Modified mitochondria and their use

Abstract

To provide a fusion protein that can be used to modify mitochondria.SOLUTION: There is provided a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a desired pharmacological protein, in order to prepare modified mitochondria with a foreign protein bound to the outer membrane of the mitochondria. There is also provided a fusion protein comprising an antibody or a fragment thereof and a mitochondrial outer membrane anchoring peptide.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

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

[0003] It has been reported that a decrease in energy production due to mitochondrial dysfunction causes various diseases. When the function of the electron transport chain reaction decreases due to mutations in the mitochondrial genome and proteome, a decrease in ATP production, excessive production of reactive oxygen species, a decrease in calcium regulation function, etc. occur. In such cases, a change occurs in the membrane permeability of mitochondria, and the apoptosis function may occur abnormally, leading to cancer and incurable diseases.

[0004] Therefore, human diseases reported to be caused by mitochondrial dysfunction include mitochondrial-related gene disorders (Wallace DC 1999), diabetes mellitus (Maechler P 2001), heart disease (Sorescu D 2002), senile dementia, such as Parkinson's disease or Alzheimer's disease (Lin MT 2006), as well as various cancers (Petros JA, 2005) and cancer metastasis (Ishikawa K, 2008). Furthermore, common features observed in over 200 different types of cancer include abnormalities in apoptosis, increased inflammatory responses, and increased abnormal metabolism. All of these processes are closely related to mitochondrial function, and there is growing interest in the relationship between cancer and mitochondria.

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

[0006] Therefore, abnormal metabolic processes in cancer cells, leading to changes in the composition of the tumor microenvironment, inhibition of apoptosis due to dysfunctional mitochondria, increased inflammatory responses, and abnormal metabolic reactions within cancer cells, all play crucial roles in cancer growth. Consequently, developing metabolism-related anticancer drugs that utilize these characteristics could be an excellent way to overcome the side effects and economic problems associated with conventional anticancer drugs.

[0007] It is known that when mitochondria present in cells are isolated and the cells are treated with them in vitro, or when mitochondria are introduced into the body, they enter the cells. This phenomenon could potentially be used to treat diseases caused by mitochondrial dysfunction by introducing normal mitochondria isolated from cells into the body, or, in particular, by using mitochondria as carriers to effectively deliver specific proteins to cells; however, there have been no reports on this yet. [Overview of the project] [Problems that the invention aims to solve]

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

[0009] To address the above issues, modified mitochondria in which an exogenous protein is bound to the mitochondrial outer membrane are provided. Furthermore, a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a desired pharmacological protein is provided for preparing the modified mitochondria. In addition, a fusion protein comprising an antibody or its fragment and a mitochondrial outer membrane anchoring peptide is provided. [Effects of the Invention]

[0010] When mitochondria bound to foreign proteins are administered to the human body, the foreign proteins can be effectively delivered into cells. Furthermore, the pharmacologically active proteins delivered into cells can repair damaged cellular function. Moreover, when mitochondria bound to foreign proteins containing pharmacologically active proteins are delivered into cells, the pharmacologically active proteins can be dissociated from the mitochondria in the cells and are expected to play useful roles. Furthermore, modified mitochondria containing antibody fragments can be effectively delivered to target cells. In particular, when antibody fragments targeting proteins present on the surface of cancer tissue are bound to the mitochondrial surface, the modified mitochondria can be effectively delivered into cancer cells. Therefore, the introduction of modified mitochondria can not only restore the damaged electron transport chain of cells, but also prevent or treat various diseases through the pharmacologically active proteins bound to the modified mitochondria. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the method for fabricating pTA-p53. [Figure 2] Figure 2 shows the method for preparing the pET15b-UB-p53 vector. [Figure 3] Figure 3 shows the expression of the UB-p53 protein in E. coli. [Figure 4] Figure 4 shows the method for preparing the pET11C-TOM70-UB-p53 vector. [Figure 5] Figure 5 shows the expression of the TOM70-UB-p53 protein in Escherichia coli. [Figure 6] Figure 6 shows the method for preparing the pET11C-TOM70-(GGGGS)3-UB-p53 vector. [Figure 7] Figure 7 shows the expression of the TOM70-(GGGGS)3-UB-p53 protein in Escherichia coli. [Figure 8] Figure 8 shows the method for preparing the pET11C-TOM70-(GGGGS)3-p53 vector. [Figure 9] Figure 9 shows the expression of TOM70-(GGGGS)3-p53 protein in Escherichia coli. [Figure 10] Figure 10 shows the method for preparing the pET15b-UB-p53-TOM7 vector. [Figure 11] Figure 11 shows the expression of UB-p53-TOM7 protein in Escherichia coli. [Figure 12] Figure 12 is a diagram showing the method for preparing the pCMV-p53-myc / His vector. [Figure 13] Figure 13 shows the expression of p53-myc / His protein in transformed CHO. [Figure 14] Figure 14 shows the result of purifying the TOM70-(GGGGS)3-p53 protein and then confirming it. [Figure 15] Figure 15 is a diagram showing the purified TOM70-(GGGGS)3-p53 protein. [Figure 16] Figure 16 shows the result of purifying the TOM70-(GGGGS)3-UB-p53 protein and then confirming it. [Figure 17] Figure 17 is a diagram showing the purified TOM70-(GGGGS)3-UB-p53 protein. [Figure 18] Figure 18 shows the result of purifying the UB-p53 protein and then confirming it. [Figure 19] Figure 19 is a diagram showing the purified UB-p53 protein. [Figure 20] Figure 20 shows the result of purifying the UB-p53-TOM7 protein and then confirming it. [Figure 21] Figure 21 is a diagram showing the purified UB-p53-TOM7 protein. [Figure 22] Figure 22 shows the method for preparing the pTA-granzymes B vector. [Figure 23] Figure 23 shows the method for preparing the pET11C-TOM70-(GGGGS)3-UB-granzymes B vector. [Figure 24]Figure 24 shows the expression of TOM70-(GGGGS)3-UB-granzyme B protein in Escherichia coli. [Figure 25] Figure 25 shows the method for preparing the pET15b-UB-granzyme B-TOM7 vector. [Figure 26] Figure 26 shows the expression of the UB-granzyme B-TOM7 protein in Escherichia coli. [Figure 27] Figure 27 shows the results of the purification of TOM70-(GGGGS)3-UB-granzyme B protein. [Figure 28] Figure 28 shows the purified UTOM70-(GGGGS)3-UB-granzyme B protein. [Figure 29] Figure 29 shows the method for preparing the pTA-RKIP vector. [Figure 30] Figure 30 shows the method for preparing the pET11C-TOM70-(GGGGS)3-UB-RKIP vector. [Figure 31] Figure 31 shows the expression of the TOM70-(GGGGS)3-UB-RKIP protein in Escherichia coli. [Figure 32] Figure 32 shows the results of the purification of the TOM70-(GGGGS)3-UB-RKIP protein. [Figure 33] Figure 33 shows the purified TOM70-(GGGGS)3-UB-RKIP protein. [Figure 34] Figure 34 shows the method for preparing the pTA-PTEN vector. [Figure 35] Figure 35 shows the method for preparing the pET11C-TOM70-(GGGGS)3-UB-PTEN vector. [Figure 36] Figure 36 shows the expression of the TOM70-(GGGGS)3-UB-PTEN protein in Escherichia coli. [Figure 37] Figure 37 shows the results of the purification of the TOM70-(GGGGS)3-UB-PTEN protein. [Figure 38] Figure 38 shows the purified TOM70-(GGGGS)3-UB-PTEN protein. [Figure 39] Figure 39 shows the results of purifying and then verifying the UB-GFP-TOM7 protein. [Figure 40] Figure 40 shows the purified UB-GFP-TOM7 protein. [Figure 41] Figure 41 shows the results of purifying and then verifying the TOM70-(GGGGS)3-UB-GFP protein. [Figure 42] Figure 42 shows the purified TOM70-(GGGGS)3-UB-GFP protein. [Figure 43] Figure 43 shows the method for preparing the pET15b-UB-scFvHER2-TOM7 vector. [Figure 44] Figure 44 shows the expression of the UB-scFvHER2-TOM7 protein in Escherichia coli. [Figure 45] Figure 45 shows the method for preparing the pCMV-scFvHER2-TOM7-myc / His vector. [Figure 46] Figure 46 shows the expression of the scFvHER2-TOM7-myc / His protein in transformed CHO cells. [Figure 47] Figure 47 shows the results of the purification of the UB-ScFvHER2-TOM7 protein. [Figure 48] Figure 48 shows the purified UB-ScFvHER2-TOM7 protein. [Figure 49] Figure 49 shows the method for preparing the pET15b-UB-scFvMEL-TOM7 vector. [Figure 50] Figure 50 shows the expression of the UB-scFvMEL-TOM7 protein in Escherichia coli. [Figure 51] Figure 51 shows the method for preparing the pCMV-scFvMEL-TOM7-myc / His vector. [Figure 52] Figure 52 shows the expression of the scFvMEL-TOM7-myc / His protein in transformed CHO cells. [Figure 53]Figure 53 shows the method for preparing the pCMV-scFvPD-L1-TOM7-myc / His vector. [Figure 54] Figure 54 shows the expression of the scFvPD-L1-TOM7-myc / His protein in transformed CHO cells. [Figure 55] Figure 55 shows the results of checking whether a fluorescent protein binds to the outer mitochondrial membrane. In this case, the mitochondria are stained red with MitoTracker CMXRos, and TOM70-UB-GFP appears green. The overlapping region of these two areas appears yellow. In this case, the magnification in Figure 55a is 200x, and the magnification in Figure 55b is 600x. [Figure 56] Figure 56 shows the results of Western blot analysis confirming the recombinant proteins TOM70-(GGGGS)3-UB-p53 and UB-p53-TOM7 bound to the exogenous mitochondrial outer membrane. [Figure 57] Figure 57 shows the results of observing the degree of intracellular introduction of mitochondria based on mitochondrial concentration after isolating foreign mitochondria and introducing them into cells, using a fluorescence microscope. [Figure 58] Figure 58 shows the effect of normal mitochondria on the proliferation of skin cancer cells. [Figure 59] Figure 59 illustrates the effect of normal mitochondria on suppressing reactive oxygen species (ROS) production in skin cancer cells. [Figure 60] Figure 60 illustrates the role of normal mitochondria in drug resistance. [Figure 61] Figure 61 illustrates the effect of normal mitochondria on the expression of antioxidant genes in cells. [Figure 62] Figure 62 shows the effect of normal mitochondria on the expression of genes involved in cancer cell metastasis. [Figure 63] Figure 63 shows a method for confirming the binding of recombinant protein p53 to the outer membrane of foreign mitochondria and the introduction of recombinant protein p53 into cells. [Figure 64]Figure 64 shows that recombinant protein p53 has bound to the outer membrane of foreign mitochondria and that p53 has been introduced into the cell. In this case, the magnification is 200x. [Figure 65] Figure 65 shows that recombinant protein p53 has bound to the outer membrane of foreign mitochondria and that p53 has been introduced into the cell. In this case, the magnification is 600x. [Figure 66] Figure 66 shows a method for confirming the apoptotic capacity of modified mitochondria bound to p53, which are introduced into cells, using a gastric cancer cell line. [Figure 67a] Figure 67a shows the confirmation of the apoptotic capacity of modified mitochondria bound to p53, introduced into gastric cancer cells, using the TUNEL assay. In this case, the magnification is 600x. [Figure 67b] Figure 67b shows the confirmation of the apoptotic capacity of modified mitochondria bound to p53, introduced into gastric cancer cells, as measured by fluorescence. [Figure 68] Figure 68 shows the effect of RKIP-bound modified mitochondria on suppressing cancer cell metastasis in MDA-MB-231 cells. [Figure 69] Figure 69 shows the expression of a single-stranded variable fragment (ScFv) antibody that targets cancer cells in the cells. [Figure 70] Figure 70 shows confirmation, using immunocytochemistry (ICC) experiments, that a single-stranded variable fragment (ScFv) antibody targeting cancer cells was expressed and bound to mitochondria present in the cells. In this case, the magnification is 200x. [Figure 71] Figure 71 shows the confirmation, using immunocytochemistry (ICC) experiments, that a single-stranded variable fragment (ScFv) antibody targeting cancer cells was expressed and bound to mitochondria present in the cells. In this case, the magnification is 600x. [Figure 72] Figure 72 compares the effects of introducing mitochondria bound to single-chain variable fragment antibodies targeting cancer cells into gastric cancer cell lines. [Figure 73] Figure 73 shows the schedule for animal experiments using modified mitochondria. [Figure 74] Figure 74 is a photograph that visually observes the growth of tumor tissue. [Figure 75] Figure 75 shows the changes in body weight of mice after administration of mitochondria and modified mitochondria. [Figure 76] Figure 76 shows the tumor size after administration of mitochondria and modified mitochondria. [Figure 77] Figure 77 shows that modified mitochondria bound to the TOM-UB-p53 protein are effective in suppressing the proliferation of A431 cells. [Figure 78] Figure 78 shows how the function of isolated mitochondria is confirmed by the amount of ATP. [Figure 79] Figure 79 shows how the function of isolated mitochondria is confirmed by membrane potential. [Figure 80] Figure 80 shows how the degree of damage to isolated mitochondria is confirmed by measuring mitochondrial ROS (mROS production). [Figure 81a] Figure 81a shows the structure of proteins located in the mitochondrial outer membrane, and the amino acid sequences of the N-terminal regions of TOM70, TOM20, or OM45. [Figure 81b] Figure 81b shows the amino acid sequences of the C-terminal regions of TOM5, TOM7, FIs1, VAMP1B, Cytb5, BCL-2, or BCL-X. [Figure 82] Figure 82 is a diagram that shows whether the desired protein is dissociated depending on the presence or absence of a linker between the outer membrane anchoring peptide and ubiquitin. [Figure 83] Figure 83 shows that the desired protein bound to the modified mitochondria is separated from the mitochondria in the cell. [Modes for carrying out the invention]

