Modified mitochondria and uses thereof

Abstract

According to one embodiment of the present invention, mitochondria modified by a targeting protein can be efficiently delivered to a target. In addition, various activities may be exhibited when a protein of interest binding to the modified mitochondrial is delivered into a cell. The modified mitochondria can effectively lead to death of cancer tissue, and thus can also be used as an anti-cancer agent. Further, the targeting protein loaded on the modified mitochondria shows various activities, so that the modified mitochondria can be applied to treatment of various diseases. In addition, according to one embodiment of the present invention, fusion proteins comprising a desired protein and fusion proteins comprising a targeting protein can be used to modify mitochondria. Moreover, the mitochondria modified by the fusion protein exhibits various effects in a target cell.

Description

[0001] This application is a divisional application of the patent application with application number: 201980028175.4, application date: April 25, 2019, and invention name: "Modified mitochondria and their applications". Technical Field

[0002] The present invention provides a fusion protein capable of modifying mitochondria, mitochondria modified by the fusion protein and a pharmaceutical composition containing the fusion protein as an active ingredient. Background Art

[0003] Mitochondria are organelles of eukaryotic cells that participate in the synthesis and regulation of adenosine triphosphate (ATP), a source of intracellular energy. Mitochondria are associated with a variety of metabolic pathways in the body, such as cell signaling, cell differentiation, cell death, and control of the cell cycle and cell growth. Mitochondria have their own genome and are organelles that play a central role in the energy metabolism of cells. Mitochondria generate energy through electron transfer and oxidative phosphorylation processes, and play an important role in participating in apoptosis signaling pathways.

[0004] It has been reported that the reduction in energy production due to decreased mitochondrial function causes a variety of diseases. When the function of the electron transport chain reaction decreases due to changes in the mitochondrial genome and proteome, a decrease in ATP production, excessive reactive oxygen production, a decrease in calcium regulation function, etc. occur. In this case, the membrane permeability of mitochondria changes, and apoptosis function may occur abnormally, leading to cancer and incurable diseases.

[0005] Therefore, human diseases caused by mitochondrial dysfunction include genetic diseases related to mitochondria (Wallace DC 1999), diabetes (Maechler P 2001), heart disease (Sorescu D 2002), senile dementia (such as Parkinson's disease or Alzheimer's disease) (Lin MT 2006), and the occurrence of various cancers (Petros JA, 2005) and cancer metastasis (Ishikawa K, 2008). In addition, common features found in more than 200 types of cancer include impaired apoptosis, enhanced inflammatory response, and increased abnormal metabolism. All of these processes are closely related to mitochondrial function, and the correlation between cancer and mitochondria is attracting attention.

[0006] On the other hand, it is known that normal cells produce 36 ATPs per glucose molecule through an electron transport system process, but cancer cells, unlike normal cells, produce 2 ATPs per glucose molecule through glycolysis (aerobic glycolysis) under sufficient oxygen conditions. Therefore, it is known that cancer cells, unlike normal cells, use the energy-inefficient glycolysis process to produce amino acids, lipids, nucleic acids, etc. required for rapid cell proliferation. For this reason, it is known that cancer cells require less oxygen than normal cells but produce a larger amount of lactic acid.

[0007] Therefore, changes in the composition of the tumor microenvironment due to abnormal metabolism in cancer cells, inhibition of apoptosis due to abnormal mitochondrial function, and enhanced inflammatory responses, as well as abnormal metabolic reactions in cancer cells, play a very important role in tumor growth. Therefore, using these characteristics to develop metabolism-related anticancer agents may be a good way to solve the side effects and economic problems of conventional anticancer agents.

[0008] It is known that when mitochondria present in cells are isolated and the cells are treated in vitro or injected into the body, mitochondria enter the cells. By utilizing this phenomenon, normal mitochondria isolated from cells can be injected into the body to treat diseases caused by mitochondrial dysfunction, or specifically, specific proteins can be effectively delivered to cells using mitochondria as carriers to treat diseases, but there has been no report. Summary of the invention

[0009] Technical issues

[0010] An object of the present invention is to provide an effective protein delivery system, showing that mitochondria can be used as a means to effectively deliver proteins that exhibit various pharmacological effects into cells. In addition, an object of the present invention is to provide a recombinant protein for effectively delivering drugs, and to provide modified mitochondria produced using the recombinant protein. In addition, an object of the present invention is to provide a pharmaceutical composition comprising the modified mitochondria as an active ingredient.

[0011] Solutions to the problem

[0012] In order to solve the above problems, a modified mitochondria is provided, wherein the exogenous protein is bound to the outer membrane of the mitochondria. In addition, in order to prepare the modified mitochondria, a fusion protein is provided, which comprises a mitochondrial outer membrane anchor peptide and a desired drug protein. In addition, a fusion protein comprising an antibody or a fragment thereof and a mitochondrial outer membrane anchor peptide is provided.

[0013] Effects of the Invention

[0014] When mitochondria combined with exogenous proteins are applied to the human body, the exogenous proteins can be effectively delivered to the cells. In addition, the damaged functions of the cells can be repaired by pharmacologically active proteins delivered to the cells. In addition, when mitochondria combined with exogenous proteins with pharmacologically active proteins are delivered to the cells, the pharmacologically active proteins are dissociated from the mitochondria in the cells, and effective effects can be expected. In addition, modified mitochondria comprising antibody fragments can be effectively delivered to target cells. Specifically, when the fragment of the antibody targeting the protein present on the surface of the cancer tissue is bound to the surface of the mitochondria, the modified mitochondria can be effectively delivered to cancer cells. Therefore, the introduction of modified mitochondria can not only restore the damaged cell electron transfer system, but also can prevent or treat various diseases by being bound to the pharmacologically active proteins of the modified mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the method for preparing pTA-p53.

[0016] Figure 2 Schematic diagram of the method for preparing pET15b-UB-p53 vector.

[0017] Figure 3 The expression of UB-p53 protein in E. coli is shown.

[0018] Figure 4 Schematic diagram of the method for preparing the pET11C-TOM70-UB-p53 vector.

[0019] Figure 5 The expression of TOM70-UB-p53 protein in E. coli is shown.

[0020] Figure 6 Schematic diagram of the method for preparing pET11C-TOM70-(GGGGS)3-UB-p53 vector.

[0021] Figure 7 The expression of TOM70-(GGGGS)3-UB-p53 protein in E. coli is shown.

[0022] Figure 8 A method for preparing the pET11C-TOM70-(GGGGS)3-p53 vector is shown.

[0023] Fig. 9 The expression of TOM70-(GGGGS)3-p53 protein in E. coli is shown.

[0024] Fig.10 A method for preparing the pET15b-UB-p53-TOM7 vector is shown.

[0025] Fig.11 The expression of UB-p53-TOM7 protein in E. coli is shown.

[0026] Fig.12 A method for preparing the pCMV-p53-myc / His vector is shown.

[0027] Fig.13 The expression of p53-myc / His protein in transformed CHO is shown.

[0028] Fig.14 Shown are the results of purification of TOM70-(GGGGS)3-p53 protein followed by identification.

[0029] Fig.15 The figure shows the purified TOM70-(GGGGS)3-p53 protein.

[0030] Fig.16 Shown are the results of purification of TOM70-(GGGGS)3-UB-p53 protein followed by identification.

[0031] Fig.17 The figure shows the purified TOM70-(GGGGS)3-UB-p53 protein.

[0032] Fig.18 Shown are the results of purification of UB-p53 protein followed by identification.

[0033] Fig.19 The figure shows the purified UB-p53 protein.

[0034] Fig. 20 Shown are the results of purification and subsequent identification of UB-p53-TOM7 protein.

[0035] Fig.21 The figure shows the purified UB-p53-TOM7 protein.

[0036] Fig. 22 A method for preparing the pTA-GranzymeB vector is shown.

[0037] Fig.23 A method for preparing the pET11C-TOM70-(GGGGS)3-UB-GranzymeB vector is shown.

[0038] Fig.24 The expression of TOM70-(GGGGS)3-UB-GranzymeB protein in E. coli is shown.

[0039] Fig.25A method for preparing the pET15b-UB-GranzymeB-TOM7 vector is shown.

[0040] Fig.26 The expression of UB-GranzymeB-TOM7 protein in E. coli is shown.

[0041] Fig. 27 The results of purification of TOM70-(GGGGS)3-UB-GranzymeB protein are shown.

[0042] Fig.28 The figure shows the purified TOM70-(GGGGS)3-UB-GranzymeB protein.

[0043] Fig.29 A method for preparing the pTA-RKIP vector is shown.

[0044] Fig.30 A method for preparing the pET11C-TOM70-(GGGGS)3-UB-RKIP vector is shown.

[0045] Fig.31 The expression of TOM70-(GGGGS)3-UB-RKIP protein in E. coli is shown.

[0046] Fig.32 The results of purification of TOM70-(GGGGS)3-UB-RKIP protein are shown.

[0047] Fig.33 The figure shows the purified TOM70-(GGGGS)3-UB-RKIP protein.

[0048] Fig.34 A method for preparing the pTA-PTEN vector is shown.

[0049] Fig.35 A method for preparing the pET11C-TOM70-(GGGGS)3-UB-PTEN vector is shown.

[0050] Fig.36 The expression of TOM70-(GGGGS)3-UB-PTE protein in E. coli is shown.

[0051] Fig.37 The results of purification of TOM70-(GGGGS)3-UB-PTEN protein are shown.

[0052] Fig.38 A diagram showing purified TOM70-(GGGGS)3-UB-PTEN protein.

[0053] Fig.39 Shown are the results of purification and subsequent identification of the UB-GFP-TOM7 protein.

[0054] Fig.40 The figure shows the purified UB-GFP-TOM7 protein.

[0055] Fig.41 The results of purifying TOM70-(GGGGS)3-UB-GFP protein and then identifying it are shown.

[0056] Fig.42 A diagram showing the purified TOM70-(GGGGS)3-UB-GFP protein.

[0057] Fig.43 A method for preparing the pET15b-UB-scFvHER2-TOM7 vector is shown.

[0058] Fig.44 The expression of UB-scFvHER2-TOM7 protein in E. coli is shown.

[0059] Fig.45 A method for preparing the pCMV-scFvHER2-TOM7-myc / His vector is shown.

[0060] Fig.46 The expression of scFvHER2-TOM7-myc / His protein in transformed CHO is shown.

[0061] Fig.47 The results of purification of UB-ScFvHER2-TOM7 protein are shown.

[0062] Fig.48 The figure shows the purified UB-ScFvHER2-TOM7 protein.

[0063] Fig.49 A method for preparing the pET15b-UB-scFvMEL-TOM7 vector is shown.

[0064] Fig.50 The expression of UB-scFvMEL-TOM7 protein in E. coli is shown.

[0065] Fig.51 A method for preparing the pCMV-scFvMEL-TOM7-myc / His vector is shown.

[0066] Fig.52 The expression of scFvMEL-TOM7-myc / His protein in transformed CHO is shown.

[0067] Fig.53 The method for preparing the pCMV-scFvPD-L1-TOM7-myc / His vector is shown.

[0068] Fig.54 The expression of scFvPD-L1-TOM7-myc / His protein in transformed CHO is shown.

[0069] Fig.55 This is a diagram to confirm whether the fluorescent protein is bound to the outer membrane of the mitochondria. In this case, the mitochondria are stained red with MitoTracker CMXRos and green with TOM70-UB-GFP. The overlapping area of ​​the two parts is shown in yellow. At this time, the magnification of part a is 200 times, while the magnification of part b is 600 times.

[0070] Fig.56 Shown are the results of Western blot analysis to identify the binding of recombinant proteins TOM70-(GGGGS)3-UB-p53 and UB-p53-TOM7 to the outer membrane of exogenous mitochondria.

[0071] Fig.57 Shown are the results of isolating exogenous mitochondria, observing the extent of intracellular injection according to the concentration of mitochondria using a fluorescence microscope, and then injecting mitochondria into cells.

[0072] Fig.58 A diagram demonstrating the effect of normal mitochondria on the proliferation of skin cancer cells.

[0073] Fig.59 A diagram demonstrating that normal mitochondria inhibit the production of reactive oxygen species (ROS) in skin cancer cells.

[0074] Fig.60 A diagram demonstrating the effect of normal mitochondria on drug resistance.

[0075] Fig.61 A diagram demonstrating the effect of normal mitochondria on antioxidant gene expression in cells.

[0076] Fig.62 A graph showing the effect of normal mitochondria on the expression of genes related to cancer cell metastasis.

[0077] Fig.63 Schematic diagram of a method for confirming that the recombinant protein p53 is loaded on the outer membrane of exogenous mitochondria and injecting the recombinant protein p53 into cells.

[0078] Fig.64 This is a picture showing that the recombinant protein p53 is loaded on the outer membrane of exogenous mitochondria and p53 is injected into cells. At this time, the magnification is 200 times.

[0079] Fig.65 This is a view to confirm that the recombinant protein p53 is loaded on the outer membrane of exogenous mitochondria and p53 is injected into cells. At this time, the magnification is 600 times.

[0080] Fig.66 Schematic representation of the method to demonstrate the apoptotic capacity of modified mitochondria loaded with p53 injected into cells using a gastric cancer cell line.

[0081] Fig.67a This is a diagram showing the apoptotic ability of modified mitochondria loaded with p53 injected into gastric cancer cells confirmed by the TUNEL method. The magnification is 600 times.

[0082] Figure 67b The figure is a diagram confirming the apoptotic ability of modified mitochondria loaded with p53 injected into gastric cancer cells by fluorescence measurement.

[0083] Fig.68 This figure demonstrates the inhibitory effect of modified mitochondria loaded with RKIP on cancer cell metastasis in MDA-MB-231 cells.

[0084] Fig.69 A diagram showing that a single-chain variable fragment (ScFv) antibody targeting cancer cells is expressed in cells.

[0085] Fig.70 This is a picture showing that the expression of a single-chain variable fragment (ScFv) antibody targeting cancer cells and binding to mitochondria present in cells was confirmed by an immunocytochemistry (ICC) experimental method. In this case, the magnification is 200 times.

[0086] Fig.71 This is a picture showing that the expression of a single-chain variable fragment (ScFv) antibody targeting cancer cells and binding to mitochondria present in cells was confirmed by an immunocytochemistry (ICC) experimental method. In this case, the magnification is 600 times.

[0087] Fig.72 The figure compares the effects of injecting mitochondria bound to a cancer cell-targeting single-chain variable fragment antibody into gastric cancer cell lines.

[0088] Fig.73 Schematic diagram of the animal experimental protocol using modified mitochondria.

[0089] Fig.74 A photograph in which an increase in tumor tissue can be observed with the naked eye.

[0090] Fig.75 The graphs demonstrate the changes in body weight of mice after administration of mitochondria and modified mitochondria.

[0091] Fig.76 Figures are provided to demonstrate tumor size after administration of mitochondria and modified mitochondria.

[0092] Fig.77 This figure demonstrates that modified mitochondria loaded with TOM-UB-p53 protein can effectively inhibit the proliferation of A431 cells.

[0093] Fig.78 The figure shows the function of isolated mitochondria confirmed by ATP content.

[0094] Fig.79 The figure shows the function of isolated mitochondria confirmed by membrane potential.

[0095] Fig.80 The figure shows the extent of damage to isolated mitochondria by measuring mitochondrial ROS (mROS production).

[0096] Fig.81a It is a diagram showing the structure of a protein present in the outer membrane of mitochondria and the amino acid sequence of the N-terminal region of TOM70, TOM20 or OM45.

[0097] Fig.81b The diagram shows the amino acid sequence of the C-terminal region of TOM5, TOM7, Fis1, VAMP1B, Cytb5, BCL-2, or BCL-X.

[0098] Fig.82 Figure 2 is a diagram showing whether the desired protein is dissociated, depending on the presence or absence of a linker between the outer membrane anchor peptide and ubiquitin.

