KR101816812B1 - A method for extending half-life of a protein - Google Patents

Abstract

The present invention relates to a method for extending a half-life of a protein by comprising substituting one or more lysine residues in an amino acid sequence of a protein, or a protein of which a half-life is extended. A protein of which lysine residues are substituted remains in the body for a long period of time and has excellent treatment effects.

Description

{A method for extending half-life of a protein}

The present invention relates to a method of increasing the half-life of a protein or (poly) peptide by substituting one or more amino acid residues of the protein or (poly) peptide. It also relates to a protein or (poly) peptide with an increased half-life produced by this method.

Intracellular protein degradation occurs through two pathways, by lysosome and proteasome. The lysosomal pathway, which degrades 10-20% of the protein, lacks substrate specificity and precise temporal control. In other words, it is a process of decomposing most extracellular or membrane proteins, just as the cell surface protein that has been introduced into the cell by endocytosis is decomposed in the lysosome. However, in order to selectively degrade proteins in eukaryotic cells, polyubiquitin chains are formed after ubiquitin is bound to the target protein by ubiquitin-binding enzyme, which is recognized and degraded by the proteasome, that is, ubiquitin-pro. It must go through the ubiquitin-proteasome pathway (UPP). More than 80 to 90% of eukaryotic proteins are degraded through this process, and the ubiquitin-proteasome pathway is responsible for protein conversion and homeostasis by controlling the degradation of most proteins present in eukaryotic cells.

Ubiquitin is a very well-preserved protein composed of 76 amino acids and is present in almost all eukaryotic cells, of which the 6, 11, 27, 29, 33, 48, 63 amino acid residues are lysine (Lysine, Lys, K), Numbers 48 and 63 play a major role in forming the polyubiquitin chain. A series of enzyme systems (E1, E2, E3) are involved in the process of labeling ubiquitin on a protein, and the labeled protein is degraded by the 26S proteasome, an ATP-dependent protease complex. The ubiquitin-proteasome pathway involves two separate successive processes, the first of which is the process of covalently labeling several ubiquitin molecules on the substrate, and the second is the process of labeling the protein labeled by ubiquitin with 26S protease. It is a process that is decomposed by the theasome complex. The binding of ubiquitin to the substrate occurs through an isopeptide bond between the lysine residue of the substrate molecule and the C-terminal glycine of ubiquitin, and by ubiquitin-activating enzyme E1, ubiquitin-binding enzyme E2, and ubiquitin ligase E3. It is formed by the formation of thiol esters between ubiquitin and the enzyme. Among them, E1 (ubiquitin-activating enzyme) activates ubiquitin through an ATP-dependent reaction. E2 (ubiquitin-conjugating enzyme) receives activated ubiquitin from E1 at a cysteine residue in the ubiquitin-conjugating domain and transfers it to E3 ligase or directly to a substrate protein. The E3 enzyme also catalyzes the stable isopeptide bond between the lysine residue of the substrate protein and the glycine residue of ubiquitin. Another ubiquitin can be linked to the C-terminal lysine residue of ubiquitin bound to the matrix protein.If this process is repeated to form a polyubiquitin chain by linking several ubiquitin molecules to the matrix protein in a branched shape, the protein is It is recognized by the 26S proteasome and is selectively degraded.

Meanwhile, various kinds of proteins and (poly) peptides having therapeutic effects in vivo are known. As such, proteins or (poly) peptides having a therapeutic effect in vivo are, for example, growth hormone releasing hormone (GHRH), growth hormone releasing peptide, interferons, interferon-α or interferon-β), interferon receptors, colony stimulating factors (CSFs), glucagon-like peptides, interleukins, interleukin receptors, Enzymes, interleukin binding proteins, cytokine binding proteins, G-protein-coupled receptors, human growth hormone (hGH), Macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, Cell necrosis glycoproteins, G-protein-coupled receptor, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor ( tumor suppressors), metastasis growth factor, alpha-1 antitrypsin, albumin (albumin), alpha-lactalbumin (alph) a-lactalbumin), apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, hemoglobin (thrombin), thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIIa, factor VIII, factor IX ( factor IX), factor XIII, plasminogen activating factor, urokinase, streptokinase, hirudin, protein C, C- C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor), epithelial growth factor, endothelial growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein , Calcitonin, insulin, atriopeptin, cartilage inducing factor, fibrin-binding p eptide), elcatonin, connective tissue activating factor, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, corpus luteum Luteinizing hormone releasing hormone, nerve growth factors, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor ( insulin-like growth factor), adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, adrenocortical hormone releasing factor ( corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens ), virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

β-trophin rapidly grows insulin-secreting pancreatic β-cells. When β-tropine is administered to type 2 diabetic patients once a month or a year, it is possible to sufficiently maintain the activity of pancreatic β-cells that constantly regulate blood sugar levels. In addition, since the human body produces its own insulin by the administration of β-tropine rather than receiving insulin, there are almost no side effects. It has been reported that transient expression of β-tropine in mouse liver promotes the proliferation of pancreatic-beta cells (Cell 153, 747-758, 2013).

Growth hormone (GH) is a type of peptide hormone that is synthesized and secreted in the anterior pituitary gland, and is involved in metabolism that promotes the decomposition of fat and protein synthesis as well as the growth of bones and cartilage in the body. Growth hormone can be used for the purpose of treating dwarfism, and dwarfism is caused by abnormal hormone secretion such as congenital heart disease, chronic lung disease, chronic kidney disease, chronic wasting disease, growth hormone deficiency, hypothyroidism, diabetes, and other hormone secretion disorders. Dwarfism, or a congenital chromosomal disease, Turner syndrome. It has been reported that growth hormone regulates the transcription of STAT (signal transducers and activators of transcription) proteins at the gene level (Oncogene, 19, 2585-2597, 2000).

Insulin regulates blood sugar in the body. Insulin can be administered to type 1 diabetic patients, whose islet cells of the pancreas are destroyed, resulting in insufficient insulin and an increase in blood sugar levels. In addition, it can be administered to type 2 diabetic patients whose blood glucose concentration is not regulated even though insulin is secreted due to resistance to the insulin receptor of somatic cells. It has been reported that insulin stimulates the phosphorylation of STAT3 in the liver and consequently regulates glucose homeostasis in the liver (Cell Metabolism 3, 267-275, 2006).

Interferons are secreted and made by immune system cells such as white blood cells, natural killer cells, fibroblasts, and epithelial cells, and are a kind of naturally occurring protein group. Interferon is classified into three types: type I, type II, and type III, and is determined according to the type of receptor that each protein delivers. The mechanism of action of interferon is complex and not fully understood, but is known to regulate the immune system's response to viruses, bacteria, cancer, and other foreign substances. On the other hand, interferon does not directly kill viral cells or cancer cells, but promotes the immune system's response and inhibits the growth of cancer cells by regulating gene functions that regulate the secretion of proteins in numerous cells. It is known that IFN-α contained in interferon type I can be used to treat hepatitis B and hepatitis C, and IFN-β can be used to treat multiple sclerosis. Interferon-α has been reported to increase STAT-1, -2 and -3 (J Immunol, 187, 2578-2585, 2011), and STAT3 protein that promotes growth by interferon-α in melanoma cells Has been reported to activate (Euro J Cancer, 45, 1315-1323, 2009). It has been reported that treatment of interferon-β in cells induces the activity of signaling processes including AKT (Pharmaceuticals (Basel), 3, 994-1015, 2010).

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release these cells into the bloodstream. Functionally, it plays the role of cytokines and hormones and is a type of colony stimulating factor. In addition to affecting the hematopoietic system, G-CSF can act as a neurotrophic factor in neurons. Its receptors are actually expressed in neurons in the brain and spinal cord, and the action of G-CSF in the central nervous system induces the development of neurons, increases neuroplasticity, and responds to predicted cell death. Due to these characteristics, G-CSF is being studied for the development of the treatment of neurological diseases such as cerebral infarction. G-CSF stimulates the production of granulocytes, a type of white blood cell. In addition, in oncology and hematology, the recombinant form of G-CSF is mainly used in cancer patients to promote recovery from neutropenia after chemotherapy. Granulocyte colony stimulating factor (G-CSF) has been reported to activate STAT3 in glioma cells and thereby participate in glioma proliferation (Cancer Biol Ther., 13(6), 389-400, 2012), It has been reported that it is expressed in ovarian epithelial cancer cells and that there is a pathological association of female uterine cancer by regulating the JAK2/STAT3 pathway (Br J Cancer, 110, 133-145, 2014).

Erythropoietin (EPO) is a glycoprotein hormone and acts with various growth factors such as interleukin 3 (IL-3), interleukin 6 (IL-6), glucocorticoids and stem cell factors. Erythropoietin is a cytokine that is present in the bone marrow as a precursor of red blood cells and is involved in the production of red blood cells. In addition, erythropoietin is also associated with vasoconstriction-dependent hypertension. It is regulated by increasing the absorption of iron ions by inhibiting the absorption of hepcidin hormone, an iron-regulatory hormone produced in the liver. . In addition, erythropoietin plays an important role in protecting nerves in the brain's response to nerve damage such as myocardial infarction or stroke. In addition, it is known to be effective in improving memory, healing wounds, and treating depression. Erythropoietin is increased in lung or blood cancer, and it has been reported that when erythropoietin is administered, the effect in hypoxia is increased by controlling cell cycle progression by phosphorylation of Erk1/2 (J Hematol Oncol., 6, 65). , 2013).

Recombinant fibroblast growth factor-1 (FGF-1) is an acidic FGF as one of fibroblast growth factors and plays a role in embryonic development, cell growth, tissue regeneration, cancer growth and metastasis. In addition, in clinical studies, it has been reported that FGF-1 induces angiogenesis in the heart (BioDrugs., 11(5), 301308, 1999). Since FGF is a substance that promotes the growth of fibroblasts in the dermis, it keeps the dermal cells healthy and strengthens skin elasticity to moisturize the skin, and the skin cells are activated to brighten the entire skin and maintain a white skin. do. In addition, it helps to quickly recover injured or damaged skin and strengthens the defense function by strengthening the skin barrier. It has been reported that treatment with HEK293 cells increases Erk 1/2 phosphorylation (Nature, 513(7518), 436-439, 2014).

Vascular endothelial growth factor A (VEGFA) is a signaling protein produced by cells that stimulate angiogenesis and angiogenesis, and plays a role in the process of storing oxygen in tissues in a hypoxic environment. Hand (Mol Cell Endocrinol., 397, 5157, 2014). In the case of bronchial asthma and diabetes, plasma concentrations of VEGF are high (Diabetes, 48(11), 22292239, 2013), and the normal function of VEGF is to develop embryos, create new blood vessels after injury, and penetrate muscles and blocked blood vessels after exercise. It functions such as the creation of new blood vessels. Overexpression of VEGF leads to disease. In the case of solid cancer, if blood is not continuously injected, it cannot grow anymore, but when VEGF is expressed, it grows more and metastasis occurs. As an important factor related to the growth and proliferation of endothelial cells, cancer cells act on angiogenesis, and it has been reported that PI3K/Akt/HIF-1α signaling is involved (Carcinogenesis, 34, 426-435, 2013). It has also been reported that VEGF induces AKT phosphorylation (Kidney Int., 68, 1648-1659, 2005).

Among the appetite suppressing proteins (Leptin) and appetite promoting hormones (Ghrelin) secreted from adipose tissue, Leptin is a 16-kDa circulating hormone in the body that plays an important role in immune, reproductive and hematopoietic functions (Cell Res., 10, 81-92, 2000), appetite promoting hormone (Ghrelin) is secreted from adipose tissue through the growth hormone secretagogue receptor (GHS-R), a gastric peptide consisting of 28 amino acids that makes you feel appetite. -peptide) (J Endocrinol., 192, 313323, 2007; Nature, 442, 656-660, 1999), formed through processing from preproghrelin (Pediatr Res., 65, 3944, 2009; J Biol Chem., 281(50), 3886738870, 2006). Appetite suppressing protein (Leptin) is a hormone that is secreted by adipose tissue and makes people feel full and no longer eats food. When a problem occurs in secretion of this hormone, the desire to eat is generated. It has been reported that fructose interferes with the secretion of insulin, lowers leptin secretion, and promotes ghrelin secretion, thereby increasing appetite (J Biol Chem., 277(7), 5667-5674, 2002; IJSN, 7(1)). , 06-15, 2016). In addition, it has been reported that the appetite suppressing protein (Leptin) increases the phosphorylation of AKT in breast cancer cells (Cancer Biol Ther., 16(8), 1220-1230, 2015). It has been reported to stimulate growth (Int J Oncol., 49(2), 847, 2016). On the other hand, it has been reported that Ghrelin regulates cell growth through the growth hormone secretagogue receptor (GHS-R) and increases STAT3 through the regulation of calcium in the body (Mol Cell Endocrinol., 285, 19-25, 2008).

Glucagon-like peptide (GLP-1) is an incretin hormone secreted from L cells in the ileum and colon and increases insulin secretion. In particular, it has been reported that hypoglycemia does not occur due to the characteristic of increasing insulin secretion depending on the glucose concentration, and it can be used for the treatment of type 2 diabetes due to this characteristic (Pharmaceuticals (Basel), 3(8), 2554-2567, 2010; Diabetologia, 36(8), 741-744, 1993). In addition, GLP-1 has the effect of lowering the movement of the upper digestive system and suppressing appetite, and can proliferate existing β cells of the pancreas (Endocr Rev., 16(3), 390-410, 1995; Endocrinology, 141(12), 4600-4605, 2000; Dig Dis Sci., 38(4), 665-673, 1993; Am J Physiol., 273(5 Pt 1), E981-988, 1997). However, since the half-life of GLP-1 in the blood is very short, about 2 minutes, it has a big disadvantage to develop as a drug. In addition, since it regulates glucose homeostasis and plays an important role in insulin resistance, it is currently used as a therapeutic agent for diabetes and has been reported to induce STAT3 activity (Biochem Biophys Res Commun., 425(2), 304-308, 2012).

Bone morphogenetic protein (BMP2) is one of the superfamily of TGF-β, a protein that plays an important role in cartilage and bone formation, and plays a pivotal role in cell growth, apoptosis, and differentiation (Genes Dev. , 10, 1580-1594, 1996; Development, 122, 3725-3734, 1996; J Biol Chem., 274, 26503-26510, 1999; J Exp Med., 189, 1139-1147, 1999). It has been reported that it can be used as a treatment for multiple myeloma as it exhibits anticancer effects that induce apoptosis due to bone disorders in myeloma patients (Blood, 96(6), 2005-2011, 2000; Leuk Lymphoma., 43(3)). ), 635-639, 2002).

Immunoglobulin G (IgG) is a type of antibody, one of the major antibodies found in blood and extracellular fluid, and controls infection of cellular tissues. IgG is a small sized monomer, which makes it easy to permeate into tissues. (Basic Histology, McGraw-Hill, ISBN 0-8385-0590-2, 2003). IgG is used to treat immunodeficiency, autoimmune diseases, and infections (Proc Natl Acad Sci US A., 107(46), 19985-19990, 2010). Please add.

Protein-related treatments related to the regulation of homeostasis in the body have side effects such as increasing the risk of cancer. For example, insulin secretion hormone degrading enzyme (DPP-4) inhibitors have been suggested to induce thyroid cancer, and insulin glargine is known to increase the risk of breast cancer. In addition, it has been reported that continuous or excessive administration of growth hormone in patients with abnormal growth hormone secretion diseases is associated with diabetes, microvascular disorders, and early death. As described above, as the necessity of developing new therapeutic agents increases due to the side effects of existing protein-related therapeutic agents, the present inventors have replaced one or more lysine residues present in the amino acid sequence of the protein, thereby degrading the protein by the ubiquitin-proteasome system. To increase the half-life of the protein was completed.

An object of the present invention is to provide a method of increasing the half-life of a protein or (poly) peptide.

Another object of the present invention is to provide a protein having an increased half-life as a protein in which one or more lysine residues present in an amino acid sequence are substituted.

In addition, an object of the present invention is to provide a pharmaceutical composition comprising a protein having an increased half-life.

In order to achieve the above object, the present invention provides a method of increasing the half-life of a protein, comprising replacing one or more lysine residues present in the amino acid sequence of the protein.

In the present invention, the lysine residue of the protein may be substituted with a conservative amino acid. In the present invention, "conservative amino acid substitution" means that an amino acid residue is substituted by another amino acid residue having a side chain having a similar, for example, charged or hydrophobic chemical property. In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. Examples of groups of amino acids having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) Acidic side chains: aspartate and glutamate 7) Sulfur-containing side chains: include cysteine and methionine.

In the present invention, the lysine residue of the protein may be substituted with arginine or histidine containing a basic side chain, preferably with an arginine residue.

According to the present invention, a protein in which at least one lysine residue in the amino acid sequence of a protein is substituted with arginine has an increased half-life and may remain in the body for a long time.

