each primer are shown in Table 2.

[0087] [Table 2]

[0088] 0.2 pmol of NdeUB primer and 0.2 pmol of T2UB primer were mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The ubiquitin (UB) gene was obtained by 25 cycles of amplification in a polymerase chain reaction (PCR) at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. The amplified ubiquitin gene was digested with restriction enzymes NdeI and SacII, and the pTA-p53 plasmid was digested with restriction enzymes SacII and XhoI. The DNA fragments of approximately 210 bp and 1200 bp were isolated by electrophoresis on a 2% agarose gel, and then inserted into the pET15b vector cleaved with the restriction enzymes NdeI and XhoI using T4 DNA ligase to obtain the plasmid pET15b-UB-p53 (Figure 2). In this case, UB-p53 was represented by the base sequence of SEQ ID NO:6.

[0089] The E. coli BL21(DE3) strain was transformed with the pET15b-UB-p53 plasmid. The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 1 mM, and the cells were then cultured with shaking for approximately 4 hours.

[0090] The E. coli cells were separated by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 3, the expression of a ubiquitin-fused p53 protein of approximately 60 kDa was confirmed. In this case, lane M in Figure 3 shows a protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after the addition of IPTG, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0091] Example 1.2.2. Construction of plasmid pET11c-TOM70-UB-p53 To prepare the outer mitochondrial membrane-associated p53 protein fused with TOM70 and ubiquitin, we constructed an expression vector capable of expressing p53 fused with TOM70 and ubiquitin. Primers NdeTOM70, TOM70-AS, TOM70UB-S, and T2UB-AS were constructed to obtain the TOM70 and ubiquitin genes. The sequences of each primer are shown in Table 3.

[0092] [Table 3]

[0093] To obtain the TOM70 gene, 0.2 pmol of NdeTOM70 primer and 0.2 pmol of TOM70-AS primer were mixed with 0.2 nM dNTPs, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The resulting mixture was then subjected to 25 cycles of amplification in a polymerase chain reaction (PCR) at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. The amplified DNA fragment was designated N-TOM70. The plasmid pET15b-UB-p53 obtained in Example 1.2.1 above was used as a template. 0.2 pmol of TOM70UB-S primer and 0.2 pmol of T2UB-AS primer were added, along with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The UB gene was isolated by 25 cycles of amplification in a polymerase chain reaction system at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. The amplified DNA fragment was designated C-UB.

[0094] The amplified DNAs N-TOM70 and C-UB were used as templates. 0.2 pmol of NdeTOM70 primer and 0.2 pmol of T2UB-AS primer were mixed with 0.2 nM dNTPs, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The amplification reaction was then carried out in a polymerase chain reaction (PCR) machine for 25 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute to obtain the amplified TOM70-UB ubiquitin gene.

[0095] The amplified TOM70-UB gene was digested with restriction enzymes NdeI and SacII, and the pTA-p53 plasmid was digested with SacII and XhoI. DNA fragments of approximately 330 bp and 1500 bp were obtained by electrophoresis on a 2% agarose gel, respectively. These fragments were then inserted into the pET11c vector digested with restriction enzymes NdeI and SalI using T4 DNA ligase to obtain the plasmid pET11c-TOM70-UB-p53 (Figure 4). In this case, TOM70-UB-p53 was represented by the nucleotide sequence of SEQ ID NO: 11.

[0096] The plasmid pET11c-TOM70-UB-p53 was used to transform E. coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium in a shaking incubator at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 1 mM, followed by shaking for an additional 4 hours.

[0097] The E. coli cells were isolated by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 5, the expression of a p53 protein fused with TOM70 and ubiquitin, approximately 62 kDa in size, was confirmed. In this case, lane M shows the protein molecular weight marker, and lane 1 shows the supernatant obtained by centrifugation of E. coli cells disrupted 4 hours after the addition of IPTG.

[0098] Example 1.2.3. Construction of plasmid pET11c-TOM70-(GGGGS)3-UB-p53 To prepare a p53 protein fused with TOM70, a linker (GGGGSGGGGSGGGGS (SEQ ID NO: 70)), and ubiquitin, which binds to the outer mitochondrial membrane, an expression vector capable of expressing a p53 protein fused with TOM70, a linker, and ubiquitin was constructed. To obtain the linker gene linked to TOM70, the following primers were constructed: TOM70(G)3-AS primer, (G)3UB-S primer, and Xp53(noT) primer. The sequences of each primer are shown in Table 4.

[0099] [Table 4]

[0100] The plasmid pET11c-TOM70-UB-p53 obtained in Example 1.2.2 above was used as a template, and 0.2 pmol of NdeTOM70 primer and 0.2 pmol of TOM70(G)3-AS primer were added. This was then mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. This was then subjected to 25 cycles of amplification in a polymerase chain reaction (PCR) at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute to obtain the TOM70 gene and the linker-linked TOM70-G3 gene. Furthermore, the plasmid pET15b-UB-p53 obtained in Example 1.2.1 above was used as a template, and 0.2 pmol of the (G)3UB-S primer and 0.2 pmol of the Xp53(noT) primer were mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase.

[0101] The amplification reaction was then carried out in a polymerase chain reaction (PCR) machine for 25 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute to obtain UB-p53, in which p53 was fused to the ubiquitin gene. The amplified TOM70-G3 gene was cleaved with the restriction enzymes NdeI and BamHI, and the amplified UB-p53 gene was cleaved with the restriction enzymes BamHI and XhoI. 100 bp and 1500 bp DNA fragments were obtained by electrophoresis on a 2% agarose gel, respectively. The fragments were then inserted into the pET11c vector cleaved with the restriction enzymes NdeI and SalI using T4 DNA ligase to obtain the plasmid pET11c-TOM70-(GGGGS)3-UB-p53 (Figure 6). In this case, TOM70-(GGGGS)3-UB-p53 was represented by the nucleotide sequence of SEQ ID NO: 15.

[0102] The plasmid pET11c-TOM70-(GGGGS)3-UB-p53 was used to transform E. coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 1 mM, followed by shaking for approximately 4 hours.

[0103] E. coli cells were isolated by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 7, the expression of a p53 protein fused with TOM70, a linker, and ubiquitin, approximately 62 kDa in size, was confirmed. In this case, lane M shows the protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after the addition of IPTG, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0104] Example 1.2.4. Construction of plasmid pET11c-TOM70-(GGGGS)3-p53 To prepare a p53 protein fused with TOM70, which binds to the outer mitochondrial membrane, and a linker (GGGGSGGGGSGGGGS (SEQ ID NO: 70)), an expression vector capable of expressing p53 fused with TOM70 and a linker was constructed. To obtain the p53 gene fused with TOM70 and a linker, a primer (B(G)3p53) was constructed. The primer sequences are shown in Table 5.

[0105] [Table 5]

[0106] The plasmid pET11c-TOM70-UB-p53 obtained in Example 1.2.2 above was used as a template. 0.2 pmol of NdeTOM70 primer and 0.2 pmol of TOM70(G)3-AS primer were added, along with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The TOM70 gene was then isolated by 25 cycles of amplification in a polymerase chain reaction system at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. The amplified DNA fragment was designated TOM70-G3.

[0107] Using the plasmid pET15b-UB-p53 obtained in Example 1.2.1 above as a template, 0.2 pmol of B(G)3p53 primer and 0.2 pmol of Xp53(noT) primer were mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. Then, 25 cycles of amplification reaction were performed in a polymerase chain reaction device at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. The amplified DNA fragment was designated G3-p53.

[0108] The amplified DNA fragment TOM70-G3 was digested with NdeI and BamHI, and the DNA fragment G3-53 was digested with restriction enzymes BamHI and XhoI. The approximately 150 bp and 1300 bp DNA fragments were then isolated by electrophoresis on a 2% agarose gel, respectively, and inserted into the pET11c vector digested with restriction enzymes NdeI and SalI using T4 DNA ligase to obtain the plasmid pET11c-TOM70-(GGGGS)3-p53 (Figure 8). In this case, TOM70-(GGGG)3-p53 was represented by the nucleotide sequence of SEQ ID NO: 17.

[0109] The plasmid pET11c-TOM70-(GGGGS)3-p53 was used to transform E. coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium in a shaking incubator at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 1 mM, followed by shaking for an additional 4 hours.

[0110] The E. coli cells were separated by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 9, the expression of a TOM70-fused p53 protein of approximately 60 kDa was confirmed. In this case, lane M shows the protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after the addition of IPTG, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0111] Example 1.2.5. pET15b-UB-p53-TOM7 To prepare p53 fused with ubiquitin and TOM70, which binds to the outer mitochondrial membrane, we constructed an expression vector capable of expressing p53 fused with ubiquitin, p53, and TOM in that order. To obtain the p53 gene fused with TOM70 and ubiquitin, we constructed the Xp53(noT) primer, XTOM7 primer, and LTOM7 primer. The sequences of each primer are shown in Table 6.

[0112] [Table 6]

[0113] The plasmid pET15b-UB-p53 obtained in Example 1.2.1 above was used as a template. 0.2 pmol of NdeUB primer and 0.2 pmol of Xp53(noT) primer were added, along with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The UB-p53 gene was then obtained by performing 25 cycles of amplification at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute in a polymerase chain reaction (PCR) machine. Furthermore, the cDNA prepared as described above was used as a template. 0.2 pmol of XTOM7 primer and 0.2 pmol of LTOM7 primer were mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase.