[0099] Fig.83 A diagram showing that the desired protein bound to the modified mitochondria is separated from the mitochondria in cells. DETAILED DESCRIPTION

[0100] Hereinafter, the present invention will be described in detail.

[0101] One aspect of the present invention provides a modified mitochondria, wherein an exogenous protein is bound to the outer membrane of the mitochondria.

[0102] The mitochondria can be obtained from mammals or humans. Specifically, the mitochondria can be isolated from cells or tissues. For example, the mitochondria can be obtained from somatic cells, germ cells or stem cells. In addition, the mitochondria can be normal mitochondria in cells, and the mitochondria in the cells have normal biological activity. In addition, the mitochondria can be cultured in vitro.

[0103] In addition, the mitochondria can be obtained from autologous, allogeneic or xenogeneic subjects. Specifically, autologous mitochondria refer to mitochondria obtained from tissues or cells of the same subject. In addition, allogeneic mitochondria refer to mitochondria obtained from a subject belonging to the same species as the subject and having a different genotype of the allele. In addition, xenogeneic mitochondria refer to mitochondria obtained from a subject belonging to a different species than the subject.

[0104] Specifically, the somatic cell can be a muscle cell, a hepatocyte, a nerve cell, a fibroblast, an epithelial cell, a fat cell, an osteocyte, a leukocyte, a lymphocyte, a platelet or a mucosal cell. In addition, the germ cell is a cell that undergoes meiosis and mitosis, and can be a sperm or an egg. In addition, the stem cell can be any one selected from mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells and tissue-derived stem cells. In this case, the mesenchymal stem cell can be any one selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta.

[0105] On the other hand, when mitochondria are isolated from specific cells, the mitochondria can be isolated by various known methods, for example, using a specific buffer or using an electric potential difference and a magnetic field, and the like.

[0106] The term "exogenous protein" as used herein refers to a protein including a desired protein that can function inside and outside a cell. In this case, the exogenous protein is a protein that does not exist in mitochondria and can be a recombinant protein. Specifically, the exogenous protein can include a mitochondrial anchor peptide and a desired protein. In addition, the exogenous protein can be a recombinant fusion protein including a mitochondrial anchor peptide and a desired protein. In this case, the exogenous protein can include a mitochondrial anchor peptide. Preferably, the mitochondrial anchor peptide can be a peptide that can be located on the outer membrane of mitochondria. Therefore, the exogenous protein can be bound to the outer membrane of the mitochondria through the mitochondrial anchor peptide. The mitochondrial anchor peptide can be a peptide that includes the N-terminal region or the C-terminal region of a protein present in a mitochondrial membrane protein, and the N-terminal region or the C-terminal region of the protein present in the mitochondrial membrane protein can be located on the outer membrane of the mitochondria. In this case, the anchor peptide can further include a mitochondrial signal sequence.

[0107] An embodiment of the protein present in the mitochondrial membrane protein may be any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. Specifically, when the mitochondrial anchoring peptide is derived from any one of the group consisting of TOM20, TOM70 and OM45, it may comprise the N-terminal region of TOM20, TOM70 or OM45. An embodiment of the mitochondrial anchoring peptide may be TOM70 derived from yeast as shown in SEQ ID NO:75, or TOM70 derived from humans as shown in SEQ ID NO:76. Another embodiment may be TOM20 derived from yeast as shown in SEQ ID NO:77, or TOM20 derived from humans as shown in SEQ ID NO:78. Another embodiment may be OM45 derived from yeast as shown in SEQ ID NO:79.

[0108] In addition, when the mitochondrial anchoring peptide is derived from any one of the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B, it may include a C-terminal region selected from any one of the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. One embodiment of the mitochondrial anchoring peptide may be TOM5 derived from yeast as shown in SEQ ID NO:80 or TOM5 derived from human as shown in SEQ ID NO:81. Another embodiment may be TOM7 derived from yeast as shown in SEQ ID NO:82, or TOM7 derived from human as shown in SEQ ID NO:83. Another embodiment may be TOM22 derived from yeast as shown in SEQ ID NO:84, or TOM22 derived from human as shown in SEQ ID NO:85. Another embodiment may be Fis1 derived from yeast as shown in SEQ ID NO:86, or Fis1 derived from human as shown in SEQ ID NO:87. Another embodiment may be Bcl-2α derived from human as shown in SEQ ID NO: 88. Another embodiment may be VAMP1 derived from yeast as shown in SEQ ID NO: 89, or VAMP1 derived from human as shown in SEQ ID NO: 90.

[0109] In this case, the desired protein capable of functioning both inside and outside the cell may be any one selected from the group consisting of an active protein exhibiting activity in the cell, a protein present in the cell, and a protein having the ability to bind to a ligand or receptor present in the cell membrane.

[0110] An embodiment of the active protein or protein present in the cell 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 factor (TRAF), 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 bound to an anchor peptide comprising the N-terminal region of TOM20, TOM70 or OM45.

[0111] This fusion protein can be combined in the following order:

[0112] N-terminus - anchor peptide comprising the N-terminal region of TOM20, TOM70 or OM45 - desired protein - C-terminus.

[0113] In addition, the exogenous protein may further comprise an amino acid sequence recognized by a proteolytic enzyme in a eukaryotic cell, or ubiquitin or a fragment thereof between an anchor peptide and a desired protein. The proteolytic enzyme in a eukaryotic cell refers to an enzyme that degrades proteins present in a eukaryotic cell. In this case, because the exogenous protein comprises an amino acid sequence recognized by an enzyme that degrades proteins, the exogenous protein bound to the outer membrane of the mitochondria can be separated into an anchor peptide and a desired protein in the cell.

[0114] In this case, the ubiquitin fragment may comprise a C-terminal Gly-Gly of an amino acid sequence as shown in SEQ ID NO: 71, and comprise 3 to 75 amino acids continuous from the C-terminus. In addition, the exogenous protein may further comprise a linker between the desired protein and ubiquitin or its fragment. In this case, the linker may be, but not limited to, composed of 1 to 150 amino acids, or composed of 10 to 100 amino acids, or composed of 20 to 50 amino acids. The linker may be composed of amino acids appropriately selected from 20 kinds of amino acids, preferably composed of glycine and / or serine. One embodiment of the linker may be composed of 5 to 50 amino acids, and the amino acids are composed of glycine and serine. One embodiment of the linker may be (G4S)n, wherein n is an integer from 1 to 10, and n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0115] In addition, the protein having the ability to bind to a ligand or receptor present in the cell membrane can be a ligand or receptor present on the surface of a tumor cell. In this case, the ligand or receptor present on the surface of the tumor cell is selected from any one of the group consisting of CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), mucin 1 cell surface associated protein (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), survivin, tyrosine-related protein 1 (tyrp1), tyrosine-related 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, and connexin 3 or a combination thereof.

[0116] In addition, the protein having the ability to bind to a ligand or receptor present in a cell membrane can be an antibody or a fragment thereof that binds to any one selected from the above group. Specifically, an antibody fragment refers to a fragment having the same complementary determining region (CDR) as an antibody. Specifically, it can be Fab, scFv, F(ab')2 or a combination thereof.

[0117] In this case, the desired protein can be bound to an anchor peptide comprising a 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 exogenous protein can be bound in the following order:

[0118] N-terminal - desired protein - 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 - C-terminal.

[0119] In addition, the exogenous 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, active protein, protein present in cells, and protein having the ability to bind to a ligand or receptor present in a cell membrane, etc. are as described above.

[0120] In one embodiment of the desired protein, the antibody or fragment thereof targeting a specific cell may be in a form bound to an anchor peptide comprising a C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. The modified mitochondria bound to the desired protein can be easily introduced into a specific target site, so that the mitochondria can effectively enter specific cells.

[0121] An embodiment of the modified mitochondria can be in a form combined with one or more desired proteins. Specifically, it can be in a form combined with a desired protein containing p53 and a desired protein containing an anti-HER-2 antibody or a fragment thereof. This modified mitochondria can effectively deliver mitochondria to cancer cells expressing HER-2. In addition, cancer cells can be effectively killed by p53 combined with the modified mitochondria.

[0122] Depending on the purpose of the modified mitochondria, a desired protein containing one or more active proteins can be constructed and bound to the mitochondria. In addition, depending on the target cell, a desired protein targeted to the cell can be constructed in various ways.

[0123] In another aspect of the present invention, a pharmaceutical composition is provided, comprising the modified mitochondria as an active ingredient. In this case, the pharmaceutical composition can be used to prevent or treat cancer. In this case, the cancer can 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.

[0124] Specifically, when active proteins, such as p53, kill tumor cells, or when proteins that inhibit proliferation bind to mitochondria, modified mitochondria bound to p53 can be used as anticancer agents. In addition, when proteins such as RKIP that can inhibit the metastasis of cancer cells bind to mitochondria, modified mitochondria bound to RKIP can be used as tumor metastasis inhibitors. When any one of 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), RAF kinase inhibitory protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1 and a combination thereof, which inhibits tumor cell proliferation or controls phosphorylation reactions in tumor cells, is selected, it binds to mitochondria, and the modified mitochondria bound to the active protein can be used as an anticancer agent.

[0125] In addition, for pharmaceutical compositions, mitochondria may be included 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. The content of the mitochondria within the above range can promote the dosage adjustment of the mitochondria after administration, and can increase the degree of improvement of the patient's disease symptoms. In this case, the dosage of the mitochondria can be determined by quantifying the membrane proteins of the isolated mitochondria for mitochondrial quantification. Specifically, the isolated mitochondria can be quantified by Bradford protein assay (paper written by James D.McCully (JVis Exp.2014; (91): 51682.).

[0126] In addition, for the pharmaceutical composition, the active protein bound to mitochondria can be, but is not limited to, a concentration of 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. The active protein contained within the above range can facilitate the dosage adjustment of the active protein after administration, and can increase the degree of improvement of the patient's disease symptoms.

[0127] In addition, for pharmaceutical compositions, the targeted protein capable of delivering mitochondria to specific cells may be, but is not limited to, a concentration of 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. The targeted protein contained within the above range can facilitate the dosage adjustment of the targeted protein when administered and can increase the degree of improvement of the patient's disease symptoms.

[0128] Specifically, the pharmaceutical composition according to the present invention can be, but is not limited to, administered to an individual taking medication at a dose of 0.01-5 mg / kg, 0.1-4 mg / kg, or 0.25-2.5 mg / kg of mitochondria each time based on body weight. That is, in terms of cell activity, it is most preferred to administer the pharmaceutical composition in a manner such that the amount of modified mitochondria falls within the above range based on the body weight of an individual with cancerous tissue. In addition, the pharmaceutical composition can be administered 1-10 times, 3-8 times, or 5-6 times, preferably 5 times. In this case, the dosing interval can be 1-7 days, or 2-5 days, preferably 3 days.

[0129] In addition, the pharmaceutical composition according to the present invention can be applied to humans or other mammals susceptible to or suffering from cancer. In addition, the pharmaceutical composition can be an injection that can be administered intravenously or an injection that can be administered topically, and can preferably be a preparation for injection.

[0130] Therefore, the pharmaceutical composition according to the present invention can be prepared into a physically or chemically highly stable injection by adjusting the pH of the composition using a buffer solution that can be used for injection preparation, such as an acidic aqueous solution or phosphate, to ensure the stability of the product during the injection distribution process.

[0131] Specifically, the pharmaceutical composition of the present invention may include water for injection. The water for injection is distilled water prepared for dissolving solid injections or diluting water-soluble injections, 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, lactic acid-Ringer's solution, phosphate buffer solution with a pH of 3.5 to 7.5, sodium dihydrogen phosphate-citrate buffer solution, etc.

[0132] In addition, the pharmaceutical composition of the present invention may contain a stabilizer or a dissolution aid. For example, the stabilizer may be sodium pyrosulfite or ethylenediaminetetraacetic acid, and the dissolution aid may be hydrochloric acid, acetic acid, sodium hydroxide, sodium bicarbonate, sodium carbonate or potassium hydroxide.

[0133] In addition, the present invention may provide a method for preventing or treating cancer, which comprises administering the above-mentioned pharmaceutical composition to an individual. Here, the individual may be a mammal, and preferably a human.

[0134] One aspect of the present invention provides a method for preparing modified mitochondria, which comprises the step of mixing isolated mitochondria with a desired protein comprising an active protein and / or a desired protein comprising a targeted protein target.

[0135] In this case, the desired protein and the mitochondria can be mixed in an appropriate ratio. For example, the desired protein: mitochondria can be mixed based on a weight ratio of 1:100 to 100:1. Specifically, they can be mixed in a ratio of 1:10, 1:5, 1:4, 1:3, 1:2 or 1:1. In addition, the ratio can be 10:1, 5:1, 4:1, 3:1 or 2:1.

[0136] In another aspect of the present invention, a method for preparing modified mitochondria from transformed cells by injecting a polynucleotide encoding the above-mentioned desired protein into a eukaryotic cell is provided. Specifically, a method for preparing the above-mentioned fusion protein is provided, which comprises the steps of transforming the above-mentioned polynucleotide into a prokaryotic cell or a eukaryotic cell without a ubiquitin degrading enzyme or a proteolytic enzyme; and the step of obtaining the fusion protein. The preparation method is applicable to the case where the desired protein does not contain an amino acid sequence recognized by a proteolytic enzyme or ubiquitin or a fragment thereof in a eukaryotic cell.

[0137] In another aspect of the present invention, the desired protein can be prepared by prokaryotic cells or prokaryotic cell extracts. In addition, a method for preparing modified mitochondria using eukaryotic cells or eukaryotic cell extracts that do not have ubiquitin degrading enzymes or proteolytic enzymes is provided.

[0138] In another aspect of the present invention, the use of mitochondria as a means of delivering exogenous proteins is provided. Specifically, the modified mitochondria can be used as a means of intracellular and extracellular delivery of exogenous proteins containing desired proteins that can function inside and outside the cell. The mitochondria can be effectively introduced into cells, and in this case, the exogenous protein desired to be delivered to the cell can be effectively delivered to the cell. In this case, the mitochondria can serve as an effective protein delivery system. The desired protein is as described above.

[0139] Another aspect of the present invention provides a fusion protein comprising a mitochondrial outer membrane anchor peptide and a desired protein. In this case, the desired protein is as described above.

[0140] As used herein, the term "mitochondrial outer membrane anchoring peptide" may be the N-terminus or C-terminus of a protein present in the mitochondrial outer membrane. The mitochondrial outer membrane anchoring peptide may have an amino acid sequence that is specifically positioned in the mitochondrial outer membrane. In this case, the mitochondrial outer membrane anchoring peptide attaches the fusion protein disclosed in the present invention to the outer membrane of the mitochondria. In this case, the mitochondrial outer membrane anchoring peptide may have the same use meaning as the mitochondrial outer membrane targeting peptide.

[0141] In addition, the mitochondrial outer membrane anchor peptide prevents the fusion protein disclosed in the present invention from entering the interior of the mitochondria. The amino terminus of the TOM (outer membrane translocator) complex present in the mitochondrial outer membrane has a mitochondrial target sequence and a single outer membrane anchor domain, and most of the carboxyl termini may have a structure exposed to the cytoplasm ( Fig.81a The carboxyl terminus of the TOM (translocator of the outer membrane) complex present in the mitochondrial outer membrane has a mitochondrial target sequence and a single outer membrane anchoring domain, and most of the amino termini may also have structures exposed to the cytoplasm ( Fig.81b ). In addition, the protein present in the mitochondrial outer membrane can be selected from the protein present in the mitochondria in eukaryotic cells. For example, it can be selected from the protein present in the mitochondrial outer membrane in yeast, animal cells or human cells.

[0142] In this case, one embodiment of the protein present in the outer membrane of mitochondria may be any one 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 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 located in the outer membrane of mitochondria.

[0143] Specifically, when the mitochondrial outer membrane anchor peptide is fused to the N-terminus of the desired protein, the mitochondrial outer membrane anchor peptide may comprise a terminal sequence of a protein selected from the group consisting of TOM20, TOM70 and OM45. Preferably, it may be an N-terminal sequence of a protein selected from the group consisting of TOM20, TOM70 and OM45. An embodiment of the mitochondrial outer membrane anchor peptide is as described above.

