NOVEL HUMAN EPIDERMAL GROWTH FACTOR-TF FUSION PROTEIN AND USE THEREOF

Abstract

The present invention relates to a novel human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein and a use thereof. More particularly, the human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein of the present invention has fused therein: a signal peptide of a Bacillus subtilis-derived xylanase; a human epidermal growth factor (hEGF); and an Escherichia coli-derived trigger factor (TF). Therefore, the present invention not only enhances the water solubility and expression rate of a target protein, but also notably enhances useful effects such as the effects of increasing skin cell growth and healing a wound, and thus may be widely used in various industries as an active ingredient for a functional cosmetic composition and a pharmaceutical composition.

Claims

1-18. (canceled)

19. A human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein, wherein the hEGF-TF fusion protein is fused with a Bacillus subtilis-derived xylanase signal peptide; an human epidermal growth factor (hEGF); and an Escherichia coli-derived trigger factor (TF).

20. The hEGF-TF fusion protein of claim 19, wherein the Bacillus subtilis-derived xylanase signal peptide consists of the amino acid sequence of SEQ ID NO: 8.

21. The hEGF-TF fusion protein of claim 19, wherein the human epidermal growth factor (hEGF) consists of the amino acid sequence of SEQ ID NO: 9.

22. The hEGF-TF fusion protein of claim 19, wherein the Escherichia coli-derived trigger factor (TF) consists of the amino acid sequence of SEQ ID NO: 10.

23. The hEGF-TF fusion protein of claim 19, wherein the hEGF-TF fusion protein consists of the amino acid sequence of SEQ ID NO: 11.

24. The hEGF-TF fusion protein of claim 19, wherein the hEGF-TF fusion protein is encoded by a gene construct for the production of human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein, wherein the gene construct comprises a gene encoding a Bacillus subtilis-derived xylanase signal peptide; a gene encoding human epidermal growth factor (hEGF), which is a target protein; and a gene encoding an Escherichia coli-derived trigger factor (TF), which is a fusion partner; and wherein the gene construct is linked by inserting a TA sequence between the gene encoding the hEGF and the gene encoding the TF.

25. The hEGF-TF fusion protein of claim 24, wherein the gene encoding the Bacillus subtilis-derived xylanase signal peptide consists of the nucleotide sequence of E. coli codon-optimized SEQ ID NO: 2.

26. The hEGF-TF fusion protein of claim 24, wherein the gene encoding the human epidermal growth factor consists of the nucleotide sequence of E. coli codon-optimized SEQ ID NO: 4.

27. The hEGF-TF fusion protein of claim 24, wherein the gene encoding the E. coli-derived trigger factor (TF) consists of the nucleotide sequence of SEQ ID NO: 5.

28. The hEGF-TF fusion protein of claim 24, wherein the gene construct consists of the nucleotide sequence of SEQ ID NO: 7.

29. A method for producing a human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein, the method comprising steps of: (a) preparing a recombinant expression vector comprising a construct fused with a gene encoding a Bacillus subtilis-derived xylanase signal peptide; a gene encoding human epidermal growth factor (hEGF), which is a target protein; and a gene encoding an Escherichia coli-derived trigger factor (TF), which is a fusion partner, wherein the gene construct is linked by inserting a TA sequence between the gene encoding the hEGF and the gene encoding the TF; (b) preparing a transformant by introducing the recombinant expression vector of the step (a) into a microorganism; and (c) culturing the transformant of the step (b) to induce the expression of human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein, thereby obtaining the same.

30. The method of claim 29, wherein the step (c) further comprises step of culturing the transformant at a high concentration and replacing the medium before inducing the expression of the human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein.

31. A method for improving skin condition, or treating or preventing a skin disease, the method comprising administering a composition comprising the human epidermal growth factor (hEGF)-TF (Trigger Factor) fusion protein of claim 19 to a subject.

32. The method of claim 31, wherein the composition is a pharmaceutical composition for treating or preventing a skin disease.

