KR101955886B1 - A method for extending half-life of angiopoietin-1 - Google Patents

Abstract

The present invention relates to a method for increasing the half-life of a protein or a protein having an increased half-life, including substituting one or more lysine residues present in the amino acid sequence of the protein, wherein the protein substituted with the lysine residue of the present invention is It remains for a long time and has excellent therapeutic effect.

Description

A method for extending half-life of angiopoietin-1

The present invention relates to a method for increasing the half-life of a protein or (poly) peptide by substituting at least one amino acid residue of the protein or (poly) peptide. Further, the present invention relates to a protein or (poly) peptide having an increased half-life produced by such a method.

Intracellular proteolysis occurs through two pathways: lysosomes and proteasomes. The lysosomal pathway, which degrades 10-20% of the protein, lacks substrate specificity and elaborate temporal regulation. In other words, it is a process of decomposing mostly extracellular or membrane proteins as the cell surface proteins incorporated into cells by endocytosis are degraded in lysosomes. However, in order for proteins to be selectively degraded in eukaryotic cells, a process in which ubiquitin is bound to a target protein by a ubiquitin-binding enzyme to form a polyubiquitin chain, which is recognized and degraded by proteasome, that is, ubiquitin- The ubiquitin-proteasome pathway (UPP). More than 80% to 90% of eukaryotic proteins are degraded through this process, and the ubiquitin-proteasome pathway regulates protein degradation and protein homeostasis by regulating protein degradation in eukaryotic cells.

Ubiquitin is a highly conserved 76 amino acid protein that is present in almost all eukaryotic cells, of which 6, 11, 27, 29, 33, 48 and 63 amino acid residues are lysine (Lysine, Lys, K) 48 and 63 play a major role in the formation of the poly ubiquitin chain. The ubiquitination of ubiquitin involves a series of enzymes (E1, E2, E3), which are degraded by the ATP-dependent protease complex 26S proteasome. The ubiquitin-proteasome pathway involves two separate, sequential processes, the first of which is the process of labeling multiple ubiquitin molecules with a covalent bond to the substrate, and the second is that the protein labeled by ubiquitin is a 26S pro It is a process that is decomposed by a teasome complex. The binding of ubiquitin and substrate takes place via an isopeptide bond between the lysine residue of the substrate molecule and the glycine at the C-terminal of ubiquitin, and the ubiquitin-activating enzyme E1, the ubiquitin-binding enzyme E2 and the ubiquitin ligase E3 The formation of thiol esters between ubiquitin and enzymes. E1 (ubiquitin-activating enzyme) activates ubiquitin by ATP-dependent reaction. E2 (ubiquitin-conjugating enzyme) takes ubiquitin activated from E1 to cysteine residue in ubiquitin-conjugated domain and transfers it to E3 ligase or directly to substrate protein. The E3 enzyme also catalyzes stable isopeptide binding between the lysine residue of the substrate protein and the glycine residue of ubiquitin. Another ubiquitin may be linked to the C-terminal lysine residue of the ubiquitin bound to the substrate protein. When this process is repeated and several ubiquitin molecules are ligated to the substrate protein in a branched form to form a polyubiquitin chain, 26S proteasome and is selectively degraded.

On the other hand, various kinds of proteins and (poly) peptides having a therapeutic effect in vivo are known. Such a protein or (poly) peptide having a therapeutic effect in vivo can be used as a therapeutic agent for the treatment of diseases such as 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, Human interleukin binding proteins, cytokine binding proteins, G-protein-coupled receptors, human growth hormone (hGH), and the like. Macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A (prot ein A), allergy inhibitor, cell necrosis glycoproteins, G-protein-coupled receptor, immunotoxin, lymphotoxin, tumor necrosis Tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, Apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, and the like. Thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factor IX, factor IX, Factor XIII, A plasminogen activating factor, a urokinase, a streptokinase, a hirudin, a protein C, a C-reactive protein, a renin inhibitor renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, , Epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin, art Atriopeptin, a cartilage inducing factor, a fibrin-binding peptide, elcatonin, a connective tissue activating factor, A tissue factor pathway inhibitor, a follicle stimulating hormone, a luteinizing hormone, a luteinizing hormone releasing hormone, a nerve growth factor, a parathyroid hormone parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecytokinin, cholesterol, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens, ns, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

Angiopoietin, an angiogenic factor, is directly involved in angiogenesis and regulates microvascular permeability, vasodilation, and vasoconstriction by signaling muscle cells surrounding the blood vessels (BMC Infect Dis., 10: 143, 2010; Cancer Lett., 328 (1): 18-26, 2013). Currently, four angiopoietins, ANGPT1, ANGPT2, ANGPT3 and ANGPT4, are known (Proc Natl Acad Sci U S A., 96 (5): 1904-1909, 1999). Among these, Angiopoietin-1 is a protein that plays an important role in angiogenesis and angiogenesis and is encoded by the ANGPT1 gene. All angiopoietin binds to endothelial cell-specific tyrosine-protein kinase receptors, which play an important role in mediating interactions between the endothelium and the surrounding matrix and mesenchyme. It also contributes to vascular maturation and stability and is involved in the early development of the heart (Cell Res., 13 (5): 309-317, 2003).