1 shows the structure of the β-tropin expression vector.
Figure 2 shows the results of PCR for the β-tropin gene.
Figure 3 shows the expression of the β-tropin plasmid gene in HEK-293T cells.
Figure 4 shows the decomposition pathway of β-tropine through ubiquitination analysis.
5 shows the degree of ubiquitination of β-tropine substituents in which lysine residues are substituted with arginine compared to wild type.
6 shows the change in half-life of β-tropine after treatment with the protein synthesis inhibitor cycloheximide (CHX).
7 shows the results for effects such as induction of JAK-STAT signal.
8 shows the structure of a growth hormone expression vector.
9 shows PCR results for growth hormone.
10 shows the expression of the growth hormone plasmid gene in HEK-293T cells.
11 shows the decomposition pathway of growth hormone through ubiquitination analysis.
12 shows the degree of ubiquitination of growth hormone substituents in which lysine residues are substituted with arginine compared to wild type.
13 shows the change in half-life of growth hormone after treatment with the protein synthesis inhibitor cycloheximide (CHX).
14 shows the results for effects such as induction of JAK-STAT signal.
15 shows the structure of the insulin expression vector.
16 shows PCR results for insulin.
17 shows the expression of insulin plasmid gene in HEK-293T cells.
18 shows the pathway of insulin degradation through ubiquitination analysis.
19 shows the degree of ubiquitination of insulin substituents in which lysine residues are substituted with arginine compared to wild type.
20 shows the change in half-life of insulin after treatment with the protein synthesis inhibitor cycloheximide (CHX).
Figure 21 shows the results for the same effect as JAK-STAT signal induction.
22 shows the structure of the interferon-α expression vector.
23 shows PCR results for the interferon-α gene.
24 shows the expression of interferon-α plasmid gene in HEK-293T cells.
25 shows the decomposition pathway of interferon-α through ubiquitination analysis.
26 shows the degree of ubiquitination of interferon-α substituents in which lysine residues are substituted with arginine compared to wild-type.
Figure 27 shows the half-life change of interferon-α after treatment with the protein synthesis inhibitor cycloheximide (CHX).
28 shows the results for effects such as induction of JAK-STAT signal.
29 shows the structure of the G-CSF expression vector.
30 shows PCR results for the G-CSF gene.
31 shows the expression of the G-CSF plasmid gene in HEK-293T cells.
Figure 32 shows the decomposition pathway of G-CSF through ubiquitination analysis.
33 shows the degree of ubiquitination of G-CSF substituents in which lysine residues are substituted with arginine compared to wild type.
34 shows the change in half-life of G-CSF after treatment with the protein synthesis inhibitor cycloheximide (CHX).
35 shows the results for the same effect as JAK-STAT signal induction.
36 shows the structure of an interferon-β expression vector.
37 shows PCR results for the interferon-β gene.
38 shows the expression of interferon-β plasmid gene in HEK-293T cells.
39 shows the pathway of decomposition of interferon-β through ubiquitination analysis.
40 shows the degree of ubiquitination of interferon-β substituents in which lysine residues are substituted with arginine compared to wild-type.
Figure 41 shows the half-life change of interferon-β after treatment with the protein synthesis inhibitor cycloheximide (CHX).
42 shows results for effects such as induction of JAK-STAT and PI3K/AKT signals.
43 shows the structure of an erythropoietin expression vector.
44 shows PCR results for the erythropoietin gene.
Figure 45 shows the expression of erythropoietin plasmid gene in HEK-293T cells.
Figure 46 shows the decomposition pathway of erythropoietin through ubiquitination analysis.
47 shows the degree of ubiquitination of erythropoietin substituents in which lysine residues are substituted with arginine compared to wild type.
48 shows the change in half-life of erythropoietin after treatment with the protein synthesis inhibitor cycloheximide (CHX).
49 shows the results for effects such as induction of MAPK/ERK signals.
50 shows the structure of the BMP2 expression vector.
51 shows PCR results for the BMP2 gene.
52 shows the expression of the BMP2 plasmid gene in HEK-293T cells.
53 shows the decomposition pathway of BMP2 through ubiquitination analysis.
54 shows the degree of ubiquitination of BMP2 substituents in which lysine residues are substituted with arginine compared to wild type.
55 shows the change in half-life of BMP2 after treatment with the protein synthesis inhibitor cycloheximide (CHX).
56 shows the results for effects such as induction of JAK-STAT signal.
57 shows the structure of a fibroblast growth factor-1 (FGF-1) expression vector.
58 shows PCR results for the fibroblast growth factor-1 (FGF-1) gene.
59 shows the expression of fibroblast growth factor-1 (FGF-1) plasmid gene in HEK-293T cells.
Figure 60 shows the decomposition pathway of fibroblast growth factor-1 (FGF-1) through ubiquitination analysis.
61 shows the degree of ubiquitination of fibroblast growth factor-1 (FGF-1) substituents in which lysine residues are substituted with arginine compared to wild type.
Figure 62 shows the half-life change of fibroblast growth factor-1 (FGF-1) after treatment with the protein synthesis inhibitor cycloheximide (CHX).
63 shows the results for effects such as induction of MAPK/ERK signals.
64 shows the structure of a leptin expression vector.
65 shows PCR results for the leptin gene.
Figure 66 shows the expression of the leptin plasmid gene in HEK-293T cells.
67 shows a pathway for degradation of leptin through ubiquitination analysis.
Figure 68 shows the degree of ubiquitination of the leptin substituent in which the lysine residue is substituted with arginine compared to the wild type.
69 shows the change in half-life of leptin after treatment with the protein synthesis inhibitor cycloheximide (CHX).
70 shows the results for the same effect as PI3K/AKT signal induction.
71 shows the structure of the vascular endothelial growth factor (VEGFA) expression vector.
72 shows PCR results for vascular endothelial growth factor (VEGFA) gene.
73 shows the expression of vascular endothelial growth factor (VEGFA) plasmid gene in HEK-293T cells.
74 shows the decomposition pathway of vascular endothelial growth factor (VEGFA) through ubiquitination analysis.
75 shows the degree of ubiquitination of vascular endothelial growth factor (VEGFA) substituents in which lysine residues are substituted with arginine compared to wild type.
76 shows changes in half-life of vascular endothelial growth factor (VEGFA) after treatment with the protein synthesis inhibitor cycloheximide (CHX).
77 shows the results for effects such as induction of JAK-STAT and PI3K/AKT signals.
Figure 78 shows the structure of the expression vector ghrelin / obestatin prepropeptide (Ghrelin / obestatin prepropeptide) (Prepro-GHRL).
79 shows PCR results for ghrelin/obestatin prepropeptide genes.
80 shows the expression of Prepro-GHRL plasmid gene in HEK-293T cells.
81 shows the pathway of decomposition of Prepro-GHRL through ubiquitination analysis.
82 shows the degree of ubiquitination of Prepro-GHRL substituents in which lysine residues are substituted with arginine compared to wild type.
83 shows the half-life change of Prepro-GHRL after treatment with the protein synthesis inhibitor cycloheximide (CHX).
84 shows results for effects such as induction of JAK-STAT signal.
85 shows the structure of the GHRL expression vector.
86 shows PCR results for the GHRL gene.
87 shows the expression of the GHRL plasmid gene in HEK-293T cells.
88 shows the pathway of degradation of GHRL through ubiquitination analysis.
89 shows the degree of ubiquitination of GHRL substituents in which lysine residues are substituted with arginine compared to wild type.
FIG. 90 shows the change in half-life of GHRL after treatment with the protein synthesis inhibitor cycloheximide (CHX).
91 shows the results for effects such as induction of JAK-STAT signal.
92 shows the structure of a glucagon-like peptide-1 (GLP-1) expression vector.
93 shows PCR results for the glucagon-like peptide-1 (GLP-1) gene.
Figure 94 shows the expression of the glucagon-like peptide-1 (GLP-1) plasmid gene in HEK-293T cells.
95 shows the decomposition pathway of glucagon-like peptide-1 (GLP-1) through ubiquitination analysis.
96 shows the degree of ubiquitination of the glucagon-like peptide-1 (GLP-1) substituent in which the lysine residue is substituted with arginine compared to the wild type.
97 shows the half-life change of glucagon-like peptide-1 (GLP-1) after treatment with the protein synthesis inhibitor cycloheximide (CHX).
98 shows the results for effects such as induction of JAK-STAT signal.
99 shows the structure of an IgG heavy chain expression vector.
100 shows PCR results for the IgG heavy chain gene.
101 shows the expression of the IgG heavy chain plasmid gene in HEK-293T cells.
Figure 102 shows the decomposition pathway of the IgG heavy chain through ubiquitination analysis.
103 shows the degree of ubiquitination of IgG heavy chain substituents in which lysine residues are substituted with arginine compared to wild type.
104 shows changes in half-life of IgG heavy chains after treatment with the protein synthesis inhibitor cycloheximide (CHX).
105 shows the structure of an IgG light chain expression vector.
106 shows PCR results for the IgG light chain gene.
107 shows the expression of the IgG light chain plasmid gene in HEK-293T cells.
108 shows the degradation pathway of the IgG light chain through ubiquitination analysis.
Figure 109 shows the degree of ubiquitination of an IgG light chain substituent in which a lysine residue is substituted with arginine compared to the wild type.
110 shows changes in half-life of an IgG heavy chain after treatment with a protein synthesis inhibitor cycloheximide (CHX).

In one embodiment of the invention, the protein is β-tropine. At least one of the lysine residues at 62, 124, 153 and 158 from the N-terminus in the amino acid sequence of β-tropine represented by SEQ ID NO: 1 is substituted with an arginine residue. As a result, there is provided a β-tropine having an increased half-life and a pharmaceutical composition for preventing and/or treating diabetes and obesity comprising the same (Cell, 153(4), 747758, 2013; Cell Metab., 18( 1), 5-6, 2013; Front Endocrinol (Lausanne), 4, 146, 2013).

In another embodiment of the present invention, the protein is growth hormone. At least one of the lysine residues at 64, 67, 96, 141, 166, 171, 184, 194 and 198 from the N-terminus in the amino acid sequence of the growth hormone represented by SEQ ID NO: 10 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for preventing or treating growth hormone with an increased half-life and dwarfism and other growth hormone deficiency diseases (Kabuki syndrome and Kearns-Sayre syndrome (KSS)) including the same (J Endocrinol Invest., 39 (6). ), 667-677, 2016; J Pediatr Endocrinol Metab., 2016, [Epub ahead of print]; Horm Res Paediatr. 2016, [Epub ahead of print]).

In another embodiment of the invention, the protein is insulin. At least one of the 53rd and 88th lysine residues from the N-terminus in the insulin amino acid sequence represented by SEQ ID NO: 17 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for preventing and/or treating diabetes including insulin having an increased half-life and the same.

In another embodiment of the invention, the protein is interferon-α. At least one of the 17, 54, 72, 93, 106, 135, 144, 154, 156, 157 and 187th lysine residues from the N-terminus in the amino acid sequence of interferon-α represented by SEQ ID NO: 22 is substituted with an arginine residue. . Accordingly, there is provided a pharmaceutical composition for preventing and/or treating cancer and/or infectious diseases including interferon-α and autoimmune including the same and/or solid cancer and hematologic cancer including the interferon-α with increased half-life (Ann Rheum Dis., 42(6), 672-676, 1983; Memo., 9, 63-65, 2016).

In another embodiment of the present invention, the protein is a granulocyte colony stimulating factor. At least one of the 11th, 46th, 53rd, 64th and 73rd lysine residues from the N-terminus in the granulocyte colony stimulating factor amino acid sequence represented by SEQ ID NO: 31 is substituted with an arginine residue. Accordingly, there is provided a granulocyte colony stimulating factor with an increased half-life and a pharmaceutical composition for the prevention and/or treatment of neutropenia comprising the same (EMBO Mol Med. 2016, [Epub ahead of print]).

In another embodiment of the invention, the protein is interferon-β. At least one of the 4, 40, 54, 66, 73, 120, 126, 129, 136, 144, 155 and 157 th lysine residues from the N-terminus in the granulocyte colony stimulating factor amino acid sequence represented by SEQ ID NO: 36 is an arginine residue. Is substituted. Accordingly, there is provided a pharmaceutical composition for the prevention and/or treatment of interferon-β with an increased half-life and multiple sclerosis, autoimmune diseases, viral infections, HIV-related diseases, hepatitis C, rheumatoid arthritis, and the like. In addition, there is provided a pharmaceutical composition for the prevention and/or treatment of immune diseases including autoimmune and/or cancer and/or infectious diseases including solid cancer and hematologic cancer (Ann Rheum Dis., 42(6), 672- 676, 1983; Memo., 9, 63-65, 2016).

In another embodiment of the invention, the protein is erythropoietin (EPO). At least one of the lysine residues at 47, 72, 79, 124, 143, 167, 179 and 181 from the N-terminus in the erythropoietin amino acid sequence represented by SEQ ID NO: 43 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for preventing and/or treating anemia, such as erythropoietin having an increased half-life and anemia due to chronic renal failure, anemia according to surgery, anemia due to cancer or chemotherapy, etc. including the same.

In another embodiment of the present invention, the protein is bone morphogenetic protein (BMP2). 32, 64, 127, 178, 185, 236, 241, 272, 278, 281, 285, 287, 290, 293, 297, 355, 358, from the N-terminus in the osteogenic protein amino acid sequence represented by SEQ ID NO: 52 At least one of the 379 and 383 lysine residues is substituted with an arginine residue. Accordingly, there is provided a osteogenic protein having an increased half-life and a pharmaceutical composition for preventing and/or treating anemia and bone disease comprising the same (Cell J., 17(2), 193-200, 2015; Clin Orthop Relat Res., 318, 222-230, 1995).

In another embodiment of the present invention, the protein is fibroblast growth factor-1 (FGF-1). At least one of the 15, 24, 25, 27, 72, 115, 116, 120, 127, 128, 133 and 143 lysine residues from the N-terminus in the fibroblast growth factor-1 amino acid sequence represented by SEQ ID NO: 61 is arginine Is substituted with a residue. Accordingly, there is provided a fibroblast growth factor-1 with an increased half-life and a pharmaceutical composition for preventing and/or treating neurological diseases comprising the same.

In another embodiment of the present invention, the protein is an appetite suppressing hormone (Leptin). At least one of the 26, 32, 36, 54, 56, 74, and 115 th lysine residues from the N-terminus in the appetite suppressing hormone (Leptin) amino acid sequence represented by SEQ ID NO: 66 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for preventing and/or treating brain disease and/or heart disease and/or obesity including the appetite suppressing hormone (Leptin) having an increased half-life. (Ann N Y Acad Sci., 1243, 1529, 2011; J Neurochem., 128(1), 162-172, 2014; Clin Exp Pharmacol Physiol., 38(12), 905-913, 2011)

In another embodiment of the present invention, the protein is vascular endothelial growth factor A (VEGFA). 22, 42, 74, 110, 127, 133, 134, 141, 142, 147, 149, 152, 154, 156, 157, 169, 180, 184, from the N-terminus in the VEGFA amino acid sequence represented by SEQ ID NO: 75 At least one of the lysine residues at positions 191 and 206 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for preventing and/or treating diseases related to anti-aging, hair growth, wound healing and angiogenesis including VEGFA having an increased half-life and the same.

In another embodiment of the present invention, the protein is an appetite promoting hormone precursor. At least one of the 39, 42, 43, 47, 85, 100, 111, and 117th lysine residues from the N-terminus in the appetite promoting hormone precursor amino acid sequence represented by SEQ ID NO: 80 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for preventing and/or treating an appetite control function disease including obesity, malnutrition, and anorexia including an appetite-promoting hormone precursor having an increased half-life.

In another embodiment of the present invention, the protein is an appetite promoting hormone (Ghrelin). At least one of the 39, 42, 43, and 47th lysine residues from the N-terminus in the appetite promoting hormone (Ghrelin) amino acid sequence represented by SEQ ID NO: 83 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for an appetite-regulating function disease including obesity, malnutrition and anorexia including the appetite promoting hormone (Ghrelin) having an increased half-life.

In another embodiment of the present invention, the protein is glucagon-like peptide (GLP-1). In the amino acid sequence of a glucagon-like peptide (GLP-1) represented by SEQ ID NO: 92, one or more of the lysine residues 117 and 125 from the N-terminus is substituted with an arginine residue. Accordingly, there is provided a glucagon-like peptide (GLP-1) having an increased half-life and a pharmaceutical composition for preventing and/or treating diabetes including the same.

In one embodiment of the invention, the protein is a heavy chain (HC) of immunoglobulin (IgG). 49, 62, 84, 95, 143, 155, 169, 227, 232, 235, 236, 240, 244, 268 from the N-terminus in the heavy chain (HC) amino acid sequence of immunoglobulin (IgG) represented by SEQ ID NO: 97 , 270, 296, 310, 312, 339, 342, 344, 348, 356, 360, 362, 382, 392, 414, 431, 436 and at least one of the 461 th lysine residues is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition of an immunoglobulin (IgG) having an increased half-life and an IgG-based antibody therapeutic agent for the treatment of various carcinomas including the same.

In one embodiment of the invention, the protein is the light chain (LC) of immunoglobulin (IgG). 61, 64, 67, 125, 129, 148, 167, 171, 191, 205, 210, 212 and 229 th lysine from the N-terminus in the amino acid sequence of the immunoglobulin (IgG) light chain (LC) represented by SEQ ID NO: 104 At least one of the residues is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition of an immunoglobulin (IgG) having an increased half-life and an IgG-based antibody therapeutic agent for the treatment of various carcinomas including the same.

In the present invention, site-directed mutagenesis was used to replace a lysine residue in the amino acid sequence of a protein with an arginine (R) residue. In this method, a primer is prepared using a DNA sequence that will induce a specific mutation, and then PCR is performed under specific conditions to produce a plasmid DNA in which a specific amino acid residue is substituted.

In the present invention, the target protein was transfected into the cell line by immunoprecipitation assay and sedimented to confirm the degree of ubiquitination, and as a result of treatment with MG132 (proteasome inhibitor) reagent, the target protein was ubiquitinated through an increase in the degree of ubiquitination. -It was confirmed that it goes through the path of degradation by the proteasome.

In the present invention, the pharmaceutical composition may be delivered to the body by various routes including oral, transcutaneous, subcutaneous, intravenous or intramuscular administration, and may be administered as an injection formulation. . In addition, the pharmaceutical composition of the present invention may be formulated according to a method well known to those skilled in the art so as to be rapidly released, delayed or slowly released after being administered according to the above method. The formulation is a tablet, a pill, a powder, a sachet, an elixir, a suspension, an emulsion, a solution, a syrup, These include aerosols, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders. Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, mannitol, xylitol, erythritol, maltitol, carbohydrates. , Gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose cellulose), polyvinyl pyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate, and mineral oil. . In addition, the formulation may further contain fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives, etc. have.

In the present invention, the singular form includes the plural form unless clearly stated otherwise. In addition, terms such as constituted or constituted in the present invention are interpreted as having a similar meaning to “including”. In the present invention, “bioactive (poly) peptide or protein” refers to a (poly) peptide or protein that exhibits useful biological activity when administered to a mammal, including humans.

Hereinafter, the present invention will be described in more detail based on examples. The following examples are for illustrative purposes only, and the present invention is not limited by the following examples.

Example 1: Analysis of ubiquitination of β-tropine protein and confirmation of half-life increase and intracellular signal transduction

1. Cloning into an expression vector and confirmation of protein expression

(1) Cloning of expression vector

In order to clone β-tropin, purified RNA was extracted from HepG2 cells (ATCC, HB-8065) using Trizol and chloroform. Next, single-stranded cDNA was synthesized using SuperScript™ First-Strand cDNA Synthesis System (Invitrogen, Grand Island, NY). The synthesized cDNA was used as a template to amplify β-tropine through polymerase chain reaction. The β-tropine DNA amplification product and pcDNA3-myc (5.6kb) were fragmented with restriction enzymes BamHI and EcoRI, conjugated and cloned (Fig. 1, β-tropine amino acid sequence: SEQ No. 1), and restriction enzyme digestion. Then, it was confirmed through agarose gel electrophoresis (Fig. 2). In addition, the part marked in bold and underlined on the nucleotide sequence of FIG. 1 is a primer set used when confirming through the polymerase chain reaction to reconfirm the cloned site, and the result was also confirmed through agarose gel electrophoresis ( Fig. 2). Polymerase chain reaction conditions were as follows; After reacting the initial denaturation at 94°C for 3 minutes, 30 seconds at 94°C for denature reaction, 30 seconds at 58°C for annealing reaction, 1 at 72°C for extension reaction Minutes were repeated for 25 cycles, and then reacted at 72° C. for 10 minutes. Western blotting of myc present in the pcDNA3-myc vector shown in the map of FIG. 1 using an anti-myc (9E10, Santa Cruz Biotechnology, sc-40) antibody in order to confirm whether the thus prepared DNA is properly expressed as a protein. The expression was confirmed through (Western blot). Through this, it was confirmed that the β-tropin protein bound to myc was well expressed, and it was confirmed that the quantification was loaded through a blot confirmed with actin (FIG. 3).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine (R) using site-directed mutagenesis, and the primer (β-tropine K62R FP 5'-AGGGACGGCTGACAAGGGCCAGGAA-) was used to induce a specific mutation. 3'(SEQ No. 2), RP 5'-CCAGGCTGTTCCTGGCCCTTGTCAGC-3' (SEQ No. 3); β-tropine K124R FP 5'-GGCACAGAGGGTGCTACGGGACAGC-3' (SEQ No. 4), RP 5'-C GTAGCACCCTCTGTGCCTGGGCCA -3' (SEQ No. 5); β-tropin K153R FP 5'-GAATTTG AGGTCTTAAGGGCTCACGC-3' (SEQ No. 6), RP 5'-CTTGTCAGCGTGAGCCCTTAAGAC CTC-3' (SEQ No. 7); β-tro After making pins K158R FP 5'-GCTCACGCTGACAGGCAGAGCCACAT-3' (SEQ No. 8), RP 5'-CCATAGGATGTGGCTCTGCCTGTCAGC-3' (SEQ No. 9), PCR was performed to prepare plasmid DNA in which specific amino acid residues were substituted. Using pcDNA3-myc-β-tropine as a template, four plasmid DNAs in which lysine residues were substituted with arginine (K→R) were prepared (Table 1).

Lysine(K) residue site Β-trophin construct in which Lysine (K) is substituted with Arginine (R) 62 pcDNA3-myc-β-trophin (K62R) 124 pcDNA3-myc-β-trophin (K124R) 153 pcDNA3-myc-β-trophin (K153R) 158 pcDNA3-myc-β-trophin (K158R)

2. In vivo ubiquitination assay

pcDNA3-myc-β-tropine WT and pMT123-HA-ubiquitin (J Biol Chem., 279(4), 2368-2376, 2004; Cell Research, 22, 873885, 2012; Oncogene, 22, 12731280, 2003; Cell , 78, 787-798, 1994) were used to infect HEK 293T cells (ATCC, CRL-3216). To confirm the degree of ubiquitination, 2 μg of pcDNA3-myc-β-tropine WT and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into cells and 24 hours later, MG132 (proteasome inhibitor, 5 Μg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 4). In addition, pcDNA3-myc-β-tropine WT, pcDNA3-myc-β-tropine substituent (K62R), pcDNA3-myc-β-tropine substituent (K124R), pcDNA3-myc-β-tropine substituent (K153R), respectively. And a plasmid encoding the pcDNA3-myc-β-tropine substituent (K158R) to transfect HEK 293T cells. To confirm the degree of ubiquitination, pcDNA3-myc-β-tropine WT, pcDNA3-myc-β-tropine substituent (K62R), pcDNA3-myc-β-tropine substituent (K124R), pcDNA3-myc-β-tro Cells were co-transfected (co-transfection) with 2 μg of each of the pin substituents (K153R) and pcDNA3-myc-β-trophin substituents (K158R) with 1 μg of pMT123-HA-ubiquitin DNA, and immunoprecipitation analysis after 24 hours Was carried out (Fig. 5).