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

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

[0014] Furthermore, mitochondria can be obtained from oneself, from allogeneic or allogeneic sources, or from different species. In particular, self-mitochondria refer to mitochondria obtained from the tissue or cells of the subject itself. Furthermore, allogeneic mitochondria refer to mitochondria obtained from a subject that belongs to the same species as the subject but has a different allele genotype. Furthermore, heterologous mitochondria refer to mitochondria obtained from a subject that belongs to a different species than the subject.

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

[0016] On the other hand, when isolating mitochondria from specific cells, they can be isolated using various known methods, such as using specific buffer solutions or utilizing potential differences and magnetic fields.

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

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

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

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

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

[0022] The aforementioned fusion proteins can be linked in the following order: N-terminus - Anchoring peptide containing the N-terminal region of TOM20, TOM70, or OM45 - Desired protein - C-terminus.

[0023] Furthermore, the foreign protein may further contain an amino acid sequence recognized by proteolytic enzymes in eukaryotic cells, or ubiquitin or a fragment thereof, between the mitochondrial anchoring peptide and the desired protein. Proteolytic enzymes in eukaryotic cells refer to enzymes present in eukaryotic cells that break down proteins. In this case, since the foreign protein contains an amino acid sequence recognized by the protein-degrading enzyme, the foreign protein bound to the mitochondrial outer membrane can be broken down into the anchoring peptide and the desired protein in the cell.

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

[0025] Furthermore, proteins capable of binding to ligands or receptors present on the cell membrane can also be ligands or receptors present on the tumor cell surface. In this case, ligands or receptors present on the tumor cell surface include CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), membrane-bound mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosine-related protein 1 (tyrp1), tyrosine-related protein 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, and UNC5H2. It may be any one selected from the group consisting of LILRB2, CEACAM, Nectin-3, and combinations thereof, but is not limited to these.

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

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

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

[0029] In one form of a desired protein, an antibody or fragment targeting specific cells may be ligated to an anchoring peptide containing any one C-terminal region selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. Since such a modified mitochondria, bound to the desired protein, can be readily introduced into a specific target, the mitochondria can be effectively introduced into a specific cell.

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

[0031] A desired protein containing one or more active proteins can be configured and bound to mitochondria, depending on the purpose of the modified mitochondria. Furthermore, a desired protein targeting cells can be configured in various ways depending on the target cell.

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

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

[0034] Furthermore, in the pharmaceutical composition, mitochondria may, but are not limited to, be included at concentrations 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. When the mitochondrial content is within these ranges, it may be easier to adjust the mitochondrial dose during administration, potentially improving the degree of improvement in the patient's disease symptoms. In this case, the mitochondrial dose can be determined by quantifying the mitochondrial content by quantifying the membrane proteins of isolated mitochondria. In particular, isolated mitochondria can be quantified by the Bradford protein assay (as described in James D. McCully's literature (J Vis Exp. 2014; (91): 51682)).

[0035] Furthermore, in the pharmaceutical composition, the active protein bound to mitochondria may, but is not limited to, be contained in concentrations 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. When the active protein content is within the above ranges, it may be easier to adjust the active protein dose during administration, and the degree of improvement in the patient's disease symptoms may be improved.

[0036] Furthermore, in the pharmaceutical composition, the targeted protein capable of delivering mitochondria to specific cells may, but is not limited to, be contained in concentrations 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. When the targeted protein content is within the above ranges, it may be easier to adjust the dose of the targeted protein during administration, and the degree of improvement in the patient's disease symptoms may be improved.

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

[0038] Furthermore, the pharmaceutical composition of the present invention may be administered to humans or other mammals who are at risk of developing cancer or who have cancer. The pharmaceutical composition may also be an injectable formulation for intravenous administration or an injectable formulation for topical administration, and is preferably an injectable formulation.

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

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

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

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

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

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

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

[0046] In another aspect of the present invention, the desired protein may be prepared using prokaryotic cells or prokaryotic cell extracts. Furthermore, a method is provided for preparing modified mitochondria using eukaryotic cells or eukaryotic cell extracts that lack ubiquitinases or proteases.

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

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

[0049] In this document, the term "mitochondrial outer membrane anchoring peptide" can refer to 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 specifically present in the mitochondrial outer membrane. In this case, the mitochondrial outer membrane anchoring peptide enables the fusion protein disclosed in this invention to bind to the mitochondrial outer membrane. In this case, the term "mitochondrial outer membrane anchoring peptide" may be used synonymously with "mitochondrial outer membrane targeting peptide."

