KR20190045080A - Modified egf protein with improved transdermal delivery, cosmetic composition for improvement of skin condition and pharmaceutical composition for preventing or treating skin diseases comprising the same - Google Patents

Abstract

The present invention relates to transformed EFG proteins with improved transdermal transmission, and a cosmetic composition for improving skin condition containing the proteins as an active ingredient. In addition, the present invention relates to a pharmaceutical composition for preventing or treating skin diseases containing the transformed EFG proteins as active ingredients. According to the present invention, the transformed EFG protein is bonded with a cell membrane to efficiently deliver EGF proteins into a cell, thereby increasing cell permeation rates and a half-life. In addition, since the shape of the transformed EGF protein is maintained intact even under harsh condition, thereby improving long-term stability. Further, the EGF protein may be effectively used in cosmetic composition and a pharmaceutical composition which have a short preserving period and are difficult to store and distribute due to protein denaturation. Therefore, the present invention may be used in a new cosmetic composition or a pharmaceutical composition with improved cell permeation rates, in vivo sustainability and stability, compared with existing EGF.

Description

TECHNICAL FIELD [0001] The present invention relates to a modified EGF protein having improved transdermal delivery rate, a cosmetic composition for improving skin condition comprising the same as an active ingredient, and a pharmaceutical composition for preventing or treating skin diseases. BACKGROUND ART MODIFIED EGF PROTEIN WITH IMPROVED TRANSDERMAL DELIVERY, COSMETIC COMPOSITION FOR IMPROVEMENT OF SKIN CONDITION AND PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING SKIN DISEASES COMPRISING THE SAME}

The present invention relates to a modified EGF protein having an improved transdermal delivery rate, and a cosmetic composition for improving skin condition comprising the EGF protein as an active ingredient. Further, the present invention relates to a pharmaceutical composition for preventing or treating skin diseases comprising the modified EGF protein as an active ingredient.

As the cosmetics industry continues to develop, high-functional cosmetics are being developed that incorporate new technologies into materials throughout the cosmetics industry. In addition, the number of consumers demanding effects such as whitening, wrinkle improvement, and skin regeneration is increasing, so that the value of functional cosmetics is increasing in the cosmetics industry, and studies for fusing various materials into cosmetics are actively underway. Recently, epidermal growth factor (EGF) has attracted much attention because of its excellent skin regeneration ability.

EGF (Epidermal Growth Factor) is a small protein of 6 kDa that acts on the nucleus to promote epidermal cell division and proliferation, thus contributing to the skin regeneration process. In addition, it regulates secretion of collagen synthesis enzyme and is effective in the recovery of corneal ulcer and damage (Young Seok Kim, 2006). The EGF is used variously in cosmetics for improving skin condition, treatment for diabetic foot ulcers, treatment for wound healing, etc. However, there are problems in that the wound area is difficult to reach and persistence is relatively low. Therefore, it is necessary to develop a new substance having increased in vivo permeability and persistence of EGF protein.

 Young Seok Kim. 2006. The effect of epidermal growth factor on scar in full thickness defect wound healing of mouse. Department of Medicine, The Graduate School, Yonsei University. pp. 28-29.

Accordingly, the inventors of the present invention have conducted studies to increase the in vivo transfer and persistence of EGF, and found that an EGF derivative (hereinafter referred to as "EGF-skin") having an amino acid sequence of SEQ ID NO: And increased intracellular permeability, thus completing the present invention.

It is an object of the present invention to provide a modified EGF protein.

Another object of the present invention is to provide a cosmetic composition for improving skin condition comprising the modified EGF protein as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating skin diseases comprising the modified EGF protein as an active ingredient.

In order to achieve the above object, the present invention provides a modified EGF protein having an amino acid sequence of SEQ ID NO: 4 with improved transdermal transmission rate.

Also provided is an expression vector comprising a polynucleotide encoding said modified EGF protein.

Also provided is a host cell transfected with the expression vector.