33. The method of claim 31, wherein the composition is a food composition for improving skin condition.

34. The method of claim 31, wherein the composition is a cosmetic composition for improving skin condition.

Description

DESCRIPTION OF DRAWINGS

[0094] FIG. 1 shows a gene construct used in this example:

[0095] hEGF-TF (A): a construct composed of a signal peptide of Bacillus subtilis-derived xylanase signal peptide; human epidermal growth factor (hEGF) and E. coli-derived trigger factor (TF),

[0096] hEGF-TF (B): a construct composed of a signal peptide of Bacillus subtilis-derived xylanase signal peptide; human epidermal growth factor (hEGF) and E. coli-derived trigger factor (TF) linked by TA.

[0097] FIG. 2 is the result of confirming the amount of intracellular protein and protein secreted into the culture medium over time by SDS-PAGE for an hEGF-TF (A) construct-inserted into E. coli BL21 (DE3) under various conditions to establish optimal conditions for increasing the production (secreted amount) of the hEGF-TF fusion protein of the present invention; FIG. 2A shows the result of culturing to 0.8 at OD600 nm and inducing expression after the addition of IPTG; and FIG. 2B shows the result of culturing to 2.5 at OD600 nm, replacing the medium and then inducing expression after the addition of IPTG.

[0098] FIG. 3 is the result of confirming the amount of intracellular protein and protein secreted into the culture medium over time by SDS-PAGE for an hEGF-TF (B) construct-inserted into E. coli BL21 (DE3) under various conditions to establish optimal conditions for increasing the production (secreted amount) of the hEGF-TF fusion protein of the present invention; FIG. 3A shows the result of culturing to 0.8 at OD600 nm and inducing expression after the addition of IPTG; and FIG. 3B shows the result of culturing to 2.5 at OD600 nm, replacing the medium and then inducing expression after the addition of IPTG.

[0099] FIG. 4 is a result of confirming the protein purified by osmotic pressure by SDS-PAGE (A) and Western blot (B).

[0100] FIG. 5 is a result of showing the cell regeneration effect of commercially available hEGF (A) and hEGF-TF (B) on fibroblasts.

[0101] FIG. 6 is a result of showing the wound healing effect of commercially available hEGF (A) and hEGF-TF (B) on fibroblasts.

MODES OF THE INVENTION

[0102] Hereinafter, it will be obvious to those of ordinary skill in the art to which the present invention pertains that the examples are only for explaining the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example 1. Manufacture of the Recombinant Expression Vector and Transformed Recombinant Microorganism for the Production of Human Epidermal Growth Factor (hEGF)-Trigger Factor (TF) Fusion Protein

[0103] The present inventors have prepared a construct, a recombinant expression vector, and a transformed recombinant microorganism for producing the novel hEGF-TF fusion protein of the present invention as follows.

[0104] First, as shown in FIG. 1, a gene encoding human epidermal growth factor (hEGF), which is a target protein and a gene encoding a trigger factor (TF) derived from E. coli, which is a fusion partner protein for producing the hEGF protein in E. coli in a water-soluble state, were linked; at this time, a TA sequence was additionally inserted between the gene encoding hEGF and the gene encoding TF; and the construct hEGF-TF (B) (SEQ ID NO: 7), to which the gene encoding a Bacillus subtilis-derived xylanase signal peptide was ligated, was designed such that the Bacillus subtilis-derived xylanase signal peptide as a signal peptide for secreting the hEGF-TF fusion protein into the culture medium was linked to the N-terminus of the hEGF-TF fusion protein.

[0105] In addition, codon-optimized sequences were used for the gene encoding the Bacillus subtilis-derived xylanase signal peptide and the gene encoding hEGF and are represented by SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0106] Here, as a control to check the effect difference depending on whether the TA sequence is inserted or not, the construct hEGF-TF (A) (SEQ ID NO: 6) in which the gene encoding hEGF and the gene encoding TF were directly linked without inserting a TA sequence was used.

[0107] The hEGF-TF construct sequence and the hEGF-TF fusion protein sequence used in this example are listed in Table 1.