The present invention aims to provide a method for increasing the half-life of a protein.

The present invention also aims to provide a protein having one or more lysine residues substituted in the amino acid sequence, wherein the protein has an increased half-life.

It is also an object of the present invention 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 for increasing the half-life of a protein, including replacing one or more lysine residues present in the amino acid sequence of the protein.

In the present invention, lysine residues of proteins may be substituted with conservative amino acids. In the present invention, " conservative amino acid substitution " means that an amino acid residue is substituted by another amino acid residue having a similar side chain, e.g., a charge or hydrophobic chemical character. In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chain: 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) Acid 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 arginine residue.

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

Figure 1 shows the structure of the ANGPT-1 expression vector.
Figure 2 shows the ANGPT-1 gene size and PCR results.
Figure 3 shows the expression of protein through the plasmid of ANGPT-1 plasmid in 3dms HEK-293T cells.
Figure 4 shows the degradation pathway of ANGPT-1 through ubiquitination assay.
Figure 5 shows the degree of ubiquitination of the ANGPT-1 substituent in which the lysine residue is substituted with arginine as compared to the wild type.
Figure 6 shows the half-life change of ANGPT-1 after treatment with the protein synthesis inhibitor cycloheximide (CHX).
Figure 7 shows the results for effects such as PI3K-AKT and MAPK / ERK signal induction.

In one embodiment of the invention, it is ANGPT-1. At least one of the 175, 216, and 414 th lysine residues from the N-terminus in the ANGPT-1 amino acid sequence shown in SEQ ID NO: 1 is substituted with an arginine residue. Accordingly, there is provided ANGPT-1 with increased half-life and a pharmaceutical composition comprising the same for the treatment of diabetes, heart disease, and / or sepsis.

In the present invention, site-directed mutagenesis was used to replace the lysine residue present in the amino acid sequence of the protein with the arginine (R) residue. In this method, a primer is prepared using a DNA sequence that induces a specific mutation, and then a PCR is carried out under specific conditions to prepare 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 analysis and precipitated to confirm the degree of ubiquitination. As a result of treatment with the MG132 (proteasome inhibitor) reagent, the degree of ubiquitination was increased, - proteasome degradation pathway.

In the present invention, the pharmaceutical composition may be delivered into the body by various routes including oral, transcutaneous, subcutaneous, intravenous or intramuscular administration and may be administered as a syringe formulation . In addition, the pharmaceutical composition of the present invention can be formulated according to methods well known to those skilled in the art, such as rapid release, delayed release or slow release after administration according to the method. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, Aerosol, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders, and the like. 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 formulations can additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives, and the like. have.

In the present invention, the singular forms include plural forms unless expressly stated otherwise. Furthermore, terms such as "comprising" in the present invention are interpreted to have a similar meaning to "including". In the present invention, " physiologically active (poly) peptide or protein " means a (poly) peptide or protein which exhibits biological activity useful when administered to a mammal including a human.

Hereinafter, the present invention will be described in more detail based on examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: ANGPT -1 protein Ubiquitination  Analysis and confirmation of increased half-life and confirmation of intracellular signaling

1. As an expression vector Cloning  And protein expression confirmation

(1) expression vector Cloning

ANGPT-1 by polymerase chain reaction The DNA amplification product and pcDNA3-myc (5.6 kb) were ligated with restriction enzymes BamHI and XhoI and then cloned (FIG. 1, ANGPT-1 amino acid sequence: SEQ No.1) , And confirmed through agarose gel electrophoresis (FIG. 2). In addition, the underlined and bolded parts on the nucleotide sequence of FIG. 1 are part of the primer set used for confirmation of the cloned site through polymerase chain reaction, and the results are shown in the agarose gel electrophoresis (Fig. 2). Polymerase chain reaction conditions were as follows; The initial denaturation was carried out at 94 ° C for 3 minutes, followed by repeated cycles of 25 cycles at 94 ° C for 30 seconds for denaturation, 30 seconds at 60 ° C for annealing, and 1 minute and 30 seconds at 72 ° C for extension reaction And then reacted at 72 ° C for 10 minutes. In order to confirm whether the DNA thus prepared properly expressed in the protein, myc present in the pcDNA3-myc vector shown in the map of FIG. 1 was Western blotted using an anti-myc (9E10, Santa Cruz Biotechnology, sc- Lt; / RTI > expression. The myc-binding ANGPT-1 protein was well expressed and the quantitation was loaded through the blot identified with actin (FIG. 3).