Also provided is a method for producing a modified EGF protein comprising the step of obtaining a modified EGF protein from said transfected host cell.

In order to accomplish the above other objects, the present invention provides a cosmetic composition for improving skin condition comprising the modified EGF protein as an active ingredient.

In order to achieve still another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating skin diseases comprising the modified EGF protein as an active ingredient.

The modified EGF protein according to the present invention binds to the cell membrane and efficiently transfers the EGF protein into the cell, thereby increasing the cell permeability and half-life. In addition, the modified EGF protein can be effectively applied to cosmetic compositions and pharmaceutical compositions having short storage periods due to protein denaturation, which are difficult to store and distribute, because EGF is maintained in an intact form even under harsh conditions to improve long-term stability. Accordingly, the present invention can be utilized as a new cosmetic composition or a pharmaceutical composition improved in cell permeability, in vivo persistence and stability compared to existing EGF.

Figure 1 is a schematic representation of the construction of a gene construct capable of expressing an EGF-skin protein.
FIG. 2 shows the results of SDS-PAGE of EGF-skin proteins expressed in 6 selected candidate cell lines of RCB (research cell bank).
FIG. 3 is a graph showing the productivity and the doubling time of the EGF-skin protein measured during the passage of the 35th passage in order to confirm the long-term passive stability of the final cell line.
4A to 4C are graphs showing the activity of EGF-skin protein over time under severe conditions. 4A shows the activity of EGF-skin protein after 3 months, FIG. 4B shows the activity after 2 months, and FIG. 4C shows activity after 3 months.
FIG. 5 is a graph showing changes in EGF-skin protein activity over time according to the number of times of frozen and thawed (F / T) repetition. At this time, the F / T process was repeated 5 times in total.
Fig. 6 shows the results of treatment of FITC-labeled PBS, EGF, EGF-skin, and EGF liposome proteins in Balb / c mice and SKH-1 hairless mice, respectively. After 6 hours and 12 hours, The permeability of the skin layer of the protein is analyzed and shown.
FIG. 7 shows blood levels of EGF in blood after treating FITC-labeled PBS, EGF, EGF-skin, and EGF liposome protein in Balb / c mice and SKH-1 hairless mice respectively. Herein, EGF (sc) refers to a group injected subcutaneously with EGF.

In one aspect, the present invention provides a modified EGF protein having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 with improved transdermal delivery.

As used herein, the term " EGF " is a protein having a molecular weight of 6 kDa and consisting of 53 amino acids (SEQ ID NO: 1). The EGF promotes the division and proliferation of epidermal cells, thereby restoring skin damage, accelerating the healing of corneal ulcers, regenerating skin, and the like, and is involved in the formation of new blood vessels, proliferation of fibroblasts, secretion of collagen synthesis enzyme Function.

As used herein, the term " modified EGF protein " may be an EGF-derivative (hereinafter EGF-skin). In the present invention, the EGF-skin may have the amino acid sequence of SEQ ID NO: 4, which may be encoded by the nucleotide sequence of SEQ ID NO: 5. Also, in one embodiment, the EGF-skin comprising the signal sequence may comprise a signal sequence and the EGF-skin comprising the signal sequence may have the amino acid sequence of SEQ ID NO: 2, which is encoded by the nucleotide sequence of SEQ ID NO: 3 . In one embodiment of the present invention, the EGF-skin has increased in vivo transmission and persistence as compared with the standard EGF, and protein activity can be maintained over a long period under severe conditions. As a result, It can be very effectively applied to cosmetic compositions and pharmaceutical compositions which are difficult to store and distribute.

In another aspect, the invention provides an expression vector comprising a polynucleotide encoding a modified EGF protein, wherein the host cell is transfected with the expression vector. Wherein the vector may be a pAD15 EGF derivative. In addition, the host cell may be a Chinese hamster ovary cell (CHO cell). In one embodiment of the present invention, an expression vector pAD15 EGF-skin expressing EGF-skin protein was prepared using an expression vector system (ProGen, Korea), and the vector was transformed into CHO (Chinese hamster ovary) -DG44 DHFR (- ) Cells. After screening two times, EGF-skin protein-expressing cell lines were selected.