Protein samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-myc (9E10) primary antibody ( Santa Cruz Biotechnology, sc-40) and incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology). The protein samples were washed twice with 2X SDS buffer, heated at 100° C. for 7 minutes, and then separated by performing SDS-PAGE The separated protein was polyvinylidene difluoride (PVDF). ) After transfer to the membrane (Millipore), anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778 ) In a weight ratio of 1:1,000 and an anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody using the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, when immunoprecipitation was performed with anti-myc (9E10, sc-40), ubiquitin was bound to pcDNA3-myc-β-tropine WT to form polyubiquitination. Smear ubiquitin was detected and the band appeared dark (Fig. 4, lanes 3 and 4) In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, polyubiquitination was formed. Is increased so that the band in which ubiquitin is detected is more It appeared very dark (Fig. 4, lane 4). These results suggest that β-tropine binds to ubiquitin and becomes polyubiquitinated through the ubiquitin-proteasome system. In addition, in the case of pcDNA3-myc-β-tropine substituent (K62R), pcDNA3-myc-β-tropine substituent (K153R), and pcDNA3-myc-β-tropine substituent (K158R), the band was softer than WT. . This indicates that less ubiquitin was detected because ubiquitin could not bind to these substituents (FIG. 5, lanes 3, 5 and 6).

3. Confirmation of the half-life of β-tropine by the protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-β-tropine WT, pcDNA3-myc-β-tropine substituent (K62R), pcDNA3-myc-β-tropine substituent (K124R), pcDNA3-myc-β-tropine substituent (K153R), and The pcDNA3-myc-β-tropine substituent (K158R) was transfected into HEK 293T cells at 2 μg each. 48 hours after transfection, the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and half-life was measured over 20 minutes, 40 minutes and 60 minutes. As a result, it was confirmed that the decomposition of human β-tropin was suppressed (FIG. 6). The half-life of human β-tropine is less than 1 hour, whereas the half-life of human β-tropine substitution (variant) (K62R) and β-tropine substitution (K158R) is 1 hour or longer than that of WT. It is represented by (Fig. 6).

4. Confirmation of signal transduction by β-tropine and β-tropine substituents in cells

It has been reported that transient expression of β-tropin in mouse liver promotes the proliferation of pancreatic-beta cells (Cell, 153, 747-758, 2013). In this example, the signal transduction process by β-tropine and β-tropine substituents in the cell was confirmed. First, PANC-1 cells (ATCC, CRL-1469) were washed 7 times with PBS, and then pcDNA3-myc-β-tropine WT, pcDNA3-myc-β-tropine substituent (K62R), pcDNA3-myc-β- PANC-1 cells were transfected using 3 μg each of the tropine substituent (K124R), the pcDNA3-myc-β-tropine substituent (K153R), and the pcDNA3-myc-β-tropine substituent (K158R). After 2 days of infection, proteins were extracted from the cells and quantified, respectively, and Western blotting was performed to confirm the intracellular signal transduction process. To this end, pcDNA3-myc-β-tropine WT, pcDNA3-myc-β-tropine substituent (K62R), pcDNA3-myc-β-tropine substituent (K124R), pcDNA3-myc-β-tropine substituent ( K153R) and pcDNA3-myc-β-tropine substituents (K158R)-infected PANC-1 cells were transferred to a polyvinylidene difluoride (PVDF) membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, cell signaling 9131S) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) 1 : A blocking solution containing at a weight ratio of 1000 and anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody. As a result, pcDNA3-myc-β-tropine substituent (K62R), pcDNA3-myc-β-tropine substituent (K124R), and pcDNA3-myc-β-tropine substituent (K153R) were pcDNA3- It showed the same or increased phospho-STAT3 signaling as that of myc-β-tropin WT (Fig. 7).

Example 2: Analysis of ubiquitination of growth hormone protein and confirmation of half-life increase and intracellular signal transduction

1. Cloning into an expression vector and confirmation of protein expression

(1) Cloning of expression vector

Growth hormone (GH) amplified through polymerase chain reaction and pCS4-flag (4.3 kb, Oncotarget., 7(12), 14441-14457, 2016) were fragmented with EcoRI, a restriction enzyme, and then conjugated and cloned ( Figure 8, GH amino acid sequence: SEQ No. 10), the results were confirmed through agarose gel electrophoresis after digestion of restriction enzymes (Fig. 9). In addition, the portion indicated in bold and underlined on the nucleotide sequence of FIG. 8 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is also confirmed through agarose gel electrophoresis. (Fig. 9). Polymerase chain reaction conditions are as follows; After the initial denaturation was reacted at 94° C. for 3 minutes, 30 seconds at 94° C. for the denaturation reaction, 30 seconds at 60° C. for the annealing reaction, and 30 seconds at 72° C. for the extension reaction were repeated in 25 cycles, Then, it was reacted at 72°C for 10 minutes. In order to confirm whether the thus-produced DNA was properly expressed as a protein, Western blotting was performed using an anti-flag (Sigma-aldrich, F3165) antibody on the flag present in the pCS4-flag vector indicated in the map of FIG. 8. As a result, it was confirmed that the growth hormone protein bound to the flag was well expressed, and the blotting confirmed by actin was quantitatively loaded (FIG. 10).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-directed mutagenesis, and the primer (GH K67R FP 5'-CCAAAGGAACAGAGGTATTCATTC-3' (SEQ No. 11) was used with the DNA sequence to induce specific mutations). , RP 5'-CAGGAATGAATACCTCTGTTCCTT-3' (SEQ No. 12); GH K141R FP 5'-GA CCTCCTAAGGGACCTAGAG-3' (SEQ No. 13), RP 5'-CTCTAGGTCCCTTAGGAGGTC-3' (SEQ No. 14); GH Plasmid DNA in which specific amino acid residues were substituted by PCR under specific conditions after making K166R FP 5'-CAGATCTTCAGGCAGACCTAC-3' (SEQ No. 15), RP 5'-GTAGGTCTGCCTGAAGATCTG-3' (SEQ No. 16) Using pcDNA3-myc-growth hormone as a template, plasmid DNA in which the lysine residue was substituted with arginine (K→R) was prepared (Table 2).

Lysine (K) residue position GH construct in which Lysine (K) is substituted with Arginine (R) 67 pCS4-flag-GH (K67R) 141 pCS4-flag-GH (K141R) 166 pCS4-flag-GH (K166R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pCS4-flag-growth hormone WT and pMT123-HA-ubiquitin DNA. In order to confirm the degree of ubiquitination, 2 µg of pCS4-flag-growth hormone WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. 24 hours after transfection, MG132 (proteasome inhibitor, 5 μg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 11). In addition, pCS4-flag-growth hormone WT, pCS4-flag-growth hormone substitute (K67R), pCS4-flag-growth hormone substitute (K141R), pCS4-flag-growth hormone substitute (K166R), and pMT123-HA-ubiquitin DNA were respectively used. HEK 293T cells were infected using the encoding plasmid. To confirm the degree of ubiquitination, pCS4-flag-growth hormone WT, pCS4-flag-growth hormone substituent (K67R), pCS4-flag-growth hormone substituent (K141R), and pCS4-flag-growth hormone substituent (K166R) each 2 Μg and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and immunoprecipitation analysis was performed 24 hours later (Fig. 12).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-flag (Sigma-aldrich, F3165) 1 After mixing with the primary antibody, it was incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. For immunoblotting, a protein sample was mixed with 2X SDS buffer, heated at 100° C. for 7 minutes, and then separated by performing SDS-PAGE The separated protein was separated by a polyvinylidene difluoride (PVDF) membrane. And then anti-flag (Sigma-aldrich, F3165), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000. The blocking solution and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody were used to develop the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). .

As a result, when immunoprecipitation was performed with anti-flag (Sigma-aldrich, F3165), ubiquitin in pCS4-flag-growth hormone WT was bound to polyubiquitin, resulting in the formation of polyubiquitin. 11, lanes 2 and 3). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 11, lane 3). In addition, in the case of pCS4-flag-growth hormone substituent (K67R), pCS4-flag-growth hormone substituent (K141R), and pCS4-flag-growth hormone substituent (K166R), the band was softer than WT, and pCS4-flag-growth hormone substituent (K67R), pCS4-flag-growth hormone substituent (K141R), and pCS4-flag-growth hormone substituent (K166R) were unable to bind to ubiquitin, and thus, less ubiquitin was detected (FIG. 12, lanes 3-5). These results show that growth hormone binds to ubiquitin and is polyubiquitinated and decomposed through the ubiquitin-proteasome system.

3. Confirmation of half-life of growth hormone by protein production inhibitor cycloheximide (CHX)

2 µg of pCS4-flag-growth hormone WT, pCS4-flag-growth hormone substitute (K67R), pCS4-flag-growth hormone substitute (K141R), and pCS4-flag-growth hormone substitute (K166R) were transfected into HEK 293T cells by 2 µg. (transfection). 48 hours after transfection, the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and half-life was measured over 1 hour, 2 hours, 4 hours, and 8 hours. As a result, it was confirmed that the decomposition of human growth hormone was suppressed (FIG. 13). The half-life of human growth hormone was less than 2 hours, whereas the half-life of human pCS4-flag-growth hormone substituent (K141R) was longer than WT by more than 8 hours, and this result is shown in a graph (FIG. 13).

4. Confirmation of signal transduction by growth hormone and growth hormone substitutes in cells

It has been reported that growth hormone regulates the transcription of STAT (signal transducers and activators of transcription) proteins at the gene level (Oncogene, 19, 2585-2597, 2000). Accordingly, in this example, the signal transduction process by growth hormone and growth hormone substitutes in the cell was confirmed. HEK293 cells were prepared by using 3 μg each of pCS4-flag-growth hormone WT, pCS4-flag-growth hormone substitute (K67R), pCS4-flag-growth hormone substitute (K141R), and pCS4-flag-growth hormone substitute (K166R). Infected. After 1 day of infection, the cells were lysed using a sonicator, and the obtained protein was treated with PANC-1 cells washed 7 times with PBS to infect, and after 2 days, PANC-1 cells Protein was extracted from and quantified respectively. Western blotting was performed to confirm the intracellular signal transduction process. PANC-1 infected with each of the pCS4-flag-growth hormone WT, pCS4-flag-growth hormone substituent (K67R), pCS4-flag-growth hormone substituent (K141R) and pCS4-flag-growth hormone substituent (K166R) in this process Protein isolated from cells was transferred to a polyvinylidene difluoride (PVDF) membrane, and then anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, Cell Signaling Technology, 9131S). ) And anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 and anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti -Mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody. As a result, pCS4-flag-growth hormone substitute (K141R) showed the same or increased phospho-STAT3 signaling as pCS4-flag-growth hormone WT in PANC-1 cells, and pCS4-flag-growth hormone substitute (K67R) Showed increased phospho-STAT3 signaling than the control group (Fig. 14).

Example 3: Ubiquitination analysis of insulin protein and confirmation of half-life increase and intracellular signal transduction

1. Cloning into an expression vector and confirmation of protein expression

(1) Cloning of expression vector

The insulin DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were fragmented with restriction enzymes BamHI and EcoRI, and then conjugated and cloned (FIG. 15, insulin amino acid sequence: SEQ No. 17). The results were confirmed through restriction enzyme digestion and agarose gel electrophoresis (FIG. 16). In addition, the part marked in bold and underlined on the nucleotide sequence of FIG. 15 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. (Fig. 16). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94° C. for 3 minutes, followed by repeating 25 cycles of 30 seconds at 94° C. for the denaturation reaction, 30 seconds at 60° C. for the annealing reaction, and 30 seconds at 72° C. for the extension reaction. Then, the reaction was performed at 72°C for 10 minutes. Western blotting of myc present in the pcDNA3-myc vector shown in the map of FIG. 15 using an anti-myc (9E10, Santa Cruz Biotechnology, sc-40) antibody in order to confirm whether the thus prepared DNA is properly expressed as a protein. Expression was confirmed through. It was confirmed that the insulin protein bound to myc was well expressed, and quantitatively loaded through a blot confirmed with actin (FIG. 17).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-directed mutagenesis, and a primer (insulin K53R FP 5'-GGCTTCTTCTACACACCCAGGACCC-3' (SEQ No. 18)) was used to induce a specific mutation. , RP 5'-CTCCCGGCGGGTCCTGGGTGTGTA-3' (SEQ No. 19); insulin K88R FP 5'-TCCCTGCAGAGGCGTGGCATTGT-3' (SEQ No. 20), RP 5'-TTGTTCCACAATGCCA CGCCTCTGCAG-3' (SEQ No. 21) was prepared. After that, PCR was performed under specific conditions to prepare a plasmid DNA in which a specific amino acid residue was substituted.PCDNA3-myc-insulin was used as a template, and a plasmid DNA in which two lysine residues were substituted with arginine (K→R) was used. Was produced (Table 3).

Lysine (K) residue position Insulin construct in which Lysine (K) is substituted for Arginine (R) 53 pcDNA3-myc-insulin (K53R) 88 pcDNA3-myc-insulin (K88R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-insulin WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 µg of pcDNA3-myc-insulin WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 μg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 18). In addition, HEK 293T cells were infected using a plasmid encoding pcDNA3-myc-insulin WT, pcDNA3-myc-insulin substituent (K53R), pcDNA3-myc-insulin substituent (K88R), and pMT123-HA-ubiquitin DNA, respectively. To confirm the degree of ubiquitination, the cells were co-coated with pcDNA3-myc-insulin WT, pcDNA3-myc-insulin substituent (K53R), and pcDNA3-myc-insulin substituent (K88R), respectively, 2 μg and 1 μg of pMT123-HA-ubiquitin DNA. Immunoprecipitation analysis was performed 24 hours after transfection (FIG. 19).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then mixed with anti-myc (9E10) primary antibody. And incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. The silver protein sample was mixed with 2X SDS buffer, heated at 100° C. for 7 minutes, and then separated by performing SDS-PAGE The separated protein was transferred to a polyvinylidenedifluoride membrane, followed by anti-myc (9E10, sc-40), anti-HA (sc-7392) and anti-β-actin (sc-47778) in a weight ratio of 1:1000 and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+) L), KPL, 074-1806) was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was performed with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin was bound to pcDNA3-myc-insulin WT to form polyubiquitin. It appeared dark (Fig. 18, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 μg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 18, lane 4). In addition, in the case of the pcDNA3-myc-insulin substituent (K53R), the band was softer than that of WT, and the pcDNA3-myc-insulin substituent (K53R) was unable to bind to ubiquitin, so less ubiquitin was detected (FIG. 19, lane 3). The above results show that insulin binds to ubiquitin and is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Confirmation of half-life of insulin by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-insulin WT, pcDNA3-myc-insulin substitutent (K53R), and pcDNA3-myc-insulin substitutent (K88R) were transfected into HEK293T cells at 2 µg each. 48 hours after transfection, the protein production inhibitor cyclohexamide (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and the half-life was measured over 2 hours, 4 hours, and 8 hours, as a result of decomposition of human insulin. It was confirmed that is suppressed (FIG. 20). As a result, the half-life of human insulin is within 30 minutes, whereas the half-life of human pcDNA3-myc-insulin substituent (K53R) is longer than WT by 1 hour or more, and this result is shown in a graph (FIG. 20).

4. Confirmation of signal transduction by insulin and insulin substitutes in cells

It has been reported that insulin stimulates the phosphorylation of STAT3 in the liver and consequently regulates glucose homeostasis in the liver (Cell Metab., 3, 267275, 2006). In this example, the signal transduction process by insulin and insulin substitutes in the cell was confirmed. First, the pcDNA3-myc-insulin WT, pcDNA3-myc-insulin substituent (K53R), and pcDNA3-myc-insulin substituent (K88R) were each used 3 μg each, and PANC-1 and HepG2 (ATCC) washed 7 times with PBS using 3 μg each. , AB-8065). After 2 days, proteins were extracted from the cells and quantified, and then Western blotting was performed to confirm the intracellular signal transduction process. In this process, proteins isolated from PANC-1 and HepG2 (ATCC, AB-8065) cells infected with pcDNA3-myc-insulin WT, pcDNA3-myc-insulin substituent (K53R) and pcDNA3-myc-insulin substituent (K88R), respectively. Transferred to PVDF membrane, then anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, Cell Signaling 9131S) and anti- Blocking solution containing β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000, anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004, and anti-mouse (Peroxidase -labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody. As a result, pcDNA3-myc-insulin substituent (K53R) showed similar or increased phospho-STAT3 signaling to pcDNA3-myc-insulin WT in PANC-1 and HepG2 (ATCC, AB-8065) cells (FIG. 21 ). .

Example 4: Analysis of ubiquitination of interferon-α protein and confirmation of half-life increase and intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

Interferon-α DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were fragmented with EcoRI, a restriction enzyme, and then conjugated and cloned (FIG. 22, interferon-α amino acid sequence: SEQ No. 22), The results were confirmed through restriction enzyme digestion and agarose gel electrophoresis (FIG. 23). In addition, the part marked in bold and underlined on the nucleotide sequence of FIG. 22 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. Was done (Fig. 23). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94° C. for 3 minutes, followed by repeating 25 cycles of 94° C. for the denaturation reaction for 30 seconds, 58° C. for the annealing reaction for 30 seconds, and 1 minute at 72° C. for the extension reaction. Then, it was reacted at 72°C for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, the myc present in the pcDNA3-myc vector shown in the map of FIG. 22 was expressed through western blotting using an anti-myc (9E10, sc-40) antibody. It was confirmed, and through this, it was confirmed that the interferon-α protein bound to myc was well expressed, and it was confirmed that it was quantitatively loaded through a blot confirmed with actin (FIG. 24).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-directed mutagenesis, and a primer (IFN-α K93R FP 5'-CTTCAGCACAAGGGACTCATC-3' (SEQ No. 23), RP 5'-CAGATGAGTCCCTTGTGCTGA-3' (SEQ No. 24); IFN-α K106R FP 5'-CTCCTAGAC AGATTCTACACT-3' (SEQ No. 25), RP 5'-AGTGTAGAATCTGTCTAGGAG-3' (SEQ No. 26); IFN-α K144R FP 5'-GCTGTGAGGAGATACTTCCAA-3' (SEQ No. 27), RP 5'-TT GGAAGTATCTCCTCACAGC-3' (SEQ No. 28); IFN-α K154R FP 5'-CTCTATCTGAGAGAG AAGAAA-3 '(SEQ No. 29), RP 5'-TTTCTTCTCTCTCAGATAGAG-3' (SEQ No. 30) was prepared, and then PCR was performed under specific conditions to prepare plasmid DNA in which specific amino acid residues were substituted. Interferon-α was used as a template, and four plasmid DNAs in which the lysine residue was substituted with arginine (K→R) were prepared (Table 4).

Lysine (K) residue position IFN-α construct in which Lysine (K) is substituted with Arginine (R) 93 pcDNA3-myc-IFN-α (K93R) 106 pcDNA3-myc-IFN-α (K106R) 144 pcDNA3-myc-IFN-α (K144R) 154 pcDNA3-myc-IFN-α (K154R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-interferon-α WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 µg of pcDNA3-myc-interferon-α WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 25). In addition, pcDNA3-myc-interferon-α WT, pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent (K106R), pcDNA3-myc-interferon-α substituent (K144R), pcDNA3-myc respectively -Interferon-α substituent (K154R), and a plasmid encoding pMT123-HA-ubiquitin DNA were used to infect HEK 293T cells. To confirm the degree of ubiquitination, pcDNA3-myc-interferon-α WT, pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent (K106R), pcDNA3-myc-interferon-α substituent (K144R) ) And pcDNA3-myc-interferon-α substituent (K154R), respectively, 2 μg and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 26 ).

Samples obtained for immunoprecipitation were dissolved in a lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then mixed with anti-myc (9E10) primary antibody. It was incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. Protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and separated by performing SDS-PAGE After transferring the separated protein to a PVDF membrane, anti-myc (9E10, Santa Cruz Biotechnology, sc- 40), a blocking solution containing an anti-HA (Santa Cruz Biotechnology, sc-7392) and an anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 and an anti-mouse (Peroxidase-labeled antibody) to mouse IgG (H+L), KPL, 074-1806) was developed with the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody.