[0114] The TOM7 gene was then isolated by 40 cycles of amplification in a polymerase chain reaction system at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. The amplified DNA fragment UB-p53 was cleaved with restriction enzymes NdeI and XhoI, and the amplified TOM7 gene was cleaved with restriction enzymes XhoI and SalI. DNA fragments of approximately 1500 bp and 150 bp, respectively, were isolated by electrophoresis on a 2% agarose gel and inserted into the pET15b vector cleaved with restriction enzymes NdeI and XhoI using T4 DNA ligase to obtain the plasmid pET15b-UB-p53-TOM7 (Figure 10). In this case, UB-p53-TOM7 was represented by the nucleotide sequence of SEQ ID NO: 21.

[0115] The plasmid pET15b-UB-p53-TOM7 was used to transform Escherichia coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 0.5 mM, followed by shaking for an additional 4 hours.

[0116] E. coli cells were isolated by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 11, the expression of a ubiquitin- and TOM7-fused p53 protein of approximately 60 kDa was confirmed. In this case, lane M shows a protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after the addition of IPTG, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0117] Example 1.2.6. Construction of mammalian expression vector pCMV-p53-myc / His An expression vector capable of expressing p53 was constructed for animal cells. To obtain the p53 gene, Rp53 primers were constructed. The primer sequences are shown in Table 7.

[0118] [Table 7]

[0119] Using the plasmid pET-UB-p53 obtained in Example 1.2.1 above as a template, 0.2 pmol of Rp53 primer and 0.2 pmol of Xp53(noT) primer were mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The p53 gene was then isolated by 25 cycles of amplification in a polymerase chain reaction system at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute.

[0120] The amplified p53 gene was digested with restriction enzymes EcoRI and XhoI, and a DNA fragment of approximately 1,300 bp was obtained by electrophoresis on a 2% agarose gel. This fragment was then inserted using T4 DNA ligase into the pcDNA3.1-myc / His A vector, which had been digested with restriction enzymes EcoRI and XhoI, to obtain the plasmid pCMV-p53-myc / His (Figure 12). In this case, p53-myc / His was represented by the nucleotide sequence of SEQ ID NO:23.

[0121] CHO cells were transformed with the pCMV-p53-myc / His plasmid, disrupted, subjected to SDS-polyacrylamide gel electrophoresis, and then detected by Western blot analysis using an anti-c-myc antibody. As shown in Figure 13, expression of a p53 protein of approximately 55 kDa was confirmed. In this case, lane M shows a protein molecular weight marker, and lane 1 shows transfection of CHO cells, disruption of the cells, SDS-polyacrylamide gel electrophoresis, and Western blot analysis using an anti-c-myc antibody.

[0122] Example 1.3. Isolation and purification of fusion proteins containing p53 Example 1.3.1. Isolation and Purification of Recombinant TOM70-(GGGGS)3-p53 Protein Derived from E. coli The E. coli BL21(DE3) production strain expressing the recombinant TOM70-(GGGGS)3-p53 protein was inoculated into LB liquid medium and cultured at 37°C. When the OD600 absorbance reached 0.4, 0.5 mM IPTG was added, and the culture was continued with shaking for an additional 4 hours to express the TOM70-(GGGGS)3-p53 protein.

[0123] After incubation, the cells were harvested by centrifugation, washed once with PBS, suspended in PBS, and then disrupted using an ultrasonic disrupter. The disrupted cells were centrifuged at high speed to collect the insoluble fraction, which was then washed three times with 50 mM Tris, 100 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0. The insoluble fraction was then dissolved in 6 M guanidine, 100 mM sodium phosphate, 10 mM Tris, pH 8.0, filtered through a 0.45 μm filter, and loaded onto a pre-packed nickel chromatography column for primary purification.

[0124] A solution containing TOM70-(GGGGS)3-p53 protein was loaded, followed by a wash using a pH 8.0 solution of 8 M urea, 50 mM sodium phosphate, 500 mM NaCl, and 10 mM imidazole until no unbound impurities were detected. The protein was then eluted using a pH 8.0 solution of 8 M urea, 50 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole, varying the imidazole concentration to 50 mM, 100 mM, 250 mM, or 500 mM (Figure 14). In this case, lane M in Figure 14 shows the protein molecular weight marker, lane 1 shows the loading sample from nickel affinity chromatography, and lane 2 shows the material that did not bind to the nickel affinity resin. Lanes 3 and 4 show the results of elution with an 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 50 mM imidazole solution. Lanes 5-7 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8-9 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 250mM imidazole solution. Lanes 10-11 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 500mM imidazole solution.

[0125] The eluate recovered from nickel chromatography was subjected to a solution exchange with PBS using the principle of osmotic pressure. After the solution exchange, the eluate was centrifuged and the supernatant was collected. The protein content of the recovered eluate was measured using a protein assay and confirmed by SDS-PAGE. As shown in Figure 15, after confirmation, the TOM70-(GGGGS)3-p53 protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M shows the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-p53 protein obtained by dialysis against PBS buffer.

[0126] Example 1.3.2. Isolation and Purification of Recombinant TOM70-(GGGGS)3-UB-p53 Protein Derived from E. coli Using E. coli expressing the TOM70-(GGGGS)3-UB-p53 recombinant protein, the TOM70-(GGGGS)3-UB-p53 protein was isolated and purified using the same method as in Example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-p53 protein was eluted (Figure 16). In this case, lane M in Figure 16 shows a protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows the sample that did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 4-7 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8 to 11 show the results of elution with a solution of 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 250 mM imidazole.

[0127] The protein content of the collected eluate was measured by protein quantification and confirmed by SDS-PAGE. After confirmation, the TOM70-(GGGGS)3-UB-p53 protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C, as shown in Figure 17. In this case, lane M in Figure 17 shows the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-p53 protein obtained by dialysis against PBS buffer.

[0128] Example 1.3.3. Isolation and Purification of Recombinant UB-p53 Protein Derived from E. coli The BL21(DE3) production strain expressing the ubiquitin-fused mature UB-p53 protein was inoculated into LB liquid medium and cultured in a shaking incubator at 37°C. When the OD600 absorbance reached 0.4, 0.5 mM IPTG was added, and the culture was continued with shaking for an additional 4 hours to express the ubiquitin-fused mature UB-p53 protein.

[0129] The UB-p53 protein was then isolated and purified using the same method as in Example 1.3.1. As a result, the UB-p53 protein was eluted (Figure 18). In this case, lane M in Figure 18 shows the protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows the sample that did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 4-6 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 7-9 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 250mM imidazole solution. Lanes 10 and 11 show the results of elution with a solution of 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 500 mM imidazole.

[0130] The protein content of the recovered eluate was measured by protein quantification and confirmed by SDS-PAGE. After confirmation, the UB-p53 protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C, as shown in Figure 19. In this case, lane M in Figure 19 shows the protein molecular weight marker, and lane 1 shows the UB-p53 protein obtained by dialysis against PBS buffer.

[0131] Example 1.3.4. Isolation and purification of recombinant UB-p53-TOM7 protein derived from E. coli The E. coli BL21(DE3) production strain expressing the mature ubiquitin-fused UB-p53-TOM7 protein was inoculated into LB liquid medium and cultured at 37°C. When the OD600 absorbance reached 0.4, 0.5 mM IPTG was added, and the culture was continued with shaking for an additional 4 hours to express the mature ubiquitin-fused UB-p53-TOM7 protein.

[0132] The UB-p53-TOM7 protein was then isolated and purified using the same method as in Example 1.3.1. As a result, the UB-p53-TOM7 protein was eluted (Figure 20). In this case, lane M in Figure 20 shows the protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows the sample that did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 10mM imidazole solution. Lane 4 shows the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5-7 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8 and 9 show the results of elution with 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 250 mM imidazole, and lanes 10 and 11 show the results of elution with 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 500 mM imidazole.

[0133] The protein content of the recovered eluate was measured by protein quantification and confirmed by SDS-PAGE. After confirmation, the UB-p53 protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C, as shown in Figure 21. In this case, lane M in Figure 21 shows the protein molecular weight marker, and lane 1 shows the UB-p53-TOM7 protein obtained by dialysis against PBS buffer.

[0134] Example 2. Preparation of fusion proteins containing granzyme B Example 2.1. Amplification of the Granzyme B Gene To express human granzyme B as a recombinant protein, total RNA was extracted from human natural killer cells and cDNA was synthesized from the RNA. Specifically, human natural killer cells (1 × 10) were cultured in 10% serum medium under conditions of 5% carbon dioxide and 37°C. 6 Then, RNA was obtained in the same manner as in Example 1.1 and used as a template for polymerase chain reaction of the granzyme B gene.

[0135] To obtain the granzyme B gene from human natural killer cells without the signal peptide sequence, primers T2GZMB, which encodes from the amino-terminal isoleucine, and XGZMB(noT), which encodes from the carboxyl-terminus, were synthesized and PCR was performed using the cDNA prepared above as a template. The sequences of these primers are listed in Table 8.

[0136] [Table 8]

[0137] Using the cDNA synthesized as described above as a template, 0.2 pmol of T2GZMB primer and 0.2 pmol of XGZMB(noT) primer were mixed with 0.2 nM dNTPs, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The amplification reaction was then carried out in a polymerase chain reaction (PCR) machine for 40 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. After the reaction, the amplified DNA fragment of approximately 700 bp was isolated by electrophoresis on a 1% agarose gel and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. Sequencing of the resulting DNA confirmed that a cDNA encoding human granzyme B protein had been obtained. The resulting granzyme B gene was designated pTA-granzyme B, and the granzyme B gene was represented by the base sequence of SEQ ID NO: 26 (FIG. 22).

[0138] Example 2.2. Construction of an E. coli expression vector for granzyme B protein Example 2.2.1. Construction of Plasmid pET11c-TOM70-(GGGGS)3-UB-Granzyme B To prepare granzyme B protein in a form fused with TOM70, a linker (GGGGSGGGGSGGGGS), and ubiquitin that binds to the outer mitochondrial membrane, an expression vector capable of expressing granzyme B protein in a form fused with TOM70, a linker, and ubiquitin was constructed.

