KR102064724B1 - A method for extending half-life of EGF - Google Patents

Abstract

The present invention relates to a method for increasing the half-life of EGF or to an increased half-life of EGF, including substituting one or more lysine residues present in the amino acid sequence of EGF, wherein the EGF substituted with the lysine residues of the present invention Remains for a long time and has a good therapeutic effect.

Description

A method for extending half-life of EGF

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

Intracellular proteolysis occurs through two pathways by lysosomes and proteasomes. Lysosomal pathways that degrade 10-20% of the protein lack substrate specificity and fine temporal control. In other words, cell surface proteins that have been incorporated into cells by endocytosis are processes that degrade most of extracellular or membrane proteins as if they were degraded in lysosomes. However, in order to selectively degrade proteins in eukaryotic cells, ubiquitin binds to the target protein by ubiquitin binding enzyme, and then a polyubiquitin chain is formed, which is recognized and degraded by the proteasome, that is, ubiquitin-pro It must go through the ubiquitin-proteasome pathway (UPP). More than 80-90% of eukaryotic proteins are degraded through this process, and the ubiquitin-proteasome pathway regulates protein degradation and homeostasis by regulating most of the proteolysis present in eukaryotic cells.

Ubiquitin is a very well-conserved protein consisting of 76 amino acids, present in almost all eukaryotic cells, of which the 6th, 11, 27, 29, 33, 48, and 63rd amino acid residues are lysine (Lysine, Lys, K), 48 and 63 play a major role in the formation of polyubiquitin chains. The process of ubiquitination of proteins with ubiquitination involves a series of enzyme systems (E1, E2, E3), which are cleaved by the 26S proteasome, an ATP-dependent protease complex. The ubiquitin-proteasome pathway involves two distinct processes, the first of which is the covalent labeling of multiple ubiquitin molecules on a substrate, and the second is the 26S pro-labeled protein ubiquitin. It is a process that is broken down by theasome complexes. The binding of ubiquitin and substrate occurs through an isopeptide bond between the lysine residue of the substrate molecule and the glycine at the C-terminus of ubiquitin, and by ubiquitin-activating enzyme E1, ubiquitin-binding enzyme E2, and ubiquitin ligase E3. This is achieved by the formation of thiol esters between ubiquitin and the enzyme. Among them, ubiquitin-activating enzyme (E1) activates ubiquitin in an ATP-dependent response. The ubiquitin-conjugating enzyme (E2) receives activated ubiquitin from E1 at cysteine residues in the ubiquitin-conjugated domain and delivers it to E3 ligase or directly to substrate proteins. The E3 enzyme also catalyzes stable isopeptide linkage between the lysine residues of the matrix protein and the glycine residues of ubiquitin. Another ubiquitin may be linked to the C-terminal lysine residue of the ubiquitin bound to the substrate protein. Repeating this process, several ubiquitin molecules are linked to the substrate protein to form a polyubiquitin chain. It is recognized by the 26S proteasome and selectively degraded.

On the other hand, various kinds of proteins and (poly) peptides having a therapeutic effect in vivo are known. Such proteins or (poly) peptides having a therapeutic effect in vivo are, for example, growth hormone releasing hormone (GHRH), growth hormone releasing peptide, interferons (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 (prot) ein A), allergy inhibitors, cell necrosis glycoproteins, G-protein-coupled receptors, immunotoxins, lymphotoxins, tumor necrosis Tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin Apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, hemoglobin, thrombin Thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factor VIII, factor IX, Factor XIII, Plasminogen activating factor, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitors ( renin inhibitors, collagenase inhibitors, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor , Endothelial growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin Lyopeptin, cartilage inducing factor, fibrin-binding peptide, elcatonin, connective tissue activating factor, tissue factor Tissue factor pathway inhibitors, follicle stimulating hormones, luteinizing hormones, luteinizing hormone releasing hormones, nerve growth factors, parathyroid hormones (parathyroid hormone), relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecytokine (cholecystokinin), pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin lactoferrin, myostatin, receptors, receptor antagonists, cell surface antige ns), virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

Epidermal growth factor (EGF) binds to receptor EGFR to promote cell growth, proliferation and differentiation, while human EGF consists of 53 amino acids (Exp Cell Res., 284 (1): 2-13, 2003 ). It is also reported that such EGF plays an important role in skin and hair regeneration (J Dermatol Sci., 72 (2): 81-86, 2013).