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

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

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

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

[0054] In this book, the term "active protein" can refer to any protein that exhibits physiological activity. One form of such an active protein may be a protein with reduced function, or a modified protein present in damaged cancer cells. Another form of an active protein may be a protein that enhances cellular activity. Such forms of active proteins are as described above.

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

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

[0057] Furthermore, enzymes known as ubiquitin proteases or UBPs (ubiquitin-specific proteases) are naturally present in eukaryotic cells and can induce the spontaneous degradation of desired proteins by cleaving the C-terminal glycine-glycine moiety of ubiquitin within the cell.

[0058] In this case, the ubiquitin fragment may contain the glycy-glycy amino acid at the C-terminus of ubiquitin, and may contain 3 to 75 consecutive amino acids from the C-terminus. In particular, examples of ubiquitin fragments may be Arg-glycy-glycy, Leu-Arg-glycy-glycy, Arg-Leu-Arg-glycy-glycy, or Leu-Arg-Leu-Arg-glycy-glycy. Furthermore, the ubiquitin fragment may have the amino acid sequence of Sequence ID No. 71.

[0059] A fusion protein containing a mitochondrial outer membrane targeting protein and a desired protein can also be called a fusion protein that modifies mitochondrial activity. Such a fusion protein may have any one of the following structures: <Structural formula 1> N-terminus - mitochondrial outer membrane anchoring peptide - desired protein - C-terminus <Structural formula 2> N-terminus - Mitochondrial outer membrane anchoring peptide - Ubiquitin or its fragment - Desired protein - C-terminus <Structural formula 3> N-terminus - Mitochondrial outer membrane anchoring peptide - Linker 1 - Ubiquitin or its fragment - Desired protein - C-terminus <Structural formula 4> N-terminus - Mitochondrial outer membrane anchoring peptide - Ubiquitin or its fragment - Linker 2 - Desired protein - C-terminus <Structural formula 5> N-terminus - Mitochondrial outer membrane anchoring peptide - Linker 1 - Ubiquitin or its fragment - Linker 2 - Desired protein - C-terminus

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

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

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

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

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

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

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

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

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

[0069] In this book, the term "target" refers to the site where modified mitochondria should be delivered. Examples of targets may include cancer cells. In particular, examples of targets may include biomarkers present on the surface of cancer cells. Specifically, targets may include tumor-associated antigens (TAAs). In this case, the tumor-associated antigen may be any one selected from the group consisting of CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), membrane-bound mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), servibin, tyrosine-related protein 1 (tyrp1), tyrosine-related protein 1 (tyrp2), brachiuri, mesothelin, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms' tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, nectin-3, and combinations thereof.

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

[0071] Examples of targeted proteins can be antibodies or fragments thereof. In particular, they can be antibodies or fragments thereof that specifically bind to tumor-associated antigens. Furthermore, the antibody fragment can be any one selected from the group consisting of Fab, Fab', scFv, and F(ab)2.

[0072] Examples of targeted proteins include scFvHER, which can bind to the epidermal growth factor receptor. Another example is scFvMEL, which can target melanoma. Yet another example is scFvPD-L1, which can bind to PD-L1, which is overexpressed on the surface of cancer cells. Yet another example is PD-1, which can bind to PDL-1, which is overexpressed on the surface of cancer cells.

[0073] One aspect of the present invention may further include ubiquitin or a fragment thereof between the targeting protein and the mitochondrial outer membrane targeting protein. A fusion protein comprising the mitochondrial 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 one of the following structures: <Structural formula 11> N-terminus - Targeted protein - Mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 12> N-terminus - Targeted protein - Ubiquitin or its fragment - Mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 13> N-terminus - Targeted protein - Linker 1 - Ubiquitin or its fragment - Mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 14> N-terminus - Targeted protein - Ubiquitin or its fragment - Linker 2 - Mitochondrial outer membrane anchoring peptide - C-terminus <Structural formula 15> N-terminus - Targeted protein - Linker 1 - Ubiquitin or its fragment - Linker 2 - Mitochondrial outer membrane anchoring peptide - C-terminus

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

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

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

[0077] One aspect of the present invention is to provide a polynucleotide encoding a fusion protein containing a targeted protein.

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

[0079] Furthermore, one aspect of the present invention provides a host cell into which a vector containing a polynucleotide encoding a fusion protein including a targeted protein has been introduced. The host cell may be a prokaryotic or eukaryotic cell. In this case, preferably, the eukaryotic cell may be a strain from which the enzyme that degrades ubiquitin has been removed.

[0080] Furthermore, one aspect of the present invention provides a method for preparing modified mitochondria from cells transformed by introducing polynucleotides encoding a fusion protein into eukaryotic cells. This disclosure relates, for example, to the following: [1] Modified mitochondria in which foreign proteins are bound to the outer membrane of the mitochondria. [2] Mitochondria are modified mitochondria as described in [1] above, isolated from a cell or tissue. [3] The modified mitochondria described in [2] above, wherein the cell is any one selected from the group consisting of somatic cells, germ cells, stem cells, and combinations thereof. [4] The modified mitochondria described in [1], wherein the foreign protein contains a desired protein capable of functioning both inside and outside the cell. [5] The modified mitochondria described in [1] above, wherein the foreign protein contains a mitochondrial anchoring peptide. [6] The modified mitochondria described in [5], wherein an exogenous protein is bound to the mitochondrial outer membrane by a mitochondrial anchoring peptide. [7] The modified mitochondria described in [6], wherein the mitochondrial anchoring peptide comprises the N-terminal or C-terminal region of a protein present in the mitochondrial membrane protein. [8] The modified mitochondria described in [7], wherein the N-terminal or C-terminal region of a protein present in the mitochondrial membrane protein is located on the mitochondrial outer membrane. [9] The modified mitochondria described in [7], wherein the protein present in the mitochondrial membrane protein is any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

[10] The modified mitochondrion according to [7], wherein the anchoring peptide comprises any one N-terminal region selected from the group consisting of TOM20, TOM70, and OM45. Mutant mitochondria.

[11] The modified mitochondrion according to [7], wherein the mitochondrial anchoring peptide comprises any one C-terminal region selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

[12] The modified mitochondrion according to [1], wherein the foreign protein is a fusion protein comprising a mitochondrial anchoring peptide and a desired protein capable of functioning both inside and outside the cell.

[13] The modified mitochondrion according to

[12] , wherein the desired protein is any one selected from the group consisting of active proteins that exhibit activity in cells, proteins present in cells, and proteins that have the ability to bind to ligands or receptors present in the cell membrane.

[14] A modified mitochondrion as described in

[13] , wherein the desired protein is any one selected from the group consisting of p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun / Fos), EGR-1, retinoblastoma (RB), phosphatase tensin homolog (PTEN), E-cadherin, neurofibromin-2 (NF-2), poly[ADP-ribose] synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis of the colon (APC), tumor necrosis factor receptor-related factor (TRAF), RAF kinase inhibitor protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, Oct3 / 4, Sox2, Klf4, and c-Myc.

[15] The modified mitochondria described in

[12] , wherein the foreign protein is a desired protein ligated to the N-terminal region of TOM20, TOM70, or OM45.

[16] Foreign proteins, N-terminus - N-terminal region of TOM20, TOM70, or OM45 - Desired protein - C-terminus The modified mitochondria described in

[15] above are linked in the order of

[15] .

[17] The modified mitochondria described in

[16] further comprising an amino acid sequence recognized by proteolytic enzymes in eukaryotic cells, or ubiquitin or a fragment thereof, between the anchoring peptide and the desired protein.

[18] The modified mitochondrion according to

[17] , wherein the ubiquitin fragment contains a C-terminal glycine-glycine of the amino acid sequence of SEQ ID NO: 71, and comprises 3 to 75 consecutive amino acids from the C-terminus.

[19] The modified mitochondrion according to

[17] , wherein the foreign protein further comprises a linker between the desired protein and ubiquitin or a fragment thereof.

[20] A modified mitochondria as described in

[19] , wherein the linker consists of 1 to 150 amino acids.

[21] The modified mitochondria described in

[20] , wherein the linker consists of 5 to 50 amino acids comprising glycine and serine.

[22] The modified mitochondrial described in

[21] above, wherein the linker is (G4S)n, where n is an integer from 1 to 10.

[23] The modified mitochondria described in

[13] , wherein a protein having the ability to bind to a ligand or receptor present on the cell membrane is a ligand or receptor present on the surface of a tumor cell.

[24] The ligands or receptors present on the surface of tumor cells include CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), membrane-bound mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosine-related protein 1 (tyrp1), tyrosine-related protein 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, and UNC5H2. A modified mitochondrion as described in

[23] , which is any one selected from the group consisting of LILRB2, CEACAM, Nectin-3, or a combination thereof.

[25] The modified mitochondrion described in

[12] , wherein an exogenous protein is ligated to any one C-terminus selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

[26] Foreign proteins, N-terminus - Desired protein - Any one C-terminal region selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B - C-terminus The modified mitochondria described in

[25] above are linked in the order of

[25] .

[27] The modified mitochondrion according to

[26] , wherein the foreign protein further comprises a linker between the desired protein and any one C-terminal region selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

[28] The modified mitochondria described in

[27] , wherein the linker consists of 1 to 150 amino acids.