[0139] The pTA-Granzyme B gene plasmid obtained in Example 2.1 above was cleaved with the restriction enzymes SacII and XhoI, and a DNA fragment of approximately 700 bp was obtained by electrophoresis on a 2% agarose gel. This fragment was then inserted into the pET11c-TOM70-(GGGGS)3-UB-(p53) vector cleaved with the restriction enzymes SacII and XhoI using T4 DNA ligase to obtain the plasmid pET11c-TOM70-(GGGGS)3-UB-Granzyme B (SEQ ID NO: 27) (Figure 23).

[0140] The plasmid pET11c-TOM70-(GGGGS)3-UB-Granzyme B was used to transform Escherichia coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium in a shaking incubator at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 1 mM, followed by shaking for an additional 4 hours.

[0141] The E. coli cell fraction was obtained by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 24, it was confirmed that a granzyme B protein of approximately 35 kDa in size, fused with TOM70, a linker, and ubiquitin, was expressed. In this case, lane M in Figure 24 shows a protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after addition of IPTG, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0142] Example 2.2.2. Construction of Plasmid pET15b-UB-Granzyme B-TOM7 To prepare granzyme B protein fused with ubiquitin and TOM7, which binds to the outer mitochondrial membrane, we constructed an expression vector capable of expressing granzyme B protein fused with ubiquitin, granzyme B, and TOM in this order.

[0143] The pTA-granzyme B gene plasmid obtained in Example 2.1 above was cleaved with the restriction enzymes SacII and XhoI, and an approximately 700 bp DNA fragment was isolated by electrophoresis on a 2% agarose gel. This fragment was inserted into the pET15b-UB-(p53)-TOM7 vector cleaved with the restriction enzymes SacII and XhoI using T4 DNA ligase to obtain the pET15b-UB-granzyme B-TOM7 plasmid (Figure 25). Here, UB-granzyme B-TOM7 is represented by the nucleotide sequence of SEQ ID NO: 28.

[0144] The plasmid pET15b-UB-Granzyme B-TOM7 was used to transform Escherichia coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 0.5 mM, followed by shaking for an additional 4 hours.

[0145] The E. coli cell fraction was obtained by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 26, it was confirmed that a granzyme B protein with a size of approximately 35 kDa, in which ubiquitin and TOM7 were fused, was expressed. In this case, lane M in Figure 26 shows a protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after addition of IPTG, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0146] Example 2.3. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-Granzyme B protein derived from E. coli TOM70-(GGGGS)3-UB-Granzyme B protein was isolated and purified using the same method as in Example 1.3.1. As a result, TOM70-(GGGGS)3-UB-Granzyme B protein was eluted (Figure 27). In this case, lane M in Figure 27 shows a protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows the sample that did not bind to the nickel affinity resin. Lanes 3 and 4 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5-7 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8-9 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 250mM imidazole solution.

[0147] The protein content of the collected eluate was measured by protein quantification and confirmed by SDS-PAGE. As shown in Figure 28, after confirmation, the TOM70-(GGGGS)3-UB-granzyme B protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 28 shows the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-granzyme B protein obtained by dialysis against PBS buffer.

[0148] Example 3. Preparation of fusion proteins containing RKIP Example 3.1. Amplification of the RKIP gene To express the human RKIP (Raf kinase inhibitory protein) gene as a recombinant protein, total RNA was extracted from human epithelial cells and cDNA was synthesized from it. Human epithelial fibroblasts (1 × 10 cells) were cultured in 10% serum medium under conditions of 5% carbon dioxide and 37°C. 6 Then, RNA was obtained in the same manner as in Example 1.1 and used as a template for polymerase chain reaction of the RKIP gene.

[0149] To obtain the RKIP gene from human skin fibroblasts without the signal peptide sequence, we synthesized the T2RKIP primer, which encodes from the amino-terminal proline, and the XRKIP(noT) primer, which encodes from the carboxyl-terminus, and performed PCR using the cDNA prepared as described above as a template. The sequences of these primers are listed in Table 9.

[0150] [Table 9]

[0151] Using the cDNA synthesized as described above as a template, 0.2 pmol of T2RKIP primer and 0.2 pmol of XRKIP(noT) primer were mixed with 0.2 nM dNTPs, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The amplification reaction was then carried out in a polymerase chain reaction (PCR) machine for 40 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. After the reaction, the amplified DNA fragment of approximately 560 bp was isolated by electrophoresis on a 1% agarose gel and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. Sequencing of the resulting DNA confirmed that a cDNA encoding human RKIP protein had been obtained. The obtained RKIP gene was designated pTA-RKIP (FIG. 29), and the base sequence of the RKIP gene was represented by the base sequence of SEQ ID NO:31.

[0152] Example 3.2. Construction of E. coli expression vector for RKIP protein Example 3.2.1. Construction of Plasmid pET11c-TOM70-(GGGGS)3-UB-RKIP To prepare RKIP protein in a form fused with TOM70, a linker (GGGGSGGGGSGGGGS), and ubiquitin that binds to the outer mitochondrial membrane, we constructed an expression vector capable of expressing RKIP in a form fused with TOM70, a linker, and ubiquitin.

[0153] The plasmid pTA-RKIP obtained in Example 3.1 above was cleaved with the restriction enzymes SacII and XhoI, and a DNA fragment of approximately 560 bp was obtained by electrophoresis on a 2% agarose gel. This fragment was then inserted using T4 DNA ligase into the pET11c-TOM70-(GGGGS)3-UB-(p53) vector that had been cleaved with the restriction enzymes SacII and XhoI to obtain the plasmid pET11c-TOM70-(GGGGS)3-UB-RKIP (Figure 30). Here, TOM70-(GGGGS)3-UB-RKIP is represented by the nucleotide sequence of SEQ ID NO: 32.

[0154] The plasmid pET11c-TOM70-(GGGGS)3-UB-RKIP was used to transform E. coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium in a shaking incubator at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 0.5 mM, followed by shaking for an additional 4 hours.

[0155] E. coli cell fractions were obtained by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 31, expression of the approximately 33 kDa RKIP protein, a fusion of TOM70, a linker, and ubiquitin, was confirmed. In this case, lane M in Figure 31 shows a protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after IPTG addition, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0156] Example 3.3. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-RKIP protein derived from E. coli The E. coli BL21(DE3) production strain expressing recombinant TOM70-(GGGGS)3-UB-RKIP was inoculated into LB liquid medium and cultured at 37°C. When the OD600 absorbance reached 0.3, the culture was placed in a refrigerator to lower the temperature, and the incubator temperature was changed to 18°C. Then, 0.5 mM IPTG was added, and the culture was further cultured with shaking for another day to express the TOM70-(GGGGS)3-UB-RKIP protein.

[0157] The TOM70-(GGGGS)3-UB-RKIP protein was then isolated and purified using the same method as in Example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-RKIP protein was eluted (Figure 32). In this case, lane M in Figure 32 shows a protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows the sample that did not bind to the nickel affinity resin. Lane 3 shows the results of elution with a 50 mM Na phosphate / 500 mM NaCl / 10 mM imidazole solution. Lanes 4-6 show the results of elution with a 50 mM Na phosphate / 500 mM NaCl / 50 mM imidazole solution. Lanes 7-8 show the results of elution with a 50 mM Na phosphate / 500 mM NaCl / 100 mM imidazole solution. Lanes 9-10 show the results of elution with 50 mM sodium phosphate / 500 mM NaCl / 175 mM imidazole solution, lanes 11-13 show the results of elution with 50 mM sodium phosphate / 500 mM NaCl / 250 mM imidazole solution, and lanes 14-16 show the results of elution with 50 mM sodium phosphate / 500 mM NaCl / 500 mM imidazole solution.

[0158] The protein content of the collected eluate was measured by protein quantification and confirmed by SDS-PAGE. As shown in Figure 33, after confirmation, the TOM70-(GGGGS)3-UB-RKIP protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 33 shows the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-RKIP protein obtained by dialysis against PBS buffer.

[0159] Example 4. Preparation of fusion proteins containing PTEN Example 4.1. PTEN gene amplification To express human PTEN (phosphatase tensin homolog) as a recombinant protein, total RNA was extracted from human epithelial cells and cDNA was synthesized from it. Fibroblasts (human dermal fibroblasts) were cultured in 10% serum medium (1 × 10 cells) under conditions of 5% carbon dioxide and 37°C. 6 Then, RNA was obtained in the same manner as in Example 1.1 and used as a template for polymerase chain reaction of the PTEN gene.

[0160] To obtain the PTEN gene from human skin fibroblasts without the signal peptide sequence, we synthesized the T2PTEN primer, which encodes from the amino-terminal threonine, and the XPTEN(noT) primer, which encodes from the carboxyl-terminus, and performed PCR using the cDNA prepared as above as a template. The sequences of each primer are listed in Table 10.

[0161] [Table 10]

[0162] Using the cDNA prepared as described above as a template, 0.2 pmol of T2PTEN primer and 0.2 pmol of XPTEN(noT) primer were mixed with 0.2 nM dNTPs, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The amplification reaction was then carried out in a polymerase chain reaction (PCR) machine for 40 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. The amplified DNA fragment of approximately 1200 bp was isolated by electrophoresis on a 1% agarose gel and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. Sequencing of the resulting DNA confirmed the cDNA encoding human RKIP protein. The obtained PTEN gene was designated pTA-PTEN (FIG. 34), and the nucleotide sequence of PTEN was represented by the nucleotide sequence of SEQ ID NO:35.