[0108] More specifically, the codon-optimized gene sequence encoding Bacillus subtilis-derived xylanase signal peptide is represented by SEQ ID NO: 2; the codon-optimized gene sequence encoding EGF is represented by SEQ ID NO: 4; the gene sequence encoding TF is represented by SEQ ID NO: 5; the construct including the signal peptide-EGF-TF gene (hEGF-TF (A)) is represented by SEQ ID NO: 6; and a construct (hEGF-TF (B)) linked by a TA sequence between the signal peptide-EGF and the TF genes is represented by SEQ ID NO: 7.

[0109] Further, in SEQ ID NOs: 6 and 7, the gene sequence encoding Bacillus subtilis-derived xylanase signal peptide is underlined; the gene sequence encoding EGF is shown in bold; and the gene sequences encoding TFs are shown in italics.

[0110] In addition, the amino acid sequence of the Bacillus subtilis-derived xylanase signal peptide is represented by SEQ ID NO: 8; the amino acid sequence of EGF is represented by SEQ ID NO: 9; the amino acid sequence of TF is represented by SEQ ID NO: 10; and the amino acid sequence of the signal peptide-EGF-TF is represented by SEQ ID NO: 11.

[0111] Further, in SEQ ID NO: 11, the sequence of Bacillus subtilis-derived xylanase signal peptide is underlined; the sequence of EGF is shown in bold; and the sequence of TF is shown in italics.

[0112] The synthesized gene constructs hEGF-TF (A) and hEGF-TF (B) were cleaved using Nde I (TaKaRa, Japan) and BamHI (TaKaRa, Japan) restriction enzymes and then were cloned into the pET11a expression vector (Novagen, USA) to construct a recombinant plasmid.

[0113] Thereafter, the prepared recombinant plasmid was transformed into E. coli BL21 (DE3) to prepare a transformed recombinant microorganism for producing the hEGF-TF fusion protein.