The present invention also provides a method for producing a modified EGF protein comprising the step of obtaining a modified EGF protein from said transfected host cell.

In another aspect, the present invention provides a cosmetic composition for improving skin condition comprising the modified EGF protein as an active ingredient.

The " skin condition improvement " is a function of a skin cell, which is selected from the group consisting of suppression of occurrence of wrinkles, inhibition of skin aging, improvement of skin elasticity, skin regeneration, wound or wound recovery, corneal regeneration, skin irritation mitigation, Protecting the skin from damage or improving the skin condition.

In addition, the present invention provides a pharmaceutical composition for preventing or treating skin diseases comprising the modified EGF protein as an active ingredient.

The "skin disease" may be selected from the group consisting of foot ulcers, compression ulcers, oral mucositis, burns, and combinations thereof.

The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier. The carrier may be present in an amount of from about 1% to about 99.99%, from 5% to 90%, from 10% to 85%, from 20% to 60% by weight, From about 90% to about 99.99% by weight.

The present invention provides a method for improving the skin condition using the cosmetic composition.

The method for improving the skin condition may include applying the cosmetic composition according to the present invention to the skin of a subject in need thereof. The subject may be a mammal, and in particular a human.

The step of applying to the skin may comprise directly applying or spraying the cosmetic composition according to the present invention to the skin according to its form. At this time, the application amount of the cosmetic composition and the number of daily use times can be appropriately set according to the user's age, sex, use, degree of symptom, etc. For example, an appropriate amount of the cosmetic composition may be administered at a frequency of 1 to 6 times a day It can be applied to the skin.

The present invention provides a method for preventing or treating skin diseases using the above pharmaceutical composition.

The method for preventing or treating skin diseases may include applying the pharmaceutical composition according to the present invention to the skin of a subject in need thereof. The subject to which the pharmaceutical composition may be applied may be a mammal, and in particular a human.

The step of applying to the skin may comprise directly applying or spraying the pharmaceutical composition according to the present invention to the skin according to its form. At this time, the application amount of the pharmaceutical composition and the number of daily use times can be appropriately set according to the user's age, sex, use, degree of symptom, etc. For example, the appropriate amount of the pharmaceutical composition may be administered once to six times a day It can be applied to the skin.

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

Example  1. Cell line development

An expression vector pAD15 EGF-skin expressing the EGF-skin protein was prepared using an expression vector system (Progen, Korea). The structure of the gene construct inserted into the expression vector is shown in FIG. The vector was transfected into CHO (Chinese hamster ovary) -DG44 DHFR (-) cells, and then a high expression monoclonal cell line was selected twice. DHFR (dihydrofolate reductase) is an enzyme essential for the synthesis of dTMP (deoxythymidine monophosphate) among the four components required for gene replication, which is a process of cell proliferation. HT (hypoxanthine & thymidine) is a precursor of dTMP, which cells can use to synthesize dTMP. Prior to transformation of the expression vector containing the DHFR gene into CHO-DG44 DHFR (-) cells, this cell growth is possible only in culture medium containing HT. Therefore, after transformation of the expression vector, the cells are stably transfected with a plasmid expressing the target gene and DHFR to survive only the cell line expressing DHFR protein by culturing in a medium containing no HT. . HT selection , The cell line expressing DHFR and EGF-skin protein was selected, and a high-expression cell line was selected with a stepwise increase in the concentration of methotrexate (MTX), a strong inhibitor of DHFR enzyme. Cell lines with productivity of 60 μg / 10 ⁶ to 80 μg / 10 ⁶ cells / day were selected at an MTX concentration of 1 μM. Six selected cell lines are shown in Table 1 below and selected as RCB candidate cell lines.