[0144] In addition, when the mitochondrial outer membrane anchor peptide is fused to the C-terminus of the desired protein, the outer membrane targeting protein may comprise a 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 a C-terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. One embodiment of the mitochondrial outer membrane anchor peptide is as described above.

[0145] The term "active protein" as used herein may be a protein that exhibits physiological activity. One embodiment of such an active protein may be a protein with reduced function or a modified protein present in damaged cancer cells. One embodiment of the active protein may be a protein that enhances cell activity. One embodiment of such an active protein is as described above.

[0146] The fusion protein can be a protein that combines the outer membrane targeting protein of the mitochondria and the desired protein from the N-terminal to the C-terminal. In this case, it can further be included in ubiquitin or its fragment with a ubiquitin protease specific cleavage site (glycine-glycine) between the outer membrane targeting protein of the mitochondria and the desired protein. In this case, in order to promote the cutting of the ubiquitin protease, it can further be included in a joint containing hydrophilic and polar amino acids (serine, glycine and threonine) between the outer membrane targeting protein of the mitochondria and the ubiquitin protein.

[0147] The term "ubiquitin" as used herein refers to a protein involved in the proteolytic process, also referred to as UB. An embodiment of the ubiquitin may be ubiquitin present in the human body or ubiquitin present in yeast. The ubiquitin in the human body consists of 76 amino acids. In this case, ubiquitin may be used in a mature form. The term "mature form" as used herein may refer to a protein in a form in which the signal peptide has been removed.

[0148] In addition, an enzyme called ubiquitin protease or UBP (ubiquitin-specific protease) exists naturally in eukaryotic cells and can induce the natural dissociation of desired proteins by cleaving the C-terminal amino acid glycine-glycine site of ubiquitin in cells.

[0149] In this case, the ubiquitin fragment may include the Gly-Gly amino acid at the C-terminus of ubiquitin, and may include 3 to 75 consecutive amino acids from the C-terminus. Specifically, one embodiment 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. In addition, the ubiquitin fragment may have an amino acid sequence as shown in SEQ ID NO:71.

[0150] A fusion protein comprising a mitochondrial outer membrane 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 of the following structures:

[0151] <Structural Formula 1>

[0152] N-terminal - mitochondrial outer membrane anchor peptide - required protein - C-terminal

[0153] <Structural Formula 2>

[0154] N-terminal - mitochondrial outer membrane anchor peptide - ubiquitin or its fragment - desired protein - C-terminal

[0155] <Structural Formula 3>

[0156] N-terminal-mitochondrial outer membrane targeting peptide-linker 1-ubiquitin or its fragment-required protein-C-terminal

[0157] <Structural Formula 4>

[0158] N-terminal-mitochondrial outer membrane anchor peptide-ubiquitin or its fragment-linker 2-required protein-C-terminal

[0159] <Structural Formula 5>

[0160] N-terminal-mitochondrial outer membrane anchor peptide-linker 1-ubiquitin or its fragment-linker 2-required protein-C-terminal

[0161] In the above structural formulas 1-5, the outer membrane anchor peptide can be a terminal sequence of a protein selected from the group consisting of TOM20, TOM70 and OM45, and the desired protein 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 factor (TRAF), RAF kinase inhibitory protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, and DKK-3PD1.

[0162] In this case, the linker 1 or 2 can be a polypeptide consisting of 1 to 100, 1 to 80, 1 to 50 or 1 to 30 amino acids, respectively, and can preferably be a polypeptide consisting of 1 to 30 amino acids, wherein the amino acid is serine, glycine or threonine alone or a combination thereof. In addition, the linker 1 or 2 can be a polypeptide consisting of 5 to 15 amino acids, respectively, and can preferably be a polypeptide consisting of 5 to 15 amino acids, wherein the amino acid is serine, glycine or threonine alone or a combination thereof. An embodiment of the linker can be (GGGGS)3 (SEQ ID NO: 70).

[0163] <Structural Formula 6>

[0164] N-terminal-required protein-mitochondrial outer membrane anchor peptide-C-terminal

[0165] <Structural Formula 7>

[0166] N-terminal-required protein-ubiquitin or its fragment-mitochondrial outer membrane anchor peptide-C-terminal

[0167] <Structural Formula 8>

[0168] N-terminal-required protein-linker 1-ubiquitin or its fragment-mitochondrial outer membrane anchor peptide-C-terminal

[0169] <Structural Formula 9>

[0170] N-terminal-required protein-ubiquitin or its fragment-linker 2-mitochondrial outer membrane anchor peptide-C-terminal

[0171] <Structural Formula 10>

[0172] N-terminal-required protein-linker 1-ubiquitin or its fragment-linker 2-mitochondrial outer membrane targeting peptide-C-terminal

[0173] In the above structural formulas 6 to 10, the outer membrane anchor 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 VAMP1B, and the desired protein can be 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), Any one of the group consisting of 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 inhibitory protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, Oct3 / 4, Sox2, Klf4 and c-Myc. In this case, the linker 1 or 2 is as described above.

[0174] One aspect of the present invention provides a polynucleotide encoding a fusion protein, which comprises a mitochondrial outer membrane anchor peptide and a desired protein.

[0175] In addition, one aspect of the present invention provides a vector carrying a polynucleotide encoding a fusion protein comprising a desired protein.

[0176] In addition, one aspect of the present invention provides a host cell into which a vector carrying a polynucleotide encoding a fusion protein containing a desired protein is introduced.

[0177] One aspect of the present invention provides a fusion protein comprising a target site targeting protein and a mitochondrial outer membrane targeting protein.

[0178] In this case, the target protein and the mitochondrial outer membrane anchor protein can be bound from the N-terminus to the C-terminus. Here, the mitochondrial outer membrane anchor 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.

[0179] As used herein, the term "target" refers to the location where the modified mitochondria should be delivered. One embodiment of the target can be a cancer cell. Specifically, one embodiment of the target can be a biomarker present on the surface of a cancer cell. Specifically, the target can be a tumor associated antigen (TAA). In this case, the tumor-associated antigen can be any one selected from the group consisting of CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), mucin 1 cell surface-associated protein (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosine-related protein 1 (tyrp1), tyrosine-related 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, connexin 3 and a combination thereof.

[0180] As used herein, the term "target targeting protein" may be a protein sequence that can bind to the above-mentioned target. In this case, one embodiment of the target targeting protein may be a protein that binds to a biomarker present on the surface of a cancer cell. In this case, an embodiment of the biomarker present on the surface of a cancer cell may be, but is not limited to, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM or connexin-3. In this case, the target targeting protein may be included in the above-mentioned exogenous protein.

[0181] One embodiment of the target targeting protein may be an antibody or a fragment thereof. Specifically, it may be an antibody or a fragment thereof that specifically binds to a tumor-associated antigen. In addition, the antibody fragment may be any one selected from the group consisting of Fab, Fab', scFv and F(ab)2.

[0182] One embodiment of the target targeting protein may be a scFvHER that can bind to epidermal growth factor receptor. Another embodiment may be a scFvMEL that can target melanoma. Another embodiment may be a scFvPD-L1 that can bind to PD-L1 overexpressed on the surface of cancer cells. Another embodiment may be a PD-1 that can bind to PDL-1 overexpressed on the surface of cancer cells.

[0183] One aspect of the present invention may further include ubiquitin or a fragment thereof between the target targeting protein and the mitochondrial outer membrane targeting protein. The fusion protein comprising the mitochondrial target targeting protein and the desired protein may be referred to as a fusion protein that modifies mitochondrial activity. Such a fusion protein may have any of the following structures:

[0184] <Structural Formula 11>

[0185] N-terminal-target protein-mitochondrial outer membrane anchor peptide-C-terminal

[0186] <Structural Formula 12>

[0187] N-terminal-target protein-ubiquitin or its fragment-mitochondrial outer membrane anchor peptide-C-terminal

[0188] <Structural Formula 13>

[0189] N-terminal-target protein-adapter 1-ubiquitin or its fragment-mitochondrial outer membrane anchor peptide-C-terminal

[0190] <Structural Formula 14>

[0191] N-terminal-target protein-ubiquitin or its fragment-linker 2-mitochondrial outer membrane anchor peptide-C-terminal

[0192] <Structural Formula 15>

[0193] N-terminal-target protein-linker 1-ubiquitin or its fragment-linker 2-mitochondrial outer membrane anchor peptide-C-terminal

[0194] In the above structural formulas 11 to 15, the outer membrane anchor 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 VAMP1B, and the target targeting protein can be selected from the group consisting of tumor associated antigen, CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), mucin 1 cell surface associated protein (MUC-1), prostatic acid phosphatase (PAP), prostate specific Any one of the group consisting of heterologous antigen (PSA), survival protein, tyrosine-related protein 1 (tyrp1), tyrosine-related 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, connexin 3 and combinations thereof. In addition, the target targeting protein can be an antibody that specifically binds to a tumor-associated antigen or a fragment thereof. In this case, the linker 1 or 2, and the amino acid sequence recognized by the proteolytic enzyme are as described above.

[0195] <Structural Formula 16>

[0196] N-terminal-mitochondrial outer membrane anchor peptide-target protein-C-terminal

[0197] <Structural Formula 17>

[0198] N-terminal-mitochondrial outer membrane anchor peptide-ubiquitin or its fragment-target protein-C-terminal

[0199] <Structural Formula 18>

[0200] N-terminal-mitochondrial outer membrane anchor peptide-linker 1-ubiquitin or its fragment-target protein-C-terminal

[0201] <Structural Formula 19>

[0202] N-terminal-mitochondrial outer membrane anchor peptide-ubiquitin or its fragment-linker 2-target protein-C-terminal

[0203] <Structural Formula 20>

[0204] N-terminal-mitochondrial outer membrane anchor peptide-linker 1-ubiquitin or its fragment-linker 2-target protein-C-terminal

[0205] In the above structural formulas 16 to 20, the outer membrane anchor peptide can be any one selected from the group consisting of TOM20, TOM70 and OM45. In addition, the target protein, ubiquitin or a fragment thereof and linker 1 or 2 are as described above.

[0206] One aspect of the present invention provides a polynucleotide encoding a fusion protein comprising a target targeting protein.

[0207] In addition, one aspect of the present invention provides a vector carrying a polynucleotide encoding a fusion protein comprising a target targeting protein.

[0208] In addition, one aspect of the present invention provides a host cell, into which a vector loaded with a polynucleotide encoding a fusion protein comprising a target targeting protein is introduced. The host cell can be a prokaryotic cell or a eukaryotic cell. In this case, preferably, the eukaryotic cell can be a strain from which an enzyme that can degrade ubiquitin has been removed.

[0209] In addition, one aspect of the present invention provides a method for preparing modified mitochondria from transformed cells by injecting a polynucleotide encoding a fusion protein into eukaryotic cells.

[0210] Example

[0211] Hereinafter, preferred embodiments will be presented to help understand the present invention. However, the following examples are provided only for easy understanding of the present invention, and the present invention is not limited to the following examples.

[0212] I. Preparation of fusion protein containing mitochondrial outer membrane anchor peptide, linker, ubiquitin and desired protein

[0213] Example 1. Preparation of fusion protein containing p53

[0214] Example 1.1. Amplification of p53 gene

[0215] In order to express human p53 as a recombinant protein, total RNA was extracted from human epithelial cells and cDNA was synthesized therefrom. Specifically, human skin fibroblasts were cultured in a 10% serum medium (1×10 6Cells). Then, the culture medium was removed and washed twice by adding PBS buffer to the cells, after which 0.5 ml of RNA extract (Trizol reagent, Thermo Fisher Scientific) was directly added. The mixture added with the RNA extract was left to stand at ambient temperature for 10 minutes, and then 0.1 ml of chloroform was added and stirred for 15 seconds, and then centrifuged at about 12,000×g for 10 minutes. Next, the separated supernatant was taken out, and the same volume of isopropanol was added and centrifuged again at 12,000×g for 10 minutes. Thereafter, the liquid was removed and washed once with 75% ethanol, and the RNA was dried at ambient temperature.

[0216] About 50 μl of nuclease-free purified distilled water was added, and the amount and purity of RNA were determined using a spectrophotometer. To synthesize cDNA, 2 μg of purified total RNA was combined with oligo dT at 70°C for 5 minutes. After that, 10× reverse transcription reaction buffer, 10 mM dNTP, nuclease inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea) were added, and cDNA synthesis reaction was performed at 42°C for 60 minutes. After that, the reverse transcriptase was inactivated by heating at 72°C for 5 minutes, and then RNase H was added to remove single-stranded RNA, which was used as a template for the polymerase chain reaction of the p53 gene.

[0217] In order to obtain the p53 gene from human skin fibroblasts without the signal peptide sequence, primers encoding glutamic acid from the amino terminal (T2p53) and primers encoding from the carboxyl terminal (Xp53) were synthesized, and PCR was performed using the cDNA prepared above as a template. The sequences of each primer are shown in Table 1 below.

[0218] [Table 1]

[0219] Primers sequence SEQ ID NO. T2p53 5'-AAA AAACCG CGG TGG TGA GGA GCC GCA GTC AGA TCC TAG-3' SEQ ID NO:1 Xp53 5'-AAA AAACTC GAG TGA GTC TGA GTC AGG CCC TTC TG-3' SEQ ID NO:2

[0220] 0.2 pmol T2p53 primer and 0.2 pmol Xp53 primer were mixed with 0.2 nM dNTP, 1×AccuPrime TaqDNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. Afterwards, the amplification reaction was carried out in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 40 cycles. After the reaction, the amplified DNA fragment of about 1.2 kbp was separated by 1% agarose gel electrophoresis, and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. The results of sequencing the obtained DNA confirmed that cDNA encoding human p53 protein was obtained. The obtained p53 gene was named pTA-p53, and its base sequence was the same as the base sequence shown in SEQ ID NO:3 ( Figure 1 ).

[0221] Example 1.2. Preparation of p53 E. coli expression vector

[0222] Example 1.2.1 Preparation of plasmid pET15b-UB-p53

[0223] In order to prepare the p53 protein fused with ubiquitin, the following expression vectors were prepared. In order to obtain the ubiquitin gene, NdeUB primers and T2UB primers were prepared. The sequences of each primer are shown in Table 2 below.

[0224] [Table 2]

[0225] Primers sequence SEQ ID NO. Nb 5'-GGA TTC CAT ATG CAA CTT TTC GTC AAA ACT CTA AC-3' SEQ ID NO:4 T2UB 5'-ATG ACC ACC GCG GAG TCT CAA CAC CAA-3' SEQ ID NO:5

[0226] 0.2 pmol NdeUB primer and 0.2 pmol T2UB primer were mixed with 0.2 nM dNTP, 1×AccuPrime TaqDNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. Afterwards, an amplification reaction was performed in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 25 cycles to obtain the ubiquitin (UB) gene. The amplified ubiquitin gene was cut with restriction enzymes NdeI and SacII, and the plasmid pTA-p53 was cut with restriction enzymes SacII and XhoI. Afterwards, DNA fragments of approximately 210 bp and 1,200 bp were obtained by 2% agarose gel electrophoresis, respectively, and inserted into the pET15b vector cut with restriction enzymes NdeI and XhoI using T4 DNA ligase, thereby obtaining the plasmid pET15b-UB-p53 ( Figure 2 ). In this case, UB-p53 is represented by the base sequence shown in SEQ ID NO:6.

[0227] The E. coli BL21 (DE3) strain was transformed using plasmid pET15b-UB-p53. The transformed strain was then cultured in a Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the obtained colonies were then cultured in an LB liquid medium at 37°C. Afterwards, when the cell density reached an absorbance of about 0.2 at OD600, IPTG was added at a final concentration of 1 mM, and then shaking culture was continued for about 4 hours.