[29] The modified mitochondria described in

[28] , wherein the linker consists of 5 to 50 amino acids comprising glycine and serine.

[30] The modified mitochondrial described in

[21] above, wherein the linker is (G4S)n, where n is an integer from 1 to 10.

[31] A pharmaceutical composition comprising a modified mitochondria as described in any of the above [1] to

[30] as an active ingredient.

[32] The pharmaceutical composition according to

[31] , wherein the pharmaceutical composition is for the prevention or treatment of cancer.

[33] The pharmaceutical composition according to

[32] , 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.

[34] The use of modified mitochondria as a means of intracellular and extracellular delivery of foreign proteins, including desired proteins capable of functioning both inside and outside cells.

[35] The use of the modified mitochondria described in

[34] , wherein the foreign protein comprises a mitochondrial outer membrane anchoring peptide, which is bound to the mitochondrial outer membrane by the outer membrane anchoring peptide, and is delivered to the inside and outside of the cell.

[36] A fusion protein containing a mitochondrial outer membrane anchoring peptide and a desired protein capable of functioning both inside and outside the cell.

[37] The fusion protein according to

[36] , wherein the mitochondrial anchoring peptide comprises the N-terminal or C-terminal sequence of a protein present in the mitochondrial outer membrane.

[38] The fusion protein according to

[36] , wherein the mitochondrial outer membrane 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.

[39] The fusion protein described in

[36] , wherein the desired protein is any one selected from the group consisting of p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun / Fos), EGR-1, retinoblastoma (RB), phosphatase tensin homolog (PTEN), E-cadherin, neurofibromin-2 (NF-2), poly[ADP-ribose] synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis of the colon (APC), tumor necrosis factor receptor-related factor (TRAF), RAF kinase inhibitor protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, Oct3 / 4, Sox2, Klf4, and c-Myc.

[40] The fusion protein according to

[36] , wherein the mitochondrial outer membrane anchoring peptide is TOM20, TOM70, or OM45, and the mitochondrial outer membrane anchoring peptide and the desired protein are linked from the N-terminus to the C-terminus.

[41] The fusion protein described in

[36] , wherein the mitochondrial outer membrane anchoring peptide is any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B, and the desired protein and the mitochondrial outer membrane anchoring peptide are linked from the N-terminus to the C-terminus.

[42] The fusion protein according to

[36] , wherein the fusion protein further comprises ubiquitin or a fragment thereof between the mitochondrial outer membrane anchoring peptide and the desired protein.

[43] The fusion protein according to

[36] , wherein the fusion protein further comprises an amino acid sequence recognized by proteolytic enzymes in eukaryotic cells between the mitochondrial outer membrane anchoring peptide and the desired protein.

[44] A polynucleotide encoding a fusion protein as described in any of the above

[36] to

[43] .

[45] The steps of introducing the polynucleotides described in

[44] into prokaryotic cells or eukaryotic cells that lack ubiquitinases or proteases; and Steps to obtain a fusion protein A method for producing a fusion protein as described in any of the above

[36] to

[43] , including the above.

[46] A fusion protein comprising a targeted protein having the ability to bind to a ligand or receptor present in the cell membrane, and a mitochondrial outer membrane anchoring peptide.

[47] The fusion protein described in

[46] , wherein the mitochondrial outer membrane anchoring peptide is any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

[48] The fusion protein described in

[46] , wherein a targeted protein having the ability to bind to a ligand or receptor present on the cell membrane and a mitochondrial outer membrane anchoring peptide are linked from the N-terminus to the C-terminus.

[49] The fusion protein according to

[48] , wherein the targeted protein having the ability to bind to a ligand or receptor present on the cell membrane is an antibody or a fragment thereof.

[50] The fusion protein according to

[49] , wherein the antibody fragment is any one selected from the group consisting of Fab, Fab', scFv, and F(ab)2.

[51] A polynucleotide encoding a fusion protein as described in any of the above

[46] to

[50] .

[52] A method for producing modified mitochondria, comprising the step of mixing isolated mitochondria with a fusion protein described in any of

[36] to

[43] and / or a fusion protein described in any of

[46] to

[50] .

[53] A method for preparing modified mitochondria from transformed cells by introducing a fusion protein described in any of

[36] to

[41] above and / or a polynucleotide encoding a fusion protein described in any of

[46] to

[50] above into eukaryotic cells. [Examples]

[0081] To aid in understanding the present invention, preferred embodiments are shown below. However, the following embodiments are provided solely to facilitate understanding of the present invention, and the present invention is not limited to these embodiments. I. Preparation of a fusion protein containing mitochondrial outer membrane anchoring peptide, linker, ubiquitin, and the desired protein. Example 1. Preparation of a fusion protein containing p53 Example 1.1. Amplification of the p53 gene To express human p53 in recombinant protein, total RNA was extracted from human epithelial cells, and cDNA was synthesized from it. Specifically, human dermal fibroblasts were cultured in 10% serum medium under conditions of 5% carbon dioxide and 37°C (1 × 10⁻¹⁶). 6 Cells). Subsequently, the culture medium was removed, the cells were washed twice with PBS buffer, and 0.5 ml of RNA extraction reagent (Trizol reagent, Thermo Fisher Scientific) was added directly. The mixture with the RNA extraction reagent was left at ambient temperature for 10 minutes, then 0.1 ml of chloroform was added, stirred for 15 seconds, and then centrifuged at approximately 12000 × g for 10 minutes. Next, the separated supernatant was taken, an equal volume of isopropyl alcohol was added, and it was centrifuged again at 12000 × g for 10 minutes. Then, the liquid was removed, washed once with 75% ethanol, and the RNA was dried at ambient temperature.

[0082] Approximately 50 μl of purified distilled water without RNAase was added, and the amount and purity of RNA were measured using a spectrophotometer. To synthesize cDNA, 2 μg of purified total RNA was subjected to a binding reaction with oligo-dT at 70°C for 5 minutes. Subsequently, 10× reverse transcription buffer, 10 mM dNTPs, an RNAse inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea) were added, and the cDNA synthesis reaction was carried out at 42°C for 60 minutes. After that, the reverse transcriptase was inactivated by heating at 72°C for 5 minutes, and then RNaseH was added to remove single-stranded RNA, which was used as a template for the polymerase chain reaction of the p53 gene.

[0083] To obtain the p53 gene from which the signal peptide sequence was removed from human dermal fibroblasts, primers encoding from the amino-terminal glutamic acid (T2p53) and primers encoding from the carboxyl-terminal (Xp53) were synthesized, and PCR was performed using the cDNA prepared as described above as a template. The sequences of each primer are shown in Table 1.

[0084] [Table 1]

[0085] 0.2 pmol of T2p53 primer and 0.2 pmol of Xp53 primer were mixed with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The mixture was then amplified in a polymerase chain reaction system for 40 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. After the reaction, an amplified DNA fragment of approximately 1.2 kbp was isolated by electrophoresis on a 1% agarose gel and then inserted into a pGEM-T easy (Promega, USA) vector using T4 DNA ligase. Sequencing of the resulting DNA confirmed the acquisition of cDNA encoding the human p53 protein. The obtained p53 gene was designated pTA-p53, and its base sequence is identical to that of Sequence ID No. 3 (Figure 1).

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

[0087] [Table 2]

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

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

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

[0091] Example 1.2.2. Preparation of plasmid pET11c-TOM70-UB-p53 To prepare a form of p53 protein fused with TOM70 and ubiquitin that binds to the mitochondrial outer membrane, an expression vector capable of expressing this fused form of p53 was constructed. NdeTOM70 primers, TOM70-AS primers, TOM70UB-S primers, and T2UB-AS primers were prepared to obtain the TOM70 and ubiquitin genes. The sequences of each primer are shown in Table 3.

[0092] [Table 3]

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

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

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

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

[0097] A portion of the E. coli cells was obtained by centrifugation, and the cells were destroyed and subjected to SDS-polyacrylamide electrophoresis. As shown in Figure 5, it was confirmed that a p53 protein of approximately 62 kDa in a fused form of TOM70 and ubiquitin was expressed. In this case, lane M shows the protein molecular weight marker, and lane 1 shows the supernatant obtained by centrifugation after E. coli was destroyed 4 hours after the addition of IPTG.

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

[0099] [Table 4]

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

[0101] Subsequently, in a polymerase chain reaction (CRR), amplification was carried out for 25 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute to obtain UB-p53, in which p53 was fused with the ubiquitin gene. The amplified TOM70-G3 gene was cleaved with restriction enzymes NdeI and BamHI, and the amplified UB-p53 gene was cleaved with restriction enzymes BamHI and XhoI. 100 bp and 1500 bp DNA fragments were obtained by electrophoresis on a 2% agarose gel, respectively. These fragments were then inserted into a pET11c vector cleaved 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 was represented by the nucleotide sequence of Sequence ID No. 15.

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

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

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

[0105] [Table 5]

[0106] Using the plasmid pET11c-TOM70-UB-p53 obtained in Example 1.2.2 above as a template, 0.2 pmol of NdeTOM70 primer and 0.2 pmol of TOM70(G)3-AS primer were added, and 0.2 nM dNTPs, 1 × AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase were mixed. Subsequently, the amplification reaction was carried out in a polymerase chain reaction apparatus for 25 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute to obtain the TOM70 gene. The amplified DNA fragment was named TOM70-G3.