Monoclonal cell line Productivity (占 퐂 / ml) (占 퐂 / 10 ⁶ cells)

MTX
(1 [mu] M) A 115 63 B 123 69 C 100 64 D 103 60 E 109 67 F 109 68

Example  2. EGF - leather  Confirmation of protein expression

SDS-PAGE was performed on the 6 cell line cultures to confirm that the EGF-skin protein was expressed well in the 6 selected RCB candidate cell lines. At this time, the culture solution loaded on each lane is shown in Table 2, and the result of SDS-PAGE for each culture solution is shown in Fig.

Lane # Sample One Standard Marker 2 CHO-DG44 DHFR (-) culture solution 3
CHO-DG44 DHFR (-) culture medium stably expressing EGF-skin protein A 4 B 5 C 6 D 7 E 8 F

As shown in Fig. 2, the molecular weight of the EGF-skin protein was confirmed to be about 62 kDa. In addition, EGF-skin proteins accounted for more than 80% of the 6 cell line cultures selected. Most of the EGF-skin proteins were in the form of monomers (62 kDa) with activity, dimers (124 kDa) in size of 5% or less, and no cleaved forms were observed.

Experimental Example  One. RCB  Long term of candidate cell line Pass  Confirm stability

For use as a cell line for pharmaceutical production, the productivity and doubling time must be kept constant within the set period during which the production is completed. When the RCB cell line selected in Example 1 is manufactured in a working cell bank (WCB) in a master cell bank (MCB), it is necessary to culture the RCB cell line at about 10 passages or more. In addition, culture of 5 or more lines is required for culturing the seed culture for 200 L or more of the main culture, and cultivation of 20 or more lines is required for the present culture. Therefore, it is necessary to maintain the productivity and doubling time of the passage 35 or more to be used for the manufacture of medicines. To confirm this, EGF-skin protein productivity and doubling time of the selected RCB candidate cell lines were measured. In order to measure the productivity, the culture solution was centrifuged and suspended at a concentration of ⁵ × 10 ⁵ cells / ml as a new culture solution. Then, 5 ml was cultured in a T-25 flask for 3 days. Then, 1 ml was taken and the cell number was measured with a 0.5 ml culture. 0.5 ml was centrifuged and the productivity per 1x10 ⁶ cells was measured by human IgG ELISA on the supernatant. The doubling time (Td) was measured as follows. In a 125 ml flask, the cells were inoculated at 0.3 × 10 ⁶ cells / ml, cultured for 3 days, and then the number of cells was measured. Td = ln2 / m (m = 1nx2-lnx1 / t2-t1). t1 and t2 are the start and end times of culture, and x1 and x2 are the number of viable cells before and after culturing. The cell lines exhibiting the best long-term passive stability among the selected RCB candidate cell lines were selected as the final cell lines for drug development, and the productivity and the doubling time measured during the passage of the 35th passage of the F cell line selected as the final cell line are shown in Table 3 3.

Passages Productivity (/ / ml) Percentage ( % ) P1-7 49.41 100 P8-14 52.64 106.54 P15-21 55.75 112.84 P22-28 65.10 131.76 P29-35 57.57 116.5

As shown in Table 3 and Fig. 3, the productivity of the final cell line was maintained in the range of 100% to 132%, and the doubling time was kept constant near 25 hours. This means that the selected final cell line can be used as a cell line for drug production.

Experimental Example  2. EGF - leather  Protein In harsh conditions  Confirm stability

In order to confirm the stability of EGF-skin protein in harsh conditions, 126 ㎍ / ㎖ of EGF-skin protein was stored for 3 months under severe condition of temperature 40 ℃ and relative humidity 75 ± 5% Were observed. The content of EGF-skin protein over time is shown in Table 4, and the activity of EGF-skin protein over time is shown in Table 5 and FIGS. 4A to 4C.