TABLE-US-00001 TABLE 1 Sequence name Sequence Bacillus ATGTTTAAGTTTAAAAAGAATTTCTTAGTTGGATTATCGGCAGCTTTAATGAGTATTAGCT subtilis TGTTTTCGGCAACCGCCTCTGCT (SEQ ID NO: 1) xylanase- SP(signal peptide) nucleotide codon- ATGTTCAAATTCAAAAAAAACTTCCTGGTCGGATTGTCAGCAGCCTTGATGTCTATCTCGC optimized SP TTTTTAGTGCAACAGCTTCGGCA(SEQ ID NO: 2) nucleotide human EGF AATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCA nucleotide TGTATATTGAAGCATTGGACAAGTATGCATGCAACTGTGTTGTTGGCTACATCGGGGAGCG ATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGC (SEQ ID NO: 3) codon- GCCAATTCAGATTCGGAGTGTCCATTATCACACGATGGTTATTGTTTACATGATGGGGTGT optimized GTATGTACATTGAGGCGTTAGACAAATACGCTTGTAATTGTGTGGTCGGCTACATTGGGGA human EGF GCGTTGCCAGTACCGTGACTTAAAGTGGTGGGAGCTTCGC (SEQ ID NO: 4) nucleotide TF ATGCAAGTTTCAGTTGAAACCACTCAAGGCCTTGGCCGCCGTGTAACGATTACTATCGCTG nucleotide CTGACAGCATCGAGACCGCTGTTAAAAGCGAGCTGGTCAACGTTGCGAAAAAAGTACGTAT TGACGGCTTCCGCAAAGGCAAAGTGCCAATGAATATCGTTGCTCAGCGTTATGGCGCGTCT GTACGCCAGGACGTTCTGGGTGACCTGATGAGCCGTAACTTCATTGACGCCATCATTAAAG AAAAAATCAATCCGGCTGGCGCACCGACTTATGTTCCGGGCGAATACAAGCTGGGTGAAGA CTTCACTTACTCTGTAGAGTTTGAAGTTTATCCGGAAGTTGAACTGCAGGGTCTGGAAGCG ATCGAAGTTGAAAAACCGATCGTTGAAGTGACCGACGCTGACGTTGACGGCATGCTGGATA CTCTGCGTAAACAGCAGGCGACCTGGAAAGAAAAAGACGGCGCTGTTGAAGCAGAAGACCG CGTAACCATCGACTTCACCGGTTCTGTAGACGGCGAAGAGTTCGAAGGCGGTAAAGCGTCT GATTTCGTACTGGCGATGGGCCAGGGTCGTATGATCCCGGGCTTTGAAGACGGTATCAAAG GCCACAAAGCTGGCGAAGAGTTCACCATCGACGTGACCTTCCCGGAAGAATACCACGCAGA AAACCTGAAAGGTAAAGCAGCGAAATTCGCTATCAACCTGAAGAAAGTTGAAGAGCGTGAA CTGCCGGAACTGACTGCAGAATTCATCAAACGTTTCGGCGTTGAAGATGGTTCCGTAGAAG GTCTGCGCGCTGAAGTGCGTAAAAACATGGAGCGCGAGCTGAAGAGCGCCATCCGTAACCG CGTTAAGTCTCAGGCGATCGAAGGTCTGGTAAAAGCTAACGACATCGACGTACCGGCTGCG CTGATCGACAGCGAAATCGACGTTCTGCGTCGCCAGGCTGCACAGCGTITCGGTGGCAACG AAAAACAAGCTCTGGAACTGCCGCGCGAACTGTTCGAAGAACAGGCTAAACGCCGCGTAGT TGTTGGCCTGCTGCTGGGCGAAGTTATCCGCACCAACGAGCTGAAAGCTGACGAAGAGCGC GTGAAAGGCCTGATCGAAGAGATGGCTTCTGCGTACGAAGATCCGAAAGAAGTTATCGAGT TCTACAGCAAAAACAAAGAACTGATGGACAACATGCGCAATGTTGCTCTGGAAGAACAGGC TGTTGAAGCTGTACTGGCGAAAGCGAAAGTGACTGAAAAAGAAACCACTTTCAACGAGCTG ATGAACCAGCAGGCGTAA (SEQ ID NO: 5) hEGF-TF(A) ATGTTCAAATTCAAAAAAAACTTCCTGGTCGGATTGTCAGCAGCCTTGATGTCTATCTCGC nucleotide TTTTTAGTGCAACAGCTTCGGCAGCCAATTCAGATTCGGAGTGTCCATTATCACACGATGG TTATTGTTTACATGATGGGGTGTGTATGTACATTGAGGCGTTAGACAAATACGCTTGTAAT TGTGTGGTCGGCTACATTGTGGGAGCGTTGCCAGTACCGTGACTTAAAGTGGTGGGAGCTT CGCATGCAAGTTTCAGTTGAAACCACTCAAGGCCTTGGCCGCCGTGTAACGATTACTATCG CTGCTGACAGCATCGAGACCGCTGTTAAAAGCGAGCTGGTCAACGTTGCGAAAAAAGTACG TATTGACGGCTTCCGCAAAGGCAAAGTGCCAATGAATATCGTTGCTCAGCGTTATGGCGCG TCTGTACGCCAGGACGTTCTGGGTGACCTGATGAGCCGTAACTTCATTGACGCCATCATTA AAGAAAAAATCAATCCGGCTGGCGCACCGACTTATGTTCCGGGCGAATAC4AGCTGGGTGA AGACTTCACTTACTCTGTAGAGTTTGAAGTTTATCCGGAAGTTGAACTGCAGGGTCTGGAA GCGATCGAAGTTGAAAAACCGATCGTTGAAGTGACCGACGCTGACGTTGACGGCATGCTGG ATACTCTGCGTAAACAGCAGGCGACCTGGAAAGAAAAAGACGGCGCTGTTGAAGCAGAAGA CCGCGTAACCATCGACTTCACCGGTTCTGTAGACGGCGAAGAGTTCGAAGGCGGTAAAGCG TCTGATTTCGTACTGGCGATGGGCCAGGGTCGTATGATCCCGGGCTTTGAAGACGGTATCA AAGGCCACAAAGCTGGCGAAGAGTTCACCATCGACGTGACCTTCCCGGAAGAATACCACGC AGAAAACCTGAAAGGTAAAGCAGCGAAATTCGCTATCAACCTGAAGAAAGTTGAAGAGCGT GAACTGCCGGAACTGACTGCAGAATTCATCAAACGTTTCGGCGTTGAAGATGGTTCCGTAG AAGGTCTGCGCGCTGAAGTGCGTAAAAACATGGAGCGCGAGCTGAAGAGCGCCATCCGTAA