[0228] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Figure 3 As shown, it was confirmed that the ubiquitin-fused p53 protein of about 60 kDa was expressed. Figure 3 Lane M in the figure shows a protein molecular weight marker, lane 1 shows a precipitate obtained by disrupting E. coli 4 hours after the addition of IPTG and centrifuging, and lane 2 shows a supernatant obtained by centrifugation after disrupting E. coli.

[0229] Example 1.2.2. Preparation of plasmid pET11C-TOM70-UB-p53

[0230] In order to prepare the p53 protein fused with TOM70 and ubiquitin that bind to the outer membrane of mitochondria, an expression vector capable of expressing p53 in the form of fusion of TOM70 and ubiquitin was prepared. In order to obtain TOM70 and ubiquitin genes, NdeTOM70 primer, TOM70-AS primer, TOM70UB-S primer and T2UB-AS primer were prepared. The sequences of various primers are shown in Table 3 below.

[0231] [Table 3]

[0232]

[0233] 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 dNTP, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit of AccuPrime Taq DNA polymerase. Then, the amplification reaction was carried out in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute for 25 cycles to obtain the TOM70 gene. The amplified DNA fragment is called The mass obtained in Example 1.2.1 is N-TOM70. The plasmid pET15b-UB-p53 was used as template and 0.2 pmol TOM70UB-S primer and 0.2 pmol T2UB-AS primer, as well as 0.2 nM dNTP, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase were mixed. After that, the amplification reaction was carried out in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute for 25 cycles to obtain the UB gene. The amplified DNA fragment is called C-UB.

[0234] The amplified DNA N-TOM70 and C-UB were used as templates, and 0.2 pmol of NdeTOM70 primer and 0.2 pmol of T2UB-AS 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. After that, the amplification reaction was carried out in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute for 25 cycles to obtain the ubiquitin gene TOM70-UB fused with the amplified TOM70.

[0235] The amplified TOM70-UB gene was cut with restriction enzymes NdeI and SacII, and plasmid pTA-p53 was cut with SacII and XhoI, and DNA fragments of 330 bp and 1,500 bp were obtained by 2% agarose gel electrophoresis, respectively. After that, it was inserted into the pET11c vector cut with restriction enzymes NdeI and SalI using T4 DNA ligase to obtain plasmid pET11c-TOM70-UB-p53 ( Figure 4 ). In this case, TOM70-UB-p53 is represented by the base sequence shown in SEQ ID NO:11.

[0236] The plasmid pET11c-TOM70-UB-p53 was used to transform the E. coli BL21 (DE3) strain. After that, the transformed strain was cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in LB liquid medium at 37°C in a shaking incubator. After that, when the cell density reached an absorbance of about 0.2 at OD600, IPTG was added at a final concentration of 1 mM, and then shaking culture was continued for about 4 hours.

[0237] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Figure 5As shown, it was confirmed that the p53 protein fused with ubiquitin of about 62 kDa was expressed. In this case, lane M shows a protein molecular weight marker, and lane 1 shows the supernatant obtained by centrifugation after the E. coli was disrupted 4 hours after the addition of IPTG.

[0238] Example 1.2.3 Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-p53

[0239] In order to prepare a p53 protein fused with TOM70, a linker (GGGGSGGGGSGGGGS (SEQ ID NO: 70)) and ubiquitin that binds to the outer membrane of mitochondria, a p53 protein capable of expressing a form fused with a TOM70 linker, a linker, and ubiquitin was prepared. In order to obtain a linker gene that binds to TOM70, a TOM70 (G) 3-AS primer, a (G) 3UB-S primer, and an Xp53 (noT) primer were prepared. The sequences of the various primers are shown in Table 4 below.

[0240] [Table 4]

[0241]

[0242]

[0243] The plasmid pET11c-TOM70-UB-p53 obtained in Example 1.2.2 was used as a template, 0.2 pmol NdeTOM70 primer and 0.2 pmol TOM70 (G) 3-AS primer were added, and mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. Afterwards, an amplification reaction was performed in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 25 cycles to obtain the TOM70-G3 gene, which is combined with the TOM70 gene and a linker. In addition, the plasmid pET15b-UB-p53 obtained in Example 1.2.1 was used as a template, and 0.2 pmol of (G)3UB-S primer and 0.2 pmol of 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.

[0244] Afterwards, an amplification reaction was performed in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 25 cycles to obtain UB-p53, which is a fusion of p53 and the ubiquitin gene. The amplified TOM70-G3 gene was cut with restriction enzymes NdeI and BamHI, and the amplified UB-p53 gene was cut with BamHI and XhoI, and DNA fragments of approximately 100 bp and 1,500 bp were obtained by 2% agarose gel electrophoresis, respectively. Afterwards, it was inserted into the pET11c vector cut with 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 is represented by the base sequence shown in SEQ ID NO:15.

[0245] The E. coli BL21 (DE3) strain was transformed using plasmid pET11c-TOM70-(GGGGS)3-UB-p53. The transformed strain was cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in LB liquid medium at 37°C. Afterwards, when the cell density reached an absorbance of about 0.2 at OD600, IPTG was added at a final concentration of 1 mM, and then shaking culture was continued for about 4 hours.

[0246] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Figure 7 As shown, it was confirmed that p53 protein of about 62 kDa fused with TOM70, linker and ubiquitin was expressed. Figure 7 Lane M in the figure shows a protein molecular weight marker, lane 1 shows a precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 shows a supernatant centrifuged after E. coli disruption.

[0247] Example 1.2.4. Preparation of plasmid pET11c-TOM70-(GGGGS)3-p53

[0248] In order to prepare the p53 protein fused with TOM70 and a linker (GGGGSGGGGSGGGGS) that binds to the outer membrane of the mitochondria, an expression vector capable of expressing the p53 protein fused with TOM70 and a linker was prepared. In order to obtain the p53 gene fused with TOM70 and a linker, a primer (B(G)3p53) was prepared. The sequences of the primers are shown in Table 5 below.

[0249] [Table 5]

[0250]

[0251] The plasmid pET11c-TOM70-UB-p53 obtained in Example 1.2.2 was used as a template, 0.2 pmol NdeTOM70 primer and 0.2 pmol TOM70 (G) 3-AS primer were added, and mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. After that, the amplification reaction was carried out in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 25 cycles to obtain the TOM70 gene. The amplified DNA fragment is called TOM70-G3.

[0252] The plasmid pET15b-UB-p53 obtained in Example 1.2.1 was used as a template, and 0.2 pmol of B(G)3p53 primer and 0.2 pmol of 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. After that, an amplification reaction was performed in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute for 25 cycles. The amplified DNA fragment was called G3-p53.

[0253] The amplified DNA fragment TOM70-G3 was cut with NdeI and BamHI, and the DNA fragment G3-53 was cut with restriction enzymes BamHI and XhoI. DNA fragments of about 150 bp and 1,300 bp were obtained by 2% agarose gel electrophoresis, respectively; then, they were inserted into the pET11c vector cut with restriction enzymes NdeI and SalI using T4 DNA ligase, thereby obtaining plasmid pET11c-TOM70-(GGGGS)3-p53( Figure 8 In this case, the plasmid pET11c-TOM70-(GGGGS)3-p53 is represented by the base sequence shown in SEQ ID NO:17.

[0254] The E. coli BL21 (DE3) strain was transformed using plasmid pET11c-TOM70-(GGGGS)3-p53. The transformed strain was cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in LB liquid medium in a shaking incubator at 37°C. Afterwards, when the cell density reached an absorbance of about 0.2 at OD600, IPTG was added at a final concentration of 1 mM, and then shaking culture was continued for about 4 hours.

[0255] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Fig. 9 As shown, it was confirmed that the p53 protein fused to TOM70 of about 60 kDa was expressed. In this case, lane M shows a protein molecular weight marker, lane 1 shows a precipitate centrifuged after E. coli was disrupted 4 hours after adding IPTG, and lane 2 shows the supernatant centrifuged after E. coli was disrupted.

[0256] Example 1.2.5. pET15b-UB-p53-TOM7

[0257] In order to prepare a p53 protein fused with ubiquitin and TOM7 bound to the outer membrane of mitochondria, an expression vector capable of expressing p53 fused with ubiquitin, p53 and TOM was prepared. In order to obtain a p53 gene fused with TOM7 and ubiquitin, Xp53 (noT) primer, XTOM7 primer and LTOM7 primer were prepared. The sequences of various primers are shown in Table 6 below.

[0258] [Table 6]

[0259]

[0260] The plasmid pET15b-UB-p53 obtained in Example 1.2.1 was used as a template, 0.2 pmol NdeUB primer and 0.2 pmol Xp53 (noT) primer were added, and mixed with 0.2 nM dNTP, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. After that, amplification reaction was performed in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 25 cycles to obtain the UB-p53 gene. In addition, the cDNA prepared above was used as a template, 0.2 pmol XTOM7 primer and 0.2 pmol LTOM7 primer were mixed with 0.2 nM dNTP, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase.

[0261] Afterwards, the amplification reaction was carried out in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 40 cycles to obtain the TOM7 gene. The amplified DNA fragment, UB-p53, was cut with restriction enzymes NdeI and XhoI, and the amplified TOM7 gene was cut with XhoI and SalI, and DNA fragments of about 1,500 bp and 150 bp were obtained by 2% agarose gel electrophoresis, respectively; then, it was inserted into the pET15b vector cut with restriction enzymes NdeI and XhoI using T4 DNA ligase, thereby obtaining the plasmid pET15b-UB-p53-TOM7 ( Fig.10 ). In this case, pET15b-UB-p53-TOM7 is represented by the base sequence shown in SEQ ID NO:21.

[0262] The E. coli BL21 (DE3) strain was transformed using plasmid pET15b-UB-p53-TOM7. The transformed strain was cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in LB liquid medium at 37°C. Thereafter, when the cell density reached an absorbance of about 0.2 at OD600, IPTG was added at a final concentration of 1 mM, and then shaking culture was continued for about 4 hours.

[0263] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Fig.11 As shown, it was confirmed that p53 protein fused with ubiquitin and TOM7 of about 60 kDa was expressed. In this case, lane M shows a protein molecular weight marker, lane 1 shows the supernatant of centrifugation after E. coli was broken 4 hours after adding IPTG, and lane 2 shows the supernatant of centrifugation after E. coli was broken.

[0264] Example 1.2.6. Construction of mammalian expression vector pCMV-p53-myc / His

[0265] An expression vector for animal cells capable of expressing p53 was prepared. In order to obtain the p53 gene, Rp53 primers were prepared. The sequences of various primers are shown in Table 7 below.

[0266] [Table 7]

[0267] Primers sequence SEQ ID NO. Rp53 5'-AAA AAA GAA TTC ATG GTC TGA GTC AGG CCC TTC TG-3' SEQ ID NO:23

[0268] The plasmid pET-UB-p53 obtained in Example 1.2.1 was used as a template, and 0.2 pmol of Rp53 primer and 0.2 pmol of 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. Then, an amplification reaction was performed in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute for 25 cycles to obtain the p53 gene.

[0269] The amplified p53 gene was cut with restriction enzymes EcoRI and XhoI, and a DNA fragment of about 1,300 bp was obtained by 2% agarose gel electrophoresis; then, it was inserted into the pcDNA3.1-myc / His A vector cut with restriction enzymes EcoRI and XhoI using T4 DNA ligase to obtain plasmid pCMV-p53-myc / His( Fig.12 ). In this case, p53-myc / His is represented by the base sequence shown in SEQ ID NO:23.

[0270] Animal CHO cells were transfected with plasmid pCMV-p53-myc / His, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis and Western blot using anti-c-myc antibody. Fig.13 As shown, it was confirmed that p53 protein of about 55 kDa was expressed. In this case, lane M shows a protein molecular weight marker, and lane 1 shows that it has been transfected into animal CHO cells, and then the cells are broken, followed by SDS-polyacrylamide electrophoresis, and Western blot is displayed by anti-c-myc antibody.

[0271] Example 1.3. Isolation and purification of fusion protein containing p53

[0272] Example 1.3.1 Isolation and purification of recombinant TOM70-(GGGGS)3-p53 protein from E. coli

[0273] 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. Thereafter, when the absorbance at OD600 reached 0.4, 0.5 mM IPTG was added, and further shake culture was performed for 4 hours to express the TOM70-(GGGGS)3-p53 protein.

[0274] After the culture is completed, the cells are recovered by centrifugation, and the recovered cells are washed once with PBS, and then the cells are suspended in a PBS solution, and the suspended cells are broken using an ultrasonic instrument. The broken cells are centrifuged using a high-speed centrifuge, and then the insoluble components are recovered, and the recovered insoluble components are washed 3 times using a solution containing 50mM Tris, 100mM ethylenediaminetetraacetic acid (EDTA), pH 8.0. Afterwards, it is dissolved in a solution containing 6M guanidine, 100mM sodium phosphate, 10mM Tris, pH 8.0, and filtered using a 0.45μm filter, and then loaded on a pre-filled nickel chromatographic column for preliminary purification.

[0275] A solution containing TOM70-(GGGGS)3-p53 protein was loaded, and then a solution containing 8 M urea, 50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 8.0 was used as the inflow wash solution until unbound impurities were not detected, and when the imidazole concentration was changed to 50 mM, 100 mM, 250 mM, 500 mM, the protein was eluted using a solution containing 8 M urea, 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0 ( Fig.14 ).in this case, Fig.14 Lane M in the figure shows protein molecular weight markers, and lane 1 shows the sample loaded on nickel affinity chromatography. Lane 2 shows that it did not bind to the nickel affinity resin. Lanes 3 to 4 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5 to 7 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8 to 9 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution. Lanes 10 to 11 show the results of elution with 8MUREA / 50mM sodium phosphate / 500mM NaCl / 500mM imidazole solution.

[0276] The eluted solution recovered from the nickel column was exchanged with PBS using the osmotic pressure principle. After the solution exchange was completed, the eluate was centrifuged to recover the supernatant, and the protein content of the recovered eluate was determined by protein quantification method and confirmed by SDS-PAGE. Fig.15 As shown, after confirmation, the TOM70-(GGGGS)3-p53 protein was quenched with liquid nitrogen and stored in a low-temperature freezer at -80°C. In this case, lane M shows a protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-p53 protein obtained after dialysis in a PBS buffer solution.

[0277] Example 1.3.2. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-p53 protein from E. coli

[0278] The same method as in Example 1.3.1 was used to separate and purify the TOM70-(GGGGS)3-UB-p53 protein using E. coli expressing the TOM70-(GGGGS)3-UB-p53 recombinant protein. As a result, the TOM70-(GGGGS)3-UB-p53 protein was eluted ( Fig.16 ).in this case, Fig.16 Lane M in the figure shows protein molecular weight markers, and lane 1 shows the sample loaded on nickel affinity chromatography. Lane 2 shows that it did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 4 to 7 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8 to 11 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution.

[0279] The protein amount of the recovered elution solution was determined by a protein quantification method and confirmed by SDS-PAGE. Fig.17 As shown, after confirmation, the TOM70-(GGGGS)3-UB-p53 protein was quenched with liquid nitrogen and stored in a low-temperature freezer at -80°C. Fig.17 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-p53 protein obtained after dialysis in PBS buffer solution.

[0280] Example 1.3.3. Isolation and purification of recombinant UB-p53 protein from E. coli

[0281] The BL21 (DE3) production strain expressing the mature UB-p53 protein fused to ubiquitin was inoculated into LB liquid medium and cultured in a shaking incubator at 37° C. When the absorbance at OD 600 reached 0.4, 0.5 mM IPTG was added and further cultured with shaking for 4 hours to express the mature form of UB-p53 protein fused to ubiquitin.

[0282] Then, the UB-p53 protein was separated and purified in the same manner as in Example 1.3.1. As a result, the UB-p53 protein ( Fig.18 ).in this case, Fig.18Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the sample loaded on the nickel affinity chromatography column. Lane 2 shows that it did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 4 to 6 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 7 to 9 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution. Lanes 10 to 11 show the results of elution with 8MUREA / 50mM sodium phosphate / 500mM NaCl / 500mM imidazole solution.