[0163] Example 4.2. Construction of E. coli expression vector for PTEN protein Example 4.2.1. Construction of Plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN To prepare PTEN protein in a form fused with TOM70, a linker (GGGGSGGGGSGGGGS), and ubiquitin that binds to the outer mitochondrial membrane, we constructed an expression vector capable of expressing the PTEN gene in a form fused with TOM70, a linker, and ubiquitin.

[0164] The pTA-PTEN plasmid obtained in Example 4.1 above was cleaved with the restriction enzymes SacII and XhoI, and a DNA fragment of approximately 1,200 bp was isolated by electrophoresis on a 2% agarose gel. The fragment was then inserted into the pET11c-TOM70-(GGGGS)3-UB-(p53) vector cleaved with the restriction enzymes SacII and XhoI using T4 DNA ligase to obtain the plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN (Figure 35). TOM70-(GGGGS)3-UB-PTEN is represented by the nucleotide sequence of SEQ ID NO: 36.

[0165] The plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN was used to transform E. coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 0.5 mM, followed by shaking for an additional 4 hours.

[0166] E. coli cell fractions were obtained by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 36, it was confirmed that an approximately 73-kDa RKIP protein fused with TOM70, a linker, and ubiquitin was expressed. In this case, lane M in Figure 36 shows a protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after IPTG addition, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0167] Example 4.3. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-PTEN protein derived from E. coli Recombinant TOM70-(GGGGS)3-UB-PTEN protein was isolated and purified using the same method as in Example 1.3.1. As a result, TOM70-(GGGGS)3-UB-PTEN protein was eluted (Figure 37). In this case, lane M in Figure 37 shows a protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows the sample that did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 10mM imidazole solution. Lane 4 shows the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5-8 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 9 and 10 show the results of elution with 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 250 mM imidazole solution, and lane 111 shows the results of elution with 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 500 mM imidazole solution.

[0168] The protein content of the recovered eluate was measured by protein quantification and confirmed by SDS-PAGE. After confirmation, the TOM70-(GGGGS)3-UB-PTEN protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C, as shown in Figure 38. In this case, lane M in Figure 38 shows the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-PTEN protein obtained by dialysis against PBS buffer.

[0169] Example 5. Preparation of a fusion protein containing a mitochondrial outer membrane protein, ubiquitin, and GFP Example 5.1. Isolation and purification of recombinant UB-GFP-TOM7 protein derived from E. coli A production strain of E. coli BL21(DE3) expressing the mature ubiquitin-fused UB-GFP-TOM7 protein was inoculated into LB liquid medium and cultured at 37°C. When the OD600 absorbance reached 0.3, the culture was placed in a refrigerator to lower the temperature, and the incubator temperature was changed to 18°C. 0.5 mM IPTG was then added, and the culture was continued with shaking for another day to express the mature ubiquitin-fused GFP-TOM7 protein.

[0170] After incubation, the cells were harvested by centrifugation, washed once with PBS, and then suspended in a solution of 50 mM sodium phosphate, 500 mM NaCl, and 10 mM imidazole, pH 8.0. The suspended cells were then disrupted using an ultrasonic disrupter. The disrupted cells were centrifuged in a high-speed centrifuge, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter and loaded onto a pre-packed nickel chromatography column for primary purification.

[0171] The disruption solution containing the mature ubiquitin-fused UB-GFP-TOM7 protein was loaded, followed by a wash using 50 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole, pH 8.0, until no unbound impurities were detected. The protein was then eluted using a gradient solution of 50 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole, pH 8.0 (Figure 39). In this case, lane M in Figure 39 shows a protein molecular weight marker, lane 1 shows the loading sample for nickel affinity chromatography, and lane 2 shows the sample that did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 20 mM imidazole. Lane 4 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 55 mM imidazole. Lane 5 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 60 mM imidazole solution. Lane 6 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 65 mM imidazole solution. Lane 7 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 70 mM imidazole solution. Lane 8 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 75 mM imidazole solution. Lane 9 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 80 mM imidazole solution. Lane 10 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 85 mM imidazole solution. Lane 11 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 90 mM imidazole solution. Lane 12 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 95 mM imidazole solution, lane 13 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 100 mM imidazole solution, and lane 14 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 105 mM imidazole solution.

[0172] To remove imidazole from the eluate, dialysis was performed using the principle of osmotic pressure in a 50 mM sodium phosphate, 500 mM NaCl, pH 8.0 solution (Figure 40). The final confirmed UB-GFP-TOM7 protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 40 shows the protein molecular weight marker, and lane 1 shows the protein obtained by dialyzing the fusion protein fractions mixed together with a 50 mM sodium phosphate / 500 mM NaCl solution.

[0173] Example 5.2. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-GFP protein derived from E. coli The E. coli BL21(DE3) production strain expressing the recombinant protein TOM70-(GGGGS)3-UB-GFP was inoculated into LB liquid medium and cultured at 37°C. When the OD600 absorbance reached 0.3, the culture was placed in a refrigerator to lower the temperature, and the incubator temperature was changed to 18°C. 0.5 mM IPTG was then added, and the culture was continued with shaking for another day to express the recombinant protein TOM70-(GGGGS)3-UB-GFP.

[0174] After incubation, the cells were harvested by centrifugation, washed once with PBS, and then suspended in a solution of 50 mM sodium phosphate, 500 mM NaCl, and 10 mM imidazole, pH 8.0. The suspended cells were then disrupted using an ultrasonic disrupter. The disrupted cells were centrifuged in a high-speed centrifuge, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter and loaded onto a pre-packed nickel chromatography column for primary purification.

[0175] The disruption solution containing the recombinant protein TOM70-(GGGGS)3-UB-GFP was loaded onto a nickel resin-packed column, followed by a wash using a 50 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole, pH 8.0 solution, until no unbound impurities were detected. The protein was then eluted using a 50 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole, pH 8.0 solution, with imidazole concentrations of 50 mM, 100 mM, 250 mM, and 500 mM (Figure 41). In this case, lane M in Figure 41 shows the protein molecular weight marker, lane 1 shows the loading sample from nickel affinity chromatography, lane 2 shows the material that did not bind to the nickel affinity resin, and lane 3 shows the results of elution with a 50 mM Na phosphate / 500 mM NaCl / 20 mM imidazole solution. Lane 4 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 50 mM imidazole solution. Lanes 5-8 show the results of elution with 50 mM Na phosphate / 500 mM NaCl / 100 mM imidazole solution. Lanes 9-11 show the results of elution with 50 mM Na phosphate / 500 mM NaCl / 250 mM imidazole solution. Lane 12 shows the results of elution with 50 mM Na phosphate / 500 mM NaCl / 500 mM imidazole solution.

[0176] The eluate recovered from nickel chromatography was subjected to a solution exchange with PBS buffer, utilizing the principle of osmotic pressure. After the solution exchange, the final recovered protein, TOM70-(GGGGS)3-UB-GFP, was confirmed by protein quantification and SDS-PAGE. As shown in Figure 42, after confirmation, the TOM70-(GGGGS)3-UB-GFP protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 42 shows the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-GFP protein obtained by dialysis against PBS buffer.

[0177] II. Preparation of fusion proteins containing mitochondrial outer membrane targeting proteins and targeting proteins Example 6. Preparation of fusion protein containing scFvHER2 Example 6.1. Synthesis of scFvHER2 gene In order to express human scFvHER2 in a recombinant protein, the scFvHER2 gene obtained by gene synthesis order from Bionics Co., Ltd. was designated pUC57-scFvHER2, and the nucleotide sequence of scFvHER2 was the same as the nucleotide sequence of SEQ ID NO:37.

[0178] Example 6.2. Construction of scFvHER2 protein expression vector Example 6.2.1. pET15b-UB-scFvHER2-TOM7 To prepare scFvHER2 protein fused with ubiquitin and TOM7, which binds to the outer mitochondrial membrane, an expression vector capable of expressing the scFvHER2 gene fused with ubiquitin and TOM7 was constructed.

[0179] The pUC57-scFvHER2 gene plasmid obtained in Example 6.1 above was cleaved with the restriction enzymes SacII and XhoI, and an approximately 750 bp DNA fragment was obtained by electrophoresis on a 2% agarose gel. This fragment was then inserted using T4 DNA ligase into the pET15b-UB-(p53)-TOM7 vector cleaved with the restriction enzymes SacII and XhoI to obtain the plasmid pET15b-UB-scFvHER2-TOM7 (Figure 39). In this case, UB-scFvHER2-TOM7 was represented by the nucleotide sequence of SEQ ID NO: 38.

[0180] The plasmid pET15b-UB-scFvHER2-TOM7 was used to transform Escherichia coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 1 mM, followed by shaking for an additional 4 hours.

[0181] The E. coli cell fraction was obtained by centrifugation, disrupted, and subjected to SDS-polyacrylamide electrophoresis. As shown in Figure 44, it was confirmed that the scFvHER2 protein, approximately 35 kDa in size, fused with ubiquitin and TOM7, was expressed. In this case, lane M in Figure 44 shows a protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after addition of IPTG, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0182] Example 6.2.2. Construction of pCMV-scFvHER2-TOM7-myc / His To prepare scFvHER2 protein fused with TOM7, which binds to the outer mitochondrial membrane, an expression vector capable of expressing scFvHER2 fused with TOM7 was constructed for animal cells. To obtain the TOM7 and scFvHER2 genes, RscFvHER2 primer and XTOM7(noT) primer were synthesized. The sequences of each primer are listed in Table 11.