CCGCGTTAAGTCTCAGGCGATCGAAGGTCTGGTAAAAGCTAACGACATCGACGTACCGGCT GCGCTGATCGACAGCGAAATCGACGTTCTGCGTCGCCAGGCTGCACAGCGTTTCGGTGGCA ACGAAAAACAAGCTCTGGAACTGCCGCGCGAACTGTTCGAAGAACAGGCTAAACGCCGCGT AGTTGTTGGCCTGCTGCTGGGCGAAGTTATCCGCACCAACGAGCTGAAAGCTGACGAAGAG CGCGTGAAAGGCCTGATCGAAGAGATGGCTTCTGCGTACGAAGATCCGAAAGAAGTTATCG AGTTCTACAGCAAAAACAAAGAACTGATGGACAACATGCGCAATGTTGCTCTGGAAGAACA GGCTGTTGAAGCTGTACTGGCGAAAGCGAAAGTGACTGAA4AAGAAACCACTTTCAACGAG CTGATGAACCAGCAGGCGTAA (SEQ ID NO: 6) hEGF-TF(B) ATGTTCAAATTCAAAAAAAACTTCCTGGTCGGATTGTCAGCAGCCTTGATGTCTATCTCGC nucleotide TTTTTAGTGCAACAGCTTCGGCAGCCAATTCAGATTCGGAGTGTCCATTATCACACGATGG TTATTGTTTACATGATGGGGTGTGTATGTACATTGAGGCGTTAGACAAATACGCTTGTAAT TGTGTGGTCGGCTACATTGGGGAGCGTTGCCAGTACCGTGACTTAAAGTGGTGGGAGCTTC GCTAATGCAAGTTTCAGTTGAAACCACTCAAGGCCTTGGCCGCCGTGTAACGATTACTATC GCTGCTGACAGCATCGAGACCGCTGTTAAAAGCGAGCTGGTCAACGTTGCGAAAAAAGTAC GTATTGACGGCTTCCGCAAAGGCAAAGTGCCAATGAATATCGTTGCTCAGCGTTATGGCGC GTCTGTACGCCAGGACGTTCTGGGTGACCTGATGAGCCGTAACTTCATTGACGCCATCATT AAAGAAAAAATCAATCCGGCTGGCGCACCGACTTATGTTCCGGGCGAATACAAGCTGGGTG AAGACTTCACTTACTCTGTAGAGTTTGAAGTTTATCCGGAAGTTGAACTGCAGGGTCTGGA AGCGATCGAAGTTGAAAAACCGATCGTTGAAGTGACCGACGCTGACGTTGACGGCATGCTG GATACTCTGCGTAAACAGCAGGCGACCTGGAAAGAAAAAGACGGCGCTGTTGAAGCAGAAG ACCGCGTAACCATCGACTTCACCGGTTCTGTAGACGGCGAAGAGTTCGAAGGCGGTAAAGC GTCTGATTTCGTACTGGCGATGGGCCAGGGTCGTATGATCCCGGGCTTTGAAGACGGTATC AAAGGCCACAAAGCTGGCGAAGAGTTCACCATCGACGTGACCTTCCCGGAAGAATACCACG CAGAAAACCTGAAAGGTAAAGCAGCGAA4TTCGCTATCAACCTGAAGAAAG7TGAAGAGCG TGAACTGCCGGAACTGACTGCAGAATTCATCAAACGTTTCGGCGTTGAAGATGGTTCCGTA GAAGGTCTGCGCGCTGAAGTGCGTAAAAACATGGAGCGCGAGCTGAAGAGCGCCATCCGTA ACCGCGTTAAGTCTCAGGCGATCGAAGGTCTGGTAAAAGCTAACGACATCGACGTACCGGC TGCGCTGATCGACAGCGAAATCGACGTTCTGCGTCGCCAGGCTGCACAGCGTTTCGGTGGC AACGAAAAACAAGCTCTGGAACTGCCGCGCGAACTGTTCGAAGAACAGGCTAAACGCCGCG TAGTTGTTGGCCTGCTGCTGGGCGAAGTTATCCGCACCAACGAGCTGAAAGCTGACGAAGA GCGCGTGAAAGGCCTGATCGAAGAGATGGCTTCTGCGTACGAAGATCCGAAAGAAGTTATC GAGTTCTACAGCAAAAACAAAGAACTGATGGACAACATGCGCAATGTTGCTCTGGAAGAAC AGGCTGTTGAAGCTGTACTGGCGAAAGCGAAAGTGACTGAAAAAGAAACCACTTTCAACGA GCTGATGAACCAGCAGGCGTAA (SEQ ID NO: 7) SP protein MFKFKKNFLVGLSAALMSISLFSATASA (SEQ ID NO: 8) human EGF NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR protein (SEQ ID NO: 9) TF protein MQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGAS VRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEA IEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKAS DFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERE LPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAA LIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEER VKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNEL MNQQA (SEQ ID NO: 10) hEGF-IF MFKFKKNFLVGLSAALMSISLFSATASAANSDSECPLSHDGYCLHDGVCMYIEALDKYACN protein CVVGYIGERCQYRDLKWWELRMQVSVETTQGLGRRVTITIAADSIETAVTKSELVNVAKKV RIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLG EDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATRKEKDGAVEAE DRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYH AENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIR NRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRR VVVGLLLGEVIRTNELKADEERYKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEE QAVEAVLAKAKVTEKETTFNELMNQQA (SEQ ID NO: 11)