Initial 1 month 2 months 3 months EGF-skin protein 100%
(126 + - 3 [mu] g / ml) 79%
(99 + -9 [mu] g / ml) 49.5%
(62 占 6 占 퐂 / ml) 32.0%
(41 3 ㎍ / ml)

standard Initial 2 months 3 months EGF-skin protein Show ED ₅₀ value 0.2 ± 0.04 ng / ml 0.22 0.05 ng / ml 0.19 + - 0.04 ng / ml

As shown in Table 4, the morphology of the EGF-skin protein gradually decreased to 79%, 49.5%, and 32% over time under the severe condition of 40 ° C, but as shown in Table 5 and Figs. 4a to 4c Similarly, most of the biological activity of the EGF-skin protein was maintained. This means that some EGF-skin degradation occurs under severe conditions, but most of the degraded EGF-skin material remains active with normal EGF. This indicates that the EGF-skin protein retains its activity almost extinguished even under severe conditions and exhibits high stability.

Experimental Example  3. EGF - leather  Formulation of proteins, and freezing and thawing

50 mM Tris-HCl at pH 7.2 ± 0.2 was used as formulation buffer for EGF-skin protein. Thereafter, it was stored frozen at a temperature of -80 캜 to -70 캜. The frozen and thawed (F / T) process of the EGF-skin protein formulated in the buffer was repeated 5 times in total. The results are shown in Table 6 and FIG.

Sample Peak 1 Peak 2 Main Peak Step-by-step Peak 2 Increase EGF - leather  Protein (control) 0.06 0.17 99.77 EGF-skin protein (1 F / T) 0.07 0.92 99.01 0.75 EGF-skin protein (2 F / T) 0.06 1.4 98.54 0.48 EGF-skin protein (3 F / T) 0.07 1.76 98.17 0.36 EGF-skin protein (4 F / T) 0.05 2.18 97.76 0.42 EGF-skin protein (5 F / T) 0.06 2.46 97.48 0.28

As shown in Table 6 and FIG. 5, as the number of freezing and thawing cycles of EGF-skin protein was increased, Peak 2 was increased and the main peak was decreased. Peak 2 showed the highest increase in the first stage of freezing and thawing, and the main peak decreased about 2% when the freezing and thawing processes were repeated 5 times in total. As a result, since the purity of about 97% or more was maintained even after 5 cycles of freezing and thawing, the EGF-skin protein retains its activity almost completely without any loss even under the severe conditions of repeated freezing and thawing, Could know.

Experimental Example  4. EGF - leather  Skin permeation analysis of protein

In order to confirm the skin permeability of the EGF-skin protein, Balb / c mice and SKH-1 hairless mice deprived as experimental animals were prepared. Using two kinds of mice, Balb / c mice that have hairless hair removed from the back skin using hair removal cream and genetically hairless hairless mice, the skin permeability of EGF-skin protein due to artificial damage of skin due to hair removal . The two kinds of mice were treated with PBS group, EGF Group, EGF-skin group, and EGF liposome (H & Pharmacam) group. At this time, each protein was labeled with fluorescence isothiocyanate (FITC) as a fluorescence labeling reagent, so that the degree of skin permeation of each protein could be confirmed. Each protein solution labeled with FITC was applied to the mouse skin of each group at a rate of 200 ng based on FITC and applied with a brush.

4.1. EGF - leather  Protein Percutaneous  relay

When the FITC-labeled protein solution was applied to the dorsal skin of the mice of each of the above-mentioned groups 6 hours and 12 hours later, mice were sacrificed and the skin tissue was removed. The skin tissue was stored in a vertical section using a low temperature maintenance apparatus, The permeability of the skin layer was analyzed through a confocal fluorescence microscope. The results are shown in Fig.

As shown in Fig. 6, FITC signals were observed in the hypodermis layer of mice belonging to the EGF-skin group 6 hours and 12 hours after transdermal delivery. This pattern was identical regardless of the species of the mouse. In addition, such skin permeability is generally similar to that of liposome, which is known to be an efficient delivery vehicle for transdermal delivery. There was a difference in percutaneous translucency in the experiments, but the overall pattern was reproducible. This means that the EGF-skin protein has high skin permeability.