[0183] [Table 11]

[0184] The plasmid pET15b-UB-scFvHER2-TOM7 obtained in Example 6.2.1 above was used as a template. 0.2 pmol of primer (RscFvHER2) and 0.2 pmol of primer (XTOM7(noT)) were mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The amplification reaction was then carried out in a polymerase chain reaction device for 25 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute to obtain the scFvHER2-TOM7 gene. The amplified scFvHER2-TOM7 gene was cleaved with the restriction enzymes EcoRI and XhoI, and DNA fragments of approximately 850 bp each were obtained by electrophoresis on a 1% agarose gel and then inserted using T4 DNA ligase into the pcDNA3.1-myc / His A vector cleaved with the restriction enzymes EcoRI and XhoI to obtain the plasmid pCMV-scFvHER2-TOM7-myc / His (Figure 45).

[0185] In this case, scFvHER2-TOM7-myc / His was represented by the nucleotide sequence of SEQ ID NO: 41. The plasmid pCMV-scFvHER2-TOM7-myc / His was transfected into CHO cells, disrupted, subjected to SDS-polyacrylamide gel electrophoresis, and then analyzed by Western blotting using an anti-c-myc antibody. As shown in Figure 46, expression of the approximately 35 kDa scFvHER2 protein fused with TOM7 was confirmed. In this case, lane M in Figure 46 shows a protein molecular weight marker, and lane 1 shows the transfection of CHO cells, disruption of the cells, SDS-polyacrylamide gel electrophoresis, and Western blotting using an anti-c-myc antibody.

[0186] Example 6.3. Isolation and purification of recombinant UB-scFvHER2-TOM7 protein derived from E. coli The UB-scFvHER2-TOM7 protein was isolated and purified using the same method as in Example 1.3.1. As a result, the UB-scFvHER2-TOM7 protein was eluted (Figure 47). In this case, lane M in Figure 47 shows a protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows the sample that did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 10mM imidazole solution. Lanes 4 and 5 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 6 and 8 show the results of elution with 8M urea / 50mM Na phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 9 and 10 show the results of elution with 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 250 mM imidazole solution, and lane 11 shows the results of elution with 8 M urea / 50 mM Na phosphate / 500 mM NaCl / 500 mM imidazole solution.

[0187] The protein content of the recovered eluate was measured by protein quantification and confirmed by SDS-PAGE. As shown in Figure 48, after confirmation, the UB-ScFvHER2-TOM7 protein was cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 48 shows the protein molecular weight marker, and lane 1 shows the UB-scFvHER2-TOM7 protein obtained by dialysis against PBS buffer.

[0188] Example 7. Preparation of fusion proteins containing scFvMEL Example 7.1. Synthesis of scFvMEL gene In order to express human scFvMEL as an antibody fragment against melanoma in a recombinant protein, the scFvMEL gene obtained by gene synthesis from Bionics Co., Ltd. was designated pUC57-scFvMEL, and the nucleotide sequence of scFvMEL was the same as the nucleotide sequence of SEQ ID NO: 42.

[0189] Example 7.2. Construction of scFvMEL protein expression vector Example 7.2.1. Construction of pET15b-UB-scFvMEL-TOM7 To prepare scFvMEL protein fused with ubiquitin and TOM7, which binds to the outer mitochondrial membrane, an expression vector capable of expressing scFvMEL fused with ubiquitin and TOM7 was constructed.

[0190] The pUC57-scFvMEL gene plasmid obtained in Example 7.1 was cleaved with the restriction enzymes SacII and XhoI, and an approximately 750 bp DNA fragment was obtained by electrophoresis on a 2% agarose gel. This fragment was then inserted using T4 DNA ligase into the pET15b-UB-(p53)-TOM7 vector that had been cleaved with the restriction enzymes SacII and XhoI to obtain the plasmid pET15b-UB-scFvMEL-TOM7 (Figure 49). In this case, UB-scFvMEL-TOM7 was represented by the nucleotide sequence of SEQ ID NO:43.

[0191] The plasmid pET15b-UB-scFvMEL-TOM7 was used to transform Escherichia coli BL21(DE3). The transformed strain was then cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the resulting colonies were cultured in LB liquid medium in a shaking incubator at 37°C. When the cell density reached an OD600 absorbance of approximately 0.2, IPTG was added to a final concentration of 1 mM, followed by shaking for an additional 4 hours.

[0192] The E. coli cell fraction was obtained by centrifugation, disrupted, and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Figure 50, it was confirmed that an approximately 35 kDa scFvMEL protein fused with ubiquitin and TOM7 was expressed. In this case, lane M in Figure 50 shows a protein molecular weight marker, lane 1 shows the precipitate obtained by centrifugation after disrupting E. coli 4 hours after addition of IPTG, and lane 2 shows the supernatant obtained by centrifugation after disrupting E. coli.

[0193] Example 7.2.2. Construction of pCMV-scFvMEL-TOM7-myc / His To prepare scFvMEL protein fused with TOM7 that binds to the outer mitochondrial membrane, an expression vector capable of expressing scFvMEL fused with TOM7 was constructed for animal cells. Primers (RscFvMEL) were synthesized to obtain the TOM7 and scFvMEL genes. The primer sequences are listed in Table 12.

[0194] [Table 12]

[0195] Using the plasmid pET15b-UB-scFvMEL-TOM7 obtained in Example 6.2.1 as a template, 0.2 pmol of RscFvMEL primer and 0.2 pmol of XTOM7(noT) primer were mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. Then, 25 cycles of amplification reaction at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute were performed in a polymerase chain reaction apparatus to obtain scFvMEL-TOM7. The amplified scFvMEL-TOM7 gene was cleaved with restriction enzymes EcoRI and XhoI, and an approximately 850 bp DNA fragment was isolated by electrophoresis on a 1% agarose gel. It was then inserted into the pcDNA3.1-myc / His A vector cleaved with restriction enzymes EcoRI and XhoI using T4 DNA ligase to obtain the plasmid pCMV-scFvMEL-TOM7-myc / His (Figure 51). Here, scFvMEL-TOM7-myc / His is SEQ ID NO: 4 It was represented by the base sequence:

[0196] The plasmid pCMV-scFvMEL-TOM7-myc / His was transfected into CHO cells, disrupted, subjected to SDS-polyacrylamide gel electrophoresis, and confirmed by Western blotting using an anti-c-myc antibody. As shown in Figure 52, expression of the approximately 35 kDa scFvMEL protein fused with TOM7 was confirmed. In this case, lane M in Figure 52 shows a protein molecular weight marker, and lane 1 shows the transfection into CHO cells, disrupted cells, subjected to SDS-polyacrylamide gel electrophoresis, and confirmed by Western blotting using an anti-c-myc antibody.

[0197] Example 8. Preparation of fusion proteins containing scFvPD-L1 Example 8.1. Synthesis of scFvPD-L1 gene To express human scFvPD-L1 in a recombinant protein, the scFvPD-L1 gene obtained by gene synthesis from Bionics Co., Ltd. was designated pUC57-scFvPD-L1, and its nucleotide sequence was the same as that of SEQ ID NO:46.

[0198] Example 8.2. Construction of scFvPD-L1 protein expression vector Example 8.2.1. Construction of pCMV-scFvPD-L1-TOM7-myc / His To prepare scFvPD-L1 protein fused with TOM7 that binds to the outer mitochondrial membrane, an expression vector for animal cells capable of expressing scFvPD-L1 fused with ubiquitin and TOM7 was constructed.

[0199] The plasmid pUC57-scFvPD-L1 was cleaved with restriction enzymes EcoRI and XhoI, and an approximately 760 bp DNA fragment was obtained by electrophoresis on a 1% agarose gel. This fragment was then inserted into the pCMV-(scFvMEL)-TOM7-myc / His vector cleaved with restriction enzymes EcoRI and XhoI using T4 DNA ligase to obtain the plasmid pCMV-scFvPD-L1-TOM7-myc / His (Figure 53). In this case, scFvPD-L1-TOM7-myc / His was represented by the nucleotide sequence of SEQ ID NO: 47.

[0200] The plasmid pCMV-scFvPD-L1-TOM7-myc / His was used to transfect CHO animal cells, and the cells were disrupted and subjected to SDS-polyacrylamide gel electrophoresis, followed by Western blotting using an anti-c-myc antibody. As shown in Figure 54, it was confirmed that a TOM7-fused scFvPD-L1 protein of approximately 35 kDa in size was expressed. In this case, lane M in Figure 54 shows the protein molecular weight marker, and lane 1 shows the transfection into CHO animal cells, followed by cell disruption and SDS-polyacrylamide gel electrophoresis, followed by Western blotting using an anti-c-myc antibody.

[0201] III. Creation of engineered mitochondria with fusion proteins Example 9. Creation of modified mitochondria To confirm whether fluorescent proteins fused with mitochondrial outer membrane binding sites bind to the mitochondrial outer membrane, we performed the following experiment. First, mitochondria were isolated from umbilical cord-derived mesenchymal stem cells (UC-MSCs) by centrifugation. They were then stained with MitoTracker CMXRos Red. They were then mixed with the recombinant protein TOM70-(GGGGS)3-UB-GFP purified from E. coli and incubated at ambient temperature for approximately 30 minutes.

[0202] Unreacted proteins were then removed by centrifugation and washed twice with PBS buffer. The mitochondria-bound fluorescent protein was then observed under a fluorescence microscope. Purified GFP protein lacking the mitochondrial outer membrane binding site was used as a control. The results confirmed that the fluorescent protein fused to the mitochondrial outer membrane binding site (TOM70-(GGGGS)3-UB-GFP) colocalized with the mitochondria of umbilical cord-derived mesenchymal stem cells (UC-MSCs) (Figures 55a and 55b).

[0203] Example 10. Confirmation of the ability of recombinant p53 protein to bind to the outer mitochondrial membrane Mitochondria isolated from umbilical cord-derived mesenchymal stem cells by centrifugation were mixed with purified recombinant protein TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 at a 1:1 ratio for 1 hour at 4°C. Mitochondria without protein were used as a control. The binding affinity between mitochondria and p53 was confirmed by Western blot analysis (Figure 56).