Example 2. Confirmation of Expression Induction of hEGF-TF Fusion Protein of the Present Invention and its Secretion into the Culture Medium

[0114] The present inventors have confirmed whether the transformed E. coli BL21 (DE3) prepared in Example 1 actually produced hEGF-TF fusion protein (hEGF-TF).

[0115] First, the transformed E. coli BL21 (DE3) prepared in Example 1 was inoculated into 5 mL of LB medium (1% tryptophan, 1% sodium chloride, and 0.5% yeast extract) containing ampicillin. Shaking culture was performed at 37° C. After culturing in 500 mL of the same medium so that the absorbance became 0.8 at 600 nm, IPTG (Isopropyl-β-D-thiolgalactoside) was added to a final concentration of 0.01 mM to induce protein expression at 20° C. In the process of inducing protein expression, 1 ml of samples were collected for each time period (0 hours, 12 hours, 24 hours, 48 hours, and 96 hours), and the cells and the culture medium were separated at 4° C. and 8,000 rpm, respectively. After the cells were completely suspended in distilled water, the cells were disrupted using a sonicator and a solution containing cellular proteins was separated.

[0116] In order to check whether the expressed proteins were discharged into the culture medium for each time, the proteins were confirmed by SDS-PAGE using the solutions and the culture solutions containing the separated cellular proteins as samples.

[0117] The amount of cellular protein and the protein secreted into the culture medium according to time after the hEGF-TF (A) construct was inserted into E. coli BL21 (DE3) is shown in FIG. 2A; and the amount of cellular protein and the protein secreted into the culture medium according to time after the hEGF-TF (B) construct was inserted into E. coli BL21 (DE3) is shown in FIG. 3A.

[0118] As a result, as shown in FIGS. 2A and 3A, it was interestingly confirmed that when protein expression was induced using a construct composed of hEGF-TF (B), that is, TA sequence was inserted between the hEGF gene and the TF gene, the production and secretion amount in the culture medium of the final product, hEGF-TF protein, was significantly increased, compared to the hEGF-TF (A) construct in which TA sequence was not inserted.