4.2. EGF - leather  Blood collection experiment for evaluation of protein safety

Each of the protein solutions labeled with FITC was applied to the dorsal skin of each of the mice of each group, and then blood EGF concentration was measured over time through eye-bleeding. The results are shown in Fig.

As shown in FIG. 7, no increase or change in blood EGF concentration was observed when compared with the PBS group, which is a negative control group, in both the EGF group, the EGF-skin group, and the EGF liposome group, Respectively. This means that the amount of EGF delivered to the blood is very low compared to the EGF concentration in the blood, and EGF is rapidly degraded by the enzyme in the body and does not affect the long-term blood EGF concentration. In addition, it was found that EGF-skin proteins can be safely used as cosmetics or pharmaceuticals.

<110> PROGEN CO., LTD. <120> MODIFIED EGF PROTEIN WITH IMPROVED TRANSDERMAL DELIVERY, COSMETIC          COMPOSITION FOR IMPROVEMENT OF SKIN CONDITION AND PHARMACEUTICAL          COMPOSITION FOR PREVENTING OR TREATING SKIN DISEASES COMPRISING          THE SAME <130> FPD / 201810-0094 <150> KR 10-2017-0137132 <151> 2017-10-23 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> EGF (epidermal growth factor) amino acid sequence <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> 323 <212> PRT <213> Artificial Sequence <220> <223> EGF-derivative amino acid sequence <400> 2 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly   1 5 10 15 Ala Val Phe Val Ser Ser Ser Ala Ser Asp Ser Glu Cys Pro              20 25 30 Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile          35 40 45 Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly      50 55 60 Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg Arg Asn  65 70 75 80 Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys Glu Glu                  85 90 95 Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln             100 105 110 Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met         115 120 125 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln     130 135 140 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 145 150 155 160 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr                 165 170 175 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly             180 185 190 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile         195 200 205 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val     210 215 220 Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val Ser 225 230 235 240 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu                 245 250 255 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro             260 265 270 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val         275 280 285 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met     290 295 300 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 305 310 315 320 Leu Gly Lys             <210> 3 <211> 969 <212> DNA <213> Artificial Sequence <220> <223> EGF-derivative nucleic acid sequence <400> 3 atggacgcca tgctgagagg cctgtgctgc gtgctgctgc tgtgcggagc cgtgttcgtg 60 agccccagcc acgccaacag cgacagcgag tgccccctga gccacgatgg ctactgcctg 120 cacgatggcg tgtgcatgta catcgaggcc ctggacaagt acgcctgcaa ctgcgtggtg 180 ggctacatcg gcgagaggtg ccagtacaga gacctgaagt ggtgggagct gagaaggaac 240 accggcagag gaggcgagga gaagaaaggc agcaaggaga aggaggagca ggaagagaga 300 gagaccaaga cacccgagtg ccccagccac acccagcccc tgggcgtgtt cctgttccct 360 cccaagccca aagacaccct gatgatcagc agaacacccg aggtgacctg cgtggtcgtg 420 gatgtgagcc aggaagatcc cgaagtgcag ttcaactggt acgtggatgg cgtggaagtg 480 cacaacgcca agaccaagcc cagagaagag cagttcaact ccacctacag agtggtgagc 540 gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgtcc 600 aacaaaggcc tgcccagctc catcgagaag accatcagca aagccaaagg ccagcccaga 660 gaaccccagg tgtacaccct gcctcccagc caggaagaga tgaccaagaa ccaggtgtcc 720 ctgacctgcc tggtgaaagg cttctacccc agcgacatcg ccgtggagtg ggaaagcaac 780 ggccagcccg agaacaatta caagacaacc cctcccgtgc tggatagcga tggcagcttc 840 tttctgtaca gcagactgac cgtggacaag agcagatggc aggaaggcaa cgtgttcagc 900 tgcagcgtga tgcacgaagc cctgcacaac cactacaccc agaagagcct gtccctgagc 960 ctgggcaag 969 <210> 4 <211> 298 <212> PRT <213> Artificial Sequence <220> <223> EGF-derivative amino acid sequence <400> 4 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 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys      50 55 60 Gly Ser Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro  65 70 75 80 Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro                  85 90 95 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys             100 105 110 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp         115 120 125 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu     130 135 140 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 145 150 155 160 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                 165 170 175 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly             180 185 190 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu         195 200 205 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr     210 215 220 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 225 230 235 240 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe                 245 250 255 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn             260 265 270 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr         275 280 285 Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys     290 295 <210> 5 <211> 894 <212> DNA <213> Artificial Sequence <220> <223> EGF-derivative nucleic acid sequence <400> 5 aacagcgaca gcgagtgccc cctgagccac gatggctact gcctgcacga tggcgtgtgc 60 atgtacatcg aggccctgga caagtacgcc tgcaactgcg tggtgggcta catcggcgag 120 aggtgccagt acagagacct gaagtggtgg gagctgagaa ggaacaccgg cagaggaggc 180 gaggagaaga aaggcagcaa ggagaaggag gagcaggaag agagagagac caagacaccc 240 gagtgcccca gccacaccca gcccctgggc gtgttcctgt tccctcccaa gcccaaagac 300 accctgatga tcagcagaac acccgaggtg acctgcgtgg tcgtggatgt gagccaggaa 360 gatcccgaag tgcagttcaa ctggtacgtg gatggcgtgg aagtgcacaa cgccaagacc 420 aagcccagag aagagcagtt caactccacc tacagagtgg tgagcgtgct gaccgtgctg 480 caccaggact ggctgaacgg caaggagtac aagtgcaagg tgtccaacaa aggcctgccc 540 agctccatcg agaagaccat cagcaaagcc aaaggccagc ccagagaacc ccaggtgtac 600 accctgcctc ccagccagga agagatgacc aagaaccagg tgtccctgac ctgcctggtg 660 aaaggcttct accccagcga catcgccgtg gagtgggaaa gcaacggcca gcccgagaac 720 aattacaaga caacccctcc cgtgctggat agcgatggca gcttctttct gtacagcaga 780 ctgaccgtgg acaagagcag atggcaggaa ggcaacgtgt tcagctgcag cgtgatgcac 840 gaagccctgc acaaccacta cacccagaag agcctgtccc tgagcctggg caag 894