It is an object of the present invention to provide a method for increasing the half-life of a protein.

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

It is also an object of the present invention to provide a pharmaceutical composition comprising a protein with increased half-life.

In order to achieve the above object, the present invention provides a method for 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, lysine residues of a protein may be substituted with conservative amino acids. In the present invention, "conservative amino acid substitutions" means that amino acid residues are substituted by other amino acid residues having side chains having similar chemical properties, for example, charge or hydrophobicity. In general, conservative amino acid substitutions do not substantially alter 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: 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, and is preferably substituted with arginine residue.

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

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

In one embodiment of the invention, the protein is EGF. At least one of the 28th and 48th lysine residues from the N-terminus is substituted with an arginine residue in the amino acid sequence of EGF represented by SEQ ID NO: 1. As a result, there is provided a pharmaceutical and / or cosmetic composition for EGF with increased half-life and cell growth, skin and hair regeneration and / or treatment comprising the same (Exp Cell Res., 284 (1): 2-13 , 2003; J Dermatol Sci., 72 (2): 81-86, 2013).

In the present invention, site-directed mutagenesis was used to replace lysine residues in the amino acid sequence of the protein with arginine (R) residues. In this method, a primer is prepared using a DNA sequence to induce a specific mutation, followed by PCR under specific conditions to prepare a plasmid DNA substituted with a specific amino acid residue.

In the present invention, the target protein was transfected into the cell line by immunoprecipitation assay and settled to confirm the degree of ubiquitination, and as a result of treating the MG132 (proteasome inhibitor) reagent, the target protein was increased by the degree of ubiquitination. It was confirmed that it passes through the degradation pathway by the proteasome.

In the present invention, the pharmaceutical composition may be delivered in various ways including oral, transcutaneous, subcutaneous, intravenous or intramuscular administration, and may be administered in an injectable preparation. . In addition, the pharmaceutical compositions of the present invention may be formulated according to methods well known to those of skill in the art such that, after administration according to the method, rapid release, delayed release or slow release. The formulations may be tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, Aerosols, 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 (starches) , Gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oils . In addition, the formulation may further include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives, and the like. have.

In the present invention, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise. Also, terms such as made having, as configured in the present invention, are to be construed as having a meaning similar to “comprising”. In the present invention, "physiologically active (poly) peptide or protein" means a (poly) peptide or protein that exhibits a 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 only for illustrating the present invention, and the present invention is not limited by the following examples.

Example 1: EGF  Protein Ubiquitination  Analysis and confirmation of half-life increase and intracellular signal transduction

1. Expression vectors Cloning  And confirm protein expression

(1) expression vectors Cloning

To clone EGF, purified RNA was extracted from HeLa cells (ATCC, CRM-CCL-2 ™) using Trizol and chloroform. Single stranded cDNA was then synthesized using the SuperScript ™ First-Strand cDNA Synthesis System (Invitrogen, Grand Island, NY). EGF was amplified by polymerase chain reaction using the synthesized cDNA as a template. EGF DNA amplification products and pcDNA3-myc (5.6kb) were fragmented with restriction enzymes BamHI and XhoI, spliced and cloned (FIG. 1, EGF amino acid sequence: SEQ No. 1), followed by agarose gel electrolysis. Confirmation was carried out through phoresis (FIG. 2). In addition, the underlined and bold letters on the nucleotide sequence of Figure 1 is a primer set used to confirm through the polymerase chain reaction to confirm the cloned site, the result was also confirmed through agarose gel electrophoresis ( 2). Polymerase chain reaction conditions were as follows; After initial denaturation at 94 ° C. for 3 minutes, 30 seconds at 94 ° C. for denature reaction, 30 seconds at 52 ° C. for annealing reaction, 30 at 72 ° C. for extension reaction The second cycle was repeated 25 cycles, and then reacted at 72 ° C. for 10 minutes. In order to confirm that the DNA thus produced is properly expressed as a protein, 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 The expression was confirmed through (Western blot). Through this, it was confirmed that the EGF protein bound to myc was well expressed, and it was confirmed that the quantitative loading was carried out through a blot identified by actin (actin) (FIG. 3).