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

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

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

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

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

[0112] [Table 6]

[0113] Using the plasmid pET15b-UB-p53 obtained in Example 1.2.1 as a template, 0.2 pmol of NdeUB primer and 0.2 pmol of Xp53(noT) primer were added, and 0.2 nM of dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase were mixed. Subsequently, the amplification reaction was carried out in a polymerase chain reaction apparatus for 25 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute to obtain the UB-p53 gene. Furthermore, using the cDNA prepared as described above as a template, 0.2 pmol of XTOM7 primer and 0.2 pmol of LTOM7 primer were mixed with 0.2 nM of dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase.

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

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

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

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

[0118] [Table 7]

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

[0120] The amplified p53 gene was digested with the restriction enzymes EcoRI and XhoI, and a DNA fragment of approximately 1300 bp was obtained by electrophoresis on a 2% agarose gel. This fragment was then inserted into the pcDNA3.1-myc / His A vector, which had been digested with the restriction enzymes EcoRI and XhoI, using T4 DNA ligase to obtain the plasmid pCMV-p53-myc / His (Figure 12). In this case, p53-myc / His was represented by the nucleotide sequence of Sequence ID No. 23.

[0121] Animal cells (CHO) were transformed using the plasmid pCMV-p53-myc / His, the cells were disrupted, and SDS-polyacrylamide electrophoresis was performed, followed by detection by Western blotting using an anti-c-myc antibody. As shown in Figure 13, the expression of a p53 protein approximately 55 kDa in size was confirmed. In this case, lane M shows the protein molecular weight marker, and lane 1 indicates that transfection of animal cells (CHO) was performed, the cells were disrupted, SDS-polyacrylamide electrophoresis was performed, and confirmation by Western blotting using an anti-c-myc antibody was performed.

[0122] Example 1.3. Isolation and purification of fusion protein containing p53 Example 1.3.1. Isolation and purification of recombinant TOM70-(GGGGS)3-p53 protein derived from Escherichia coli. A recombinant E. coli BL21(DE3) strain expressing the TOM70-(GGGGS)3-p53 protein was seeded in LB liquid medium and cultured at 37°C. When the OD600 absorbance reached 0.4, 0.5 mM IPTG was added, and the culture was shaken for a further 4 hours to express the TOM70-(GGGGS)3-p53 protein.

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

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

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

[0126] Example 1.3.2. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-p53 protein derived from Escherichia coli. Using Escherichia coli expressing the TOM70-(GGGGS)3-UB-p53 recombinant protein, the TOM70-(GGGGS)3-UB-p53 protein was isolated and purified using the same method as in Example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-p53 protein was eluted (Figure 16). In this case, lane M in Figure 16 shows the protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows samples that 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 4-7 show the results of elution with 8M urea / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8-11 show the elution results using an 8M urea / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution.

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

[0128] Example 1.3.3. Isolation and purification of recombinant UB-p53 protein derived from Escherichia coli. BL21(DE3) strains expressing the mature UB-p53 protein fused with ubiquitin were seeded in LB liquid medium and cultured in a 37°C shaking incubator. When the OD600 absorbance reached 0.4, 0.5 mM IPTG was added, and the culture was continued for another 4 hours under shaking conditions to express the mature UB-p53 protein fused with ubiquitin.

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

[0130] The protein amount of the recovered eluate was measured by a protein quantification method and confirmed by SDS-PAGE. As shown in Fig. 19, after confirmation, the UB-p53 protein was cooled with liquid nitrogen and stored in an ultra-low temperature freezer at -80°C. In this case, lane M in Fig. 19 indicates the protein molecular weight marker, and lane 1 indicates the UB-p53 protein obtained by dialysis with PBS buffer solution.

[0131] Example 1.3.4. Isolation and Purification of Recombinant UB-p53-TOM7 Protein Induced from Escherichia coli A production strain of Escherichia coli BL21(DE3) expressing the mature UB-p53-TOM7 protein fused with ubiquitin was inoculated into LB liquid medium and cultured under the condition of 37°C. When the absorbance at OD600 reached 0.4, 0.5 mM IPTG was added, and further shaking culture was carried out for 4 hours to express the mature form of UB-p53-TOM7 protein fused with ubiquitin.

[0132] Thereafter, the UB-p53-TOM7 protein was isolated 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, lane M in Fig. 20 indicates the protein molecular weight marker, lane 1 indicates the loading sample of nickel affinity chromatography. Lane 2 indicates those not bound to nickel affinity resin. Lane 3 shows the elution result with 8M urea / 50mM sodium phosphate / 500mM NaCl / 10mM imidazole solution. Lane 4 shows the elution result with 8M urea / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5-7 show the elution results with 8M urea / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8-9 show the elution results with 8M urea / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution. Lanes 10-11 show the elution results with 8M urea / 50mM sodium phosphate / 500mM NaCl / 500mM imidazole solution.

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

[0134] Example 2. Preparation of a fusion protein containing granzyme B Example 2.1. Amplification of the granzyme B gene To express human granzyme B in recombinant proteins, total RNA was extracted from human natural killer cells, and cDNA was synthesized from it. Specifically, human natural killer cells were cultured in 10% serum medium under conditions of 5% carbon dioxide and 37°C (1 × 10⁻¹⁶). 6 (Cells). Subsequently, RNA was obtained using the same method as in Example 1.1, and it was used as a template for the polymerase chain reaction of the granzyme B gene.

[0135] To obtain the granzyme B gene from which the signal peptide sequence was removed from human natural killer cells, T2GZMB primers encoding from the amino-terminal isoleucine and XGZMB(noT) primers encoding from the carboxyl terminus were synthesized, and PCR was performed using the cDNA prepared as described above as a template. The sequences of each primer are shown in Table 8.

[0136] [Table 8]

[0137] Using the cDNA synthesized as described above as a template, 0.2 pmol of T2GZMB primer and 0.2 pmol of XGZMB(noT) primer were mixed with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. Subsequently, the amplification reaction was carried out for 40 cycles in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute. After the reaction, an amplified DNA fragment of approximately 700 bp was isolated by electrophoresis on a 1% agarose gel and then inserted into a pGEM-T easy (Promega, USA) vector using T4 DNA ligase. Sequencing of the resulting DNA confirmed that cDNA encoding human granzyme B protein was obtained. The obtained granzyme B gene was named pTA-granzyme B, and the granzyme B gene was represented by the nucleotide sequence of sequence number 26 (Figure 22).

[0138] Example 2.2. Preparation of an E. coli expression vector for granzyme B protein Example 2.2.1. Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-granzyme B To prepare a granzyme B protein in which TOM70, which binds to the mitochondrial outer membrane, a linker (GGGGSGGGGSGGGGS), and ubiquitin are fused, we created an expression vector capable of expressing this granzyme B protein in its fused form.

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

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

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

[0142] Example 2.2.2. Preparation of plasmid pET15b-UB-granzyme B-TOM7 To prepare a granzyme B protein in which ubiquitin and TOM7, which binds to the mitochondrial outer membrane, were fused, an expression vector was created that could express a granzyme B protein in which ubiquitin, granzyme B, and TOM were fused in this order.

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

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

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

[0146] Example 2.3. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-granzyme B protein derived from Escherichia coli. TOM70-(GGGGS)3-UB-granzyme B protein was isolated and purified using the same method as in Example 1.3.1. As a result, TOM70-(GGGGS)3-UB-granzyme B protein was eluted (Figure 27). In this case, lane M in Figure 27 shows the protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows samples that 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 5-7 show the results of elution with 8M urea / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 8-9 show the results of elution with 8M urea / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution.

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

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

[0149] To obtain the gene of RKIP with the signal peptide sequence removed from human skin fibroblasts, primers T2RKIP encoding from the amino-terminal proline and XRKIP(noT) encoding from the carboxyl-terminal were synthesized, and PCR was performed using the cDNA prepared as described above as a template. The sequences of each primer are listed in Table 9.

[0150]

Table 9

[0151] Using the cDNA synthesized as described above as a template, 0.2 pmol of T2RKIP primer and 0.2 pmol of XRKIP(noT) primer were mixed with 0.2 nM dNTP, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. Then, in a polymerase chain reaction apparatus, an amplification reaction at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute was performed for 40 cycles. After the reaction, an amplified DNA fragment of about 560 bp was isolated by electrophoresis on a 1% agarose gel and then inserted into the pGEM-T easy (Promega, USA) vector using T4 DNA ligase. As a result of sequencing the DNA obtained in this way, it was confirmed that a cDNA encoding human RKIP protein was obtained. The obtained RKIP gene was designated pTA-RKIP (Figure 29), and the nucleotide sequence of the RKIP gene was represented by the nucleotide sequence of SEQ ID NO: 31.

[0152] Example 3.2. Preparation of Escherichia coli expression vector for RKIP protein Example 3.2.1. Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-RKIP To prepare the RKIP protein in a fused form of TOM70, a linker (GGGGSGGGGSGGGGS), and ubiquitin, which binds to the mitochondrial outer membrane, we created an expression vector capable of expressing RKIP in this fused form.