(2) Lysine  (Lysine, K) Residue  substitution

The lysine residue was substituted with arginine using site-directed mutagenesis and the primer (ANGPT-1 K175R FP 5'-ACCTACAAGCTAGAGAGGCAACTTCTTCAA-3 '(SEQ. (SEQ ID NO: 2), RP 5'-TTGAAGAAGTTGCCTCTCTAGCTTGTAGGT-3 '(SEQ ID No. 3), ANGPT-1 K216R FP 5'-ACCTTAAAGGAAGAGAGAGAGAACCTTCAA-3' (SEQ ID No. 4), RP 5'-TTGAAGGTTCTCTCTCTTCCTTTAAGGT- (SEQ. No. 6) and RP 5'-GCTGCTCTGTCTTCCTGCTGTCCC-3 '(SEQ No. 7), and then PCR was carried out under specific conditions to obtain a specific amino acid residue Three plasmid DNAs were prepared using pcDNA3-myc-ANGPT-1 as a template and replacing the lysine residue with arginine (K → R) (Table 1).

Lysine (K) residue position ANGPT-1 constructs in which Lysine (K) was replaced with Arginine (R) 175 pcDNA3-myc-ANGPT-1 (K175R) 216 pcDNA3-myc-ANGPT-1 (K216R) 414 pcDNA3-myc-ANGPT-1 (K414R)

2. In vivo Ubiquitination  analysis

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-ANGPT-1 WT and pMT123-HA-ubiquitin DNA. To confirm the ubiquitination process, 3 μg of pcDNA3-myc-ANGPT-1 WT and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into the cells. After 24 hours, MG132 (proteasome inhibitor, 5 μg / Ml, Sigma-Aldrich) for 6 hours, followed by immunoprecipitation analysis (FIG. 4). In order to compare the degree of ubiquitination between the WT and the substituent, the pcDNA3-myc-ANGPT-1 WT, pcDNA3-myc-ANGPT-1 substituent (K175R), pcDNA3-myc- ANGPT-1 substituent (K216R) 3 μg of each of the ANGPT-1 substituent (K414R) was co-transfected with 1 μg of pMT123-HA-ubiquitin DNA in HEK 293T cells (ATCC, CRL-3216) and subjected to immunoprecipitation analysis after 24 hours (Fig. 4).

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 mixed with anti-myc (9E10) And incubated overnight at 4 ° C. Immunoprecipitates were separated by reaction with protein A / G beads (Santa Cruz Biotechnology) at 4 ° C. for 2 hours and then washed twice with lysis buffer. The protein samples were mixed with 2X SDS buffer, heated at 100 ° C for 7 minutes, and separated by SDS-PAGE. The separated proteins were transferred to a PVDF membrane and then subjected to anti-myc (9E10, Santa Cruz Biotechnology, blocking solution containing anti-beta-actin (sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti- beta -actin (Santa Cruz Biotechnology, labeled antibody to mouse IgG (H + L), KPL, 074-1806) using an ECL system Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was carried out with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), pcDNA3-myc-ANGPT-1 WT was found to bind to ubiquitin and polyubiquitinization, The band appeared thick (Fig. 4, lanes 3 and 4). In addition, treatment with MG132 (protease inhibitor, 5 / / ml) for 6 hours resulted in an increase in polyubiquitin formation and a darker band in which ubiquitin was detected (Fig. 4, lane 4). In the case of the pcDNA3-myc-ANGPT-1 substituent (K175R), pcDNA3-myc-ANGPT-1 substituent (K216R) and pcDNA3-myc-ANGPT-1 substituent (K414R) The ubiquitin was little detected because the ANGPT-1 substituent (K175R), pcDNA3-myc-ANGPT-1 substituent (K216R) and pcDNA3-myc-ANGPT-1 substituent (K414R) failed to bind to ubiquitin (Fig. ). These results indicate that ANGPT-1 binds to ubiquitin and is degraded by polyubiquitination through the ubiquitin-proteasome system.