[0283] The protein amount of the recovered elution solution was determined by a protein quantification method and confirmed by SDS-PAGE. Fig.19 As shown, after confirmation, the UB-p53 protein was quenched with liquid nitrogen and stored in a -80°C low-temperature freezer. Fig.19 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the UB-p53 protein obtained after dialysis in PBS buffer solution.

[0284] Example 1.3.4. Isolation and purification of recombinant UB-p53-TOM7 protein from E. coli

[0285] The E. coli BL21 (DE3) production strain expressing the mature form of UB-p53-TOM7 protein fused to ubiquitin was inoculated into LB liquid medium and cultured at 37° C. When the absorbance at OD600 reached 0.4, 0.5 mM IPTG was added, and further shaking culture was performed for 4 hours to express the mature form of UB-p53-TOM7 protein fused to ubiquitin.

[0286] Then, the UB-p53-TOM7 protein was separated and purified in the same manner as in Example 1.3.1. As a result, the UB-p53-TOM7 protein was eluted ( Fig. 20 ).in this case, Fig. 20Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the sample loaded on the nickel affinity chromatography column. Lane 2 shows that it did not bind to the nickel affinity resin. Lane 3 shows the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 10mM imidazole solution. Lane 4 shows the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5 to 7 show the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8 to 9 show the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution. Lanes 10 to 11 show the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 500mM imidazole solution.

[0287] The protein amount of the recovered elution solution was determined by a protein quantification method and confirmed by SDS-PAGE. Fig.21 As shown, after confirmation, the UB-p53 protein was quenched with liquid nitrogen and stored in a -80°C low-temperature freezer. Fig.21 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the UB-p53-TOM7 protein obtained after dialysis in PBS buffer solution.

[0288] Example 2. Preparation of fusion protein containing granzyme B

[0289] Example 2.1 Amplification of Granzyme B Gene

[0290] In order to express human granzyme B as a recombinant protein, total RNA was extracted from human natural killer cells and cDNA was synthesized therefrom. Specifically, human natural killer cells were cultured in a 10% serum medium (1×10 6 cells). Thereafter, RNA was obtained in the same manner as in Example 1.1 and then used as a template for polymerase chain reaction of the granzyme B gene.

[0291] In order to obtain the granzyme B gene from human natural killer cells with the signal peptide sequence removed, primers T2GZMB encoding isoleucine from the amino terminus and primer XGZMB (noT) encoding from the carboxyl terminus were synthesized, and then PCR was performed using the cDNA prepared above as a template. The sequences of various primers are shown in Table 8 below.

[0292] [Table 8]

[0293]

[0294] The cDNA prepared above was used as a template, and 0.2 pmol T2GZMB primer and 0.2 pmol XGZMB (noT) primer were mixed with 0.2 nM dNTP, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. Afterwards, an amplification reaction was performed in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 40 cycles. After the reaction, the amplified DNA fragment of about 700 bp was separated by 1% agarose gel electrophoresis, and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. The results of sequencing the obtained DNA confirmed that the cDNA encoding human granzyme B protein was obtained. The obtained granzyme B gene was named pTA-Granzyme B, and the granzyme B gene is represented by the base sequence shown in SEQ ID NO: 26 ( Fig. 22 ).

[0295] Example 2.2. Preparation of E. coli expression vector for granzyme B protein

[0296] Example 2.2.1. Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-Granzyme B

[0297] In order to prepare a granzyme B protein fused with TOM70 that binds to the outer membrane of mitochondria, a linker (GGGGSGGGGSGGGGS) and ubiquitin, an expression vector capable of expressing granzyme B fused with TOM70, a linker and ubiquitin was prepared.

[0298] The plasmid pTA-Granzyme B gene obtained in Example 2.1 was cut with restriction enzymes SacII and XhoI, and a DNA fragment of about 700 bp was obtained by 2% agarose gel electrophoresis. Then, it was inserted into the pET11c-TOM70-(GGGGS)3-UB-(p53) vector cut with restriction enzymes SacII and XhoI using T4 DNA ligase to obtain plasmid pET11c-TOM70-(GGGGS)3-UB-Granzyme B (SEQ ID NO: 27) ( Fig.23 ).

[0299] The E. coli BL21 (DE3) strain was transformed using plasmid pET11c-TOM70-(GGGGS)3-UB-Granzyme B. After that, the transformed strain was cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in LB liquid medium in a shaking incubator at 37°C. After that, when the cell density reached an absorbance of about 0.2 at OD 600, IPTG was added at a final concentration of 0.5 mM, and then shaking culture was continued for about 4 hours.

[0300] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Fig.24 As shown, it was confirmed that the granzyme B protein of about 35 kDa fused with TOM70, linker and ubiquitin was expressed. Fig.24 Lane M in the figure shows a protein molecular weight marker, lane 1 shows a precipitate obtained by disrupting E. coli and centrifuging 4 hours after the addition of IPTG, and lane 2 shows a supernatant obtained by centrifugation after the disruption of E. coli.

[0301] Example 2.2.2. Preparation of plasmid pET15b-UB-Granzyme B-TOM7

[0302] In order to prepare Granzyme B protein fused with ubiquitin and TOM7 bound to the mitochondrial outer membrane, an expression vector capable of expressing Granzyme B protein in a form fused with ubiquitin, Granzyme B and TOM7 was prepared.

[0303] The plasmid pTA-Granzyme B gene obtained in Example 2.1 was cut with restriction enzymes SacII and XhoI, and a DNA fragment of about 700 bp was obtained by 2% agarose gel electrophoresis. Then, it was inserted into the pET15b-UB-(p53)-TOM7 vector cut with restriction enzymes SacII and XhoI using T4 DNA ligase to obtain plasmid pET15b-UB-GranzymeB-TOM7 ( Fig.25 ). Here, UB-GranzymeB-TOM7 is represented by the base sequence shown in SEQ ID NO:28.

[0304] The E. coli BL21 (DE3) strain was transformed using plasmid pET15b-UB-GranzymeB-TOM7. Thereafter, the transformed strain was cultured in a Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in an LB liquid medium at 37°C. Thereafter, when the cell density reached an absorbance of about 0.2 at OD600, IPTG was added at a final concentration of 0.5 mM, and then shaking culture was continued for about 4 hours.

[0305] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Fig.26 As shown, it was confirmed that Granzyme B protein of about 35 kDa fused with ubiquitin and TOM70 was expressed. Fig.26 Lane M in the figure shows a protein molecular weight marker, lane 1 shows a precipitate obtained by disrupting E. coli and centrifuging 4 hours after the addition of IPTG, and lane 2 shows a supernatant obtained by centrifugation after the disruption of E. coli.

[0306] Example 2.3. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-Granzyme B protein from E. coli

[0307] The TOM70-(GGGGS)3-UB-GranzymeB protein was isolated and purified by the same method as in Example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-GranzymeB protein was eluted ( Fig. 27 ).in this case, Fig. 27 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the sample loaded on the nickel affinity chromatography column. Lane 2 shows that it did not bind to the nickel affinity resin. Lanes 3 and 4 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5 to 7 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8 to 9 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution.

[0308] The protein amount of the recovered elution solution was determined by a protein quantification method and confirmed by SDS-PAGE. Fig.28 As shown, after confirmation, the TOM70-(GGGGS)3-UB-granzyme B protein was quenched with liquid nitrogen and stored in a low-temperature freezer at -80°C. Fig.28Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-GranzymeB protein obtained after dialysis in PBS buffer solution.

[0309] Example 3. Preparation of fusion protein containing RKIP

[0310] Example 3.1 Amplification of the RKIP gene

[0311] In order 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 therefrom. Human skin fibroblasts (1×10 6 cells). Thereafter, RNA was obtained in the same manner as in Example 1.1, and then used as a template for polymerase chain reaction of the RKIP gene.

[0312] In order to obtain the RKIP gene from human skin fibroblasts with the signal peptide sequence removed, T2RKIP primers encoding proline from the amino terminal and XRKIP (noT) primers encoding from the carboxyl terminal were synthesized, and PCR was performed using the cDNA prepared above as a template. The sequences of various primers are shown in Table 9 below.

[0313] [Table 9]

[0314]

[0315] The cDNA prepared above was used as a template, and 0.2 pmol T2RKIP primer and 0.2 pmol XRKIP (noT) primer were mixed with 0.2 nM dNTP, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. Afterwards, the amplification reaction was carried out in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 40 cycles. After the reaction, the amplified DNA fragment of about 560 bp was separated by 1% agarose gel electrophoresis, and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. The results of sequencing the obtained DNA confirmed that the cDNA encoding the human RKIP protein was obtained. The obtained RKIP gene was named pTA-RKIP ( Fig.29 ), and the base sequence of the RKIP gene is represented by the base sequence shown in SEQ ID NO:31.

[0316] Example 3.2. Preparation of E. coli expression vector for RKIP protein

[0317] Example 3.2.1. Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-RKIP

[0318] In order to prepare an RKIP protein fused with TOM70 that binds to the outer membrane of mitochondria, a linker (GGGGSGGGGSGGGGS), and ubiquitin, an expression vector capable of expressing RKIP fused with TOM70, a linker, and ubiquitin was prepared.

[0319] The plasmid pTA-RKIP gene obtained in Example 3.1 was cut with restriction enzymes SacII and XhoI, and a DNA fragment of about 560 bp was obtained by 2% agarose gel electrophoresis, which was then inserted into the pET11c-TOM70-(GGGGS)3-UB-(p53) vector cut with restriction enzymes SacII and XhoI using T4 DNA ligase to obtain the plasmid pET11-TOM70-(GGGGS)3-UB-RKIP( Fig.30 Here, TOM70-(GGGGS)3-UB-RKIP is represented by the base sequence shown in SEQ ID NO:32.

[0320] The plasmid pET11c-TOM70-(GGGGS)3-UB-RKIP was used to transform the E. coli BL21 (DE3) strain. After that, the transformed strain was cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in LB liquid medium in a shaking incubator at 37°C. After that, when the cell density reached an absorbance of about 0.2 at OD 600, IPTG was added at a final concentration of 0.5 mM, and then the shaking culture was continued for about 4 hours.

[0321] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Fig.31 As shown, it was confirmed that the RKIP protein of about 33 kDa fused with TOM70, linker and ubiquitin was expressed. Fig.31 Lane M in the figure shows a protein molecular weight marker, lane 1 shows a precipitate obtained by disrupting E. coli and centrifuging 4 hours after the addition of IPTG, and lane 2 shows a supernatant obtained by centrifugation after the disruption of E. coli.

[0322] Example 3.3. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-RKIP protein from E. coli

[0323] 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 absorbance at OD600 reached 0.3, it was placed in a refrigerator to lower the temperature of the culture solution, and the temperature of the incubator was changed to 18° C., and then 0.5 mM IPTG was added and shaken for 1 day to express the TOM70-(GGGGS)3-UB-RKIP protein.

[0324] Then, the TOM70-(GGGGS)3-UB-RKIP protein was isolated and purified in the same manner as in Example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-RKIP protein was eluted ( Fig.32 ).in this case, Fig.32 Lane M in shows protein molecular weight markers, and lane 1 shows the sample loaded on the nickel affinity chromatography column. Lane 2 shows that it did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 10mM imidazole. Lanes 4 to 6 show the results of elution with 50mM sodium phosphate / 500mM NaCl / 50mM imidazole. Lanes 7 to 8 show the results of elution with 50mM sodium phosphate / 500mM NaCl / 100mM imidazole. Lanes 9 to 10 show the results of elution with 50mM sodium phosphate / 500mM NaCl / 175mM imidazole. Lanes 11 to 13 show the results of elution with 50mM sodium phosphate / 500mM NaCl / 250mM imidazole. Lanes 14 to 16 show the results of elution with 50mM sodium phosphate / 500mM NaCl / 500mM imidazole.

[0325] The protein amount of the recovered elution solution was determined by a protein quantification method and confirmed by SDS-PAGE. Fig.33 As shown, after confirmation, the TOM70-(GGGGS)3-UB-RKIP protein was quenched with liquid nitrogen and stored in a -80°C freezer. Fig.33 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-RKIP protein obtained after dialysis in PBS buffer solution.

[0326] Example 4. Preparation of fusion protein containing PTEN

[0327] Example 4.1. Amplification of the PTEN gene

[0328] In order to express human PTEN (phosphatase and tenascin homolog) as a recombinant protein, total RNA was extracted from human epithelial cells and cDNA was synthesized therefrom. Fibroblasts (human skin fibroblasts) were cultured in 10% serum medium (1×10 6 Thereafter, RNA was obtained in the same manner as in Example 1.1 and then used as a template for polymerase chain reaction of the PTEN gene.

[0329] In order to obtain the PTEN gene with the signal peptide sequence removed from human skin fibroblasts, T2PTEN primers encoding threonine from the amino terminal and XPTEN (noT) primers encoding from the carboxyl terminal were synthesized, and PCR was performed using the cDNA prepared above as a template. The sequences of each primer are shown in Table 10 below.

[0330] [Table 10]

[0331]

[0332] The cDNA prepared above was used as a template, and 0.2 pmol T2RKIP primer and 0.2 pmol XRKIP (noT) primer were mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. Afterwards, the amplification reaction was carried out in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 40 cycles. After the reaction, the amplified DNA fragment of about 1,200 bp was separated by 1% agarose gel electrophoresis, and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. The results of sequencing the obtained DNA confirmed that the cDNA encoding the human RKIP protein was obtained. The obtained PTEN gene was named pTA-PTEN ( Fig.34 ), and the base sequence of PTEN is represented by the base sequence shown in SEQ ID NO:35.

[0333] Example 4.2 Preparation of E. coli expression vector for PTEN protein

[0334] Example 4.2.1 Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN

[0335] In order to prepare a PTEN protein fused with TOM70 that binds to the outer membrane of mitochondria, a linker (GGGGSGGGGSGGGGS), and ubiquitin, an expression vector capable of expressing the PTEN gene fused with TOM70, a linker, and ubiquitin was prepared.

[0336] The plasmid pTA-PTEN gene obtained in Example 4.1 was cut with restriction enzymes SacII and XhoI, and a DNA fragment of about 1,200 bp was obtained by 2% agarose gel electrophoresis. Then, it was inserted into the pET11c-TOM70-(GGGGS)3-UB-(p53) vector cut with restriction enzymes SacII and XhoI using T4 DNA ligase to obtain plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN( Fig.35 Here, TOM70-(GGGGS)3-UB-PTEN is represented by the base sequence shown in SEQ ID NO:36.

[0337] The E. coli BL21 (DE3) strain was transformed using the plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN. The transformed strain was then cultured in a Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and the obtained colonies were then cultured in an LB liquid medium at 37°C. When the cell density reached an absorbance of about 0.2 at OD 600, IPTG was added at a final concentration of 0.5 mM, and then shaking culture was continued for about 4 hours.

[0338] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Fig.36 As shown, it was confirmed that a PTEN protein of about 73 kDa fused with TOM70, linker and ubiquitin was expressed. Fig.36 Lane M in the figure shows a protein molecular weight marker, lane 1 shows a precipitate obtained by disrupting E. coli and centrifuging 4 hours after the addition of IPTG, and lane 2 shows a supernatant obtained by centrifugation after the disruption of E. coli.

[0339] Example 4.3. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-PTEN protein from E. coli

[0340] The TOM70-(GGGGS)3-UB-PTEN protein was isolated and purified in the same manner as in Example 1.3.1. Fig.37 ).in this case, Fig.37Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the sample loaded on the nickel affinity chromatography column. Lane 2 shows that it did not bind to the nickel affinity resin. Lane 3 shows the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 10mM imidazole solution. Lane 4 shows the result of elution with 8MUREA / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5 to 8 show the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 9 to 10 show the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution. Lane 11 shows the result of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 500mM imidazole solution.

[0341] The protein amount of the recovered elution solution was determined by a protein quantification method and confirmed by SDS-PAGE. Fig.38 As shown, after confirmation, the TOM70-(GGGGS)3-UB-PTEN protein was quenched with liquid nitrogen and stored in a -80°C freezer. Fig.38 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-PTEN protein obtained after dialysis in PBS buffer solution.