As a result, when immunoprecipitation was performed with anti-myc (9E10, sc-40), ubiquitin was bound to pcDNA3-myc-interferon-α WT to form polyubiquitin. (Figure 25, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 25, lane 4). In addition, in the case of pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent (K106R), pcDNA3-myc-interferon-α substituent (K144R) and pcDNA3-myc-interferon-α substituent (K154R) , The band was softer than WT, pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent (K106R), pcDNA3-myc-interferon-α substituent (K144R) and pcDNA3-myc-interferon- Since the α substituent (K154R) could not bind to ubiquitin, less ubiquitin was detected (FIG. 26, lanes 3 and 6). The above results show that interferon-α binds to ubiquitin and is polyubiquitinated and decomposed through the ubiquitin-proteasome system.

3. Confirmation of half-life of interferon-α by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-interferon-α WT, pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent (K106R), pcDNA3-myc-interferon-α substituent (K144R) and pcDNA3-myc-interferon The -α substituent (K154R) was transfected into HEK 293T cells by 2 µg each. 48 hours after transfection, the protein production inhibitor cyclohexamid (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and the half-life was measured over 1 and 2 days. As a result, degradation of human interferon-α was inhibited. It was confirmed that it became (FIG. 27). The half-life of human interferon-α is within 1 day, while that of human pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent (K144R) and pcDNA3-myc-interferon-α substituent (K154R). The half-life was longer than that of WT by more than 2 days, and this result was shown as a graph (FIG. 27).

4. Confirmation of signal transduction by interferon-α and interferon-α substituents in cells

It has been reported that interferon-α increases STAT-1, -2 and -3 (J Immunol., 187, 2578-2585, 2011), and in melanoma cells, interferon-α activates STAT3 protein, which promotes growth. Has been reported (Eur J Cancer, 45, 1315-1323, 2009). In this example, the signaling process by interferon-α and interferon-α substituents in the cell was confirmed. After washing THP-1 cells (ATCC, TIB-202) 7 times with PBS, pcDNA3-myc-interferon-α WT, pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent ( K106R), pcDNA3-myc-interferon-α substituent (K144R), and pcDNA3-myc-interferon-α substituent (K154R) were each used in 3 μg each to infect THP-1 cells and then sequentially after 1 and 2 days. After each quantification by extracting the protein from the cells, Western blot was performed to confirm the intracellular signal transduction process. In this process, pcDNA3-myc-interferon-α WT, pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent (K106R), pcDNA3-myc-interferon-α substituent (K144R), pcDNA3- After transferring the protein isolated from THP-1 cells infected with the myc-interferon-α substituent (K154R) to the PVDF membrane, anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc -21876), anti-phospho-STAT3 (Y705, cell signaling 9131S) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 and a blocking solution containing anti-rabbit (goat anti- rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody using ECL system (Western blot detection kit , ABfrontier, Seoul, Korea). As a result, pcDNA3-myc-interferon-α substituent (K93R), pcDNA3-myc-interferon-α substituent (K106R), pcDNA3-myc-interferon-α substituent (K144R), pcDNA3-myc-interferon-α substituent (K154R) Showed the same or increased phospho-STAT3 signaling as pcDNA3-myc-interferon-α WT in THP-1 cells (ATCC, TIB-202) (FIG. 28 ).

Example 5: Granulocyte colony Stimulus factor (G- CSF ) Of protein Ubiquitination Analysis and confirmation of half-life increase and intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

Granulocyte colony stimulating factor (G-CSF) DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were fragmented with EcoRI, a restriction enzyme, and then conjugated and cloned (Figure 29, G-CSF amino acid sequence: SEQ No. 31), the results were confirmed through agarose gel electrophoresis after digestion with restriction enzymes (FIG. 30). In addition, the portion marked in bold and underlined on the nucleotide sequence of FIG. 29 is the primer cent used when confirming through the polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. (Fig. 30). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94° C. for 3 minutes, followed by repeating 25 cycles of 30 seconds at 94° C. for the denaturation reaction, 30 seconds at 58° C. for the annealing reaction, and 1 minute at 72° C. for the extension reaction. Then, it was reacted at 72° C. for 10 minutes. Western blotting of myc present in the pcDNA3-myc vector shown in the map of FIG. 29 using an anti-myc (9E10, Santa Cruz Biotechnology, sc-40) antibody in order to confirm whether the thus produced DNA is properly expressed as a protein. Expression was confirmed through. It was confirmed that the granulocyte colony stimulating factor (G-CSF) protein bound to myc was well expressed and quantitatively loaded through a blot confirmed with actin (FIG. 31).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-directed mutagenesis, and a primer (G-CSF K46R FP 5'-AGCTTCCTGCTCAGGTGCTTAGAG-3' (SEQ No. 32), RP 5'-TTGCTCTAAGCACCTGAGCAGGAA-3' (SEQ No. 33), G-CSF K73R FP 5'- TGTGCCACCTACAGGCTGTGCCAC-3' (SEQ No. 34), RP 5'-GGGGTGGCACAGCCTGTA GGTGGC-3' (SEQ No. 35) was prepared, and then PCR was performed under specific conditions to prepare a plasmid DNA in which a specific amino acid residue was substituted. Two plasmid DNAs substituted (K→R) were prepared (Table 5).

Lysine (K) residue position G-CSF construct in which Lysine (K) is substituted with Arginine (R) 46 pcDNA3-myc-G-CSF (K46R) 73 pcDNA3-myc-G-CSF (K73R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-granular colony stimulating factor (G-CSF) WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 µg of pcDNA3-myc-granular colony stimulating factor (G-CSF) WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of infection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 32). In addition, pcDNA3-myc-granular colony stimulating factor (G-CSF) WT, pcDNA3-myc-granular colony stimulating factor (G-CSF) substituent (K46R), pcDNA3-myc-granular colony stimulating factor (G-CSF) (K73R) ) And the plasmid encoding pMT123-HA-ubiquitin DNA were used to infect HEK 293T cells. To confirm the degree of ubiquitination, pcDNA3-myc-granular colony stimulating factor (G-CSF) WT, pcDNA3-myc-granular colony stimulating factor (G-CSF) substituent (K46R), pcDNA3-myc-granular colony stimulating factor (G -CSF) (K73R) was co-transfected into cells with 2 µg, respectively, and 1 µg of pMT123-HA-ubiquitin DNA, and immunoprecipitation analysis was performed 24 hours later (Fig. 33).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then mixed with anti-myc (9E10) primary antibody. And incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. The silver protein sample was mixed with 2X SDS buffer, heated at 100° C. for 7 minutes, and then separated by performing SDS-PAGE The separated protein was transferred to a PVDF membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 blocking solution and anti-mouse (Peroxidase- Labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody.

As a result, when immunoprecipitation was performed with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin was bound to the pcDNA3-myc-granular colony stimulating factor (G-CSF) WT and spread as polyubiquitin was formed. Shaped ubiquitin was detected and the band appeared dark (FIG. 32, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 32, lane 4). In addition, in the case of pcDNA3-myc-granular colony stimulating factor (G-CSF) (K73R), the band was softer than that of WT, and pcDNA3-myc-granular colony stimulating factor (G-CSF) (K73R) was unable to bind to ubiquitin. Less of this was detected (Fig. 33, lane 4). The above results show that the granulocyte colony stimulating factor (G-CSF) binds to ubiquitin and is polyubiquitinated and decomposed through the ubiquitin-proteasome system.

3. Confirmation of half-life of granulocyte colony stimulating factor (G-CSF) by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-granular colony stimulating factor (G-CSF) WT, pcDNA3-myc-granular colony stimulating factor (G-CSF) substituent (K46R), pcDNA3-myc-granular colony stimulating factor (G-CSF) (K73R) Each 2 μg of HEK 293T cells were transfected. 48 hours after transfection, the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and the half-life was measured over 4 hours, 8 hours and 16 hours. As a result, it was confirmed that the decomposition of the human granulocyte colony stimulating factor (G-CSF) was inhibited (FIG. 34). The half-life of human granulocyte colony stimulating factor (G-CSF) was 4 hours, whereas the half-life of human pcDNA3-myc-granular colony stimulating factor (G-CSF) (K73R) was 16 hours or longer than that of WT. Shown (Fig. 34).

4. In the cell Granulocyte colony Stimulus factor (G- CSF )Wow Granulocyte colony Stimulus factor (G- CSF ) Confirmation of signal transmission by substituents

Granulocyte colony stimulating factor (G-CSF) has been reported to activate STAT3 in glioma cells and thereby participate in glioma proliferation (Cancer Biol Ther., 13(6), 389-400, 2012), It has been reported that it is expressed in ovarian epithelial cancer cells and that there is a pathological association of female uterine cancer by regulating the JAK2/STAT3 pathway (Br J Cancer, 110, 133-145, 2014). In this example, the signaling process by the granulocyte colony stimulating factor (G-CSF) and granulocyte colony stimulating factor (G-CSF) substituents was confirmed in the cell. First, THP-1 cells (ATCC, TIB-202) were washed 7 times with PBS, and then pcDNA3-myc-granular colony stimulating factor (G-CSF) WT, pcDNA3-myc-granular colony stimulating factor (G-CSF) substituent (K46R) and pcDNA3-myc-granular colony stimulating factor (G-CSF) substituents (K73R) were used to infect THP-1 cells (ATCC, TIB-202) using 3 µg each. After 1 day of infection, proteins were extracted from the cells and quantified, respectively, and Western blotting was performed to confirm the intracellular signal transduction process. In this process, pcDNA3-myc-granular colony stimulating factor (G-CSF) WT, pcDNA3-myc-granular colony stimulating factor (G-CSF) substituent (K46R), pcDNA3-myc-granular colony stimulating factor (G-CSF) substituent (K73R)-infected THP-1 cells (ATCC, TIB-202) were transferred to a polyvinylidene difluoride (PVDF) membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc). -40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, cell signaling 9131S) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1,000 Blocking solution and anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) The secondary antibody was developed with the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, pcDNA3-myc-granular colony stimulating factor (G-CSF) substituent (K46R), pcDNA3-myc-granular colony stimulating factor (G-CSF) substituent (K73R) is THP-1 cells (ATCC, TIB-202) Within the pcDNA3-myc-granulocyte colony stimulating factor (G-CSF) WT showed the same or increased phospho-STAT3 signaling (FIG. 35).

Example 6: Analysis of ubiquitination of interferon-β protein and confirmation of half-life increase and intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

Interferon-β DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were fragmented with EcoRI, a restriction enzyme, and then cloned by conjugation (FIG. 36, interferon-β amino acid sequence: SEQ No. 36), The results were confirmed through restriction enzyme digestion and agarose gel electrophoresis (FIG. 37). In addition, the portion marked in bold and underlined on the nucleotide sequence of FIG. 36 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. (Fig. 37). Polymerase chain reaction conditions are as follows; After reacting the initial denaturation at 94° C. for 3 minutes, 25 cycles were repeated for 30 seconds at 94° C. for the denaturation reaction, 30 seconds at 58° C. for the annealing reaction, and 50 seconds at 72° C. for the extension reaction. After that, it was reacted for 10 minutes at 72°C. In order to confirm whether the thus-produced DNA is properly expressed as a protein, the myc present in the pcDNA3-myc vector shown in the map of FIG. 36 was expressed through western blotting using an anti-myc (9E10, sc-40) antibody. It was confirmed, and through this, it was confirmed that the interferon-β protein bound to myc was well expressed, and it was found that the quantification was loaded through the blot confirmed with actin (FIG. 38). In addition, in the case of interferon-β, two types of expression bands appeared due to glycosylation in cells, and 500 units of PNGase F (New England Biolabs Inc., P0704S) blocking this process were added at 37°C. After treatment for a period of time, it was confirmed that only one type of expression band was visible (FIG. 38).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine (R) using site-directed mutagenesis, and the primer (IFN-β K40R FP 5'-CAGTGTCAGAGGCTCCTGTGG-3) was used to induce a specific mutation. '(SEQ No. 37), RP 5'-CCACAGGAGCCTCTGACACTG-3' (SEQ No. 38); IFN-β K126R FP 5'-CT GGAAGAAAGACTGGAGAAA-3' (SEQ No. 39), RP 5'-TTTCTCCAGTCTTTCTTCCAG-3 '(SEQ No. 40); IFN-β K155R FP 5'-CATTACCTGAGGGCCAAGGAG-3' (SEQ No. 41), RP 5'-CTCCTTGGCCCTCAGGTAATG-3' (SEQ No. 42) was prepared, and then PCR under specific conditions. Plasmid DNA in which a specific amino acid residue was substituted was prepared by using pcDNA3-myc-interferon-β as a template, and plasmid DNA in which the lysine residue was substituted with arginine (K→R) was prepared (Table 6).

Lysine (K) residue position IFN-β construct in which Lysine (K) is substituted with Arginine (R) 40 pcDNA3-myc-IFN-β (K40R) 126 pcDNA3-myc-IFN-β (K126R) 155 pcDNA3-myc-IFN-β (K155R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-interferon-β WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 µg of pcDNA3-myc-interferon-β WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 39). In addition, pcDNA3-myc-interferon-β WT, pcDNA3-myc-interferon-β substituent (K40R), pcDNA3-myc-interferon-β substituent (K126R), pcDNA3-myc-interferon-β substituent (K155R) and pMT123-HA- HEK 293T cells were infected using plasmids each encoding ubiquitin DNA. To confirm the degree of ubiquitination, pcDNA3-myc-interferon-β WT, pcDNA3-myc-interferon-β substituent (K40R), pcDNA3-myc-interferon-β substituent (K126R), pcDNA3-myc-interferon-β substituent (K155R) ), respectively, 2 μg and 1 μg of pMT123-HA-ubiquitin DNA were cotransfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 40).

Samples obtained for immunoprecipitation were dissolved in a lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then mixed with anti-myc (9E10) primary antibody. It was incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. Protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and then separated by performing SDS-PAGE The separated protein was transferred to a PVDF membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 blocking solution and anti-mouse (Peroxidase- Labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody.

As a result, when immunoprecipitation was performed with anti-myc (9E10, sc-40), ubiquitin was bound to pcDNA3-myc-interferon-β WT to form polyubiquitin. (Figure 39, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 39, lane 4). In addition, in the case of pcDNA3-myc-interferon-β substituent (K40R), pcDNA3-myc-interferon-β substituent (K126R), and pcDNA3-myc-interferon-β substituent (K155R), the band was softer than WT, and pcDNA3-myc- Interferon-β substituent (K40R), pcDNA3-myc-interferon-β substituent (K126R), and pcDNA3-myc-interferon-β substituent (K155R) were unable to bind to ubiquitin, resulting in less ubiquitin detection (Fig. 40, lanes 3 and 5). ). The above results show that interferon-β binds to ubiquitin and is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Confirmation of half-life of interferon-β by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-interferon-β WT, pcDNA3-myc-interferon-β substituent (K40R), pcDNA3-myc-interferon-β substituent (K126R), pcDNA3-myc-interferon-β substituent (K155R) in HEK by 2 μg each 293T cells were transfected. 48 hours after transfection, the protein production inhibitor cyclohexamide (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and the half-life was measured over 4 hours and 8 hours. As a result, it was confirmed that the decomposition of human interferon-β was suppressed (Fig. 41). The half-life of human interferon-β is 4 hours, whereas the half-life of human pcDNA3-myc-interferon-β substituent (K126R) and pcDNA3-myc-interferon-β substituent (K155R) is 8 hours, which is longer than that of WT. It is represented by (Fig. 41).

4. Confirmation of signaling by interferon-β and interferon-β substituents in cells

It has been reported that treatment of interferon in cells induces the activity of signaling processes including AKT (Pharmaceuticals (Basel), 3, 994-1015, 2010). In this example, the signaling process by interferon-β and interferon-β substituents in the cell was confirmed. pcDNA3-myc-interferon-β WT, pcDNA3-myc-interferon-β substituent (K40R), pcDNA3-myc-interferon-β substituent (K126R), pcDNA3-myc-interferon-β substituent (K155R) were each used by 3 μg. To infect HEK293 cells. After 1 day of infection, the cells were lysed using a sonicator, and the obtained protein was treated with HepG2 cells (ATCC, AB-8065) washed 7 times with PBS to infect, and after 2 days, HepG2 Proteins were extracted from the cells and quantified respectively. Western blot was performed to confirm the intracellular signal transduction process. In this process, they were infected with pcDNA3-myc-interferon-β WT, pcDNA3-myc-interferon-β substituent (K40R), pcDNA3-myc-interferon-β substituent (K126R), and pcDNA3-myc-interferon-β substituent (K155R), respectively. After moving the protein isolated from HepG2 cells to the PVDF membrane, anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, cell signaling 9131S), anti-AKT (H-136, Santa Cruz Biotechnology, sc-8312), anti-phospho-AKT (S473, cell signaling 9271S) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 blocking solution and anti-rabbit ( goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody using ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, pcDNA3-myc-interferon-β substituents (K40R), pcDNA3-myc-interferon-β substituents (K126R), and pcDNA3-myc-interferon-β substituents (K155R) are pcDNA3-myc-interferon-β in HepG2 cells. It showed the same or increased phospho-AKT signaling as WT (Fig. 42).

Example 7: of erythropoietin protein Ubiquitination Analysis and confirmation of half-life increase and intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

The erythropoietin DNA amplification product by polymerase chain reaction and the pcDNA3-myc expression vector (5.6 kb) were fragmented with EcoRI, a restriction enzyme, and then conjugated and cloned (Fig. 43, erythropoietin amino acid sequence: SEQ No. 43). ), the result was confirmed through agarose gel electrophoresis after digestion of the restriction enzyme (FIG. 44). In addition, the part marked in bold and underlined on the nucleotide sequence of FIG. 43 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. (Fig. 44). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94° C. for 3 minutes, followed by 25 cycles of 30 seconds at 94° C. for the denaturation reaction, 30 seconds at 58° C. for the annealing reaction, and 1 minute at 72° C. for the extension reaction. Then, it was reacted at 72° C. for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, western blotting of myc present in the pcDNA3-myc vector shown in the map of FIG. 43 was performed using an anti-myc (9E10, sc-40) antibody. As a result, it was confirmed that the erythropoietin protein bound to myc was well expressed, and it was found that the quantification was loaded through the blot confirmed with actin (FIG. 45).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-directed mutagenesis, and a primer (EPO K124R FP 5'-GCATGTGGATAGAGCCGTCAGTGC-3' (SEQ No. 44) using the DNA sequence to induce a specific mutation) was used. , RP 5'-GCACTGACGGCTCTATCCACATGC-3' (SEQ No. 45); EPO K167R FP 5'-T GACACTTTCCGCAGACTCTTCCGAGTCTAC-3' (SEQ No. 46), RP 5'-GTAGACTCGGAAG AGTCTGCGGAAAGTGTCA-3' (SEQ No. 47); EPO K179R FP 5'-CTCCGGGGAAGGCTG AAGCTG-3' (SEQ No. 48), RP 5'-CAGCTTCAGCCTTCCCCGGAG-3' (SEQ No. 49); EPO K181R FP 5'-GGAAAGCTGAGGCTGTACACAGG-3' (SEQ No. 50), After preparing RP 5'-CCTGTGTACAG CCTCAGCTTTCC-3' (SEQ No. 51), PCR was performed under specific conditions to produce plasmid DNA in which specific amino acid residues were substituted.PCDNA3-myc-erythropoietin was used as a template. Then, plasmid DNA in which the lysine residue was substituted with arginine (K→R) was prepared (Table 7).