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

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

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

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

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

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

[0159] Example 4. Preparation of a fusion protein containing PTEN Example 4.1. Amplification of the PTEN gene To express human PTEN (phosphatase tensin homolog) in recombinant proteins, total RNA was extracted from human epithelial cells, and cDNA was synthesized from it. Fibroblasts (human dermal fibroblasts) were cultured in 10% serum medium under conditions of 5% carbon dioxide and 37°C (1 × 10⁻¹⁶). 6 (Cells). Subsequently, RNA was obtained using the same method as in Example 1.1, and it was used as a template for the polymerase chain reaction of the PTEN gene.

[0160] To obtain the PTEN gene from which the signal peptide sequence was removed from human dermal fibroblasts, T2PTEN primers encoding from the amino-terminal threonine and XPTEN(noT) primers encoding from the carboxyl-terminal were synthesized, and PCR was performed using the cDNA prepared as described above as a template. The sequences of each primer are shown in Table 10.

[0161] [Table 10]

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

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

[0164] The pTA-PTEN gene plasmid obtained in Example 4.1 above was cleaved with restriction enzymes SacII and XhoI, and a DNA fragment of approximately 1200 bp was obtained by electrophoresis on a 2% agarose gel. Then, using T4 DNA ligase, it was inserted into the pET11c-TOM70-(GGGGS)3-UB-(p53) vector, which had been cleaved with restriction enzymes SacII and XhoI, to obtain the plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN (Figure 35). Here, TOM70-(GGGGS)3-UB-PTEN was represented by the nucleotide sequence of Sequence ID No. 36.

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

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

[0167] Example 4.3. Isolation and purification of recombinant TOM70-(GGGGS)3-UB-PTEN protein derived from Escherichia coli. Recombinant TOM70-(GGGGS)3-UB-PTEN protein was isolated and purified using the same method as in Example 1.3.1. As a result, TOM70-(GGGGS)3-UB-PTEN protein was eluted (Figure 37). In this case, lane M in Figure 37 shows the protein molecular weight marker, and lane 1 shows the loading sample for nickel affinity chromatography. Lane 2 shows samples that 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 solution. Lane 4 shows the results of elution with 8M urea / 50mM sodium phosphate / 500mM NaCl / 50mM imidazole solution. Lanes 5-8 show the results of elution with 8M urea / 50mM sodium phosphate / 500mM NaCl / 100mM imidazole solution. Lanes 9-10 show the elution results with an 8M urea / 50mM sodium phosphate / 500mM NaCl / 250mM imidazole solution. Lanes 111 show the elution results with an 8M urea / 50mM sodium phosphate / 500mM NaCl / 500mM imidazole solution.

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

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

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

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

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

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

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

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

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

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

[0178] Example 6.2. Preparation of scFvHER2 protein expression vector Example 6.2.1. pET15b-UB-scFvHER2-TOM7 To prepare the scFvHER2 protein in a fused form of ubiquitin and TOM7, which binds to the mitochondrial outer membrane, we created an expression vector capable of expressing the scFvHER2 gene in this fused form.

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

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

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

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

[0183] [Table 11]

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

[0185] In this case, scFvHER2-TOM7-myc / His was represented by the nucleotide sequence of Sequence ID No. 41. This plasmid pCMV-scFvHER2-TOM7-myc / His was transfected into animal cells (CHO), the cells were disrupted, and SDS-polyacrylamide electrophoresis was performed, followed by Western blotting using an anti-c-myc antibody. As shown in Figure 46, it was confirmed that an scFvHER2 protein of approximately 35 kDa in a fused form with TOM7 was expressed. In this case, lane M in Figure 46 shows the protein molecular weight marker, and lane 1 shows that transfection into animal cells (CHO) was performed, the cells were disrupted, SDS-polyacrylamide electrophoresis was performed, and confirmation was performed by Western blotting using an anti-c-myc antibody.

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

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

[0188] Example 7. Preparation of a fusion protein containing scFvMEL Example 7.1. Synthesis of the scFvMEL gene To express human scFvMEL as an antibody fragment against melanoma in recombinant protein, the scFvMEL gene obtained from Bionics Co., Ltd. through a gene synthesis request was named pUC57-scFvMEL, and the base sequence of scFvMEL was the same as the base sequence of Sequence ID No. 42.

[0189] Example 7.2. Preparation of scFvMEL protein expression vector Example 7.2.1. Preparation of pET15b-UB-scFvMEL-TOM7 To prepare the scFvMEL protein in a fused form of ubiquitin and TOM7, which binds to the mitochondrial outer membrane, we created an expression vector capable of expressing the scFvMEL in this fused form.

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

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

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

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

[0194] [Table 12]

[0195] Using the plasmid pET15b-UB-scFvMEL-TOM7 obtained in Example 6.2.1 as a template, 0.2 pmol of RscFvMEL primer and 0.2 pmol of XTOM7(noT) primer were mixed with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. Subsequently, the amplification reaction was carried out in a polymerase chain reaction apparatus for 25 cycles at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute to obtain scFvMEL-TOM7. The amplified scFvMEL-TOM7 gene was cleaved with restriction enzymes EcoRI and XhoI, and DNA fragments of approximately 850 bp were obtained by electrophoresis on a 1% agarose gel. Next, this was inserted into a pcDNA3.1-myc / His A vector, which had been cleaved with restriction enzymes EcoRI and XhoI using T4 DNA ligase, to obtain the plasmid pCMV-scFvMEL-TOM7-myc / His (Figure 51). Here, scFvMEL-TOM7-myc / His is sequence number 4. It was represented by the base sequence.

[0196] This was transfected into animal cells (CHO) using the plasmid pCMV-scFvMEL-TOM7-myc / His, the cells were disrupted, and SDS-polyacrylamide electrophoresis was performed, followed by Western blotting using an anti-c-myc antibody. As shown in Figure 52, it was confirmed that an scFvMEL protein of approximately 35 kDa in a fused form with TOM7 was expressed. In this case, lane M in Figure 52 shows the protein molecular weight marker, and lane 1 shows that transfection into animal cells (CHO) was performed, the cells were disrupted, SDS-polyacrylamide electrophoresis was performed, and confirmation was performed by Western blotting using an anti-c-myc antibody.

[0197] Example 8. Preparation of a fusion protein containing scFvPD-L1 Example 8.1. Synthesis of the scFvPD-L1 gene To express human scFvPD-L1 in recombinant protein, the scFvPD-L1 gene obtained from Bionics Co., Ltd. through a gene synthesis request was designated pUC57-scFvPD-L1, and its base sequence was identical to that of sequence number 46.

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

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

[0200] This was transfected into animal cells (CHO) using the plasmid pCMV-scFvPD-L1-TOM7-myc / His, the cells were disrupted, and SDS-polyacrylamide electrophoresis was performed, followed by Western blotting using an anti-c-myc antibody. As shown in Figure 54, it was confirmed that the scFvPD-L1 protein, approximately 35 kDa in size and fused with TOM7, was expressed. In this case, lane M in Figure 54 shows the protein molecular weight marker, and lane 1 indicates that transfection into animal cells (CHO) was performed, the cells were disrupted, SDS-polyacrylamide electrophoresis was performed, and confirmation was performed by Western blotting using an anti-c-myc antibody.

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

[0202] Subsequently, unreacted proteins were removed by centrifugation, and the samples were washed twice with PBS buffer. The fluorescent proteins bound to mitochondria were then observed using a fluorescence microscope. A purified GFP protein without the mitochondrial outer membrane binding site was used as a control group. As a result, it was confirmed that the fluorescent protein fused with 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) (Figures 55a and 55b).

[0203] Example 10. Confirmation of the ability of recombinant protein p53 to bind to the foreign mitochondrial outer membrane. 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 bound in a 1:1 ratio for 1 hour under reaction conditions of 4°C. Mitochondria that were not mixed with the protein were used as a control group. The binding affinity between mitochondria and p53 was confirmed by Western blotting (Figure 56).

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

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

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

[0207] Proliferation of cutaneous epidermal cancer cells and production of reactive oxygen species (ROS) were observed 24, 48, and 72 hours after introduction. The results confirmed that when mitochondria obtained from normal cells derived from various organs were introduced into cancer cells, they had a concentration-dependent inhibitory effect on cancer cell proliferation. Furthermore, it was confirmed that ROS production in cancer cells was suppressed in a concentration-dependent manner with normal mitochondria (Figures 58 and 59).

[0208] Example 13. Confirmation of the effect of normal mitochondria on drug resistance. The effects of introducing mitochondria derived from normal cells into cancer cells on characteristic cancer cell traits such as drug resistance, antioxidant gene expression, and cancer metastasis were investigated using the following method. First, normal hepatocytes (WRL-68) were used as mitochondrial donor cells, and mitochondria were isolated from the cells by centrifugation. HepG2 cells, a liver cancer cell line, were used as the cancer cell line to be used as the mitochondrial recipient cells. Mitochondria were delivered to liver cancer cells using centrifugal force according to concentration, and as a result of observing drug resistance to the anticancer drug doxorubicin, it was confirmed that the cancer cell line that received mitochondria showed higher drug sensitivity (Figure 60).