[0204] First, mitochondria and p53 protein were allowed to bind, and then the mixture was centrifuged at 13,000 rpm for 10 minutes to obtain mitochondria or p53-bound mitochondria in the form of a precipitate. Proteins that were not bound to mitochondria were removed by washing twice with PBS. The washed precipitate was subjected to protein electrophoresis (SDS-PAGE) and then Western blotting. Rabbit anti-p53 antibody was used as the primary antibody, and anti-rabbit IgG-HRP was used as the secondary antibody. A band of 60 kDa, the expected molecular weight, was confirmed in the test groups of mitochondria bound to TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7, compared with the control group containing unbound mitochondria alone (Figure 56).

[0205] IV. Confirmation of activity of modified mitochondria with active proteins bound Example 11. Isolation and intracellular introduction of exogenous mitochondria Mitochondria were isolated from umbilical cord-derived mesenchymal stem cells (UC-MSCs) by centrifugation. The isolated mitochondria were stained with Mitotracker CMX Ros, and the concentration and total amount of isolated mitochondria were confirmed by BCA quantification. 0 μg, 1 μg, 5 μg, 10 μg, 50 μg, and 100 μg of mitochondria were then introduced into gastric cancer cell line SNU-484 cells by centrifugation. Fluorescence microscopy confirmed that the degree of mitochondrial introduction into cells was concentration-dependent (Figure 57).

[0206] Example 12. Confirmation of the effect of normal mitochondria on cancer cells To investigate the effect of mitochondria derived from normal cells on cancer cell proliferation and ROS production, we conducted the following experiment. First, hepatocytes (WRL-68), fibroblasts, and umbilical cord-derived mesenchymal stem cells (UC-MSC) were selected as mitochondrial donor cells. Mitochondria were isolated from the cells by centrifugal fractionation. The cancer cells used as mitochondrial recipient cells were the A431 epidermal carcinoma cell line. In this case, mitochondria were delivered to the epidermal carcinoma cells using centrifugal force according to their concentration (Korean Patent Application No. 10-2017-0151526).

[0207] The proliferation of skin epidermal cancer cells and the production of reactive oxygen species (ROS) were monitored 24, 48, and 72 hours after transfection. The results confirmed that the introduction of mitochondria isolated from normal cells derived from various organs into cancer cells had a concentration-dependent inhibitory effect on cancer cell proliferation. Furthermore, the suppression of ROS production in cancer cells was confirmed to be concentration-dependent (Figures 58 and 59).

[0208] Example 13. Confirmation of the effect of normal mitochondria on drug resistance When mitochondria derived from normal cells were introduced into cancer cells, we investigated how they affected drug resistance, antioxidant gene expression, and cancer metastasis, which are characteristics of cancer cells, using the following method. First, normal liver cells (WRL-68) were used as mitochondrial donor cells, and mitochondria were isolated from the cells by centrifugal fractionation. The liver cancer cell line, HepG2, was used as the cancer cell line to serve as the mitochondrial recipient. Mitochondria were delivered to the liver cancer cells using centrifugal force according to their concentration, and we observed drug resistance to the anticancer drug doxorubicin, confirming that the cancer cell line that received mitochondria exhibited higher drug sensitivity (Figure 60).

[0209] Example 14. Confirmation of the effect of normal mitochondria on antioxidant effects When mitochondria isolated from normal cells were introduced into the liver cancer cell line HepG2 cells at various concentrations, it was confirmed that the expression of the antioxidant enzyme catalase and SOD-2 (superoxide dismutase 2) genes was increased in the cancer cells (Figure 61).

[0210] Example 15. Confirmation of the effect of normal mitochondria on cancer cell metastasis Regarding metastasis, we examined the expression of the α-smooth muscle actin (α-SMA) gene, one of the genes involved in EMT (epithelial-mesenchymal transition). In this case, we found that in liver cancer cells that received mitochondria, α-SMA protein expression was significantly reduced in a mitochondria concentration-dependent manner compared to liver cancer cells that did not receive mitochondria. On the other hand, E-cadherin protein, a cell adhesion protein, increased in a mitochondria concentration-dependent manner (Figure 62). This confirmed that normal mitochondria introduced into cancer cells caused changes in proteins known to be involved in cancer metastasis, and therefore also affected cancer cell metastasis.

[0211] Example 16. Confirmation of binding of recombinant p53 protein to the outer membrane of foreign mitochondria and its introduction into cells Mitochondria were isolated from umbilical cord-derived mesenchymal stem cells by centrifugation, stained with Mitotracker CMX Ros, and mixed with purified recombinant proteins TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 at a 1:1 ratio. The mixture was incubated at 4°C for 1 hour. The unreacted proteins were then removed by centrifugation. After washing twice with PBS buffer, the p53-bound mitochondria were then introduced into gastric cancer cell line SNU-484 cells by centrifugation (Figure 63). Control groups included a group without mitochondria and a group with mitochondria alone. After one day of culture, p53 bound to the introduced mitochondria was observed by immunocytochemistry (ICC) using a fluorescent microscope.

[0212] Rabbit anti-p53 antibody was used as the primary antibody, and goat anti-rabbit IgG Alexa Fluor 488 was used as the secondary antibody. The results confirmed that TOM70-(GGGGS)3-UB-p53 (stained green) or UB-p53-TOM7 (stained green) proteins bound to foreign mitochondria (stained red) were located in the cytoplasm of the cells into which the foreign mitochondria had been introduced during cell transduction (Figure 64, 200x magnification, and Figure 65, 400x magnification). These results demonstrate that the recombinant proteins were easily introduced into cells by mitochondria.

[0213] Example 17. Confirmation of p53-bound mitochondrial activity in cancer cell lines Example 17.1. Confirmation of the apoptotic potential of p53-binding exogenous mitochondria introduced into cells using gastric cancer cell lines Mitochondria isolated from umbilical cord-derived mesenchymal stem cells by centrifugation were mixed with recombinant proteins TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 purified from E. coli and allowed to bind at a 1:1 ratio for 1 hour at 4°C. As controls, UB-p53 protein without TOM70 and ubiquitin-free TOM70-(GGGGS)3-p53 were used. Unbound proteins were removed by centrifugation and washing with PBS. The protein-bound mitochondria were then introduced by centrifugation into the gastric cancer cell line SNU-484, which lacks p53 activity due to a mutation in the p53 gene (Figure 66). After 1 day of culture, the cells were fixed with 4% paraformaldehyde for 1 hour, and then permeabilized with a permeabilization solution (0.1% Triton-X-100 in 0.1% sodium citrate buffer, pH 7.4) and incubated with TUNEL solution (in situ cell death detection kit, TMR RED, Roche) at 37°C for 1 hour.

[0214] In TUNEL analysis, areas where nucleic acid fragmentation (DNA fragmentation) occurred stained red, indicating apoptosis. Compared to the control group, cells transfected with mitochondria bound to TOM70-(GGGGS)3-ub-p53 or p53-TOM7 showed more red staining, indicating that apoptosis was induced by mitochondria bound to TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7. In particular, the induction of apoptosis by mitochondria bound to the TOM70-(GGGGS)3-UB-p53 protein was greater (Figure 67a).

[0215] Example 17.2. Luciferase binding confirms the apoptotic potential of p53-bound exogenous mitochondria To confirm whether the biological activity of the delivered TOM70-(GGGGS)3-UB-p53 protein in recipient cells was maintained after delivery of the mitochondrial-bound form of TOM70-(GGGGS)3-UB-p53 protein obtained in Example 5.2 into recipient cells, a cell-based analysis using a reporter gene was performed. Because the p53 protein is a transcription factor, a gene containing six repeats of the nucleotide sequence RRRCWWGYYY (where R represents G or A, W represents A or T, and Y represents C or T) to which the p53 transcription factor can bind was synthesized with the following sequence: The base sequence of P53-promoter-S is as follows: (5'-GGG CAT GCT CGG GCA TGC CCG GGC ATG CTC GGG CAT GCC CGG GCA TGC TCG GGC ATG CCC-3') (SEQ ID NO: 91), and the base sequence of P53-promoter-AS is as follows: (5'-GGG CAT GCC CGA GCA TGC CCG GGC ATG CCC GAG CAT GCC CGG GCA TGC CCG AGC ATG CCC-3') (SEQ ID NO: 92).

[0216] Five micrograms of the synthetic gene P53-promoter-S and 5 micrograms of the synthetic gene P53-promoter-AS were incubated at 70°C for 20 minutes to promote the synthesis of the double-stranded gene, followed by phosphorylation using polynucleotide T4 kinase. The phosphorylated double-stranded gene was inserted into pGL3 vector cleaved with the restriction enzyme SmaI. A gene containing six repeats of the p53 transcription factor-binding sequence (RRRCWWGYYY) was ligated to the luciferase reporter gene to generate the plasmid p6xp53-Luc. The plasmid p6xp53-Luc and the β-galactosidase expression vector pRSVb-gal were transfected into HEK293 human kidney cells using the Lipofectamine method.

[0217] After 6 hours, HEK293 cells were treated with 10 μg of mitochondria bound to 5 μg, 10 μg, or 20 μg of TOM70-(GGGGS)3-UB-p53 protein. As a control, cells were treated with PBS or 10 μg of p53-bound mitochondria. After 18 hours of incubation, the treated cells were analyzed by measuring luciferase activity. To correct for the effects of transfection, the luciferase value was divided by the value obtained by measuring β-galactosidase activity to determine the corrected luciferase value.

[0218] The luciferase activity was high in cells treated with 10 μg of mitochondria and 5 μg, 10 μg, or 20 μg of TOM70-(GGGGS)3-UB-p53 protein, confirming that the p53 protein was intracellularly active (Figure 67b).