Lysine (K) residue position EPO construct in which Lysine (K) is substituted with Arginine (R) 124 pcDNA3-myc-EPO (K124R) 167 pcDNA3-myc-EPO (K167R) 179 pcDNA3-myc-EPO (K179R) 181 pcDNA3-myc-EPO (K181R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-erythropoietin WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 μg of pcDNA3-myc-erythropoietin WT and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours, MG132 (proteasome inhibitor, 5 Μg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 46). In addition, pcDNA3-myc-erythropoietin WT, pcDNA3-myc-erythropoietin substituent (K124R), pcDNA3-myc-erythropoietin substituent (K167R), pcDNA3-myc-erythropoietin substituent (K179R), pcDNA3-myc HEK 293T cells were infected with a plasmid encoding -erythropoietin substituent (K181R) and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, pcDNA3-myc-erythropoietin WT, pcDNA3-myc-erythropoietin substituent (K124R), pcDNA3-myc-erythropoietin substituent (K167R), pcDNA3-myc-erythropoietin substituent (K179R) ), pcDNA3-myc-erythropoietin substituent (K181R), respectively, 2 μg, and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 47).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then mixed with anti-myc (9E10) primary antibody. And incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. Silver protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and then separated by performing SDS-PAGE The separated protein was transferred to a PVDF membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 containing a blocking solution and anti-mouse secondary antibody Using the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) As a result, when immunoprecipitation was performed with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), pcDNA3-myc-erythro As ubiquitin binds to poietin WT and polyubiquitinization is formed, ubiquitin in a smeared shape is detected, and the band is dark (Fig. 46, lanes 3 and 4) In addition, MG132 (proteasome inhibitor, 5 µg/ml) was used. In the case of treatment for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected became more intense (Fig. 46, lane 4) In addition, the pcDNA3-myc-erythropoietin substituent (K181R) was unable to bind to ubiquitin and thus ubiquitin Little was detected (Fig. 47, lane 6). It is shown that poietin binds to ubiquitin and is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Confirmation of half-life of erythropoietin by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-erythropoietin WT, pcDNA3-myc-erythropoietin substituent (K124R), pcDNA3-myc-erythropoietin substituent (K167R), pcDNA3-myc-erythropoietin substituent (K179R), pcDNA3-myc-erythro The poietin substitute (K181R) was transfected into HEK 293T cells by 2 µg each. After 48 hours, the protein production inhibitor cyclohexamide (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and the half-life was measured over 2 hours, 4 hours, and 8 hours. As a result, the degradation of human erythropoietin was It was confirmed that it was suppressed (FIG. 48). As a result, the half-life of human erythropoietin was within 4 hours, whereas the half-life of human pcDNA3-myc-erythropoietin substitute (K124R) was longer than WT at 8 hours, and this result is shown in a graph (FIG. 48).

4. Confirmation of signal transduction by erythropoietin and erythropoietin substituents in cells

It has been reported that erythropoietin is increased in lung or blood cancer, and that when erythropoietin is administered, the effect in hypoxia is increased by controlling cell cycle progression by phosphorylation of Erk1/2 (J Hematol Oncol., 6, 65). , 2013). In this example, the signal transduction process by erythropoietin and erythropoietin substituents in the cell was confirmed. First, after starving HepG2 (ATCC, AB-8065) cells for 8 hours, pcDNA3-myc-erythropoietin WT, pcDNA3-myc-erythropoietin substituent (K124R), pcDNA3-myc-erythropoietin substituent (K167R) , pcDNA3-myc-erythropoietin substitutent (K179R) and pcDNA3-myc-erythropoietin substitutent (K181R) were respectively used to infect HepG2 cells using 3 µg. After 2 days of infection, proteins were extracted from the cells and quantified, respectively, and Western blotting was performed to confirm the intracellular signal transduction process. In this process, pcDNA3-myc-erythropoietin WT, pcDNA3-myc-erythropoietin substituent (K124R), pcDNA3-myc-erythropoietin substituent (K167R), pcDNA3-myc-erythropoietin substituent (K179R), and The protein isolated from HepG2 cells infected with pcDNA3-myc-erythropoietin substituent (K181R) was transferred to the PVDF membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-Erk1/2 (9B3, Abfrontier LF-MA0134), anti-phospho-Erk1/2 (Thr202/Tyr204, Abfrontier LF-PA0090) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 and a blocking solution containing Using anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibodies It was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, pcDNA3-myc-erythropoietin substituent (K124R), pcDNA3-myc-erythropoietin substituent (K167R), pcDNA3-myc-erythropoietin substituent (K179R), pcDNA3-myc-erythropoietin substituent (K181R) Showed the same or increased phospho-Erk1/2 signaling with pcDNA3-myc-erythropoietin WT in HepG2 cells (FIG. 49).

Example 8: Bone morphogenetic protein ( BMP2 )of Ubiquitination Analysis and confirmation of increased half-life and intracellular signal transduction

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

The bone morphogenetic protein (BMP2) DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were fragmented with restriction enzymes EcoRI and XhoI, and then conjugated and cloned (FIG. 50, BMP2 amino acid sequence: SEQ No. 52), the results were confirmed through agarose gel electrophoresis after digestion with restriction enzymes (FIG. 51). In addition, the portion indicated in bold and underlined on the nucleotide sequence of FIG. 50 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is through agarose gel electrophoresis. Confirmed (Fig. 51). Polymerase chain reaction conditions are as follows; After reacting the initial denaturation at 94°C for 3 minutes, repeating 25 cycles for 30 seconds at 94°C for the denaturation reaction, 30 seconds at 58°C for the annealing reaction, and 1 minute 30 seconds at 72°C for the extension reaction Then, it was reacted at 72° C. for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, the myc present in the pcDNA3-myc vector shown in the map of FIG. 50 was expressed through western blotting using an anti-myc (9E10, sc-40) antibody. Confirmed. Through this, it was confirmed that the bone-forming protein bound to myc was well expressed, and it was found that it was quantified through a blot confirmed with actin (FIG. 52).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-directed mutagenesis, and the primer (BMP2 K293R FP 5'-GAAACGCCTTAGGTCCAGCTGTAAGAGAC-3' (SEQ No. 53) was used using the DNA sequence to induce a specific mutation. , RP 5'-GTCTCTTACAGCTGGACCTAAGGCGTTTC-3' (SEQ No. 54); BMP2 K297R FP 5'-TTAAGTCCAGCTGTAGGAGACACCCTTTGT-3' (SEQ No. 55), RP 5'-ACAAAGGGTGTCTCCTACAGCTGGACTTAA-3' (SEQ No. 56); FP 5'-GTTAACTCTAGGATTCCTAAGGC-3' (SEQ No. 57), RP 5'-GC CTTAGGAATCCTAGAGTTAAC-3' (SEQ No. 58); BMP2 K383R FP 5'- GGTTGTATTAAGGAACTATCAGGAC-3' (SEQ No. 59), RP 5 After making'-GTCCTGATAGTTCCTTAAT ACAACC-3' (SEQ No. 60), PCR was performed under specific conditions to produce plasmid DNA in which specific amino acid residues were substituted, pcDNA3-myc-osteogenesis protein (BMP2) as a template. Was used, and a plasmid DNA in which the lysine residue was substituted with arginine (K→R) was prepared (Table 8).

Lysine (K) residue position BMP2 construct in which Lysine (K) is substituted with Arginine (R) 293 pcDNA3-myc-BMP2 (K293R) 297 pcDNA3-myc-BMP2 (K297R) 355 pcDNA3-myc-BMP2 (K355R) 383 pcDNA3-myc-BMP2 (K383R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-osteogenic protein WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 µg of pcDNA3-myc-osteogenic protein WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 53). In addition, each pcDNA3-myc-osteoplastic protein WT, pcDNA3-myc-osteoplastic protein substitute (K293R), pcDNA3-myc-osteoplastic protein substitute (K297R), pcDNA3-myc-osteoplastic protein substitute (K355R), pcDNA3- HEK 293T cells were infected with a plasmid encoding myc-osteogenic protein substitute (K383R) and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, pcDNA3-myc-osteogenesis protein WT, pcDNA3-myc-osteogenesis protein substitutent (K293R), pcDNA3-myc-osteogenesis protein substitutent (K297R), pcDNA3-myc-osteogenesis protein substitutent (K355R) ), pcDNA3-myc-osteoplastic protein substitute (K383R), respectively, 2 μg, and 1 μg of pMT123-HA-ubiquitin DNA were cotransfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 54). Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-myc (9E10, Santa Cruz Biotechnology, sc -40) Mixed with the primary antibody and incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology). For immunoblotting, protein samples were mixed with 2X SDS buffer, heated at 100° C. for 7 minutes, and then separated by performing SDS-PAGE The separated protein was transferred to a PVDF membrane and then anti- Blocking comprising myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 Solution and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody was used to develop with the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was performed with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin in the pcDNA3-myc-osteogenesis protein WT was combined with ubiquitin to form polyubiquitin, resulting in a smeared ubiquitin. The band appeared dark (FIG. 53, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 μg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 53, lane 4). In addition, in the case of pcDNA3-myc-bone morphogenetic protein substituent (K293R), pcDNA3-myc-bone morphogenetic protein substituent (K297R), and pcDNA3-myc-bone morphogenetic protein substituent (K355R), the band was softer than WT, and pcDNA3-myc- The bone morphogenetic protein substituent (K293R), pcDNA3-myc-bone morphogenetic protein substituent (K297R), and pcDNA3-myc-bone morphogenetic protein substituent (K355R) were unable to bind to ubiquitin, and thus less ubiquitin was detected (Fig. 54, lanes 3 and 5). ). The above results show that the osteogenic protein binds to ubiquitin and is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Confirmation of half-life of bone morphogenetic protein by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-osteoplastic protein WT, pcDNA3-myc-osteoplastic protein substitute (K293R), pcDNA3-myc-osteoplastic protein substitute (K297R), pcDNA3-myc-osteoplastic protein substitute (K355R), pcDNA3-myc-bone HEK 293T cells were transfected with 2 µg each of the forming protein substitutes (K383R). 48 hours after transfection, the protein production inhibitor cyclohexamid (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and half-life was measured over 2 hours, 4 hours and 8 hours. As a result, it was confirmed that the decomposition of human bone morphogenetic protein was suppressed (FIG. 55). The half-life of human bone morphogenetic protein is within 2 hours, whereas the half-life of human pcDNA3-myc-bone morphogenetic protein substituent (K297R) and pcDNA3-myc-bone morphogenetic protein substitute (K355R) is more than 4 hours, which is longer than that of WT. It is shown as a graph (FIG. 55).

4. Confirmation of signal transduction by bone morphogenetic protein and bone morphogenetic protein substitutes in cells

It has been reported that osteogenic proteins cause apoptosis by inactivating STAT3 in various myeloma cells (Blood, 96, 2005-2011, 2000). In this example, the signal transduction process by the osteogenic protein and the osteogenic protein substitutes in the cell was confirmed. First, after starving HepG2 (ATCC, AB-8065) cells for 8 hours, pcDNA3-myc-osteoplastic protein WT, pcDNA3-myc-osteoplastic protein substitute (K293R), pcDNA3-myc-osteoplastic protein substitute (K297R) , pcDNA3-myc-osteoplastic protein substitutent (K355R) and pcDNA3-myc-osteoplastic protein substitutent (K383R) were each used to infect HepG2 cells using 3 µg each. After 2 days of infection, proteins were extracted from the cells and quantified, respectively, and Western blotting was performed to confirm the intracellular signal transduction process. In this process, pcDNA3-myc-osteogenesis protein substituent (K293R), pcDNA3-myc-osteogenesis protein substituent (K297R), pcDNA3-myc-osteogenesis protein substituent (K355R) and pcDNA3-myc-osteogenesis protein substituent (K383R), respectively. ), and then transfer the protein isolated from the infected HepG2 cells to the PVDF membrane, and then anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 ( Y705, cell signaling 9131S) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) using a blocking solution containing a weight ratio of 1:1000 and anti-rabbit and anti-mouse secondary antibodies (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, pcDNA3-myc-osteogenesis protein substituent (K293R), pcDNA3-myc-osteogenesis protein substituent (K297R), pcDNA3-myc-osteogenesis protein substituent (K355R), pcDNA3-myc-osteogenesis protein substituent (K383R) Showed reduced phospho-STAT3 signaling in HepG2 cells compared to the control group in the same manner as pcDNA3-myc-osteogenic protein WT (FIG. 56).

Example 9: Fibroblast growth factor-1 ( FGF -1) of protein Ubiquitination Analysis and confirmation of half-life increase and intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

The fibroblast growth factor-1 DNA amplification product by polymerase chain reaction and pCMV3-C-myc (6.1 kb) were fragmented with restriction enzymes KpnI and XbaI, then conjugated and cloned (Fig. 57, fibroblast growth factor- 1 amino acid sequence: SEQ No. 61), the result was confirmed through agarose gel electrophoresis after digestion with restriction enzymes (FIG. 58). In addition, the portions marked in bold and underlined on the nucleotide sequence of FIG. 57 are primer sets used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. (Figure 58). Polymerase chain reaction conditions are as follows; After the initial denaturation was reacted at 94°C for 3 minutes, 30 seconds at 94°C for the denaturation reaction, 30 seconds at 58°C for the annealing reaction, and 30 seconds at 72°C for the extension reaction were repeated in 25 cycles, Then, it was reacted at 72°C for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, myc present in the pCMV3-C-myc vector shown in the map of FIG. 57 was subjected to western blotting using an anti-myc (9E10, sc-40) antibody. Expression was confirmed. It was confirmed that the fibroblast growth factor-1 protein bound to myc was well expressed and quantitatively loaded through blotting confirmed with actin (FIG. 59).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-specific mutagenesis, and primers (FGF-1 K27R FP 5'-AAGAAGCCCAGACTCCTCTAC-3' (SEQ No. 62), RP 5') using the DNA sequence to induce specific mutations. -GTAGAGGAGTCTGGGCTTCT T-3' (SEQ No. 63); FGF-1 K120R FP 5'-CAT GCAGAGAGGAATTGGTTT-3' (SEQ No. 64), RP 5'-AAACCAATTCCTCTCTGCATG-3' (SEQ No. 65) was prepared. After that, PCR was performed under specific conditions to prepare a plasmid DNA in which a specific amino acid residue was substituted, using pCMV3-C-myc-FGF-1 DNA as a template, and a plasmid in which the Lysine residue was substituted with arginine (K→R). DNA was prepared (Table 9).

Lysine (K) residue position FGF-1 construct in which Lysine (K) is substituted with Arginine (R) 27 pCMV3-C-myc-FGF-1 (K27R) 120 pCMV3-C-myc-FGF-1 (K120R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pCMV3-C-myc-fibroblast growth factor-1 WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 µg of pCMV3-C-myc-fibroblast growth factor-1 WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of infection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 60). In addition, pCMV3-C-myc-fibroblast growth factor-1 WT, pCMV3-C-myc-fibroblast growth factor-1 substituent (K27R), pCMV3-C-myc-fibroblast growth factor-1 substituent (K120R), respectively, HEK 293T cells were infected with a plasmid encoding pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, pCMV3-C-myc-fibroblast growth factor-1 WT, pCMV3-C-myc-fibroblast growth factor-1 substitute (K27R), pCMV3-C-myc-fibroblast growth factor-1 (K120R) 2 μg, respectively, and 1 μg of pMT123-HA-ubiquitin DNA were cotransfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 61).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-myc (9E10, Santa Cruz Biotechnology, sc -40) Mixed with the primary antibody and incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology). For immunoblotting, protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and then separated by performing SDS-PAGE The separated protein was transferred to a PVDF membrane and then anti- Blocking comprising myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 The solution and anti-mouse secondary antibody were used to develop with the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was performed with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin was bound to pCMV3-C-myc-fibroblast growth factor-1 WT, resulting in the formation of polyubiquitination. The ubiquitin of was detected and the band appeared dark (FIG. 60, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 60, lane 4). In addition, in the case of pCMV3-C-myc-fibroblast growth factor-1 substituent (K27R) and pCMV3-C-myc-fibroblast growth factor-1 substituent (K120R), the band was softer than that of WT, and pCMV3-C-myc- The fibroblast growth factor-1 substituent (K27R) and the pCMV3-C-myc-fibroblast growth factor-1 substituent (K120R) were unable to bind to ubiquitin, and thus less ubiquitin was detected (FIG. 61, lanes 3 and 4). The above results show that fibroblast growth factor-1 binds to ubiquitin and is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Confirmation of the half-life of fibroblast growth factor-1 by the protein production inhibitor cycloheximide (CHX)

pCMV3-C-myc-fibroblast growth factor-1 WT, pCMV3-C-myc-fibroblast growth factor-1 substitution (K27R), pCMV3-C-myc-fibroblast growth factor-1 substitution (K120R) respectively After 48 hours of transfection into HEK 293T cells by μg, the protein production inhibitor cyclohexamide (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and the half-life was measured over 24 hours and 36 hours. It was confirmed that the degradation of fibroblast growth factor-1 was inhibited (FIG. 62). The half-life of human fibroblast growth factor-1 is within 1 day, whereas that of human pCMV3-C-myc-fibroblast growth factor-1 substituent (K27R) and pCMV3-C-myc-fibroblast growth factor-1 substituent (K120R). The half-life was longer than that of WT by more than 1 day, and this result was shown as a graph (FIG. 62).

4. Confirmation of signal transduction by fibroblast growth factor-1 and fibroblast growth factor-1 substituents in cells

It has been reported that treatment with recombinant fibroblast growth factor-1 on HEK293 cells increases Erk 1/2 phosphorylation (Nature, 513(7518), 436-439, 2014). In this example, the signal transduction process by fibroblast growth factor-1 and fibroblast growth factor-1 substituents in the cell was confirmed. First, HepG2 (ATCC, AB-8065) cells were long ?E for 8 hours, then pCMV3-C-myc-fibroblast growth factor-1 WT, pCMV3-C-myc-fibroblast growth factor-1 substitution (K27R) ), pCMV3-C-myc-fibroblast growth factor-1 substitution (K120R) was infected with HepG2 cells using 3 µg each, and after 2 days, proteins were extracted from the cells and quantified respectively, and intracellular signal transduction process Western blotting was performed to confirm. In this process, pCMV3-C-myc-fibroblast growth factor-1 WT, pCMV3-C-myc-fibroblast growth factor-1 substitution (K27R), pCMV3-C-myc-fibroblast growth factor-1 substitution (K120R) The protein isolated from the infected HepG2 cells was transferred to the PVDF membrane, and then anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-Erk1/2 (9B3, Abfrontier LF-MA0134), and anti-phospho-Erk1 A blocking solution containing /2 (Thr202/Tyr204, Abfrontier LF-PA0090) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 and anti-rabbit IgG-HRP , Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) using secondary antibody ECL system (Western blot detection kit, ABfrontier, Seoul) , Korea). As a result, pCMV3-C-myc-fibroblast growth factor-1 substitute (K27R) and pCMV3-C-myc-fibroblast growth factor-1 substitute (K120R) were used to grow pCMV3-C-myc-fibroblasts in HepG2 cells. It showed the same or increased phospho-ERK1/2 signaling as factor-1 WT (Fig. 63).

Example 10: Appetite suppressing hormone ( Leptin ) Of protein Ubiquitination Analysis and confirmation of half-life increase and intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

Appetite suppressing hormone (Leptin) by polymerase chain reaction The DNA amplification product and pCMV3-C-myc (6.1 kb) were fragmented with restriction enzymes KpnI and XbaI, then conjugated and cloned (FIG. 64, leptin amino acid sequence: SEQ No. 66), and the result was after restriction enzyme digestion. , It was confirmed through agarose gel electrophoresis (FIG. 65). In addition, the portion marked in bold and underlined on the nucleotide sequence of FIG. 64 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. (Figure 65). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94° C. for 3 minutes, followed by 25 cycles of 30 seconds at 94° C. for the denaturation reaction, 30 seconds at 58° C. for the annealing reaction, and 45 seconds at 72° C. for the extension reaction. Then, it was reacted at 72° C. for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, myc present in the pCMV3-C-myc vector shown in the map of FIG. 64 is subjected to western blotting using an anti-myc (9E10, sc-40) antibody. Expression was confirmed. Appetite suppressing hormone (Leptin) bound to myc It was confirmed that the protein was well expressed, and it was found that it was quantitatively loaded through a blot confirmed with actin (FIG. 66).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-specific mutagenesis, and primers (Leptin K26R FP 5'-CCCATCCAAAAGGTCCAAGAT-3' (SEQ No. 67), RP 5'-ATCTTGGACCTTTTGGATGGG) using the DNA sequence to induce specific mutations. -3' (SEQ No. 68); Leptin K32R FP 5'-GA TGACACCAAGACCCTCATC-3' (SEQ No. 69), RP 5'-GATGAGGGTCTTGGTGTCATC-3' (SEQ No. 70); Leptin K36R FP 5'-ACCCTCATCAGGACAATTGTC -3' (SEQ No. 71), RP 5'-GACAATTGTCCTGATGAGGGT-3' (SEQ No. 72); Leptin K74R FP 5'-ACCTTATCCAG GATGGACCAG-3' (SEQ No. 73), RP 5'-CTGGTCCATCCTGGATAAGGT-3 '(SEQ No. 74) was prepared, and then PCR was performed under specific conditions to prepare a plasmid DNA in which a specific amino acid residue was substituted. pCMV3-C-myc-appetite suppressing hormone (Leptin) DNA was used as a template, and plasmid DNA in which the lysine residue was substituted with arginine (K→R) was prepared (Table 10).