(2) Lysine  (Lysine, K) Residue  substitution

Site-directed mutagenesis was used to replace lysine residues with arginine (R), and the primer (EGF K28R FP 5'-GAAGCATTGGACAGGTATGCATGCAAC-3 '( SEQ No. 2), RP 5'-GTTGCATGCATACCTGTCCAATGCTTC-3 '(SEQ No. 3); EGF K48R FP 5'-TACCGAGACCTGAGGTGGTGGGAACTG-3' (SEQ No. 4), RP 5'-CAGTTCCCACCACCTCAGGTCTCGGTA-3 '(SEQ No. 5), and then PCR was performed to prepare plasmid DNA substituted with specific amino acid residues Two plasmid DNAs using pcDNA3-myc-EGF as a template and lysine residues substituted with arginine (K → R) Was prepared (Table 1).

Lysine (K) residue site EGF construct with Lysine (K) substituted with Arginine (R) 28 pcDNA3-myc-EGF (K28R) 48 pcDNA3-myc-EGF (K48R) 28 + 48 pcDNA3-myc-EGF (K28R + K48R)

2. In vivo Ubiquitination  analysis

pcDNA3-myc-EGF 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) was used to infect HEK 293T cells (ATCC, CRL-3216). To confirm the ubiquitination process, 3 μg of pcDNA3-myc-EGF WT and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into the cells and after 24 hours, MG132 (proteasome inhibitor, 5 μg / ml , Sigma-Aldrich) was treated for 6 hours, followed by immunoprecipitation analysis (FIG. 4). In addition, to compare the degree of ubiquitination between WT and the substituent, 3 μg of each of the pcDNA3-myc-EGF WT, pcDNA3-myc-EGF substituent (K28R), and pcDNA3-myc-EGF substituent (K48R) was determined as pMT123-HA-ubiquitin DNA 1 HEK 293T cells (ATCC, CRL-3216) with μg were co-transfected and immunoprecipitation assayed 24 hours later (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), followed by anti-myc (9E10) primary antibody ( Santa Cruz Biotechnology, sc-40) and incubated overnight at 4 ° C. The immunoprecipitates were separated by reaction for 2 hours at 4 ° C. using protein A / G beads (Santa Cruz Biotechnology). The sample was washed twice with buffer The protein sample was mixed with 2X SDS buffer and heated at 100 ° C. for 7 minutes, and then separated by SDS-PAGE The isolated protein was polyvinylidene difluoride (PVDF). ) Transfer to membrane (Millipore), 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 ) Blocking solution and anti-mouse (Peroxida) Se-labeled antibody to mouse IgG (H + L), KPL, 074-1806) was developed using an ECL system (Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, in case of immunoprecipitation with anti-myc (9E10, sc-40), as ubiquitin binds to pcDNA3-myc-EGF WT, polyubiquitination is formed, smeared ubiquitin is detected and the band is darkened. (Figure 4, lanes 3 and 4). In addition, treatment with MG132 (proteasome inhibitor, 5 μg / ml) for 6 hours resulted in increased polyubiquitination formation, resulting in a thicker band where ubiquitin is detected (FIG. 4, lane 4). These results suggest that EGF binds to ubiquitin and is polyubiquitinated via the ubiquitin-proteasome system. In addition, in the case of pcDNA3-myc-EGF substituent (K28R), the band appeared lighter than WT. This indicates that ubiquitin could not be bound to this substituent, so that less ubiquitin was detected (FIG. 5, lane 3).