[0219] Example 18. Confirmation of the ability of RKIP-binding exogenous mitochondria introduced into cells to reduce metastasis of cancer cell lines Mitochondria isolated from umbilical cord-derived mesenchymal stem cells by centrifugation were mixed with purified recombinant protein TOM70-(GGGGS)3-UB-RKIP at a 1:1 ratio and allowed to bind for 1 hour at 4°C. The protein-bound mitochondria were then introduced by centrifugation into the breast cancer cell line MDA-MB-231, whose metastatic potential is known to be enhanced by reduced RKIP protein levels.

[0220] To confirm the metastatic potential of cancer cells, a cell invasion assay using a transwell plate was performed. The upper chamber of a transwell with a pore size of 8 μm was coated with Matrigel for 30 minutes at 37°C. MDA-MB-231 cells transfected with single mitochondria and MDA-MB-231 cells transfected with mitochondria bound to RKIP protein were used as test groups. 1 × 10 cells of each type were placed in the upper chamber of the transwell containing serum-free medium. 5The cells were placed in the lower chamber, and medium containing 10% bovine serum was placed in the lower chamber. After culturing at 37°C for 12 hours, the cells were fixed with 4% paraformaldehyde for 1 hour, and the cells that had passed through the Matrigel were stained with 1% crystal violet.

[0221] Microscopic observation revealed that purple-stained cells were observed in the membrane below the upper chamber, indicating the process of cell migration. The number of purple-stained cells was confirmed to be reduced in the test groups treated with mitochondria alone and RKIP-bound mitochondria compared to the control group. Four sections were randomly selected, and the number of stained cells was measured and plotted on a graph (Figure 68).

[0222] IV. Confirmation of delivery rate of modified mitochondria conjugated with targeting proteins Example 19. Confirmation of intracellular expression of single-chain variable fragment (ScFv) antibodies targeting cancer cells and confirmation of their binding to mitochondria in cells To express pCMV-ScFv-HER2-TOM7, pCMV-ScFv-MEL-TOM7, or pCMV-ScFv-PD-L1-TOM7 in animal cells, DNA was transfected into CHO cells using Lipofectamine LTX and PLUS or Lipofectamine 2000. GFP-TOM7 DNA was used as a control. To confirm intracellular expression and mitochondrial binding within the same cells, cytosol and mitochondria were isolated from transfected cells by centrifugation, adjusted to the same protein content using a BCA assay, and subjected to PAGE electrophoresis. The results were then analyzed by Western blot. A monoclonal c-myc antibody was used as the primary antibody, and anti-mouse IgG HRP was used as the secondary antibody.

[0223] The ScFv-HER2-TOM7 or ScFv-MEL-TOM7 protein bands were confirmed at the expected size of 35 kDa. All were confirmed in the mitochondrial layer, suggesting that the transfected and expressed proteins were bound to mitochondria in cells via TOM7 (Figure 69).

[0224] Next, to confirm that the target proteins expressed in the cells bound to mitochondria in the same cells, the expressed ScFv-HER2-TOM7, scFv-MEL-TOM7, or pCMV-PD-L1-TOM7 proteins in the cells were observed under a fluorescent microscope using immunocytochemistry (ICC). A monoclonal c-myc antibody was used as the primary antibody, and goat anti-mouse IgG Alexa Fluor 488 was used as the secondary antibody. Mitochondria in the cells were stained with Mitotracker CMX Ros. The results confirmed that the expressed ScFv-HER2-TOM7, ScFv-MEL-TOM7, or ScFv-PD-L1-TOM7 proteins colocalized with and bound to mitochondria in the cells (Figures 70 and 71).

[0225] Example 20. Isolation of mitochondria bound by single-chain variable fragment antibodies targeting cancer cells and comparison of mitochondrial transfection in gastric cancer cell lines Mitochondria were isolated from CHO cells transfected with pCMV-ScFv-HER2-TOM7 or pCMV-ScFv-PD-L1-TOM7. Mitochondria isolated from untransfected CHO cells served as a control. Mitochondria isolated from each cell line were stained with Mitotracker CMX Ros. The gastric cancer cell line SNU-484 was treated with the same amount of mitochondria, and the following day, the degree of mitochondrial transduction was compared using a fluorescent microscope. Compared to the control group, mitochondria bound to ScFv-HER2-TOM7 or ScFv-PD-L1-TOM7 were confirmed to be transfected into cancer cells to a greater extent than mitochondria from the control group (Figure 72). Therefore, it was demonstrated that mitochondria bound to target proteins are more easily transfected into cancer cells than mitochondria alone.

[0226] VI. Confirmation of in vivo activity of modified mitochondria with bound active proteins Example 21. Generation of a xenograft model (SNU-484) and administration of test materials Example 21.1. Preparation of Cancer Cells On the day of the experiment, gastric cancer cell line SNU-484 cells were inoculated into mice at 5 × 10 6 The cells were prepared for cell culture. The cell culture medium was removed, and PBS was added to wash the cells. The cells were dissociated with trypsin-EDTA solution, placed in a 50 mL tube, washed twice with PBS buffer, and then 20 mL of PBS was added to examine the cell count and viability. Based on the measured cell count, the cell number per mouse was set at 5 × 10 6 The cells were prepared by adjusting the volume of the cells and dividing them into groups. The transplant volume per mouse was adjusted to be the same, 100 μL. As a control, a group receiving 100 μL of cancer cells alone was prepared.

[0227] Example 21.2. Preparation of test materials Mitochondria isolated from umbilical cord blood mesenchymal stem cells as described above were prepared for transplantation at a dose of 50 μg per mouse based on protein concentration. For the group receiving single mitochondria, mitochondria were prepared by thoroughly mixing them with 100 μL of PBS containing cancer cells. For the modified mitochondria group, the prepared amount of mitochondria was mixed with TOM70-(GGGGS)3-UB-p53 protein at a 1:1 concentration ratio in an Eppendorf tube before mixing with cancer cells and incubated at ambient temperature for 1 hour. After the incubation period, the supernatant was removed by centrifugation at 20,000 × g for 10 minutes, and a pellet of protein-bound mitochondria (MT + TOM70-(GGGGS)3-UB-p53) was obtained. After washing twice with PBS buffer, p53-bound mitochondria (MT + TOM70-(GGGGS)3-UB-p53) was prepared by thoroughly mixing them with 100 μL of PBS containing cancer cells.

[0228] Example 21.3. Preparation of Test Animals and Implantation of Test Materials For the transplant samples prepared by group, Matrigel (BD) was added in the same volume as PBS and gently mixed with the cells to prepare 200 μL of test material per mouse. All procedures were performed on ice. To create the model, Balb / c nude mice (female, 7 weeks old) were purchased from RAONBIO. They were anesthetized with isoflurane inhalation for cancer cell transplantation, and the right dorsal area (based on the animal) was disinfected with an alcohol swab. Then, 200 μL of the injection solution was subcutaneously administered to the right dorsal area of ​​the test animals using a 1 ml syringe. After administration, the animals' weights and tumor sizes were measured twice a week, and observations were continued for up to three weeks to analyze the results (Figure 73).

[0229] Example 21.4. Confirmation of tumor formation The tumor volume was calculated by measuring the long and short axis lengths of the tumor and applying the following formula: <Formula 1> Long axis x short axis x short axis x 0.5 = tumor volume (mm3)

[0230] Example 21.5. Observation of physiological and morphological changes To observe physiological and morphological changes in mice due to the administration of anticancer drug candidates, changes in volume and tumor size were measured twice a week from the time of administration of cancer cells and test materials (FIG. 74).

[0231] The mice were weighed on a scale, and changes in body weight were analyzed by group (Figure 75). It was confirmed that there was no significant difference in body weight change over the three weeks between the group not injected with mitochondria, the group administered mitochondria alone, and the group injected with modified mitochondria. The tumor's major axis (length) and minor axis (width) were measured with calipers and tumor size was calculated by applying the formula 1 above. Changes in body weight were analyzed by group (Figure 76). While tumor size significantly increased over time in the group not treated with mitochondria, the increase in tumor size over time was slowed in the mice administered mitochondria. Furthermore, it was confirmed that the increase in tumor size was significantly slowed in the group administered mitochondria bound to p53 protein compared to the group administered mitochondria alone (Figure 76).

[0232] Example 22. Confirmation of the effect of modified mitochondria on the inhibition of skin cancer cell proliferation The p53-bound mitochondria obtained as described above were delivered to A431 skin cancer cells by centrifugation, and cell proliferation was monitored. Saline was used as a control, and an equal amount of mitochondria without p53 protein was used as a control. Mitochondria bound to p53 protein, a protein that induces apoptosis, were found to significantly suppress A431 cell proliferation compared to the control group and the group containing mitochondria alone (Figure 76).

[0233] V. Confirmation of activity of isolated mitochondria Example 23. Confirmation of isolated mitochondrial function: ATP content To isolate intracellular mitochondria from umbilical cord-derived mesenchymal stem cells (UC-MSCs), cells were homogenized using a syringe and subsequently centrifuged to obtain mitochondria. To confirm the functionality of the isolated mitochondria, the mitochondrial protein concentration of the isolated mitochondria was quantified by BCA assay, and 5 μg of mitochondria were prepared. The amount of ATP in the mitochondria was confirmed using the CellTiter-Glo Luminescence Kit (Promega, Madison, WI).

[0234] The prepared mitochondria were mixed with 100 μl of PBS and placed in a 96-well plate. This was compared with 100 μl of PBS without mitochondria as a control. 100 μl of the test solution included in the kit was added in the same manner, and the mixture was mixed thoroughly in a shaker for 2 minutes, followed by 10 minutes at ambient temperature. The ATP content was then measured using a luminescence microplate reader. The ATP content was higher in the samples containing mitochondria than in the control group, confirming mitochondrial function (Figure 78).