Claims (10)

A modified EGF protein having the amino acid sequence of SEQ ID NO: 4 with improved transdermal delivery. The method according to claim 1,
Wherein the modified EGF protein having the amino acid sequence of SEQ ID NO: 4 is encoded by the nucleotide sequence of SEQ ID NO: 5. An expression vector comprising a polynucleotide encoding the modified EGF protein of claim 1. A host cell transfected with the expression vector of claim 3. 5. The method of claim 4,
Wherein the host cell is a Chinese hamster ovary cell (CHO cell). A method for producing a modified EGF protein, comprising obtaining a modified EGF protein from the transfected host cell of claim 4. A cosmetic composition for improving skin condition comprising the modified EGF protein according to claim 1 as an active ingredient. 8. The method of claim 7,
Wherein the skin condition improvement is selected from the group consisting of suppression of wrinkle formation, skin aging inhibition, skin elasticity improvement, skin regeneration, wound or wound healing, corneal regeneration, skin irritation mitigation, Wherein the composition is to protect the skin or improve the skin condition from degradation or loss. A pharmaceutical composition for preventing or treating a skin disease comprising the modified EGF protein according to claim 1 as an active ingredient. 10. The method of claim 9,
Wherein the skin disease is selected from the group consisting of foot ulcers, compression ulcers, oral mucositis, burns, and combinations thereof.

Images