3. Protein Production Inhibitors to cycloheximide (CHX)  by Of EGF  Half life check

3 μg of pcDNA3-myc-EGF WT, pcDNA3-myc-EGF substituent (K28R), pcDNA3-myc-EGF substituent (K48R), and pcDNA3-myc-EGF substituent (K28R + K48R) were transfected into HEK 293T cells (3 μg each). transfection). 48 hours after transfection, the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 μg / ml) was treated and the half-life was measured over 1 hour, 2 hours, and 4 hours. As a result, it was confirmed that degradation of human EGF was suppressed (FIG. 6). The half-life of human EGF is less than 2 hours, whereas the half-life of human EGF substituent (K28R), EGF substituent (K48R), and EGF substituent (K28R + K48R) is longer than WT over 4 hours. 6).

4. within cells EGF and On EGF substituents  Signal transmission confirmation by

EGF is reported to activate Erk1 / 2 by Ras-Raf-MEK1 / 2-Erk signaling as a growth factor (Journal of Cell Science 117, 4619-4628, 2004).

This example confirmed the signaling process by EGF and EGF substituents in the cell. First, HeLa cells were transfected using 3 μg of pcDNA3-myc-EGF WT, pcDNA3-myc-EGF substituent (K28R), and pcDNA3-myc-EGF substituent (K48R). After 2 days of infection, proteins were extracted from the cells and quantified, and Western blotting was performed to confirm intracellular signaling. To this end, proteins isolated from HeLa cells infected with pcDNA3-myc-EGF WT, pcDNA3-myc-EGF substituent (K28R) and pcDNA3-myc-EGF substituent (K48R), respectively, were polyvinylidene difluoride (PVDF). ) Transfer to membrane, 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), 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) blocking solution and anti-rabbit (goat anti-rabbit IgG-HRP) in a weight ratio of 1: 1000-1: 3000 , Santa Cruz Biotechnology, sc-2004) and ECL system (Western blot detection kit, ABfrontier, Seoul) using anti-mouse (Peroxidase-labeled antibody to mouse IgG (H + L), KPL, 074-1806) secondary antibody , Korea). As a result, the pcDNA3-myc-EGF substituent (K28R) and pcDNA3-myc-EGF substituent (K48R) are the same or increased phospho-STAT3, phospho-AKT and phospho-Erk1 / in the HeLa cells. Signaling of 2 was shown (FIG. 7).

According to the present invention, EGF with increased half-life is provided. Therefore, the present invention relates to EGF that can be used as a therapeutic agent, and may be usefully used in the pharmaceutical industry and the cosmetic industry.

<110> UbiProtein. Corp <120> A method for extending half-life of EGF <130> UBPRN17P0001P <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Epidermal growth factor <400> 1 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His   1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn              20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys          35 40 45 Trp Trp Glu Leu Arg      50 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 gaagcattgg acaggtatgc atgcaac 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gttgcatgca tacctgtcca atgcttc 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 taccgagacc tgaggtggtg ggaactg 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 cagttcccac cacctcaggt ctcggta 27

Claims (5)

Epidermis with increased half-life, wherein at least one of the lysine residues at positions 28 and 48 from the N-terminus of the epidermal growth factor (EGF) having the amino acid sequence of SEQ ID NO: 1 is substituted with arginine Growth factor. delete A cosmetic composition comprising the epidermal growth factor of claim 1. (a) a promoter; (b) a base sequence encoding the epidermal growth factor of claim 1; And an expression vector comprising any linker, wherein the promoter and the nucleotide sequence are operably linked. A host cell comprising the expression vector of claim 4.

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