Lysine (K) residue position Leptin construct in which Lysine (K) is substituted with Arginine (R) 26 pCMV3-C-myc-Leptin (K26R) 32 pCMV3-C-myc-Leptin (K32R) 36 pCMV3-C-myc-Leptin (K36R) 74 pCMV3-C-myc-Leptin (K74R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pCMV3-C-myc-appetite suppressing protein WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 6 µg of pCMV3-C-myc-appetite suppressing protein WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and 24 hours later, MG132 (proteasome inhibitor, 5 µg/ml). ) Was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 67). In addition, pCMV3-C-myc-appetite suppressing protein WT, pCMV3-C-myc-appetite suppressing protein substitute (K26R), pCMV3-C-myc-appetite suppressing protein substitute (K32R), pCMV3-C-myc-appetite suppressing protein, respectively HEK 293T cells were infected using a plasmid encoding a substituent (K36R), pCMV3-C-myc-appetite suppressing protein substituent (K74R), and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, pCMV3-C-myc-appetite suppressing protein WT, pCMV3-C-myc-appetite suppressing protein substitute (K26R), pCMV3-C-myc-appetite suppressing protein substitute (K32R), pCMV3-C -myc-appetite suppressing protein substitute (K36R), pCMV3-C-myc-appetite suppressing protein substitute (K74R) 6 µg, and pMT123-HA-ubiquitin DNA 1 µg were co-transfected into cells, and immunoprecipitation analysis 24 hours later Was carried out (FIG. 68).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then mixed with anti-myc (9E10) primary antibody. And incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. Silver protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and then separated by performing SDS-PAGE The separated protein was transferred to a PVDF membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 blocking solution and anti-mouse (Peroxidase- Labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody.

As a result, when immunoprecipitation was performed with anti-myc (9E10, sc-40), ubiquitin in pCMV3-C-myc-appetite suppressing protein WT was bound to form polyubiquitin, resulting in ubiquitin in a smeared shape. It appeared dark (Fig. 67, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 67, lane 4). In addition, pCMV3-C-myc-appetite-suppressing protein substituent (K26R), pCMV3-C-myc-appetite-suppressing protein substituent (K36R), and pCMV3-C-myc-appetite-suppressing protein substituent (K74R) have a longer band than WT. And pCMV3-C-myc-appetite suppressing protein substitutent (K26R), pCMV3-C-myc-appetite suppressing protein substitutent (K36R), and pCMV3-C-myc-appetite suppressing protein substitutent (K74R) failed to bind to ubiquitin. Less of this was detected (Figure 68, lanes 3, 5 and 6). The above results show that the appetite suppressing protein binds to ubiquitin and is polyubiquitinated and decomposed through the ubiquitin-proteasome system.

3. Confirmation of half-life of appetite suppressing protein (Leptin) by protein production inhibitor cycloheximide (CHX)

pCMV3-C-myc-appetite suppressing protein WT, pCMV3-C-myc-appetite suppressing protein substitutent (K26R), pCMV3-C-myc-appetite suppressing protein substitutent (K32R), pCMV3-C-myc-appetite suppressing protein substitutent ( K36R), pCMV3-C-myc-appetite suppressing protein substitute (K74R) was transfected into HEK 293T cells at 6 μg each, and 48 hours later, protein production inhibitor cyclohexamid (CHX) (Sigma-Aldrich) (100 μg/ Ml), and then measuring the half-life over 2 hours, 4 hours and 8 hours, it was confirmed that the decomposition of the human appetite suppressing protein was suppressed (FIG. 69). As a result, the half-life of human appetite suppressing protein was 4 hours, whereas the half-life of human pCMV3-C-myc-appetite suppressing protein substitute (K26R) and pCMV3-C-myc-appetite suppressing protein substitute (K36R) was more than 8 hours. It was lengthened, and this result was shown graphically (Fig. 69).

4. Confirmation of signal transduction by appetite-suppressing protein and appetite-suppressing protein substitutes in cells

It has been reported that appetite suppressing proteins increase the phosphorylation of AKT in breast cancer cells (Cancer Biol Ther., 16(8), 1220-1230, 2015). In uterine cancer, it is said that it stimulates cancer cell growth by PI3K/AKT signaling. Has been reported (Int J Oncol., 49(2), 847, 2016). In this example, the signaling process by the appetite-suppressing protein and the appetite-suppressing protein substitutes in the cell was confirmed. First, after starving HepG2 (ATCC, AB-8065) cells for 8 hours, pCMV3-C-myc-appetite suppressing protein WT, pCMV3-C-myc-appetite suppressing protein substitute (K26R), pCMV3-C-myc-appetite HepG2 cells were infected with 6 μg each of the inhibitory protein substituents (K32R), pCMV3-C-myc-appetite suppressing protein substituents (K36R), and pCMV3-C-myc-appetite suppressing protein substituents (K74R). After 2 days of infection, proteins were extracted from the cells and quantified, respectively, and Western blot was performed to confirm the intracellular signal transduction process. In this process, pCMV3-C-myc-appetite suppressing protein WT, pCMV3-C-myc-appetite suppressing protein substituent (K26R), pCMV3-C-myc-appetite suppressing protein substituent (K32R), pCMV3-C-myc-appetite, respectively. Protein isolated from HepG2 cells infected with inhibitory protein substituent (K36R) and pCMV3-C-myc-appetite suppressing protein substituent (K74R) was transferred to PVDF membrane, followed by anti-myc (9E10, sc-40), anti-AKT (H-136, Santa Cruz Biotechnology, sc-8312), anti-phospho-AKT (S473, Cell Signaling 9271S) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 Blocking solution and anti-rabbit and anti-mouse secondary antibodies were used and developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, pCMV3-C-myc-appetite suppressing protein substitutent (K26R), pCMV3-C-myc-appetite suppressing protein substitutent (K32R), pCMV3-C-myc-appetite suppressing protein substitutent (K36R), pCMV3-C-myc -Appetite suppressing protein substitute (K74R) showed remarkably increased phospho-AKT signaling in HepG2 cells compared to the control group in the same manner as pCMV3-C-myc-appetite suppressing protein WT (FIG. 70).

Example 11: Vascular endothelial factor A ( VEGFA ) Of protein Ubiquitination Analysis and confirmation of half-life increase and intracellular signaling

1. Cloning into an expression vector and confirmation of protein expression

(1) Cloning of expression vector

The vascular endothelial factor A DNA amplification product by polymerase chain reaction and pCMV3-C-myc (6.1 kb) were fragmented with restriction enzymes KpnI and XbaI, and then conjugated and cloned (Fig. 71, vascular endothelial factor A amino acid). Sequence: SEQ No. 75), the result was confirmed through agarose gel electrophoresis after digestion with restriction enzymes (FIG. 72). In addition, the part marked in bold and underlined on the nucleotide sequence of FIG. 71 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. (Fig. 72). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94° C. for 3 minutes, followed by repeating 25 cycles of 94° C. for the denaturation reaction for 30 seconds, 58° C. for the annealing reaction for 30 seconds, and 1 minute at 72° C. for the extension reaction. Then, it was reacted at 72°C for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, the myc present in the pCMV3-C-myc vector shown in the map of FIG. 71 was westernized using an anti-myc (9E10, Santa Cruz Biotechnology, sc-40) antibody. Expression was confirmed through blotting. Through this, it was confirmed that the VEGFA protein bound to myc was well expressed, and it was found that it was quantitatively loaded through a blot confirmed with actin (FIG. 73).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-specific mutagenesis, and primers (VEGFA K127R FP 5'-TACAGCACAACAGATGTGAATGCAGACC-3' (SEQ No. 76), RP 5'-GGTCTGCATTCA) using the DNA sequence to induce specific mutations. After making CATCTGTTGTGCTGTA-3' (SEQ No. 77); VEGFA K180R FP 5'-ATCCGCAGACGTGTAG ATGTTCCTGCA-3' (SEQ No. 78), RP 5'-TG CAGGAACATCTACACGTCTGCGGAT-3' (SEQ No. 79), and then specific Plasmid DNA in which a specific amino acid residue was substituted was prepared by performing PCR under the conditions.PCMV3-C-myc-VEGFA DNA was used as a template, and a plasmid DNA in which the Lysine residue was substituted with arginine (K→R) was prepared ( Table 11).

Lysine (K) residue position VEGFA construct in which Lysine (K) is substituted with Arginine (R) 127 pCMV3-C-myc-VEGFA (K127R) 180 pCMV3-C-myc-VEGFA (K180R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pCMV3-C-myc-VEGFA WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 6 µg of pCMV3-C-myc-VEGFA WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (Fig. 74). In addition, HEK 293T using a plasmid encoding pCMV3-C-myc-VEGFA WT, pCMV3-C-myc-VEGFA substituent (K127R), pCMV3-C-myc-VEGFA substituent (K180R) and pMT123-HA-ubiquitin DNA, respectively. The cells were infected. To confirm the degree of ubiquitination, pCMV3-C-myc-VEGFA WT, pCMV3-C-myc-VEGFA substituent (K127R) and pCMV3-C-myc-VEGFA substituent (K180R) 6 μg, respectively, and pMT123-HA-ubiquitin DNA 1 µg was co-transfected into cells and immunoprecipitation analysis was performed 24 hours later (Fig. 75).

Samples obtained for immunoprecipitation were dissolved in a lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-myc (9E10, sc-40) primary It was mixed with the antibody and incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with a lysis buffer. For immunoblotting, protein samples were mixed with 2X SDS buffer, heated at 100° C. for 7 minutes, and then separated by performing SDS-PAGE The separated protein was transferred to a PVDF membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 containing a blocking solution and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody.

As a result, when immunoprecipitation was performed with anti-myc (9E10, sc-40), ubiquitin was bound to pCMV3-C-myc-VEGFA WT to form polyubiquitin. (Fig. 74, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected became more intense (FIG. 74, lane 4). In addition, in the case of pCMV3-C-myc-VEGFA substituent (K127R) and pCMV3-C-myc-VEGFA substituent (K180R), the band was softer than WT, and pCMV3-C-myc-VEGFA substituent (K127R), pCMV3-C- The myc-VEGFA substituent (K180R) was unable to bind to ubiquitin, so less ubiquitin was detected (FIG. 75, lanes 3 and 4). The above results show that VEGFA binds to ubiquitin and is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Confirmation of the half-life of VEGFA by the protein production inhibitor cycloheximide (CHX)

The pCMV3-C-myc-VEGFA WT, pCMV3-C-myc-VEGFA substituent (K127R), and pCMV3-C-myc-VEGFA substituent (K180R) were each transfected into HEK 293T cells by 6 μg. 48 hours after transfection, the protein production inhibitor cyclohexamid (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and half-life was measured over 2 hours, 4 hours, and 8 hours. As a result, degradation of human VEGFA It was confirmed that is suppressed (Fig. 76). The half-life of human VEGFA was within 2 hours, whereas the half-life of human pCMV3-C-myc-VEGFA substituent (K127R) and pCMV3-C-myc-VEGFA substituent (K180R) was longer than WT by more than 4 hours. It is represented by (Fig. 76).

4. Confirmation of signal transduction by VEGFA and VEGFA substituents in cells

VEGFA is an important factor related to endothelial cell growth and proliferation, and it has been reported that PI3K/Akt/HIF-1α signaling is involved in the formation of angiogenesis in cancer cells (Carcinogenesis, 34, 426-435. , 2013). It has also been reported that VEGF induces AKT phosphorylation (Kidney Int., 68, 1648-1659, 2005). In this example, the signaling process by VEGFA and VEGFA substituents in the cell was confirmed. First, after starving HepG2 (ATCC, AB-8065) cells for 8 hours, pCMV3-C-myc-VEGFA WT, pCMV3-C-myc-VEGFA substituent (K127R) and pCMV3-C-myc-VEGFA substituent (K180R) HepG2 cells were infected with each of 6 μg. After 2 days of infection, proteins were extracted from the cells and quantified, respectively, and Western blotting was performed to confirm the intracellular signal transduction process. In this process, proteins isolated from HepG2 cells infected with pCMV3-C-myc-VEGFA WT, pCMV3-C-myc-VEGFA substituent (K127R), and pCMV3-C-myc-VEGFA substituent (K180R) were transferred to the PVDF membrane, respectively. Then, anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, cell signaling 9131S), anti-AKT (H- 136, Santa Cruz Biotechnology, sc-8312), anti-phospho-AKT (S473, cell signaling 9271S) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 and Using anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibodies It was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, pCMV3-C-myc-VEGFA substituent (K127R) and pCMV3-C-myc-VEGFA substituent (K180R) were the same or increased phospho-STAT3 and phospho- as pCMV3-C-myc-VEGFA WT in HepG2 cells. It showed AKT signaling (Fig. 77).

Example 12: Appetite promoting hormone precursor ( Ghrelin / Obestatin Preprohormone ; prepro - GHRL In) Ubiquitination Analysis and confirmation of half-life increase and intracellular signaling

1. Cloning into an expression vector and confirmation of protein expression

(1) Cloning of expression vector

Appetite-promoting hormone precursor by polymerase chain reaction (Ghrelin/Obestatin Preprohormone; prepro-GHRL) The DNA amplification product and pCMV3-C-myc (6.1 kb) were fragmented with restriction enzymes KpnI and XbaI, and then conjugated and cloned (Fig. 78, prepro-GHRL amino acid sequence: SEQ No. 80), and the result is a restriction enzyme. After cutting, it was confirmed through agarose gel electrophoresis (FIG. 79). In addition, the part marked in bold and underlined on the nucleotide sequence of FIG. 78 is a primer set used when confirming through the polymerase chain reaction to confirm the cloned site once again, and the result was confirmed through agarose gel electrophoresis. (Fig. 79). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94° C. for 3 minutes, followed by 25 cycles of 30 seconds at 94° C. for the denaturation reaction, 30 seconds at 58° C. for the annealing reaction, and 30 seconds at 72° C. for the extension reaction. Then, it was reacted at 72° C. for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, the myc present in the pCMV3-C-myc vector shown in the map of FIG. 78 was used as an anti-myc (9E10, Santa Cruz Biotechnology, sc-40) antibody. Expression was confirmed through blot. Through this, it was confirmed that an appetite-promoting hormone precursor (Ghrelin/Obestatin Preprohormone; prepro-GHRL) protein bound to myc was well expressed, and it was found that the protein was quantitatively loaded through a blot confirmed with actin (FIG. 80).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-specific mutagenesis, and primers (prepro-GHRL K100R FP 5'-GCCCTGGGGAGGTTTCTTCAG-3' (SEQ No. 81), RP 5') using the DNA sequence to induce specific mutations. -CTGAAGAAACCTCCCCAGGGC-3' (SEQ No. 82) was prepared, and then PCR was performed under specific conditions to produce plasmid DNA in which specific amino acid residues were replaced: pCMV3-C-myc-appetite promoting hormone precursor (Ghrelin/Obestatin Preprohormone) ; prepro-GHRL) DNA was used as a template, and a plasmid DNA in which the Lysine residue was substituted with arginine (K→R) was prepared (Table 12).

Lysine (K) residue position Ghrelin construct in which Lysine (K) is substituted with Arginine (R) 100 pCMV3-C-myc-prepro-GHRL (K100R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pCMV3-C-myc-prepro-GHRL WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 6 µg of pCMV3-C-myc-prepro-GHRL WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 81). HEK 293T cells were infected with a plasmid encoding pCMV3-C-myc-prepro-GHRL WT, pCMV3-C-myc-prepro-GHRL substituent (K100R) and pMT123-HA-ubiquitin DNA, respectively. To confirm the degree of ubiquitination, pCMV3-C-myc-prepro-GHRL WT, and pCMV3-C-myc-prepro-GHRL substituent (K100R), respectively, 6 μg, and pMT123-HA-ubiquitin DNA 1 μg were co-transformed into cells. Immunoprecipitation analysis was performed 24 hours after infection (FIG. 82).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-myc (9E10, sc-40) 1 After mixing with the primary antibody, it was incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. For immunoblotting, protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and then separated by performing SDS-PAGE The separated protein was transferred to a PVDF membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 containing a blocking solution and anti- Mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody.

As a result, when immunoprecipitation was performed with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin was bound to pCMV3-C-myc-prepro-GHRL WT to form polyubiquitination, resulting in smear (smear). ) Ubiquitin was detected and the band appeared dark (FIG. 81, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 81, lane 4). In addition, in the case of the pCMV3-C-myc-prepro-GHRL substituent (K100R), the band was softer than that of WT, and pCMV3-C-myc-prepro-GHRL Since the substituent (K100R) could not bind to ubiquitin, less ubiquitin was detected (Fig. 82, lane 3). The above results show that the appetite promoting protein binds to ubiquitin and is polyubiquitinated and decomposed through the ubiquitin-proteasome system.

3. Confirmation of the half-life of the appetite promoting protein (Ghrelin) by the protein production inhibitor cycloheximide (CHX)

The pCMV3-C-myc-prepro-GHRL WT and pCMV3-C-myc-prepro-GHRL substituents (K100R) were transfected into HEK 293T cells at 6 μg each. 48 hours after transfection, the protein production inhibitor cyclohexamid (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and the half-life was measured over 2 hours, 4 hours and 8 hours. It was confirmed that decomposition was inhibited (FIG. 83). As a result, the half-life of the human appetite-promoting hormone precursor (Ghrelin/Obestatin Preprohormone; prepro-GHRL) is within 2 hours, whereas the half-life of the human pCMV3-C-myc-prepro-GHRL substituent (K100R) is more than 2 hours, which is longer than WT. This result is shown in a graph (FIG. 83).

4. Appetite-promoting hormone precursor in cells ( Ghrelin / Obestatin Preprohormone ; prepro - GHRL ) And appetite promoting hormone precursors (Ghrelin/Obestatin Preprohormone; prepro-GHRL)

It has been reported that appetite-stimulating hormone regulates cell growth through the growth hormone secretagogue receptor (GHS-R) and increases STAT3 through calcium regulation in the body (Mol Cell Endocrinol., 285, 19-25). , 2008). In this example, the signaling process by the appetite-promoting hormone precursor (Ghrelin/Obestatin Preprohormone; prepro-GHRL) and the appetite-promoting hormone precursor (Ghrelin/Obestatin Preprohormone; prepro-GHRL) substituents was confirmed in the cells. First, HepG2 (ATCC, AB-8065) cells were starved for 8 hours, and then pCMV3-C-myc-prepro-GHRL WT and pCMV3-C-myc-prepro-GHRL substituents (K100R) were each used by 6 μg of HepG2. The cells were infected. After 2 days of infection, proteins were extracted from the cells and quantified, and then Western blotting was performed to confirm the intracellular signal transduction process. In this process, the protein isolated from HepG2 (ATCC, AB-8065) cells infected with pCMV3-C-myc-prepro-GHRL WT and pCMV3-C-myc-prepro-GHRL substituent (K100R) was converted to polyvinylidenedifluoride ( polyvinylidene difluoride, PVDF) membrane, anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, cell signaling 9131S) ), and a blocking solution containing an anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 and an anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) Anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) was developed with an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a secondary antibody. As a result, pCMV3-C-myc-prepro-GHRL substituent (K100R) showed the same or increased phospho-STAT3 signaling as pCMV3-C-myc-prepro-GHRL WT in HepG2 cells (FIG. 84).

Example 13: Appetite promoting hormone ( Ghrelin In) Ubiquitination Analysis and confirmation of half-life increase and intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

Appetite-promoting hormone (Ghrelin) by polymerase chain reaction The DNA amplification product and pcDNA3-myc (5.6kb) were fragmented with restriction enzymes BamHI and XhoI, and then conjugated and cloned (Fig. 85, Ghrelin amino acid sequence: SEQ No. 83). It was confirmed through rose gel electrophoresis (FIG. 86). In addition, the part marked in bold and underlined on the nucleotide sequence of FIG. 85 is a primer set used when confirming through polymerase chain reaction to confirm the cloned site once again, and the result is confirmed through agarose gel electrophoresis. (Fig. 86). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94°C for 3 minutes, followed by repeating 25 cycles of 30 seconds at 94°C for the denaturation reaction, 30 seconds at 58°C for the annealing reaction, and 20 seconds at 72°C for the extension reaction. Then, it was reacted at 72° C. for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, the myc present in the pcDNA3-myc vector shown in the map of FIG. 85 was expressed through western blotting using an anti-myc (9E10, sc-40) antibody. Confirmed. Through this, an appetite promoting hormone (Ghrelin) bound to myc It was confirmed that the protein was well expressed, and it was found that the protein was quantitatively loaded through a blot confirmed with actin (FIG. 87).