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

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

[0211] Example 16. Confirmation of binding of recombinant protein p53 to the outer membrane of foreign mitochondria and introduction into cells. Mitochondria were isolated from umbilical cord-derived mesenchymal stem cells by centrifugation, stained with Mitotracker CMX Ros, and mixed with purified recombinant protein TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 in a 1:1 ratio. The mixture was incubated at 4°C for 1 hour under reaction conditions, then centrifuged to remove unreacted protein. After washing twice with PBS buffer, mitochondria bound to p53 protein were introduced into gastric cancer cell line SNU-484 cells by centrifugation (Figure 63). In this case, the control groups were set as a group without mitochondria and a group using mitochondria alone. After 1 day of culture, the p53 protein bound to the exogenous mitochondria introduced into the cells was observed by fluorescence microscopy using immunocytochemistry (ICC).

[0212] Rabbit anti-p53 antibody was used as the primary antibody, and goat anti-rabbit IgG Alexa Fluor 488 was used as the secondary antibody. As a result, it was confirmed that the TOM70-(GGGGS)3-UB-p53 (stained green) or UB-p53-TOM7 (stained green) proteins bound to foreign mitochondria (stained red) were located in the cytoplasm of the introduced cells during introduction into the cells (Figure 64, 200x, and Figure 65, 400x). Consequently, it was found that the recombinant proteins were readily introduced into the cells by mitochondria.

[0213] Example 17. Confirmation of p53-binding mitochondria activity in cancer cell lines. Example 17.1. Confirmation of the apoptotic capacity of p53-bound exogenous mitochondria introduced into gastric cancer cells using a gastric cancer cell line. 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 bound in a 1:1 ratio for 1 hour under reaction conditions of 4°C. As control groups, UB-p53 protein without TOM70 and TOM70-(GGGGS)3-p53 without ubiquitin were used. Unbound proteins were removed by centrifugation and washing with PBS, and the protein-bound mitochondria were introduced by centrifugation into gastric cancer cell line SNU-484, which lacks p53 ability due to a mutation in the p53 gene (Figure 66). After 1 day of culture, cells were fixed with 4% paraformaldehyde for 1 hour, then osmoticized using a osmotic solution (0.1% sodium citrate buffer containing 0.1% Triton-X-100, pH 7.4), and reacted with TUNEL solution (in situ cell death detection kit, TMR RED, Roche) at 37°C for 1 hour.

[0214] In TUNEL analysis, regions where nucleic acid fragmentation (DNA fragmentation) has occurred are stained red, indicating that apoptosis has occurred. Compared to the control group, cells into which mitochondria bound to TOM70-(GGGGS)3-ub-p53 or p53-TOM7 were introduced showed a greater number of red-stained regions, unlike the control group, indicating that apoptosis was induced by mitochondria bound to TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7. In particular, it was confirmed that the induction of apoptosis by mitochondria bound to the TOM70-(GGGGS)3-UB-p53 protein was greater (Figure 67a).

[0215] Example 17.2. Confirmation of apoptotic capacity of p53-bound exogenous mitochondria by luciferase binding. To confirm whether the biological activity of the TOM70-(GGGGS)3-UB-p53 protein delivered to recipient cells is maintained after delivery to recipient cells of the mitochondrial-bound form of the TOM70-(GGGGS)3-UB-p53 protein obtained in Example 5.2, cell-based analysis using a reporter gene was performed. Since the p53 protein is a transcription factor, a gene containing six repeats of the nucleotide sequence RRRCWWGYYY (where R represents G or A, W represents A or T, and Y represents C or T), to which the p53 transcription factor can bind, was synthesized with the following sequence. The nucleotide sequence of P53-promoter-S is as follows (5'-GGG CAT GCT CGG GCA TGC CCG GGC ATG CTC GGG CAT GCC CGG GCA TGC TCG GGC ATG CCC-3') (Sequence ID 91), and the nucleotide sequence of P53-promoter-AS is as follows (5'-GGG CAT GCC CGA GCA TGC CCG GGC ATG CCC GAG CAT GCC CGG GCA TGC CCG AGC ATG CCC-3') (Sequence ID 92).

[0216] 5 μg of the synthetic gene P53-promoter-S and 5 μg of the synthetic gene P53-promoter-AS were incubated at 70°C for 20 minutes to promote the synthesis of the double helix gene, and then phosphorylation was induced using a polynucleotide T4 kinase enzyme. The phosphorylated double helix gene was inserted into a pGL3 vector cleaved with the restriction enzyme SmaI, and a gene containing six repeats of the nucleotide sequence (RRRCWWGYYY) to which the p53 transcription factor can bind was conjugated to the reporter gene luciferase to construct plasmid p6xp53-Luc. Plasmid p6xp53-Luc and the β-galactosidase expression vector plasmid pRSVb-gal were introduced into human kidney cells (HEK293 cells) using the lipofectamine method.

[0217] Six hours later, HEK293 cells were treated with 10 μg of mitochondria and combinations of 5 μg, 10 μg, and 20 μg of TOM70-(GGGGS)3-UB-p53 protein, respectively. As a control group, cells were treated with either PBS or 10 μg of mitochondria conjugated with p53 protein. After culturing the treated cells for 18 hours, luciferase activity was measured and analyzed. In this case, to correct for the effect of transduction, the corrected luciferase value was determined by dividing the luciferase value by the value obtained from measuring β-galactosidase activity.

[0218] Luciferase levels were elevated in cells treated with combinations of 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 exhibited activity (Figure 67b).

[0219] Example 18. Confirmation of the ability of RKIP-bound exogenous mitochondria introduced into cells to reduce metastasis of cancer cell lines. Mitochondria isolated from umbilical cord-derived mesenchymal stem cells by centrifugation were mixed with the purified recombinant protein TOM70-(GGGGS)3-UB-RKIP and bound in a 1:1 ratio for 1 hour under reaction conditions of 4°C. The protein-bound mitochondria were introduced by centrifugation into the breast cancer cell line MDA-MB-231, which is known to have increased metastatic potential due to a decrease in RKIP protein.

[0220] To confirm the metastatic potential of cancer cells, a cell invasion assay using Transwell plates was performed. Transwell upper chambers with a pore size of 8 μm were coated with Matrigel at 37°C for 30 minutes. The test groups consisted of MDA-MB-231 cells introduced with single mitochondria and MDA-MB-231 cells introduced with mitochondria bound to RKIP protein. Each cell was placed in a Transwell upper chamber containing serum-free medium at a rate of 1 × 10⁶ 5Cells were placed in individual cells and culture medium containing 10% bovine serum was placed in the lower chamber. After incubation at 37°C for 12 hours, the cells were fixed with 4% paraformaldehyde for 1 hour, and then the cells that passed through Matrigel were stained with 1% crystal violet.

[0221] Microscopic observation revealed purple-stained cells in the membrane beneath the upper chamber, indicating a process of cell translocation. A reduction in purple-stained cells was observed in both the group treated with single mitochondria and the group treated with RKIP-bound mitochondria compared to the untreated control group. Four randomly selected areas were analyzed, the number of stained cells measured, and plotted on a graph (Figure 68).

[0222] IV. Confirmation of the delivery rate of modified mitochondria bound to targeted proteins. Example 19. Confirmation of intracellular expression of single-stranded variable fragment (ScFv) antibodies targeting cancer cells, and confirmation of their binding to mitochondria in cells. To express pCMV-ScFv-HER2-TOM7, pCMV-ScFv-MEL-TOM7, or pCMV-ScFv-PD-L1-TOM7 in animal cells, DNA was transfected into CHO cells using lipofectamine LTX and PLUS or lipofectamine 2000. GFP-TOM7 DNA was used as a control group. To confirm that these genes were expressed in cells and bound to mitochondria within the same cells, cytosol and mitochondria were isolated from transfected cells by centrifugation, adjusted to the same protein levels using a BCA assay, and subjected to PAGE electrophoresis. The results were observed by Western blotting. Monoclonal c-myc antibody was used as the primary antibody, and anti-mouse IgG HRP was used as the secondary antibody.

[0223] Bands of the ScFv-HER2-TOM7 or ScFv-MEL-TOM7 proteins were confirmed at the expected size of 35 kDa. Based on the fact that all were confirmed in the mitochondrial layer, it could be concluded that in cells, the transfected and expressed proteins bound to mitochondria in the cell via TOM7 (Figure 69).

[0224] Next, to confirm that the target proteins expressed in cells bound to mitochondria in the same cells, expressed ScFv-HER2-TOM7, scFv-MEL-TOM7, or pCMV-PD-L1-TOM7 proteins in cells were observed using a fluorescence microscope with immunocytochemistry (ICC). Monoclonal c-myc antibody was used as the primary antibody, and goat anti-mouse IgG Alexa Fluor 488 was used as the secondary antibody. Mitochondria in 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 proteins were located in the same location as mitochondria in the cells and bound to mitochondria (Figures 70 and 71).

[0225] Example 20. Isolation of mitochondria bound to single-chain variable fragment antibodies targeting cancer cells, and comparison of mitochondria introduction into gastric cancer cell lines. Mitochondria were isolated from CHO cells transfected with pCMV-ScFv-HER2-TOM7 or pCMV-ScFv-PD-L1-TOM7. Mitochondria isolated from untransformed CHO cells were used as a control group. Mitochondria isolated from each cell group were stained with Mitotracker CMX Ros. Gastric cancer cell line SNU-484 was treated with the same amount of mitochondria, and the degree of mitochondrial translocation was compared and confirmed the following day using a fluorescence microscope. Compared to the control group, mitochondria bound to ScFv-HER2-TOM7 or ScFv-PD-L1-TOM7 were translocated into cancer cells more readily than mitochondria obtained from the control group (Figure 72). Therefore, it was found that mitochondria bound to the target protein are translocated into cancer cells more easily than mitochondria alone.