[0342] Example 5. Preparation of fusion protein containing mitochondrial outer membrane protein, ubiquitin and GFP

[0343] Example 5.1. Isolation and purification of recombinant UB-GFP-TOM7 protein from E. coli

[0344] The E. coli BL21 (DE3) production strain expressing the mature UB-GFP-TOM7 protein fused with ubiquitin was inoculated into LB liquid medium and cultured at 37° C. When the absorbance at OD600 reached 0.3, it was placed in a refrigerator to lower the temperature of the culture solution, and the temperature of the incubator was changed to 18° C., and then 0.5 mM IPTG was added, and shaking culture was performed for 1 day to express the mature form of GFP-TOM7 protein fused with ubiquitin.

[0345] After the culture is completed, the cells are recovered by centrifugation, washed once with PBS, and then suspended in a solution containing 50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 8.0, and the suspended cells are broken using an ultrasonic device. The broken cells are centrifuged using a high-speed centrifuge, and then the supernatant is recovered, and the recovered supernatant is filtered using a 0.45 μm filter and then loaded on a pre-loaded nickel column for preliminary purification.

[0346] The fragmentation solution containing the mature form of UB-GFP-TOM7 protein fused with ubiquitin was loaded, and then a solution containing 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 8.0 was flowed in as a washing solution until unbound impurities were no longer detected, and the protein was eluted using a solution containing 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0 according to a concentration gradient ( Fig.39 ).in this case, Fig.39 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the sample loaded on the nickel affinity chromatography column. Lane 2 shows that it did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 50mM sodium phosphate / 500mMNaCl / 20mM imidazole. Lane 4 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 55mM imidazole. Lane 5 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 60mM imidazole. Lane 6 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 65mM imidazole. Lane 7 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 70mM imidazole. Lane 8 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 75mM imidazole. Lane 9 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 80mM imidazole. Lane 10 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 85mM imidazole. Lane 11 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 90mM imidazole. Lane 12 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 95mM imidazole. Lane 13 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 100mM imidazole. Lane 14 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 105mM imidazole.

[0347] In order to remove imidazole in the elution solution, dialysis was performed in a solution containing 50 mM sodium phosphate, 500 mM NaCl, pH 8.0 using the osmotic pressure principle ( Fig.40). The final identified UB-GFP-TOM7 protein was quenched with liquid nitrogen and stored in a low-temperature freezer at -80°C. Fig.40 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the protein obtained after dialysis in a 50 mM sodium phosphate / 500 mM NaCl solution.

[0348] Example 5.2. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-GFP protein from E. coli

[0349] 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 absorbance at OD600 reached 0.3, it was placed in a refrigerator to lower the temperature of the culture solution, and the temperature of the incubator was changed to 18° C., and then 0.5 mM IPTG was added, and shaking culture was performed for 1 day to express the recombinant protein TOM70-(GGGGS) 3-UB-GFP.

[0350] After the culture is completed, the cells are recovered by centrifugation, washed once with PBS, and then suspended in a solution containing 50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 8.0, and the suspended cells are broken using an ultrasonic device. The broken cells are centrifuged using a high-speed centrifuge, and then the supernatant is recovered and filtered using a 0.45 μm filter, and then loaded on a pre-loaded nickel column for preliminary purification.

[0351] The fragmented solution containing the recombinant protein TOM70-(GGGGS)3-UB-GFP was loaded onto a chromatographic column containing nickel resin, and then a solution containing 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 8.0 was flowed in as a washing solution until unbound impurities were not detected. Then, when the concentration of imidazole was changed to 50 mM, 100 mM, 250 mM, and 500 mM, the protein was eluted using a solution of 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0 ( Fig.41 ).in this case, Fig.41Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the sample loaded on the nickel affinity chromatography column. Lane 2 shows that it did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 20mM imidazole. Lane 4 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 50mM imidazole. Lanes 5 to 8 show the results of elution with 50mM sodium phosphate / 500mM NaCl / 100mM imidazole. Lanes 9 to 11 show the results of elution with 50mM sodium phosphate / 500mM NaCl / 250mM imidazole. Lane 12 shows the results of elution with 50mM sodium phosphate / 500mM NaCl / 500mM imidazole.

[0352] Using the osmotic pressure principle, the eluted solution recovered from the nickel chromatography was exchanged with the PBS buffer solution. After the solution exchange was completed, protein quantification and SDS-PAGE were used to identify the final recovered protein TOM70-(GGGGS)3-UB-GFP. Fig.42 As shown, after identification, the TOM70-(GGGGS)3-UB-GFP protein was quenched with liquid nitrogen and stored in a -80°C freezer. Fig.42 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-GFP protein obtained after dialysis in PBS buffer solution.

[0353] II. Preparation of fusion protein containing mitochondrial outer membrane targeting protein and target site targeting protein

[0354] Example 6. Preparation of fusion protein containing scFvHER2

[0355] Example 6.1 Synthesis of scFvHER2 gene

[0356] In order to express human scFvHER2 as a recombinant protein, gene synthesis was requested from Bionics Co., Ltd. The obtained scFvHER2 gene was named pUC57-scFvHER2, and the base sequence of scFvHER2 was the same as that of SEQ ID NO:37.

[0357] Example 6.2. Preparation of scFvHER2 protein expression vector

[0358] Example 6.2.1. pET15b-UB-scFvHER2-TOM7

[0359] In order to prepare the scFvHER2 protein fused with ubiquitin and TOM7 that binds to the outer membrane of mitochondria, an expression vector capable of expressing the scFvHER2 gene fused with ubiquitin and TOM7 was prepared.

[0360] The plasmid pUC57-scFvHER2 gene obtained in Example 6.1 was cut with restriction enzymes SacII and XhoI, and a DNA fragment of about 750 bp was obtained by 2% agarose gel electrophoresis, which was then inserted into the pET15b-UB-(p53)-TOM7 vector cut with restriction enzymes SacII and XhoI using T4 DNA ligase to obtain plasmid pET15b-UB-scFvHER2-TOM7 ( Fig.39 ). In this case, UB-scFvHER2-TOM7 is represented by the base sequence shown in SEQ ID NO:38.

[0361] The plasmid pET15b-UB-scFvHER2-TOM7 was used to transform the E. coli BL21 (DE3) strain. Thereafter, the transformed strain was cultured in a Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in an LB liquid medium at 37°C. Then, when the cell density reached an absorbance of about 0.2 at OD600, IPTG was added at a final concentration of 1 mM, and then shaking culture was continued for about 4 hours.

[0362] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Fig.44 As shown, it was confirmed that the scFvHER2 protein fused with ubiquitin and TOM7 of about 35 kDa was expressed. Fig.44 Lane M in the figure shows a protein molecular weight marker, lane 1 shows a precipitate obtained by disrupting E. coli and centrifuging 4 hours after the addition of IPTG, and lane 2 shows a supernatant obtained by centrifugation after the disruption of E. coli.

[0363] Example 6.2.2 Preparation of pCMV-scFvHER2-TOM7-myc / His

[0364] In order to prepare scFvHER2 protein fused with TOM7 bound to the outer membrane of mitochondria, an expression vector of animal cells capable of expressing scFvHER2 fused with TOM7 was prepared. In order to obtain TOM7 and scFvHER2 genes, RscFvHER2 primers and XTOM7 (noT) primers were prepared. The sequences of various primers are shown in Table 11 below.

[0365] [Table 11]

[0366]

[0367]

[0368] The plasmid pET15b-UB-scFvHER2-TOM7 obtained in Example 6.2.1 was used as a template, and 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. After that, an amplification reaction was performed in a polymerase chain reaction instrument at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 25 cycles to obtain the gene scFvHER2-TOM7. The amplified scFvHER2-TOM7 gene was cut with restriction enzymes EcoRI and XhoI, and DNA fragments of about 850 bp were obtained by 1% agarose gel electrophoresis, and then inserted into pcDNA3.1-myc / His A cut with restriction enzymes EcoRI and XhoI using T4 DNA ligase to obtain plasmid pCMV-scFvHER2-TOM7-myc / His ( Fig.45 ).

[0369] In this case, scFvHER2-TOM7-myc / His is represented by the base sequence shown in SEQ ID NO: 41. Plasmid pCMV-scFvHER2-TOM7-myc / His was used to transfect animal CHO cells, and the cells were disrupted, followed by SDS-polyacrylamide electrophoresis, and displayed by Western blot using an anti-c-myc antibody. Fig.46 As shown, it was confirmed that the scFvHER2 protein fused to TOM7 of about 35 kDa was expressed. Fig.46 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows that the protein was transfected into animal CHO cells, the cells were broken, and then SDS-polyacrylamide electrophoresis was performed, and then Western blot detection was performed using an anti-c-myc antibody to confirm.

[0370] Example 6.3. Isolation and purification of recombinant UB-ScFvHER2-TOM7 protein from E. coli

[0371] The UB-ScFvHER2-TOM7 protein was separated and purified in the same manner as in Example 1.3.1. As a result, the UB-ScFvHER2-TOM7 protein was eluted ( Fig.47 ).in this case, Fig.47Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the sample loaded on the nickel affinity chromatography column. Lane 2 shows that it did not bind to the nickel affinity resin. Lane 3 shows the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 10mM imidazole. Lanes 4 to 5 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole. Lanes 6 to 8 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole. Lanes 9 to 10 show the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole. Lane 11 shows the results of elution with 8M UREA / 50mM sodium phosphate / 500mM NaCl / 500mM imidazole.

[0372] The protein amount of the recovered elution solution was determined by a protein quantification method and confirmed by SDS-PAGE. Fig.48 As shown, after confirmation, the UB-ScFvHER2-TOM7 protein was quenched with liquid nitrogen and stored in a low-temperature freezer at -80°C. Fig.48 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows the UB-ScFvHER2-TOM7 protein obtained after dialysis in PBS buffer solution.

[0373] Example 7. Preparation of fusion protein containing scFvMEL

[0374] Example 7.1. Synthesis of scFvMEL gene

[0375] In order to express human scFvMEL as an anti-melanoma antibody fragment as a recombinant protein, gene synthesis was requested from Bionics Co., Ltd. The obtained scFvMEL gene was named pUC57-scFvMEL, and the base sequence of scFvMEL was the same as that of SEQ ID NO:42.

[0376] Example 7.2. Preparation of scFvMEL protein expression vector

[0377] Example 7.2.1. Preparation of pET15b-UB-scFvMEL-TOM7

[0378] In order to prepare a scFvMEL protein fused with ubiquitin and TOM7 that binds to the outer membrane of mitochondria, an expression vector capable of expressing scFvMEL fused with ubiquitin and TOM7 was prepared.

[0379] The plasmid pUC57-scFvMEL gene obtained in Example 7.1 was cut with restriction enzymes SacII and XhoI, and a DNA fragment of about 750 bp was obtained by 2% agarose gel electrophoresis, which was then inserted into the pET15b-UB-(p53)-TOM7 vector cut with restriction enzymes SacII and XhoI using T4 DNA ligase to obtain plasmid pET15b-UB-scFvMEL-TOM7 ( Fig.49 ). In this case, UB-scFvMEL-TOM7 is represented by the base sequence shown in SEQ ID NO:43.

[0380] The plasmid pET15b-UB-scFvMEL-TOM7 was used to transform the E. coli BL21 (DE3) strain. After that, the transformed strain was cultured in Luria-Bertani (LB) solid medium supplemented with the antibiotic ampicillin, and then the obtained colonies were cultured in LB liquid medium in a shaking incubator at 37°C. After that, when the cell density reached an absorbance of about 0.2 at OD600, IPTG was added at a final concentration of 1 mM, and then the shaking culture was continued for about 4 hours.

[0381] A portion of the E. coli cells was obtained by centrifugation, and then the cells were broken and subjected to SDS-polyacrylamide electrophoresis. Fig.50 As shown, it was confirmed that the scFvMEL protein of about 35 kDa fused with ubiquitin and TOM7 was expressed. Fig.50 Lane M in the figure shows a protein molecular weight marker, lane 1 shows a precipitate obtained by disrupting E. coli and centrifuging 4 hours after the addition of IPTG, and lane 2 shows a supernatant obtained by centrifugation after the disruption of E. coli.

[0382] Example 7.2.2 Preparation of pCMV-scFvMEL-TOM7-myc / His

[0383] In order to prepare the scFvMEL protein fused with TOM7 bound to the outer membrane of mitochondria, an expression vector of animal cells capable of expressing scFvMEL fused with TOM7 was prepared. In order to obtain TOM7 and scFvMEL genes, primers (RscFvMEL) were prepared. The sequences of each primer are shown in Table 12 below.

[0384] [Table 12]

[0385] Primers sequence SEQ ID NO. RscFvMEL 5'-AAA AAA GAA TTC ATG AAA ACA AGT AAC CCA GGA GTG-3' SEQ ID NO:44

[0386] The plasmid pET15b-UB-scFvMEL-TOM7 obtained in Example 6.2.1 was used as a template, and 0.2 pmol RscFvMEL primer and 0.2 pmol XTOM7 (noT) primer were mixed with 0.2 nM dNTP, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit AccuPrime Taq DNA polymerase. After that, in a polymerase chain reaction instrument, an amplification reaction was performed at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute, and 25 cycles to obtain scFvMEL-TOM7. The amplified scFvMEL-TOM7 gene was cut with restriction enzymes EcoRI and XhoI, and a DNA fragment of about 850 bp was obtained by 1% agarose gel electrophoresis. Then, T4 DNA ligase was used to insert it into the pcDNA3.1-myc / His A vector cut with restriction enzymes EcoRI and XhoI to obtain plasmid pCMV-scFvMEL-TOM7-myc / His ( Fig.51 ). Here, scFvMEL-TOM7-myc / His is represented by the base sequence shown in SEQ ID NO:45.

[0387] The plasmid pCMV-scFvMEL-TOM7-myc / His was used to transfect animal CHO cells, and the cells were broken and then subjected to SDS-polyacrylamide electrophoresis and displayed by Western blot using anti-c-myc antibody. Fig.52 As shown, it was confirmed that the scFvMEL protein fused with TOM7 of about 35 kDa was expressed. Fig.52 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows that the protein was transfected into animal CHO cells, the cells were disrupted, and then subjected to SDS-polyacrylamide electrophoresis, and then confirmed by Western blot detection using an anti-c-myc antibody.

[0388] Example 8. Preparation of fusion protein containing scFvPD-L1

[0389] Example 8.1 Synthesis of scFvPD-L1 gene

[0390] In order to express human scFvPD-L1 as a recombinant protein, gene synthesis was requested from Bionics Co., Ltd. The obtained scFvPD-L1 gene was named pUC57-scFvPD-L1, and its base sequence was the same as the base sequence shown in SEQ ID NO:46.

[0391] Example 8.2. Preparation of scFvPD-L1 protein expression vector

[0392] Example 8.2.1. Preparation of pCMV-scFvPD-L1-TOM7-myc / His

[0393] In order to prepare the scFvPD-L1 protein fused with TOM7 that binds to the outer membrane of mitochondria, an expression vector for animal cells capable of expressing the scFvPD-L1 fused with ubiquitin and TOM7 was prepared.

[0394] The plasmid pUC57-scFvPD-L1 was cut with restriction enzymes EcoRI and XhoI, and a DNA fragment of about 760 bp was obtained by 1% agarose gel electrophoresis. Then, it was inserted into the pCMV-(scFvMEL)-TOM7-myc / His vector cut with restriction enzymes EcoRI and XhoI using T4 DNA ligase to obtain plasmid pCMV-scFvPD-L1-TOM7-myc / His ( Fig.53 ). In this case, scFvPD-L1-TOM7-myc / His is represented by the base sequence shown in SEQ ID NO:47.