3. Protein production inhibitors cycloheximide  ( CHX )On by ANGPT Determine the half-life of -1

3 g of each of the pcDNA3-myc-ANGPT-1 WT, pcDNA3-myc-ANGPT-1 substituent (K175R), pcDNA3-myc-ANGPT-1 substituent (K216R) Cells were transfected. After 48 hours of transfection, the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 [mu] g / ml) was treated and half-life was measured over 30 min, 60 min and 90 min. As a result, it was confirmed that the degradation of human ANGPT-1 was inhibited (FIG. 6). The half-life of human ANGPT-1 was within 20 minutes, whereas the half-life of human pcDNA3-myc-ANGPT-1 substituent (K216R) was longer than 30 minutes and the result was plotted (Figure 6).

4. Intracellular ANGPT - 1 and ANGPT -1 substituents for signal transduction

It has been reported that ANGPT-1 activates MAPK and PI3K / AKT signal transduction by intracellular ANGPTTIE signal transduction and then is involved in endothelial cell interaction and cell growth (Nat Rev Cancer, 10 (8): 575-585 , 2010).

In this example, signal transduction by ANGPT-1 and ANGPT-1 substituents was observed in the cells. HeLa cells were infected with 5 μg each of pcDNA3-myc-ANGPT-1 substituent (K175R), pcDNA3-myc-ANGPT-1 substituent (K216R) and pcDNA3-myc-ANGPT-1 substituent (K414R). After 2 days of infection, proteins were extracted from the cells, quantified, and Western blotting was carried out to confirm intracellular signal transduction. Proteins isolated from HeLa cells infected with pcDNA3-myc-ANGPT-1 substituent (K175R), pcDNA3-myc-ANGPT-1 substituent (K216R), and pcDNA3-myc-ANGPT-1 substituent (K414R) (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705), anti- , cell signaling 9131S), anti-AKT (H-136, Santa Cruz Biotechnology, sc-8312), anti-phospho-AKT (S473, cell signaling 9271S), anti-Erk1 / 2 (9B3, Abfrontier LF-MAO134) Blocking solution containing anti-phospho-Erk1 / 2 (Thr202 / Tyr204, Abfrontier LF-PA0090) and anti-beta-actin (Santa Cruz Biotechnology, sc- 47778) at a weight ratio of 1: 1000 to 1: (ECL) system using a second antibody, anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, (Western blot detection kit, AB Frontier, Se oul, Korea). As a result, pcDNA3-myc-ANGPT-1 substituent (K175R), pcDNA3-myc-ANGPT-1 substituent (K216R), and pcDNA3-myc- ANGPT-1 substituent (K414R) Showed the same or increased phospho-AKT and phospho-Erk1 / 2 signaling as WT (Fig. 7).

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 which can be used as a therapeutic agent, and may be usefully used in the pharmaceutical industry.

&Lt; 110 > UbiProtein. Corp <120> A method for extending half-life of angiopoietin-1 <130> UBPRN17P-0008 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 498 <212> PRT <213> Artificial Sequence <220> <223> Angiopoietin-1 <400> 1 Met Thr Val Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His   1 5 10 15 Ile Gly Cys Ser Asn Gln Arg Arg Ser Ser Glu Asn Ser Gly Arg Arg              20 25 30 Tyr Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro          35 40 45 Glu His Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr      50 55 60 Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser  65 70 75 80 Gln Lys Leu Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp                  85 90 95 Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met             100 105 110 Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu         115 120 125 Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys     130 135 140 Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155 160 Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln                 165 170 175 Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser             180 185 190 Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu         195 200 205 Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr     210 215 220 Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala 225 230 235 240 Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp                 245 250 255 Thr Val His Asn Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu             260 265 270 Lys Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp         275 280 285 Val Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile     290 295 300 Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn 305 310 315 320 Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp                 325 330 335 Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser             340 345 350 Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln         355 360 365 Arg Gln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg     370 375 380 Ala Tyr Ser Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn 385 390 395 400 Tyr Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser                 405 410 415 Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn             420 425 430 Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp         435 440 445 Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala     450 455 460 Gly Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys 465 470 475 480 Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu                 485 490 495 Asp Phe         <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 acctacaagc tagagaggca acttcttcaa 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ttgaagaagt tgcctctcta gcttgtaggt 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 accttaaagg aagagagaga gaaccttcaa 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 ttgaaggttc tctctctctt cctttaaggt 30 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gggacagcag gaagacagag cagc 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gctgctctgt cttcctgctg tccc 24

Claims (4)

Angiopoietin having an increased half-life, wherein the lysine residue at position 216 from the N-terminus of angiopoietin-1 (ANGPT-1, Angiopoietin-1) having the amino acid sequence of SEQ ID NO: This is Tin-1. delete (a) a promoter; And (b) an expression vector comprising the base sequence encoding the angiopoietin-1 of claim 1, wherein the promoter and the base sequence are operatively linked. A host cell comprising the expression vector of claim 3.

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