(2) Substitution of lysine (K) residues

The lysine residue was substituted with arginine using site-specific mutagenesis, and primers (Ghrelin K39R FP 5'-AGTCCAGCAGAGAAGGGAGTCGAAGAAGCCA-3' (SEQ No. 84), RP 5'-TGGCTTCTTCGACTCCCTTCTCTGCTGGACT) using the DNA sequence to induce specific mutations. -3' (SEQ No. 85); Ghrelin K42R FP 5'-AGAAAGGAGTCGAGGAAGCCACCAGCCAAGC-3' (SEQ No. 86), RP 5'-GCTTGGCTGGTGGCTTCCTCGACTCCTTTCT-3' (SEQ No. 87); Ghrelin K43R FP 5'-AGGCCAAGGAGTCGAAG 3'(SEQ No. 88), RP 5'-GC TTGGCTGGTGGCCTCTTCGACTCCTTTCT-3' (SEQ No. 89); Ghrelin K47R FP 5'-AAGA AGCCACCAGCCAGGCTGCAGCCCCGA-3' (SEQ No. 90), RP 5'-TCGGGGCTGCAGCCT GGCTGGTGGCTTCTT After preparing 3'(SEQ No. 91), a plasmid DNA was prepared in which a specific amino acid residue was substituted by performing PCR under specific conditions: pcDNA3-myc-appetite promoting hormone (Ghrelin) DNA was used as a template, and plasmid DNA in which the lysine residue was substituted with arginine (K→R) was prepared (Table 13).

Lysine (K) residue position Ghrelin construct in which Lysine (K) is substituted with Arginine (R) 39 pcDNA3-myc-Ghrelin (K39R) 42 pcDNA3-myc-Ghrelin (K42R) 43 pcDNA3-myc-Ghrelin (K43R) 47 pcDNA3-myc-Ghrelin (K47R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-appetite promoting hormone WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 ㎍ of pcDNA3-myc-appetite promoting hormone WT and 1 ㎍ of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 88). In addition, pcDNA3-myc-appetite promoting hormone WT, pcDNA3-myc-appetite promoting hormone substituent (K39R), pcDNA3-myc-appetite promoting hormone substituent (K42R), pcDNA3-myc-appetite promoting hormone (K43R), pcDNA3-myc- HEK 293T cells were infected using a plasmid encoding an appetite promoting hormone substitute (K47R) and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, pcDNA3-myc-appetite promoting hormone WT, pcDNA3-myc-appetite promoting hormone substituent (K39R), pcDNA3-myc-appetite promoting hormone substituent (K42R), pcDNA3-myc-appetite promoting hormone substituent (K43R) ), pcDNA3-myc-appetite promoting hormone substitute (K47R), respectively, 2 μg, and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 89 ).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM phenylmethanesulfonyl fluoride), and then anti-myc (9E10, Santa Cruz Biotechnology, sc -40) Mixed with the primary antibody and incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology). For immunoblotting, protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and then separated by performing SDS-PAGE. , PVDF) membrane, then anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778 ) In a weight ratio of 1:1000 and an anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody using an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was performed with anti-myc (9E10, sc-40), ubiquitin was bound to pcDNA3-myc-appetite promoting protein WT to form polyubiquitin, resulting in smear ubiquitin being detected. Appeared dark (Fig. 88, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 μg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 88, lane 4). In addition, pcDNA3-myc-appetite promoting hormone substituent (K39R), pcDNA3-myc-appetite promoting hormone substituent (K42R), pcDNA3-myc-appetite promoting hormone substituent (K43R), pcDNA3-myc-appetite promoting hormone substituent (K47R) , The band was softer than WT, pcDNA3-myc-appetite promoting hormone substituent (K39R), pcDNA3-myc-appetite promoting hormone substituent (K42R), pcDNA3-myc-appetite promoting hormone substituent (K43R), pcDNA3-myc-appetite promoting The hormone substitutent (K47R) was unable to bind to ubiquitin, so less ubiquitin was detected (FIG. 89, lanes 3 and 6). The above results show that the appetite promoting hormone binds to ubiquitin and is polyubiquitinated and decomposed through the ubiquitin-proteasome system.

3. Confirmation of the half-life of the appetite promoting hormone (Ghrelin) by the protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-appetite promoting hormone WT, pcDNA3-myc-appetite promoting hormone substituent (K39R), pcDNA3-myc-appetite promoting hormone substituent (K42R), pcDNA3-myc-appetite promoting hormone substituent (K43R), pcDNA3-myc-appetite HEK 293T cells were transfected with 2 µg each of the promoting hormone substitutes (K47R), and 48 hours later, protein production inhibitor cyclohexamide (CHX) (Sigma-Aldrich) (100 ug/ml) was treated, followed by 12 hours and 24 hours. , As a result of measuring the half-life over 36 hours, it was confirmed that the decomposition of human appetite promoting hormone was suppressed (FIG. 90). As a result, the half-life of human appetite-promoting hormone (Ghrelin) was less than 15 hours, whereas the half-life of human pcDNA3-myc-appetite-promoting hormone substitute (K39R) and pcDNA3-myc-appetite-promoting hormone (K47R) was longer than WT. This result is shown in a graph (FIG. 90).

4. Appetite promoting hormone in cells ( Ghrelin ) And appetite promoting hormone ( Ghrelin ) Confirmation of signal transmission by substituents

It has been reported that appetite-stimulating hormone regulates cell growth through the growth hormone secretagogue receptor (GHS-R) and increases STAT3 through the regulation of calcium in the body (Mol Cell Endocrinol., 285, 19-25). , 2008). In this example, the signal transduction process by the appetite-promoting hormone (Ghrelin) and the appetite-promoting hormone (Ghrelin) substituents was confirmed in the cell. First, after starving HepG2 (ATCC, AB-8065) cells for 8 hours, pcDNA3-myc-appetite-promoting hormone WT, pcDNA3-myc-appetite-promoting hormone substitute (K39R), pcDNA3-myc-appetite-promoting hormone substitute (K42R), HepG2 cells were infected with 3 μg each of pcDNA3-myc-appetite-promoting hormone substitute (K43R) and pcDNA3-myc-appetite-promoting hormone substitute (K47R). After 2 days of infection, proteins were extracted from the cells and quantified, respectively, and Western blotting was performed to confirm the intracellular signal transduction process. In this process, pcDNA3-myc-appetite promoting hormone WT, pcDNA3-myc-appetite promoting hormone substituent (K39R), pcDNA3-myc-appetite promoting hormone substituent (K42R), pcDNA3-myc-appetite promoting hormone substituent (K43R), and The protein isolated from HepG2 cells infected with pcDNA3-myc-appetite promoting hormone substitute (K47R) was transferred to a polyvinylidene difluoride (PVDF) membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc- 40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, cell signaling 9131S), and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 Blocking solution and anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) The secondary antibody was developed with the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, pcDNA3-myc-appetite-promoting hormone substitute (K39R) showed increased phospho-STAT3 signaling in HepG2 cells than pcDNA3-myc-appetite-promoting hormone WT (FIG. 91).

Example 14: Glucagon-like peptide (GLP-1) protein Ubiquitination Analysis and confirmation of half-life increase and intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

Glucagon-like peptide (GLP-1) DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were fragmented with restriction enzymes BamHI and XhoI, and then conjugated and cloned (Fig. 92, GLP-1 amino acid sequence : SEQ No. 92), the result was confirmed through agarose gel electrophoresis after digestion with restriction enzymes (FIG. 93). In addition, the part marked in bold and underlined on the nucleotide sequence of FIG. 92 is a primer set used when confirming through the polymerase chain reaction to reconfirm the cloned site, and the result was confirmed through agarose gel electrophoresis ( Figure 93). Polymerase chain reaction conditions are as follows; The initial denaturation was reacted at 94°C for 3 minutes, followed by 25 cycles of 30 seconds at 94°C for the denaturation reaction, 30 seconds at 58°C for the annealing reaction, and 20 seconds at 72°C for the extension reaction. Then, it was reacted at 72°C for 10 minutes. In order to confirm whether the thus-produced DNA is properly expressed as a protein, the myc present in the pcDNA3-myc vector shown in the map of FIG. 92 was expressed through western blotting using an anti-myc (9E10, sc-40) antibody. Confirmed. Through this, it was confirmed that the glucagon-like peptide (GLP-1) protein bound to myc was well expressed, and it was found that it was quantitatively loaded through a blot confirmed with actin (FIG. 94).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-specific mutagenesis, and primers (GLP-1 K117R FP 5'-AAGCTGCCAGGGAATTCA-3' (SEQ No. 93), RP 5') using the DNA sequence to induce specific mutations. -TGAATTCCCTGGCAGCTT-3' (SEQ No. 94); GLP-1 K125R FP 5'-TTGGC TGGTGAGAGGCC-3' (SEQ No. 95), RP 5'-GGCCTCTCACCAGCCAA-3' (SEQ No. 96) , Plasmid DNA was prepared in which specific amino acid residues were substituted by performing PCR under specific conditions, pcDNA3-myc-glucagon-like peptide (GLP-1) DNA was used as a template, and lysine residues were substituted with arginine (K→R) Plasmid DNA was prepared (Table 14).

Lysine (K) residue position GLP-1 construct in which Lysine (K) is substituted with Arginine (R) 117 pcDNA3-myc-GLP-1 (K117R) 125 pcDNA3-myc-GLP-1 (K125R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-glucagon-like peptide (GLP-1) WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 µg of pcDNA3-myc-glucagon-like peptide (GLP-1) WT and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 95). In addition, pcDNA3-myc-glucagon-like peptide (GLP-1) WT, pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K117R), pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K125R), respectively, HEK 293T cells were infected with a plasmid encoding pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, pcDNA3-myc-glucagon-like peptide (GLP-1) WT, pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K117R), pcDNA3-myc-glucagon-like peptide (GLP-1) Substituents (K125R) 2 μg and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 96).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-myc (9E10, Santa Cruz Biotechnology, sc -40) Mixed with the primary antibody and incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology). For immunoblotting, protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and then separated by performing SDS-PAGE. , PVDF) membrane, then anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778 ) In a weight ratio of 1:1000 and an anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody using an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was performed with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin was bound to pcDNA3-myc-glucagon-like peptide (GLP-1) WT, resulting in the formation of polyubiquitination. The ubiquitin of was detected and the band appeared dark (FIG. 95, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 μg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 95, lane 4). In addition, in the case of pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K117R) and pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K125R), the band was softer than WT, and pcDNA3-myc-glucagon-like Peptide (GLP-1) substituent (K117R), pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K125R) was unable to bind to ubiquitin, and thus less ubiquitin was detected (FIG. 96, lanes 3 and 4). The above results show that glucagon-like peptide (GLP-1) binds to ubiquitin and is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Confirmation of half-life of glucagon-like peptide (GLP-1) by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-glucagon-like peptide (GLP-1) WT, pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K117R), pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K125R) respectively. Each μg was transfected into HEK 293T cells. 48 hours after transfection, the protein production inhibitor cyclohexamid (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and half-life was measured over 2 hours, 4 hours and 8 hours. As a result, human glucagon-like peptide ( It was confirmed that the decomposition of GLP-1) was inhibited (FIG. 97). As a result, the half-life of human glucagon-like peptide (GLP-1) is 2 hours, whereas human pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K117R), pcDNA3-myc-glucagon-like peptide (GLP-1) substituent ( K125R) had a half-life of 4 hours or longer than that of WT, and these results were compared graphically (FIG. 97).

4. In the cell Glucagon-like peptide (GLP- 1) and Glucagon-like peptide Confirmation of signal transduction by (GLP-1) substituents

Since glucagon-like peptide (GLP-1) regulates glucose homeostasis and plays an important role in insulin resistance, it is currently used as a diabetes treatment and has been reported to induce STAT3 activity (Biochem Biophys Res Commun., 425(2), 304- 308, 2012). In this example, the signaling process by the glucagon-like peptide (GLP-1) and glucagon-like peptide (GLP-1) substituents in the cell was confirmed. First, HepG2 (ATCC, AB-8065) cells were starved for 8 hours, then pcDNA3-myc-glucagon-like peptide (GLP-1) WT, pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K117R), pcDNA3- HepG2 cells were infected with 6 μg each of the myc-glucagon-like peptide (GLP-1) substituents (K125R). After 2 days of infection, proteins were extracted from the cells and quantified, respectively, and Western blotting was performed to confirm the intracellular signal transduction process. In this process, pcDNA3-myc-glucagon-like peptide (GLP-1) WT, pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K117R) and pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K125R), respectively. ), the protein isolated from the infected HepG2 cells was transferred to a polyvinylidene difluoride (PVDF) membrane, followed by anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology). , sc-21876), anti-phospho-STAT3 (Y705, cell signaling 9131S), and anti-β-actin (Santa Cruz Biotechnology, sc-47778) in a weight ratio of 1:1000 containing a blocking solution and anti-rabbit ( goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody using ECL system (Western blot detection kit, ABfrontier, Seoul, Korea). As a result, the pcDNA3-myc-glucagon-like peptide (GLP-1) substituent (K117R) was increased in HepG2 (ATCC, AB-8065) cells compared to the control group in the same manner as pcDNA3-myc-glucagon-like peptide (GLP-1) WT. Showed phospho-STAT3 signaling (Fig. 98).

Example 15: Analysis of ubiquitination of immunoglobulin (IgG) heavy chain (HC) and confirmation of half-life increase

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

For the immunoglobulin (IgG) heavy chain (HC), refer to the patent specification (Patent No. EP1308455 B9, Patent Name: A composition comprising anti-HER2 antibodies, p. 24) for a breast cancer treatment called Herceptin that has expired among patents held by Roche. In order to facilitate protein expression in mammalian cells, codon optimization was performed to synthesize the unique DNA sequence used in this study. After that, a fragment was made using the restriction enzymes EcoRI and XhoI to produce pcDNA3-myc (5.6kb). ) Of EcoRI and XhoI were conjugated and cloned (FIG. 99, immunoglobulin (IgG) heavy chain amino acid sequence: SEQ No. 97), and the product through this process was digested with restriction enzymes, followed by agarose gel electrophoresis. It was confirmed again (FIG. 100). Western blotting of myc present in the pcDNA3-myc vector shown in the map of FIG. 99 using an anti-myc (9E10, Santa Cruz Biotechnology, sc-40) antibody in order to confirm whether the thus prepared DNA is properly expressed as a protein. Expression was confirmed through. Through this, it was confirmed that the heavy chain (HC) protein of immunoglobulin (IgG) bound to myc was well expressed, and it was found that it was quantitatively loaded through a blot confirmed with actin (FIG. 101).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-specific mutagenesis, and primers (IgG HC K235R FP 5'-ACAAAGGTGGACAGGAAGGTGGAGCCCAAG-3' (SEQ No. 98), RP 5'-) using the DNA sequence to induce specific mutations. CTTGGGCTCC ACCTTCCTGTCCACCTTTGT-3' (SEQ No. 99); IgG HC K344R FP 5'-GAGTATAAGTGC AGGGTGTCCAATAAGGCCCTGC-3' (SEQ No. 100), RP 5'-GCAGGGCCTTATTGGACAC CCTGCACTTATACTC-3' (SEQ No. 101); IgG HC K431R After making FP 5'-CTTTCTGTATAGCAGG CTGACCGTGGATAAGTCC-3' (SEQ No. 102), RP 5'-GGACTTATCCACGGTCAGCCTGC TATACAGAAAG-3' (SEQ No. 103), a plasmid in which a specific amino acid residue was substituted by performing PCR under specific conditions DNA was prepared: pcDNA3-myc-IgG HC DNA was used as a template, and plasmid DNA in which the lysine residue was substituted with arginine (K→R) was prepared (Table 15).

Lysine (K) residue position IgG HC construct in which Lysine (K) is substituted with Arginine (R) 235 pcDNA3-myc-IgG HC (K235R) 344 pcDNA3-myc-IgG HC (K344R) 431 pcDNA3-myc-IgG HC (K431R)

2. In vivo Ubiquitination analysis

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-immunoglobulin (IgG)-HC WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, 2 µg of WT of pcDNA3-myc-immunoglobulin (IgG)-HC and 1 µg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and 24 hours later, MG132 (proteasome inhibitor, 5 µg /Ml) was treated for 6 hours, and then immunoprecipitation analysis was performed (FIG. 102). In addition, pcDNA3-myc-immunoglobulin (IgG)-HC WT, pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K235R), pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K344R), pcDNA3-myc, respectively. HEK 293T cells were infected with a plasmid encoding -immunoglobulin (IgG)-HC substituent (K431R) and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, pcDNA3-myc-immunoglobulin (IgG)-HC WT, pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K235R), pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K344R) ), pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K431R), respectively, 2 μg and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 103 ).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-myc (9E10, sc-40) 1 After mixing with the primary antibody, it was incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. For immunoblotting, a protein sample was mixed with 2X SDS buffer, heated at 100° C. for 7 minutes, and then separated by performing SDS-PAGE The separated protein was separated by a polyvinylidene difluoride (PVDF) membrane. And then anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) 1: Using a blocking solution containing a weight ratio of 1000 and an anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody, the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was performed with anti-myc (9E10, sc-40), ubiquitin was bound to pcDNA3-myc-immunoglobulin (IgG)-HC WT to form polyubiquitin, resulting in smear ubiquitin. Was detected, and the band appeared dark (FIG. 102, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 102, lane 4). In addition, in the case of pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K431R), the band was softer than that of WT, and pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K431R) could not bind ubiquitin and thus had less ubiquitin. Was detected (FIG. 103, lane 5). The above results show that immunoglobulin (IgG)-HC is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Identification of half-life of immunoglobulin (IgG)-HC by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-immunoglobulin (IgG)-HC WT, pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K235R), pcDNA3-myc-immunoglobulin (IgG)-HC substituent (K344R), and pcDNA3-myc- Immunoglobulin (IgG)-HC substituent (K431R) was transfected into HEK 293T cells at 2 μg each, and 48 hours later, protein production inhibitor cyclohexamide (CHX) (Sigma-Aldrich) (100 μg/ml) was treated. Next, as a result of measuring the half-life over 2 hours, 4 hours and 8 hours, it was confirmed that the degradation of human immunoglobulin (IgG)-HC was suppressed (FIG. 104). As a result, the half-life of human immunoglobulin (IgG)-HC is less than 2 hours, whereas the half-life of human pcDNA3-myc-immunoglobulin (IgG)-HC substitute (K431R) is about 4 hours longer than that of WT. (Figure 104).

Example 16: Analysis of ubiquitination of immunoglobulin (IgG) light chain (LC) and confirmation of half-life increase

1. Cloning into expression vector and confirmation of protein expression

(1) Cloning of expression vector

The light chain (LC) of immunoglobulin (IgG) is a patent specification (patent number EP1308455 B9, patent name: A composition comprising anti-HER2 antibodies, p. 23) for a breast cancer treatment called Herceptin that has expired among patents held by Roche. For reference, codon optimization was performed to facilitate protein expression in mammalian cells to synthesize the unique DNA sequence used in this study. After that, a fragment was made using the restriction enzymes EcoRI and XhoI, and the EcoRI of pcDNA3-myc (5.6kb). And a fragment using XhoI and cloned (Fig. 105, light chain (LC) amino acid sequence of immunoglobulin (IgG): SEQ No. 104), and the product through this process was digested with restriction enzymes, followed by agarose gel electrophoresis. It was reconfirmed through (FIG. 106). Western blotting using anti-myc (9E10, Santa Cruz Biotechnology, sc-40) antibody to myc present in the pcDNA3-myc vector shown in the map of FIG. 105 in order to confirm whether the thus produced DNA is properly expressed as a protein. Expression was confirmed through. Through this, it was confirmed that the light chain (LC) protein of immunoglobulin (IgG) bound to myc was well expressed, and it was found that it was quantitatively loaded through a blot confirmed with actin (FIG. 107).