[0395] Plasmid pCMV-scFvPD-L1-TOM7-myc / His was used to transfect animal CHO cells, the cells were broken, and then subjected to SDS-polyacrylamide electrophoresis and Western blot display using anti-c-myc antibody. Fig.54 As shown, it was confirmed that the scFvPD-L1 protein fused with TOM7 was expressed, and its size was about 35 kDa. In this case, Fig.54 Lane M in the figure shows a protein molecular weight marker, and lane 1 shows that the protein was transfected into animal CHO cells, the cells were disrupted, and then subjected to SDS-polyacrylamide electrophoresis, and then confirmed by Western blot detection using an anti-c-myc antibody.

[0396] III. Preparation of modified mitochondria bound to fusion proteins

[0397] Example 9. Preparation of modified mitochondria

[0398] The following experiment was performed to confirm whether the fluorescent protein fused to the mitochondrial outer membrane binding site binds to the outer membrane of the mitochondria. First, mitochondria were isolated from umbilical cord-derived mesenchymal stem cells (UC-MSC) by centrifugation. Afterwards, they were stained with MitoTracker CMXRos Red. They were mixed with the recombinant protein TOM70-(GGGGS)3-UB-GFP purified from E. coli and incubated at ambient temperature for about 30 minutes.

[0399] Afterwards, unreacted proteins were removed by centrifugation and washed twice with PBS buffer. Afterwards, fluorescent proteins bound to mitochondria were observed using a fluorescence microscope. Purified GFP protein that did not contain the mitochondrial outer membrane binding site was used as a control group. As a result, it was confirmed that the fluorescent protein fused to the mitochondrial outer membrane binding site (TOM70-(GGGGS)3-UB-GFP) was located in the same position as the mitochondria of umbilical cord-derived mesenchymal stem cells (UC-MSCs) ( Fig.55 Part A of Fig.55 b) of the

[0400] Example 10. Confirmation of the ability of recombinant protein p53 to bind to the outer membrane of exogenous mitochondria

[0401] Mitochondria isolated from umbilical cord-derived mesenchymal stem cells by centrifugation were mixed with purified recombinant proteins TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 and combined at a ratio of 1:1 for 1 hour at 4°C. Mitochondria not mixed with proteins were used as a control group. The binding ability between mitochondria and p53 was confirmed by Western blot experimental method ( Fig.56 ).

[0402] First, mitochondria and p53 protein were bound, and then centrifuged at 13,000 rpm for 10 minutes to obtain mitochondria or mitochondrial precipitates bound to p53. Proteins not bound to mitochondria were removed by two PBS washing processes, and the precipitates obtained by washing were subjected to protein electrophoresis (SDS-PAGE) and then Western blot. Rabbit anti-p53 antibody was used as the primary antibody and anti-rabbit IgG HRP was used as the secondary antibody. It was confirmed that the band was located at the same position as the size of 60 kDa, which was the expected molecular weight that mitochondria in the experimental group were indeed bound to TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 compared to individual mitochondria without protein binding ( Fig.56 ).

[0403] IV. Confirmation of Activity of Modified Mitochondria Binding to Active Proteins

[0404] Example 11. Isolation and intracellular injection of exogenous mitochondria

[0405] Mitochondria were isolated from umbilical cord-derived mesenchymal stem cells (UC-MSCs) using centrifugation. The isolated mitochondria were stained with Mitotracker CMX Ros, and the concentration and total amount of isolated mitochondria were determined by BCA quantification method. 0μg, 1μg, 5μg, 10μg, 50μg, and 100μg of mitochondria were injected into SNU-484 cells, a gastric cancer cell line, using centrifugation. The results of the experiment confirmed by fluorescence microscopy that the concentration of mitochondria injected into the cells depends on the amount of mitochondria ( Fig.57 ).

[0406] Example 12. Confirmation of the effect of normal mitochondria on cancer cells

[0407] The following experiment was conducted to study how mitochondria from normal cells affect the proliferation of cancer cells and the generation of ROS. First, hepatocytes (WRL-68), fibroblasts, and umbilical cord-derived mesenchymal stem cells (UC-MSC) were selected as mitochondrial donor cells. Mitochondria were separated from cells by centrifugation. The cancer cells used as mitochondrial recipient cells were skin epidermal cancer cell A431 cell lines. In this case, mitochondria were delivered to skin epidermal cancer cells using centrifugal force according to concentration (see Korean Patent Application No. 10-2017-0151526).

[0408] 24, 48 and 72 hours after the introduction, the proliferation of skin epidermal cancer cells and the generation of reactive oxygen species (ROS) were observed. As a result, it was confirmed that when mitochondria obtained from normal cells of various origins were injected into cancer cells, there was a concentration-dependent inhibitory effect on the proliferation of cancer cells. In addition, it was confirmed that the generation of ROS in cancer cells was inhibited, which depended on the concentration of normal mitochondria ( Fig.58 and 59 ).

[0409] Example 13. Confirmation of the influence of normal mitochondria on drug resistance

[0410] The following method was used to study how the drug resistance, expression of antioxidant genes, and cancer cell metastasis (metastasis) are affected when mitochondria derived from normal cells are injected into cancer cells, which are all characteristics of cancer cells. First, normal liver cells (WRL-68) were set as mitochondrial donor cells, and mitochondria were separated from the cells by centrifugation, and cancer cells using the liver cancer cell line HepG2 cells as mitochondrial recipient cells. Mitochondria were delivered to liver cancer cells using centrifugal force according to the concentration, and then it was confirmed that the cancer cell line that received the mitochondria showed higher drug sensitivity by observing the resistance to the anticancer drug doxorubicin ( Fig.60 ).

[0411] Example 14. Demonstration of the influence of normal mitochondria on anti-oxidation

[0412] Mitochondria isolated from normal cells were injected into the liver cancer cell line HepG2 cells, and the expression of catalase, antioxidant protein and SOD-2 (superoxide dismutase-2) genes in cancer cells was confirmed to increase according to the concentration ( Fig.61 ).

[0413] Example 15. Confirmation of the effect of normal mitochondria on cancer cell metastasis

[0414] For metastasis, it has been confirmed whether there is expression of the α-smooth muscle actin (α-SMA) gene, which is one of the genes involved in EMT (epithelial to mesenchymal transition). In this case, it was found that the expression of α-SMA protein in liver cancer cells that received mitochondria was significantly reduced according to the concentration of mitochondria compared with liver cancer cells that did not receive mitochondria. In contrast, it was found that E-cadherin, one of the cell adhesion proteins, increased according to the concentration of mitochondria ( Fig.62 ). They demonstrated that changes in proteins known to be involved in cancer metastasis were caused by normal mitochondria injected into cancer cells, thus also affecting the metastasis of cancer cells.

[0415] Example 16. Confirmation that the recombinant protein p53 is loaded into the outer membrane of exogenous mitochondria and injected into cells

[0416] Mitochondria were isolated from umbilical cord-derived mesenchymal stem cells by centrifugation, stained with Mitotracker CMXRos, mixed with purified recombinant protein TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7, and incubated at a ratio of 1:1 for 1 hour at 4°C. Unreacted proteins were then removed by centrifugation, washed twice with buffer solution PBS, and then injected into gastric cancer cell line SNU-484 cells with p53 protein by centrifugation. Fig.63 In this case, a control group was set up for a group without mitochondria and a group with only mitochondria. After one day of culture, exogenous mitochondria loaded with p53 protein injected into cells were observed using immunocytochemistry (ICC) with a fluorescence microscope.

[0417] Rabbit anti-p53 antibody was used as the primary antibody, and goat anti-rabbit IgG Alexa Fluor 488 was used as the secondary antibody. As a result, it was confirmed that TOM70-(GGGGS)3-UB-p53 (green) or UB-p53-TOM7 (green) proteins loaded on exogenous mitochondria (red) were located in the cytoplasm of cells injected with exogenous mitochondria (red). Fig.64 , magnified 200 times, Fig.65 , magnified 400 times). As a result, it was found that the recombinant protein was easily injected into cells through mitochondria.

[0418] Example 17. Confirmation of the activity of p53-loaded mitochondria in cancer cell lines

[0419] Example 17.1. Demonstration of the apoptotic capacity of p53-loaded exogenous mitochondria injected into cells using gastric cancer cell lines

[0420] 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 ratio of 1:1 for 1 hour at 4°C. UB-p53 protein without TOM70 and TOM70-(GGGGS)3-p53 without ubiquitin served as controls. Unbound proteins were removed by centrifugation and PBS washing, and the protein-bound mitochondria were injected by centrifugation into the gastric cancer cell line SNU-484 ( ) that lacks p53 ability due to p53 gene mutation. Fig.66 After culturing for 1 day, cells were fixed with 4% paraformaldehyde for 1 hour, and then permeabilized with a permeabilization solution (0.1% sodium citrate buffer containing 0.1% Triton-X-100, pH 7.4) and reacted with a TUNEL solution (In Situ Cell Death Detection Kit, TMR RED, Roche) at 37°C for 1 hour.

[0421] In the TUNEL analysis method, the part where nucleic acid fragmentation (DNA fragmentation) occurred was stained red, indicating that apoptosis occurred. Compared with the control group, a large amount of red parts were found in the cells injected with mitochondria bound to TOM70-(GGGGS)3-ub-p53 or p53-TOM7, indicating that apoptosis was due to mitochondria bound to TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7. Specifically, it was confirmed that more apoptosis occurred in mitochondria bound to the TOM70-(GGGGS)3-UB-p53 protein ( Fig.67a ).

[0422] Example 17.2 Demonstration of the apoptotic capacity of luciferase-bound, p53-loaded exogenous mitochondria

[0423] In order to confirm whether the delivered TOM70-(GGGGS)3-UB-p53 protein bound to the mitochondria obtained in Example 5.2 above maintains biological activity in the recipient cells after delivery to the recipient cells, a cell-based analysis was performed using a reporter gene. Since the p53 protein is a transcription factor, a gene with a base sequence of RRRCWWGYYY (where R represents G or A, W represents A or T, and Y represents C or T) repeated 6 times to which the p53 transcription factor can bind was synthesized according to the following sequence. The base sequence of P53-promoter-S is as follows (5'-GGG CAT GCT CGG GCA TGC CCG GGC ATG CTCGGG 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 TGCCCG AGC ATG CCC-3') (SEQ ID NO: 92).

[0424] 5 μg of synthetic gene P53-promter-S and 5 μg of synthetic gene P53-promter-AS were incubated at 70°C for 20 minutes to promote the synthesis of double helix genes, and then polynucleotide T4 kinase was used to induce phosphorylation. The double helix gene induced phosphorylation was inserted into the pGL3 vector cut by restriction enzyme Sma I, and the gene with a base sequence (RRRCWWGYYY) to which the p53 transcription factor can bind was repeated 6 times and combined with the reporter gene luciferase to prepare plasmid p6xp53-Luc. Plasmid p6xp53-Luc and β-galactosidase expression vector plasmid pRSVb-gal were transformed into human kidney cell HEK293 cells by lipofection amine method.

[0425] Then, after 6 hours, HEK293 cells were treated with 10 μg of mitochondria and 5 μg, 10 μg and 20 μg of TOM70-(GGGGS)3-UB-p53 protein, respectively. In this case, as a control group, cells were treated with 10 μg of mitochondria combined with PBS or p53 protein, respectively. The treated cells were cultured for 18 hours, and then the luciferase activity was measured and analyzed. In this case, in order to correct the conversion efficiency, the luciferase value was divided by the value obtained by measuring the activity of β-galactosidase as the corrected luciferase value.

[0426] It was confirmed that the luciferase value increased in cells treated with 10 μg of mitochondria and 5 μg, 10 μg, and 20 μg of TOM70-(GGGGS)3-UB-p53 protein, respectively. Therefore, it was confirmed that the p53 protein entered the cells and showed activity ( Figure 67b ).

[0427] Example 18. Confirmation of the ability of exogenous mitochondria loaded with RKIP injected into cells to reduce the 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 and combined at a ratio of 1:1 for 1 hour at 4°C. The protein-bound mitochondria were injected into the breast cancer cell line MDA-MB-231 by centrifugation, which has enhanced metastatic ability due to the reduction of RKIP protein.

[0428] To confirm the metastatic ability of cancer cells, a cell invasion experiment was performed using a transwell plate. The upper chamber of a transwell with a pore size of 8 μm was coated with matrigel at 37°C for 30 minutes. As a test group, MDA-MB-231 cells injected with only mitochondria and MDA-MB-231 cells injected with mitochondria bound to RKIP protein were used. 1×10 5 10 cells were placed in the upper chamber of the transwell containing serum-free medium, and medium containing 10% fetal 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 then the cells through the matrix gel were stained with 1% crystal violet.

[0429] As a result of microscopic observation, purple-stained cells were observed in the membrane below the upper chamber, which can be said to be the process of cell transfer. It can be confirmed that the purple-stained cells in the experimental group treated with only mitochondria and the experimental group treated with mitochondria combined with RKIP decreased compared with the control group treated with blank treatment. Four sections were randomly selected, and the number of stained cells was measured and plotted on a graph ( Fig.68 ).

[0430] IV. Confirmation of delivery rate of modified mitochondria bound to target protein

[0431] Example 19. Demonstration of intracellular expression of single-chain variable fragment (ScFv) antibodies for targeting cancer cells and demonstration of binding to mitochondria in cells

[0432] To express pCMV-ScFv-HER2-TOM7 or 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 group. To confirm its expression in cells and binding to mitochondria in the same cells, cytosol and mitochondria were separated from transfected cells by centrifugation and adjusted to the same protein amount by BCA analysis, followed by PAGE electrophoresis, and then observed by Western blot. Monoclonal c-myc antibody was used as the primary antibody, and anti-mouse IgG HRP was used as the secondary antibody.

[0433] The bands of ScFv-HER2-TOM7 or ScFv-MEL-TOM7 proteins were identified, with the expected size of 35 kDa. Based on the fact that all of these were identified at the mitochondrial level, it can be expected that the transfected and expressed proteins bind to mitochondria in cells through TOM7 ( Fig.69 ).

[0434] Next, in order to confirm the binding of the targeted protein expressed in the cell to the mitochondria in the same cell, the ScFv-HER2-TOM7, ScFv-MEL-TOM7 or ScFv-PD-L1-TOM7 protein expressed in the cell was observed under a fluorescence microscope using an immunocytochemistry (ICC) experimental method. Monoclonal c-myc antibody was used as the primary antibody, and goat anti-mouse IgG Alexa Fluor488 was used as the secondary antibody. The mitochondria in the cells were stained with Mitotracker CMX Ros. As a result, it was confirmed that the expressed ScFv-HER2-TOM7, ScFv-MEL-TOM7 or ScFv-PD-L1-TOM7 protein co-localized with the mitochondria and bound to the mitochondria in the cell ( Fig.70 and 71 ).

[0435] Example 20. Isolation of mitochondria bound to single-chain variable fragment antibodies targeting cancer cells and comparison of mitochondrial injection in gastric cancer cell lines

[0436] Mitochondria were isolated from CHO cells transfected with pCMV-ScFv-HER2-TOM7 or pCMV-ScFv-PD-L1-TOM7. Mitochondria of untransformed CHO cells were isolated as a control group. Mitochondria isolated from each cell were stained with MitotrackerCMX Ros. The gastric cancer cell line SNU-484 was treated with the same amount of mitochondria, and the next day, the concentration of mitochondria injected into the cells was compared and confirmed by fluorescence microscopy. It can be confirmed that compared with the control group, the mitochondria injected into the cancer cells with ScFv-HER2-TOM7 or ScFv-PD-L1-TOM7 binding were more than those obtained in the control group ( Fig.72 ). Therefore, it was found that mitochondria bound to the targeting protein were easier to inject into cancer cells when used alone.