[0235] Example 24. Confirmation of isolated mitochondrial function: membrane potential JC-1 dye (Molecular Probes, catalog number 1743159) was used to confirm the membrane potential of isolated mitochondria. Prepared mitochondria were mixed with 50 μl of PBS and arranged in a 96-well plate. A control group (50 μl of PBS without mitochondria) and a CCCP (R&D Systems, CAS 555-60-2) treatment group were prepared. CCCP, a mitochondrial ionophore, inhibits mitochondrial function by depolarizing the mitochondrial membrane potential. The CCCP group was reacted with isolated mitochondria at room temperature for 10 minutes at 50 μM.

[0236] The dye was then reacted with JC-1 dye (2 μM) in the same manner, and the absorbance was measured, taking advantage of the fact that it exhibits different spectra depending on the concentration, resulting from changes in membrane potential. At low concentrations, it exists as a monomer and emits green fluorescence, while at high concentrations, the dye aggregates (J-aggregates) and emits red fluorescence. The mitochondrial membrane potential was analyzed by calculating the ratio of green absorbance to red absorbance. After the reaction was completed, the mitochondrial membrane potential was measured using a fluorescence microplate reader (monomer: excitation 485 / emission 530, J-aggregates: excitation 535 / emission 590). The results are shown in Figure 79.

[0237] Example 25. Confirmation of the degree of damage to isolated mitochondria by measuring mROS production To confirm whether 5 μg of mitochondria prepared as described above were damaged, we used MitoSOX red indicator dye (Invitrogen, catalog number M36008), which can analyze mitochondrial reactive oxygen species in isolated mitochondria. The prepared mitochondria were mixed with 50 μl of PBS and placed in a 96-well plate. Comparison was made with 50 μl of PBS without mitochondria as a control. MitoSOX red dye was mixed with 50 μl of PBS to a concentration of 10 μM, placed in the 96-well plate (final concentration: 5 μM), and incubated in a CO2 incubator at 37°C for 20 minutes. After the reaction, the amount of ROS in the mitochondria was measured using a microplate reader (excitation 510 / emission 580). The results are shown in Figure 80.

[0238] VI. Confirmation of release of desired protein bound to mitochondrial outer membrane protein outside and inside the cell Example 26. Confirmation of dissociation of a desired protein bound to a mitochondrial outer membrane protein outside the cell To obtain the desired protein in free form when the active protein bound to mitochondria was introduced into cells, a fusion protein (TOM70-UB-p53 or TOM-UB-GFP) was prepared from E. coli in which the ubiquitin protein was inserted between the outer mitochondrial membrane protein and the desired protein. To confirm whether the ubiquitin sequence could be cleaved by the ubiquitin cleavage enzyme UBP1, the recombinant fusion protein TOM70-UB-p53 was reacted with UBP1 enzyme at 37°C for 1 hour.

[0239] Subsequent analysis by SDS-PAGE electrophoresis confirmed that UBP1 did not cleave the ubiquitin protein from the fusion protein. This was thought to be due to structural interference with the mitochondrial outer membrane protein. Therefore, a linker protein consisting of the amino acids glycine and serine was inserted between the mitochondrial outer membrane protein and the ubiquitin protein. The new fusion proteins (TOM70-(GGGGS)3-UB-p53 or TOM70-(GGGGS)3-UB-GFP) were purified from E. coli and reacted with UBP1 enzyme at 37°C for 1 hour as described above. SDS-PAGE electrophoresis confirmed that the 3' end of ubiquitin was cleaved by UBP1 enzyme, resulting in the release of only the p53 protein, as expected (Figure 82).

[0240] Example 26. Confirmation of dissociation of a desired protein bound to a mitochondrial outer membrane protein inside the cell We investigated whether the fusion proteins obtained in the above examples (TOM70-(GGGGS)3-UB-p53 or TOM70-(GGGGS)3-UB-GFP) bound to mitochondria and then entered cells, resulting in the release of the active protein by the ubiquitin cleavage enzyme present in the cells. First, mitochondria from umbilical cord blood mesenchymal cells were incubated with the fusion protein TOM70-(GGGGS)3-UB-GFP in a microtube for 1 hour to allow binding. Unbound fusion protein was then removed by centrifugation and washed twice with PBS buffer. A fusion protein from which ubiquitin had been removed (TOM70-(GGGGS)3-p53) was used as a control.

[0241] The mitochondrial-associated proteins were then introduced into MDA-MB-231 cells, a breast cancer cell line, by centrifugation. One day later, the MDA-MB-231 cells were disrupted and fractionated into mitochondrial and cytosolic fractions based on their weight differences. SDS-PAGE electrophoresis and Western blot analysis revealed that for fusion proteins containing ubiquitin, GFP protein dissociated from the outer mitochondrial membrane protein, the linker protein, and ubiquitin were predominantly detected in the cytosolic fraction. For fusion proteins lacking ubiquitin, GFP protein bound to the outer mitochondrial membrane protein and the linker protein was predominantly detected in the mitochondrial fraction (Figure 83).

[0242] As a result, when a mitochondrial outer membrane protein-linker-ubiquitin-active protein bound to mitochondria was introduced into cells, the linkage between ubiquitin and the active protein was cleaved, and the dissociated active protein was released into the cytoplasm. Therefore, it was found that mitochondria can be used as a delivery vehicle as one of the means for effectively delivering useful proteins into cells.

Claims

1. A modified mitochondria in which a fusion protein is bound to the outer membrane of the mitochondria, The fusion protein is a fusion protein comprising a targeted protein having the ability to bind to a ligand or receptor present in the cell membrane, and a mitochondrial outer membrane anchoring peptide. The target-targeting protein is an antibody or a fragment of the antibody having the same CDR as the complementarity-determining region (CDR) of the antibody. The antibody fragment is scFv. Modified mitochondria.

2. The modified mitochondria according to claim 1, wherein the mitochondria are isolated from a cell or tissue.

3. The modified mitochondrion according to claim 2, wherein the cell is any one selected from the group consisting of somatic cells, germ cells, stem cells, and combinations thereof.

4. The modified mitochondrion according to claim 1, wherein the targeted protein is a protein that binds to a biomarker present on the surface of tumor cells.

5. The targeted proteins include CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), membrane-bound mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosine-related protein 1 (tyrp1), tyrosine-related protein 2 (tyrp2), brachyury, mesothelin, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms' tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, and UNC5H2. A modified mitochondrion according to claim 4, which is a protein capable of binding to any one selected from the group consisting of LILRB2, CEACAM, Nectin-3, and combinations thereof.

6. The modified mitochondrion according to claim 5, wherein the targeted protein is scFvHER2 that binds to human epidermal growth factor receptor 2, scFvMEL that binds to melanoma antigen E, or scFvPD-L1 that binds to PD-L1.

7. The modified mitochondrial according to claim 1, wherein the mitochondrial outer membrane anchoring peptide is any one selected from the group consisting of TOM5, TOM7, Fis1, VAMP1B, Cytb5, Bcl-2, and Bcl-x, or their C-terminal regions.

8. The modified mitochondrion according to claim 1, wherein the targeting protein and the mitochondrial outer membrane anchoring peptide are linked from the N-terminus to the C-terminus.

9. The modified mitochondrion according to claim 1, wherein the fusion protein further comprises a linker between the targeting protein and the mitochondrial outer membrane anchoring peptide.

10. The fusion protein, N-terminus - Targeted protein - Mitochondrial outer membrane anchoring peptide - C-terminus; N-terminus - Targeted protein - Ubiquitin or its fragment - Mitochondrial outer membrane anchoring peptide - C-terminus; N-terminus - Targeted protein - Linker 1 - Ubiquitin or its fragment - Mitochondrial outer membrane anchoring peptide - C-terminus; N-terminus - Targeted protein - Ubiquitin or its fragment - Linker 2 - Mitochondrial outer membrane anchoring peptide - C-terminus; or N-terminus - Targeted protein - Linker 1 - Ubiquitin or its fragment - Linker 2 - Mitochondrial outer membrane anchoring peptide - C-terminus; A modified mitochondria according to claim 8 or 9, having any of the structures.

11. The modified mitochondrion according to claim 9, wherein the linker consists of 1 to 150 amino acids.

12. The modified mitochondrion according to claim 11, wherein the linker consists of 5 to 50 amino acids comprising glycine and serine.

13. The modified mitochondrion according to claim 12, wherein the linker is (G4S)n, where n is an integer from 1 to 10.

14. A pharmaceutical composition comprising the modified mitochondria described in claim 1 as an active ingredient.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition is for the prevention or treatment of cancer.

16. The pharmaceutical composition according to claim 15, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

17. A modified mitochondria according to claim 1, for the prevention or treatment of cancer.

18. Use of the modified mitochondria described in claim 1 in the manufacture of a pharmaceutical product for the prevention or treatment of cancer.

19. A method for producing modified mitochondria, comprising the step of mixing isolated mitochondria with a fusion protein, The fusion protein is a fusion protein comprising a targeted protein having the ability to bind to a ligand or receptor present in the cell membrane, and a mitochondrial outer membrane anchoring peptide. The target-targeting protein is an antibody or a fragment of the antibody having the same CDR as the CDR of the antibody. A method wherein the antibody fragment is scFv.

20. A method for preparing modified mitochondria from transformed cells by introducing polynucleotides encoding a fusion protein, The fusion protein is a fusion protein comprising a targeted protein having the ability to bind to a ligand or receptor present in the cell membrane, and a mitochondrial outer membrane anchoring peptide. The target-targeting protein is an antibody or a fragment of the antibody having the same CDR as the CDR of the antibody. A method wherein the antibody fragment is scFv.

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