(2) Substitution of lysine (K) residues

The lysine residue was replaced with arginine using site-specific mutagenesis, and primers (IgG LC K67R FP 5'-CCTGGCAAGGCCCCAAGGCTGCTGATCTAC-3' (SEQ No. 105), RP 5'- using the DNA sequence to induce a specific mutation), RP 5'- GTAGATCAG CAGCCTTGGGGCCTTGCCAGG-3' (SEQ No. 106); IgG LC K129R FP 5'-ACAAAGGT GGAGATCAGGAGGACCGTGGCC-3' (SEQ No. 107), RP 5'-GGCCACGGTCCTCCTGAT CTCCACCTTTGT-3' (SEQ No. 108); IgG LC K171R After making FP 5'-GCCAAGGTGCAGTGGAGG GTGGATAACGCC-3' (SEQ No. 109), RP 5'-GGCGTTATCCACCCTCCACTGCACCTTGG C-3' (SEQ No. 110), a plasmid in which a specific amino acid residue was substituted by performing PCR under specific conditions DNA was prepared Using pcDNA3-myc-IgG LC DNA as a template, plasmid DNA in which the lysine residue was substituted with arginine (K→R) was prepared (Table 16).

Lysine (K) residue position IgG LC construct in which Lysine (K) is substituted with Arginine (R) 67 pcDNA3-myc-IgG LC (K67R) 129 pcDNA3-myc-IgG LC (K129R) 171 pcDNA3-myc-IgG LC (K171R)

2. In vivo ubiquitination assay

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-immunoglobulin (IgG)-LC WT and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, cells were co-transfected with 2 μg of WT of pcDNA3-myc-immunoglobulin (IgG)-LC and 1 μg of pMT123-HA-ubiquitin DNA. After 24 hours of transfection, MG132 (proteasome inhibitor, 5 µg/ml) was treated for 6 hours, followed by immunoprecipitation analysis (FIG. 108). In addition, pcDNA3-myc-immunoglobulin (IgG)-LC WT, pcDNA3-myc-immunoglobulin (IgG)-LC substituent (K67R), pcDNA3-myc-immunoglobulin (IgG)-LC substituent (K129R), pcDNA3-myc, respectively. HEK 293T cells were infected with a plasmid encoding -immunoglobulin (IgG)-LC substituent (K171R) and pMT123-HA-ubiquitin DNA. To confirm the degree of ubiquitination, pcDNA3-myc-immunoglobulin (IgG)-LC WT, pcDNA3-myc-immunoglobulin (IgG)-LC substituent (K67R), pcDNA3-myc-immunoglobulin (IgG)-LC substituent ( K129R), pcDNA3-myc-immunoglobulin (IgG)-LC substitute (K171R), respectively, 2 μg and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into cells, and immunoprecipitation analysis was performed 24 hours later (FIG. 109 ). .

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride)), and then anti-myc (9E10, sc-40) 1 After mixing with the secondary antibody, it was incubated overnight at 4° C. The immunoprecipitates were separated by reacting for 2 hours at 4° C. using protein A/G beads (Santa Cruz Biotechnology), and then washed twice with lysis buffer. For immunoblotting, protein samples were mixed with 2X SDS buffer, boiled for 7 minutes at 100° C., and then separated by performing SDS-PAGE The separated protein was separated by polyvinylidene difluoride (PVDF) membrane. And then anti-myc (9E10, Santa Cruz Biotechnology, sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) 1: Using a blocking solution containing a weight ratio of 1000 and an anti-mouse (Peroxidase-labeled antibody to mouse IgG (H+L), KPL, 074-1806) secondary antibody, the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was performed with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin was bound to pcDNA3-myc-immunoglobulin (IgG)-LC WT to form polyubiquitination. Ubiquitin was detected and the band appeared dark (Fig. 108, lanes 3 and 4). In addition, when MG132 (proteasome inhibitor, 5 μg/ml) was treated for 6 hours, the formation of polyubiquitin was increased, and the band in which ubiquitin was detected was more intense (FIG. 108, lane 4). In addition, in the case of pcDNA3-myc-immunoglobulin (IgG)-LC substituent (K171R), the band was softer than that of WT, and pcDNA3-myc-immunoglobulin (IgG)-LC substituent (K171R) did not bind to ubiquitin, resulting in less ubiquitin. Was detected (Fig. 109, lane 5). The above results show that immunoglobulin (IgG)-LC is polyubiquitinated and degraded through the ubiquitin-proteasome system.

3. Confirmation of half-life of immunoglobulin (IgG)-LC by protein production inhibitor cycloheximide (CHX)

pcDNA3-myc-immunoglobulin (IgG)-LC WT, pcDNA3-myc-immunoglobulin (IgG)-LC substituent (K67R), pcDNA3-myc-immunoglobulin (IgG)-LC substituent (K129R), pcDNA3-myc-immunity Globulin (IgG)-LC substituent (K171R) was transfected into HEK 293T cells at 2 μg each, and 48 hours later, protein production inhibitor cyclohexamid (CHX) (Sigma-Aldrich) (100 μg/ml) was treated and 2 As a result of measuring the half-life over time, 4 hours and 8 hours, it was confirmed that the degradation of human immunoglobulin (IgG)-LC was suppressed (FIG. 110). As a result, the half-life of human immunoglobulin (IgG)-LC was less than 1 hour, whereas the half-life of human pcDNA3-myc-immunoglobulin (IgG)-LC substitute (K171R) was 2 hours or longer than that of WT. Represented by (Fig. 110).

According to the present invention, a protein or (poly) peptide with an increased half-life is provided. Accordingly, the present invention relates to a protein or (poly) peptide that can be used as a therapeutic agent, and may be usefully used in the pharmaceutical industry.

<110> UbiProtein. Corp <120> A method for extending half-life of a protein <130> UBPRN16P02KR <150> KR 10-2015-0160728 <151> 2015-11-16 <160> 110 <170> KoPatentIn 3.0 <210> 1 <211> 198 <212> PRT <213> Artificial Sequence <220> <223> Human beta trophin <400> 1 Met Pro Val Pro Ala Leu Cys Leu Leu Trp Ala Leu Ala Met Val Thr 1 5 10 15 Arg Pro Ala Ser Ala Ala Pro Met Gly Gly Pro Glu Leu Ala Gln His 20 25 30 Glu Glu Leu Thr Leu Leu Phe His Gly Thr Leu Gln Leu Gly Gln Ala 35 40 45 Leu Asn Gly Val Tyr Arg Thr Thr Glu Gly Arg Leu Thr Lys Ala Arg 50 55 60 Asn Ser Leu Gly Leu Tyr Gly Arg Thr Ile Glu Leu Leu Gly Gln Glu 65 70 75 80 Val Ser Arg Gly Arg Asp Ala Ala Gln Glu Leu Arg Ala Ser Leu Leu 85 90 95 Glu Thr Gln Met Glu Glu Asp Ile Leu Gln Leu Gln Ala Glu Ala Thr 100 105 110 Ala Glu Val Leu Gly Glu Val Ala Gln Ala Gln Lys Val Leu Arg Asp 115 120 125 Ser Val Gln Arg Leu Glu Val Gln Leu Arg Ser Ala Trp Leu Gly Pro 130 135 140 Ala Tyr Arg Glu Phe Glu Val Leu Lys Ala His Ala Asp Lys Gln Ser 145 150 155 160 His Ile Leu Trp Ala Leu Thr Gly His Val Gln Arg Gln Arg Arg Glu 165 170 175 Met Val Ala Gln Gln His Arg Leu Arg Gln Ile Gln Glu Arg Leu His 180 185 190 Thr Ala Ala Leu Pro Ala 195 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human beta trophin <400> 2 agggacggct gacaagggcc aggaa 25 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Human beta trophin <400> 3 ccaggctgtt cctggccctt gtcagc 26 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human beta trophin <400> 4 ggcacagagg gtgctacggg acagc 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human beta trophin <400> 5 cgtagcaccc tctgtgcctg ggcca 25 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Human beta trophin <400> 6 gaatttgagg tcttaagggc tcacgc 26 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Human beta trophin <400> 7 cttgtcagcg tgagccctta agacctc 27 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Human beta trophin <400> 8 gctcacgctg acaggcagag ccacat 26 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Human beta trophin <400> 9 ccataggatg tggctctgcc tgtcagc 27 <210> 10 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Human growth hormone <400> 10 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe 210 215 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human growth hormone <400> 11 ccaaaggaac agaggtattc attc 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human growth hormone <400> 12 caggaatgaa tacctctgtt cctt 24 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human growth hormone <400> 13 gacctcctaa gggacctaga g 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human growth hormone <400> 14 ctctaggtcc cttaggaggt c 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human growth hormone <400> 15 cagatcttca ggcagaccta c 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human growth hormone <400> 16 gtaggtctgc ctgaagatct g 21 <210> 17 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Human insulin <400> 17 Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu 1 5 10 15 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly 20 25 30 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe 35 40 45 Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly 50 55 60 Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu 65 70 75 80 Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys 85 90 95 Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 100 105 110 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human insulin <400> 18 ggcttcttct acacacccag gaccc 25 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human insulin <400> 19 ctcccggcgg gtcctgggtg tgta 24 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Human insulin <400> 20 tccctgcaga ggcgtggcat tgt 23 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Human insulin <400> 21 ttgttccaca atgccacgcc tctgcag 27 <210> 22 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> Human interferon alpha <400> 22 Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys 1 5 10 15 Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gln Thr His Ser Leu 20 25 30 Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser 35 40 45 Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu 50 55 60 Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His 65 70 75 80 Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 85 90 95 Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr 100 105 110 Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val 115 120 125 Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys 130 135 140 Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro 145 150 155 160 Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu 165 170 175 Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu 180 185 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon alpha <400> 23 cttcagcaca agggactcat c 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon alpha <400> 24 cagatgagtc ccttgtgctg a 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon alpha <400> 25 ctcctagaca gattctacac t 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon alpha <400> 26 agtgtagaat ctgtctagga g 21 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon alpha <400> 27 gctgtgagga gatacttcca a 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon alpha <400> 28 ttggaagtat ctcctcacag c 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon alpha <400> 29 ctctatctga gagagaagaa a 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon alpha <400> 30 tttcttctct ctcagataga g 21 <210> 31 <211> 207 <212> PRT <213> Artificial Sequence <220> <223> Human G-CSF <400> 31 Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro 20 25 30 Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 35 40 45 Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys 50 55 60 Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 65 70 75 80 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 85 90 95 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 100 105 110 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 115 120 125 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 130 135 140 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 145 150 155 160 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 165 170 175 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 180 185 190 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 195 200 205 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human G-CSF <400> 32 agcttcctgc tcaggtgctt agag 24 <210> 33 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human G-CSF <400> 33 ttgctctaag cacctgagca ggaa 24 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human G-CSF <400> 34 tgtgccacct acaggctgtg ccac 24 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human G-CSF <400> 35 ggggtggcac agcctgtagg tggc 24 <210> 36 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> Human interferon beta <400> 36 Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg 20 25 30 Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45 Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60 Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile 65 70 75 80 Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser 85 90 95 Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val 100 105 110 Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125 Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140 Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser 145 150 155 160 His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175 Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn 180 185 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon beta <400> 37 cagtgtcaga ggctcctgtg g 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon beta <400> 38 ccacaggagc ctctgacact g 21 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon beta <400> 39 ctggaagaaa gactggagaa a 21 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon beta <400> 40 tttctccagt ctttcttcca g 21 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon beta <400> 41 cattacctga gggccaagga g 21 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human interferon beta <400> 42 ctccttggcc ctcaggtaat g 21 <210> 43 <211> 193 <212> PRT <213> Artificial Sequence <220> <223> Human erythropoietin <400> 43 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human erythropoietin <400> 44 gcatgtggat agagccgtca gtgc 24 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human erythropoietin <400> 45 gcactgacgg ctctatccac atgc 24 <210> 46 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Human erythropoietin <400> 46 tgacactttc cgcagactct tccgagtcta c 31 <210> 47 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Human erythropoietin <400> 47 gtagactcgg aagagtctgc ggaaagtgtc a 31 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human erythropoietin <400> 48 ctccggggaa ggctgaagct g 21 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human erythropoietin <400> 49 cagcttcagc cttccccgga g 21 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Human erythropoietin <400> 50 ggaaagctga ggctgtacac agg 23 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Human erythropoietin <400> 51 cctgtgtaca gcctcagctt tcc 23 <210> 52 <211> 396 <212> PRT <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 52 Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val 1 5 10 15 Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys 20 25 30 Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu 35 40 45 Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys 50 55 60 Gln Arg Pro Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu 65 70 75 80 Asp Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp 85 90 95 His Arg Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe 100 105 110 His His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr 115 120 125 Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe 130 135 140 Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala 145 150 155 160 Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile 165 170 175 Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu 180 185 190 Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp 195 200 205 Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His 210 215 220 Gly Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser 225 230 235 240 Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser 245 250 255 Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys 260 265 270 Gly His Pro Leu His Lys Arg Glu Lys Arg Gln Ala Lys His Lys Gln 275 280 285 Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr Val Asp 290 295 300 Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr 305 310 315 320 His Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His 325 330 335 Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val 340 345 350 Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala 355 360 365 Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn 370 375 380 Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg 385 390 395 <210> 53 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 53 gaaacgcctt aggtccagct gtaagagac 29 <210> 54 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 54 gtctcttaca gctggaccta aggcgtttc 29 <210> 55 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 55 ttaagtccag ctgtaggaga caccctttgt 30 <210> 56 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 56 acaaagggtg tctcctacag ctggacttaa 30 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 57 gttaactcta ggattcctaa ggc 23 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 58 gccttaggaa tcctagagtt aac 23 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 59 ggttgtatta aggaactatc aggac 25 <210> 60 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human bone morphogenetic protein-2 <400> 60 gtcctgatag ttccttaata caacc 25 <210> 61 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> Human fibroblast growth factor-1 <400> 61 Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30 Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu 50 55 60 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human fibroblast growth factor-1 <400> 62 aagaagccca gactcctcta c 21 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human fibroblast growth factor-1 <400> 63 gtagaggagt ctgggcttct t 21 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human fibroblast growth factor-1 <400> 64 catgcagaga ggaattggtt t 21 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human fibroblast growth factor-1 <400> 65 aaaccaattc ctctctgcat g 21 <210> 66 <211> 167 <212> PRT <213> Artificial Sequence <220> <223> Human Leptin <400> 66 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu 1 5 10 15 Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys 20 25 30 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr 35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro 50 55 60 Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala 65 70 75 80 Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln 85 90 95 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala 100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu 115 120 125 Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val 130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys 165 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human Leptin <400> 67 cccatccaaa aggtccaaga t 21 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human Leptin <400> 68 atcttggacc ttttggatgg g 21 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human Leptin <400> 69 gatgacacca agaccctcat c 21 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human Leptin <400> 70 gatgagggtc ttggtgtcat c 21 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human Leptin <400> 71 accctcatca ggacaattgt c 21 <210> 72 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human Leptin <400> 72 gacaattgtc ctgatgaggg t 21 <210> 73 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human Leptin <400> 73 accttatcca ggatggacca g 21 <210> 74 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human Leptin <400> 74 ctggtccatc ctggataagg t 21 <210> 75 <211> 209 <212> PRT <213> Artificial Sequence <220> <223> Human vascular endothelial growth factor A <400> 75 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135 140 Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Pro 145 150 155 160 Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro 165 170 175 Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala 180 185 190 Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg 195 200 205 Arg <210> 76 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Human vascular endothelial growth factor A <400> 76 tacagcacaa cagatgtgaa tgcagacc 28 <210> 77 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Human vascular endothelial growth factor A <400> 77 ggtctgcatt cacatctgtt gtgctgta 28 <210> 78 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Human vascular endothelial growth factor A <400> 78 atccgcagac gtgtagatgt tcctgca 27 <210> 79 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Human vascular endothelial growth factor A <400> 79 tgcaggaaca tctacacgtc tgcggat 27 <210> 80 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Human prepro-GHRL <400> 80 Met Pro Ser Pro Gly Thr Val Cys Ser Leu Leu Leu Leu Gly Met Leu 1 5 10 15 Trp Leu Asp Leu Ala Met Ala Gly Ser Ser Phe Leu Ser Pro Glu His 20 25 30 Gln Arg Val Gln Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu 35 40 45 Gln Pro Arg Ala Leu Ala Gly Trp Leu Arg Pro Glu Asp Gly Gly Gln 50 55 60 Ala Glu Gly Ala Glu Asp Glu Met Glu Val Arg Phe Asn Ala Pro Phe 65 70 75 80 Asp Val Gly Ile Lys Leu Ser Gly Val Gln Tyr Gln Gln His Ser Gln 85 90 95 Ala Leu Gly Lys Phe Leu Gln Asp Ile Leu Trp Glu Glu Ala Lys Glu 100 105 110 Ala Pro Ala Asp Lys 115 <210> 81 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human prepro-GHRL <400> 81 gccctgggga ggtttcttca g 21 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human prepro-GHRL <400> 82 ctgaagaaac ctccccaggg c 21 <210> 83 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 83 Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg 20 25 <210> 84 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 84 agtccagcag agaagggagt cgaagaagcc a 31 <210> 85 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 85 tggcttcttc gactcccttc tctgctggac t 31 <210> 86 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 86 agaaaggagt cgaggaagcc accagccaag c 31 <210> 87 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 87 gcttggctgg tggcttcctc gactcctttc t 31 <210> 88 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 88 agaaaggagt cgaagaggcc accagccaag c 31 <210> 89 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 89 gcttggctgg tggcctcttc gactcctttc t 31 <210> 90 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 90 aagaagccac cagccaggct gcagccccga 30 <210> 91 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human appetite stimulating hormone (Ghrelin) <400> 91 tcggggctgc agcctggctg gtggcttctt 30 <210> 92 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Human glucagon-like peptide-1 (GLP-1) <400> 92 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 <210> 93 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Human glucagon-like peptide-1 (GLP-1) <400> 93 aagctgccag ggaattca 18 <210> 94 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Human glucagon-like peptide-1 (GLP-1) <400> 94 tgaattccct ggcagctt 18 <210> 95 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Human glucagon-like peptide-1 (GLP-1) <400> 95 ttggctggtg agaggcc 17 <210> 96 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Human glucagon-like peptide-1 (GLP-1) <400> 96 ggcctctcac cagccaa 17 <210> 97 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> Human IgG heavy chain <400> 97 Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly 1 5 10 15 Val Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro Gly Leu Glu 465 470 <210> 98 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human IgG heavy chain <400> 98 acaaaggtgg acaggaaggt ggagcccaag 30 <210> 99 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human IgG heavy chain <400> 99 cttgggctcc accttcctgt ccacctttgt 30 <210> 100 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Human IgG heavy chain <400> 100 gagtataagt gcagggtgtc caataaggcc ctgc 34 <210> 101 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Human IgG heavy chain <400> 101 gcagggcctt attggacacc ctgcacttat actc 34 <210> 102 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Human IgG heavy chain <400> 102 ctttctgtat agcaggctga ccgtggataa gtcc 34 <210> 103 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Human IgG heavy chain <400> 103 ggacttatcc acggtcagcc tgctatacag aaag 34 <210> 104 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Human IgG light chain <400> 104 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Leu Glu 225 230 235 <210> 105 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human IgG light chain <400> 105 cctggcaagg ccccaaggct gctgatctac 30 <210> 106 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human IgG light chain <400> 106 gtagatcagc agccttgggg ccttgccagg 30 <210> 107 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human IgG light chain <400> 107 acaaaggtgg agatcaggag gaccgtggcc 30 <210> 108 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human IgG light chain <400> 108 ggccacggtc ctcctgatct ccacctttgt 30 <210> 109 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human IgG light chain <400> 109 gccaaggtgc agtggagggt ggataacgcc 30 <210> 110 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Human IgG light chain <400> 110 ggcgttatcc accctccact gcaccttggc 30

Claims (5)

An insulin with an increased half-life, wherein the lysine residue at position 53 from the N-terminus of the insulin having SEQ ID NO: 17 is substituted with arginine. delete A pharmaceutical composition for preventing, treating, or preventing and treating diabetes, comprising the insulin of claim 1 and a pharmaceutically acceptable excipient. (a) a promoter; (b) a nucleotide sequence encoding the insulin of claim 1; And an optional linker, wherein the promoter and the base sequence are operatively linked. A host cell comprising the expression vector of claim 4.

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