[0437] VI. Confirmation of in vivo activity of modified mitochondria bound to active proteins

[0438] Example 21. Construction of xenograft model (SNU-484) and administration of test substances

[0439] Example 21.1. Preparation of cancer cells

[0440] On the day of the experiment, SNU-484 cells (a gastric cancer cell line) were prepared at 5 × 10 per mouse. 6 cells. Remove the culture medium of the cells and then add PBS to wash the cells. Dissociate the cells using trypsin-EDTA solution, then place the cells in a 50 mL tube and wash twice with PBS buffer solution, then add 20 mL PBS and measure the cell number and viability. Based on the measured cell number, the cell number was adjusted to 5 × 10 per mouse. 6 cells and prepare the cells by dividing into several groups. Adjust the transplant volume to the same 100 μL for each mouse. Prepare 100 μL of a separate cancer cell group as a control group.

[0441] Example 21.2 Preparation of test substances

[0442] As described above, 50 μg of mitochondria isolated from umbilical cord-derived mesenchymal stem cells were prepared for transplantation per mouse according to the protein concentration. In the group to which only mitochondria were administered, mitochondria were prepared by mixing wells with 100 μL of PBS mixed with cancer cells. In the modified mitochondria group, TOM70-(GGGGS)3-UB-p53 protein was mixed with mitochondria prepared in an Eppendorf tube at a concentration ratio of 1:1 before mixing with cancer cells, and then allowed to stand at ambient temperature for 1 hour. After the reaction time was over, the supernatant was removed after centrifugation at 20,000×g for 10 minutes to obtain pellets of mitochondria bound to the protein (MT+TOM70-(GGGGS)3-UB-p53). After washing twice with PBS buffer solution, mitochondria bound to the p53 protein (MT+TOM70-(GGGGS)3-UB-p53) were prepared by mixing with 100 μL of PBS mixed with cancer cells.

[0443] Example 21.3 Preparation of experimental animals and transplantation of test substances

[0444] For transplant samples prepared by group, Matrigel (BD) was added in the same amount as PBS and gently mixed with the cells to prepare 200 μL of the test substance per mouse. In this case, all operations were performed on ice. For model construction, Balb / c nude mice (female, 7 weeks old) were purchased from RAONBIO and anesthetized by inhalation of isoflurane for transplantation of cancer cells, and then disinfected with an alcohol swab in the right rear area (based on the animal). After that, 200 μL was subcutaneously injected into the right rear area of ​​the experimental animal using a 1 mL syringe filled with injection solution. After administration, the body weight and tumor size of the animals were measured twice a week, and the results were analyzed while observing for up to 3 weeks ( Fig.73 ).

[0445] Example 21.4 Demonstration of tumor formation

[0446] The volume of the tumor was calculated by measuring the major and minor axis lengths of the tumor and applying them to the following equation.

[0447] <Mathematical equation 1>

[0448] Long axis × short axis × short axis × 0.5 = tumor volume (mm^3)

[0449] Example 21.5 Observation of Physiological and Morphological Changes

[0450] In order to observe the physiological and morphological changes of mice administered with anticancer candidates, changes in body weight and tumor size were measured twice a week starting from the administration of cancer cells and test substances ( Fig.74 ).

[0451] The body weight of the mice was measured using a weighing scale, and the values ​​measured twice a week were used to analyze the changes in each group ( Fig.75 ). It was confirmed that there was no significant difference in weight change over 3 weeks between the group without mitochondria injection, the group with mitochondria injection alone, and the group with modified mitochondria injection. The size of the tumor was calculated by measuring the length of the major axis (length) and the minor axis (width) of the tumor using a caliper and then applying it to the equation of the above mathematical equation 1. The changes in each group were analyzed using the twice-weekly measurements ( Fig.76 ). It has been found that in the group not treated with mitochondria, the size of the tumor increased significantly over time, while in the mice given mitochondria, the growth of the tumor size over time was significantly slowed down. In addition, it has been confirmed that the increase in tumor size in the group administered with p53 protein-loaded mitochondria was significantly reduced compared to the group administered with mitochondria alone ( Fig.76 ).

[0452] Example 22. Confirmation of the effect of modified mitochondria in inhibiting the proliferation of skin cancer cells

[0453] The p53-binding mitochondria obtained above were delivered to skin cancer cell A431 cells by centrifugation, and then the proliferation of A431 cells was observed. In this case, physiological saline was used as a control group, and an equal amount of mitochondria without p53 protein fusion was used as a test control group. It has been confirmed that mitochondria loaded with apoptosis-inducing protein p53 protein can significantly inhibit the proliferation of A431 cells compared with the control group and the group using only mitochondria ( Fig.76 ).

[0454] V. Confirmation of the Activity of Isolated Mitochondria

[0455] Example 23. Confirmation of isolated mitochondrial function: ATP content

[0456] In order to separate intracellular mitochondria from umbilical cord derived mesenchymal stem cells (UC-MSC), a syringe was used for homogenization to destroy cells, and then continuous centrifugation was performed to obtain mitochondria. In order to confirm the function of isolated mitochondria, the mitochondrial protein concentration of the mitochondria separated quantitatively was analyzed by BCA to prepare 5 μg of mitochondria. The amount of ATP in mitochondria was confirmed using CellTiter-Glo luminescent kit (Promega, Madison, WI).

[0457] The prepared mitochondria were mixed in 100 μl of PBS and then prepared in a 96-well plate and compared with 100 μl of PBS containing no mitochondria as a control group. 100 μL of the test solution contained in the kit was added in the same manner, reacted and mixed well for 2 minutes in a stirrer, and then reacted at ambient temperature for 10 minutes, after which the amount of ATP was measured using a luminescent microplate reader. It was confirmed that when mitochondria were included, ATP increased compared to the control group, and the function of mitochondria was confirmed ( Fig.78 ).

[0458] Example 24. Confirmation of isolated mitochondrial function: membrane potential

[0459] In order to confirm the membrane potential of isolated mitochondria, JC-1 dye (Molecular Probes, catalog number 1743159) was used. The prepared mitochondria were mixed with 50 μL PBS and then prepared in a 96-well plate. A PBS (50 μL) group without mitochondria was prepared as a control group, and CCCP (R&D systems, CAS 555-60-2) was prepared as a treatment group. CCCP is an ion carrier of mitochondria that inhibits mitochondrial function by depolarizing the mitochondrial membrane potential. The CCCP group was reacted with isolated mitochondria at a concentration of 50 μM at room temperature for 10 minutes.

[0460] Afterwards, it was reacted with JC-1 dye (2 μM) in the same way, and then the absorbance was measured using the properties of different spectra according to the concentration produced by the change in membrane potential. At low concentrations, isolated mitochondria exist as monomers and show green fluorescence, and at high concentrations, dye aggregates (J aggregates) show 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 fluorescent microplate reader (monomer: Ex485 / Em 530, J-aggregate: Ex 535 / Em 590). The results are shown in Figure 2. Fig.79 shown.

[0461] Example 25. Confirmation of the extent of damage to isolated mitochondria by confirming the production of mROS

[0462] To confirm whether the 5 μg of mitochondria prepared as described above were damaged, the MitoSOX red indicator (Invitrogen, catalog number M36008) capable of analyzing mitochondrial reactive oxygen species in isolated mitochondria was used. The prepared mitochondria were mixed with 50 μL PBS and then prepared in a 96-well plate and compared with 50 μL PBS without mitochondria as a control group. The MitoSOX red dye was mixed with 50 μL PBS to a concentration of 10 μM and placed in a 96-well plate (final concentration of 5 μM) and then incubated at 37°C in CO 2The reaction was allowed to proceed in an incubator for 20 minutes. After the reaction was completed, the amount of ROS in the mitochondria was measured using an ELISA reader (Ex 510 / Em 580). Fig.80 shown.

[0463] VI. Demonstration of dissociation of desired proteins bound to extracellular and intracellular mitochondrial outer membrane proteins

[0464] Example 26. Demonstration of dissociation of desired protein bound to mitochondrial outer membrane protein outside the cell

[0465] In order to obtain the desired protein in free form when the active protein bound to mitochondria is injected into cells, a fusion protein (TOM70-UB-p53 or TOM-UB-GFP) in which ubiquitin protein is inserted between the mitochondrial outer membrane protein and the desired protein is prepared from E. coli. In order to confirm whether the ubiquitin cleavage enzyme UBP1 cleaves the ubiquitin sequence, the recombinant fusion protein TOM70-UB-p53 is reacted with the UBP1 enzyme at 37°C for 1 hour.

[0466] Afterwards, the results of the analysis by SDS-PAGE electrophoresis confirmed that the dissociation of the ubiquitin protein from the fusion protein by UBP1 did not occur at all. This is considered to be a phenomenon of structural interference with the mitochondrial outer membrane protein, so a linker protein composed of amino acids glycine and serine was inserted between the mitochondrial outer membrane protein and the ubiquitin protein, and a new fusion protein (TOM70-(GGGGS)3-UB-p53 or TOM70-(GGGGS)3-UB-GFP) was purified from E. coli and then reacted with the UBP1 enzyme at 37°C for 1 hour as described above. As a result, it was confirmed by SDS-PAGE electrophoresis that the 3' end of ubiquitin was cleaved by the UBP1 enzyme, and only the p53 protein dissociated as expected ( Fig.82 ).

[0467] Example 26. Demonstration of dissociation of desired protein bound to mitochondrial outer membrane protein in cells

[0468] When the fusion protein (TOM70-(GGGGS)3-UB-p53 or TOM70-(GGGGS)3-UB-GFP) obtained in the above example enters the cell in a state bound to mitochondria, it is observed whether its active protein is dissociated by the ubiquitin cleavage enzyme present in the cell. First, the mitochondria obtained from umbilical cord-derived mesenchymal cells are reacted with the fusion protein TOM70-(GGGGS)3-UB-GFP in a microtube for 1 hour to bind them, and then the unbound fusion protein is removed by centrifugation, and then washed twice with PBS buffer solution. In this case, the fusion protein (TOM70-(GGGGS)3-GFP) from which ubiquitin has been removed is used as a control group.

[0469] Afterwards, the mitochondrial-bound proteins were injected into the breast cancer cell line MDA-MB-231 cells by centrifugation. One day later, the MDA-MB-231 cells were broken up and separated into mitochondrial and cytoplasmic parts by gravity of differentiation. The results of SDS-PAGE electrophoresis and Western blot analysis showed that in the fusion protein containing ubiquitin, the GFP protein dissociated from the mitochondrial outer membrane protein, and the adaptor protein and ubiquitin were mainly detected in the cytoplasmic part. It was found that in the fusion protein without ubiquitin, the GFP protein bound to the mitochondrial outer membrane protein and the adaptor protein was mainly detected in the mitochondrial part ( Fig.83 ).

[0470] The results showed that when the mitochondrial outer membrane protein-linker-ubiquitin-active protein bound to mitochondria was injected into cells, the connection site between ubiquitin and the active protein was cleaved, and the dissociated active protein was released into the cytoplasm; and it was found that through this method, mitochondria can be used as a delivery tool as one of the methods to effectively deliver useful proteins into cells.

Claims

1. An isolated modified mitochondrion, wherein: The exogenous protein is bound to the outer membrane of the mitochondria; Wherein, the exogenous protein is a fusion protein, the fusion protein comprises a mitochondrial anchor peptide and a target targeting protein, and the target targeting protein can bind to a ligand or receptor present in a cell membrane; Wherein, the exogenous protein is bound to the outer membrane of the mitochondria via a mitochondrial anchor peptide.

2. The isolated modified mitochondria of claim 1, wherein The mitochondria are isolated from eukaryotic cells, tissues or platelets.

3. The isolated modified mitochondria of claim 1, wherein The mitochondrial anchoring peptide comprises an N-terminal region or a C-terminal region of a protein present in a mitochondrial membrane protein.

4. The isolated modified mitochondria according to claim 3, characterized in that The N-terminal region or the C-terminal region of the protein present in the mitochondrial membrane protein is located on the outer membrane of the mitochondria.

5. The isolated modified mitochondria according to claim 3, characterized in that The mitochondrial anchoring peptide is any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

6. The isolated modified mitochondria according to claim 3, characterized in that The mitochondrial anchoring peptide comprises an N-terminal region of any one selected from the group consisting of TOM20, TOM70 and OM45.

7. The isolated modified mitochondria according to claim 3, characterized in that The mitochondrial anchoring peptide comprises a C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

8. The isolated modified mitochondria of claim 1, wherein The exogenous protein is a target targeting protein, and the target targeting protein binds to the N-terminal region of TOM20, TOM70 or OM45.

9. The isolated modified mitochondria of claim 1, wherein The target protein is a ligand or receptor present on the surface of tumor cells.

10. The isolated modified mitochondria of claim 9, wherein The ligand or receptor present on the surface of tumor cells is any one selected from the group consisting of CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), mucin 1 cell surface associated protein (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), survivin, tyrosine-related protein 1 (tyrp1), tyrosine-related 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, connexin 3 or a combination thereof.

11. The isolated modified mitochondria of claim 1, wherein The exogenous protein is a target targeting protein, which binds to the C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

12. The isolated modified mitochondria of claim 1, wherein The exogenous protein further comprises at least one linker.

13. The isolated modified mitochondria of claim 12, wherein The linker consists of 1 to 150 amino acids.

14. The isolated modified mitochondria of claim 12, wherein The linker consists of 5 to 50 amino acids, and the amino acids are glycine and serine.

15. The isolated modified mitochondria of claim 14, wherein The linker is (G4S)n, wherein n is an integer from 1 to 10.

16. The isolated modified mitochondria of claim 12, wherein The fusion protein has any of the following structures: 1) N-terminus - target protein - mitochondrial anchor peptide - C-terminus; 2) N-terminus - target protein - ubiquitin or its fragment - mitochondrial anchor peptide - C-terminus; 3) N-terminus - target protein - linker 1 - ubiquitin or its fragment - mitochondrial anchor peptide - C-terminus; 4) N-terminus - target protein - ubiquitin or ubiquitin fragment - linker 2 - mitochondrial anchor peptide - C-terminus; 5) N-terminus - target protein - linker 1 - ubiquitin or its fragment - linker 2 - mitochondrial anchor peptide - C-terminus; 6) N-terminus - mitochondrial anchor peptide - target protein - C-terminus; 7) N-terminus - mitochondrial anchor peptide - ubiquitin or its fragment - target protein - C-terminus; 8) N-terminus - mitochondrial anchor peptide - linker 1 - ubiquitin or its fragment - target protein - C-terminus; 9) N-terminus - mitochondrial anchor peptide - ubiquitin or its fragment - linker 2 - target protein - C-terminus; and 10) N-terminus - mitochondrial anchor peptide - linker 1 - ubiquitin or its fragment - linker 2 - C-terminus of target protein.

17. A pharmaceutical composition comprising the isolated modified mitochondria according to claim 1 as an active ingredient.

18. The pharmaceutical composition according to claim 17, wherein The pharmaceutical composition is used for preventing or treating cancer.

19. The pharmaceutical composition according to claim 18, 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.

20. Use of the isolated modified mitochondria of claim 1 as a means for delivering exogenous proteins intracellularly and extracellularly, wherein the exogenous proteins comprise target targeting proteins capable of binding to ligands or receptors present in cell membranes, The exogenous protein is bound to the outer membrane of the mitochondria via a mitochondrial anchor peptide.

21. A fusion protein comprising a target-targeting protein capable of binding to a ligand or receptor present in a cell membrane and a mitochondrial outer membrane anchoring peptide.

22. The fusion protein according to claim 21, wherein The mitochondrial outer membrane anchored peptide is any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

23. The fusion protein of claim 21, wherein The target targeting protein and the mitochondrial anchoring peptide are combined from the N-terminus to the C-terminus.

24. The fusion protein of claim 21, wherein The target protein is an antibody or a fragment thereof.

25. The fusion protein of claim 24, wherein The antibody fragment is any one selected from the group consisting of Fab, Fab', scFv and F(ab)2.

26. A polynucleotide encoding the fusion protein of claim 21.

27. A method for preparing isolated modified mitochondria, comprising the step of mixing isolated mitochondria with the fusion protein of claim 21.

28. A method for preparing isolated modified mitochondria from transformed cells by injecting a polynucleotide encoding the fusion protein of claim 21 into eukaryotic cells.

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