[0226] VI. Confirmation of in vivo activity of modified mitochondria bound to active proteins. Example 21. Preparation of xenograft model (SNU-484) and administration of test material. Example 21.1. Preparation of cancer cells On the day of the experiment, the gastric cancer cell line SNU-484 was administered to each mouse at a rate of 5 × 10⁶ 6 The cells were prepared. The cell culture medium was removed, and the cells were washed with PBS. The cells were dissociated with trypsin-EDTA solution, placed in a 50 mL tube, washed twice with PBS buffer, and then 20 mL of PBS was added to examine the number and viability of the cells. Based on the measured number of cells, the number of cells per mouse was determined to be 5 × 10⁶. 6 The cells were prepared by adjusting their structure and dividing them into groups. The transplant volume per mouse was adjusted to be the same at 100 μL. A control group consisting of 100 μL of cancer cells alone was prepared.

[0227] Example 21.2. Preparation of test materials As described above, mitochondria isolated from umbilical cord blood mesenchymal stem cells were prepared for transplantation at a dose of 50 μg per mouse based on protein concentration. In the group receiving single mitochondria, the mitochondria were prepared by thoroughly mixing them with 100 μL of PBS mixed with cancer cells. In the group receiving modified mitochondria, the prepared amount of mitochondria was mixed with TOM70-(GGGGS)3-UB-p53 protein in a 1:1 ratio in an Eppendorf tube before mixing with cancer cells, and left at ambient temperature for 1 hour. After the reaction time, the supernatant was removed by centrifugation at 20000 × g for 10 minutes to obtain a pellet of protein-bound mitochondria (MT+TOM70-(GGGGS)3-UB-p53). After washing this pellet twice with PBS buffer, the p53 protein-bound mitochondria (MT+TOM70-(GGGGS)3-UB-p53) were prepared by thoroughly mixing them with 100 μL of PBS mixed with cancer cells.

[0228] Example 21.3. Preparation of test animals and implantation of test materials For the transplant samples prepared by group, Matrigel (BD) was added in the same amount as PBS and gently mixed with the cells to prepare 200 μL of test material per mouse. In this case, all operations were performed on ice. For model preparation, Balb / c nude mice (female, 7 weeks old) were purchased from RAONBIO, anesthetized by isoflurane inhalation for cancer cell transplantation, and the right dorsal region (animal-based) was disinfected with an alcohol swab. Then, 200 μL of the injection solution was subcutaneously administered into the right dorsal region of the test animals using a 1 ml syringe containing the injection solution. After administration, the animals' body weight and tumor size were measured twice a week, and observations were continued for up to 3 weeks before the results were analyzed (Figure 73).

[0229] Example 21.4. Confirmation of tumor formation The tumor volume was calculated by measuring the long and short axis lengths of the tumor and applying them to the following formula. <Formula 1> Long axis x short axis x short axis x 0.5 = tumor volume (mm 3 )

[0230] Example 21.5. Observation of physiological and morphological changes To observe the physiological and morphological changes in mice induced by the administration of candidate anticancer drugs, changes in volume and tumor size were measured twice a week from the time of administration of cancer cells and test materials (Figure 74).

[0231] Mouse body weight was measured using a scale, and group-specific changes were analyzed using values ​​measured twice a week (Figure 75). No significant difference in body weight change was observed over a three-week period among the group that did not receive mitochondria injection, the group that received mitochondria alone, and the group that received modified mitochondria injection. Tumor size was calculated by measuring the long axis length (length) and short axis length (width) of the tumor using a caliper and applying these values ​​to Equation 1 above. Group-specific changes were analyzed using values ​​measured twice a week (Figure 76). In the group not treated with mitochondria, tumor size increased significantly over time, but in mice treated with mitochondria, the increase in tumor size over time slowed. Furthermore, in the group treated with mitochondria bound to the p53 protein, the increase in tumor size was significantly slower compared to the group treated with mitochondria alone (Figure 76).

[0232] Example 22. Confirmation of the effect of modified mitochondria on inhibiting the proliferation of skin cancer cells. Mitochondria bound to p53, obtained as described above, were delivered to A431 cells, a type of skin cancer cell, by centrifugation, and the proliferation of A431 cells was observed. In this case, physiological saline was used as the control group, and an equal amount of mitochondria without p53 protein fusion was used as the control test group. It was confirmed that mitochondria bound to the p53 protein, which induces apoptosis, could significantly suppress the proliferation of A431 cells compared to the control group and the group using mitochondria alone (Figure 76).

[0233] V. Confirmation of the activity of isolated mitochondria Example 23. Confirmation of function of isolated mitochondria: ATP levels To isolate intracellular mitochondria from umbilical cord-derived mesenchymal stem cells (UC-MSCs), cells were homogenized using a syringe to disrupt them, and then obtained by continuous centrifugation. To confirm the function of the isolated mitochondria, the mitochondrial protein concentration of the isolated mitochondria was quantified by a BCA assay, and 5 μg of mitochondria was prepared. The amount of ATP in the mitochondria was confirmed using a CellTiter-Glo luminescence kit (Promega, Madison, WI).

[0234] The prepared mitochondria were mixed with 100 μl of PBS and prepared in a 96-well plate, and compared with a control group of 100 μl of PBS without mitochondria. 100 μl of the test solution included in the kit was added in the same manner, mixed thoroughly in a stirrer for 2 minutes, and then allowed to react at ambient temperature for 10 minutes. Afterward, the amount of ATP was measured using a luminescent microplate reader. It was confirmed that the amount of ATP was higher in the group with mitochondria compared to the control group, confirming mitochondrial function (Figure 78).

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

[0236] Subsequently, it was reacted with JC-1 dye (2 μM) in the same manner, and its absorbance was measured by utilizing its property that it exhibits different spectra depending on the concentration caused by the change in membrane potential. At low concentrations, it exists as a monomer and emits green fluorescence, while at high concentrations, the dye aggregates (J-aggregates) and emits red fluorescence. The mitochondrial membrane potential was analyzed by calculating the ratio of green absorbance to red absorbance. After the reaction was complete, the mitochondrial membrane potential was examined using a fluorescence microplate reader (monomer: excited 485 / fluorescent 530, J-aggregates: excited 535 / fluorescent 590). The results are shown in Figure 79.

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

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

[0239] Subsequently, analysis by SDS-PAGE electrophoresis confirmed that no dissociation of the ubiquitin protein from the fusion protein by UBP1 occurred. This is thought to be a structural interference phenomenon of the mitochondrial outer membrane protein. Therefore, a linker protein consisting of the amino acids glycine and serine was inserted between the mitochondrial outer membrane protein and the ubiquitin protein, and a new fusion protein (TOM70-(GGGGS)3-UB-p53 or TOM70-(GGGGS)3-UB-GFP) was obtained by purification from E. coli and reacted with the UBP1 enzyme at 37°C for 1 hour as described above. As a result, SDS-PAGE electrophoresis confirmed that the 3' end of ubiquitin was cleaved by the UBP1 enzyme, and only the p53 protein was dissociated as expected (Figure 82).

[0240] Example 26. Confirmation of the dissociation of a desired protein bound to a mitochondrial outer membrane protein inside a cell. We observed whether the active protein was dissociated by ubiquitin-cleaving enzymes present in cells when the fusion protein (TOM70-(GGGGS)3-UB-p53 or TOM70-(GGGGS)3-UB-GFP) obtained in the above examples entered cells bound to mitochondria. First, mitochondria obtained from umbilical cord blood mesenchymal cells and the fusion protein TOM70-(GGGGS)3-UB-GFP were reacted in a microtube for 1 hour to bind, and then the unbound fusion protein was removed by centrifugation and washed twice with PBS buffer. In this case, the fusion protein from which ubiquitin had been removed (TOM70-(GGGGS)3-p53) was used as the control group.

[0241] Next, the mitochondria-bound protein was introduced into the breast cancer cell line MDA-MB-231 by centrifugation. After one day, the MDA-MB-231 cells were disrupted and fractionated into mitochondrial and cytosolic regions based on their weight differences. Analysis by SDS-PAGE electrophoresis and Western blot revealed that in the case of fusion proteins containing ubiquitin, GFP protein dissociated from mitochondrial outer membrane proteins, linker proteins, and ubiquitin were detected in large quantities in the cytosolic region. In the case of fusion proteins without ubiquitin, GFP protein bound to mitochondrial outer membrane proteins and linker proteins was detected in large quantities in the mitochondrial fraction (Figure 83).

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

Claims

1. A fusion protein comprising a targeted protein having the ability to bind to a ligand or receptor present in the cell membrane, and a mitochondrial outer membrane anchoring peptide, The target-targeting protein is an antibody or a fragment of the antibody having the same CDR as the complementarity-determining region (CDR) of the antibody. The antibody fragment is scFv, The mitochondrial outer membrane anchoring peptide is any one selected from the group consisting of TOM5, TOM7, Fis1, Bcl-2, and VAMP1B. A fusion protein in which a targeting protein and a mitochondrial outer membrane anchoring peptide are linked from the N-terminus to the C-terminus.

2. A polynucleotide encoding the fusion protein described in claim 1.

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