Example 3. Confirmation of Optimal Conditions for Increasing the Production (Secretion Amount) of the hEGF-TF Fusion Protein of the Present Invention

[0119] In order to establish optimal conditions for increasing the production (secretion amount) of the hEGF-TF fusion protein, the present inventors have measured the secretion level of the hEGF-TF fusion protein by applying conditions according to high-concentration culture and medium replacement of recombinant microorganisms prior to induction of expression.

[0120] The transformed E. coli BL21 (DE3) prepared in Example 1 was inoculated into 5 mL of LB medium (1% tryptophan, 1% sodium chloride, and 0.5% yeast extract) containing ampicillin. Shaking culture was performed at 37° C. After culturing in 500 mL of the same medium so that the absorbance became 2.5 at 600 nm, centrifugation was performed at 4° C. and 8,000 rpm for 10 minutes. Then, the culture medium was discarded and the precipitated cells were suspended in the same amount of medium, and then IPTG (Isopropyl-β-D-thiolgalactoside) was added to a final concentration of 0.01 mM to induce gene expression. Thereafter, while cultured at 20° C., cells and culture medium were separated at 4° C. and 8,000 rpm for 12 hours, 24 hours, 48 hours, and 96 hours, respectively. After the cells were completely suspended in distilled water, the cells were disrupted using a sonicator, and a solution containing cellular proteins was separated.

[0121] In order to check whether the expressed proteins are discharged into the culture medium, the solutions and the culture medium containing cellular proteins separated at 12 hours, 24 hours, 48 hours, and 96 hours, respectively, were used as samples, and the protein is confirmed by SDS-PAGE.

[0122] The amount of cellular protein and the protein secreted into the culture medium according to time after the hEGF-TF (A) construct was inserted into E. coli BL21 (DE3) is shown in FIG. 2B; and the amount of cellular protein and the protein secreted into the culture medium according to time after the hEGF-TF (B) construct was inserted into E. coli BL21 (DE3) is shown in FIG. 3B.

[0123] As a result, as shown in FIG. 3B, it was confirmed that at 96 hours of culture, 1300.6 mg of hEGF-TF per liter of culture was secreted, which was significantly increased by nearly 30% compared to the result of FIG. 3A in Example 2, in which 1087.6 mg of hEGF-TF per 1 liter of culture was discharged at 96 hours of culture.

[0124] This suggests that the secretion of hEGF-TF protein may be excellently improved when the medium is replaced while culturing the transformed microorganism at a high concentration before induction of expression.

[0125] That is, optimal conditions for increasing hEGF-TF fusion protein output have been established by demonstrating that the present invention shows a synergistic effect of increasing the secretion of an hEGF-TF fusion protein according to high-concentration culture and medium replacement conditions of recombinant microorganisms before induction of expression.

[0126] Therefore, it may be usefully used for mass production of high-quality human epidermal growth factor (EGF)-E. coli-derived trigger factor (TF) fusion protein.

[0127] Accordingly, the following examples were performed by using hEGF-TF, which was produced by introducing the hEGF-TF (B) construct in which the TA sequence was inserted between the hEGF gene and the TF gene.

Example 4. Quantitative Analysis of hEGF-TF Fusion Protein of the Present Invention

[0128] In order to check the amount of hEGF-TF fusion protein secreted into the culture medium, the present inventors have loaded 1, 4, and 8 μg of standard protein onto an SDS-PAGE gel, and created a calibration curve using the ImageJ program. The amount of protein corresponding to the epidermal growth factor-trigger factor fusion protein band among the proteins secreted into the culture medium in Example 3-3 was confirmed.

[0129] As a result, as shown in Table 2 below, it was confirmed that the amount of secreted protein was increased according to the culture time when the hEGF-TF protein secreted for each time period of FIG. 3B was quantitatively analyzed.

TABLE-US-00002 TABLE 2 Culture time 12 h 24 h 48 h 96 h Amount of epidermal growth 0 139.2 1171.1 1300.6 factor-Trigger factor (mg/L)

Example 5. Purification and Confirmation of hEGF-TF Fusion Protein of the Present Invention

[0130] For the purification of hEGF-TF fusion protein, the present inventors have inoculated E. coli BL21 (DE3) containing hEGF-TF (B) construct in 5 mL of LB broth (1% tryptophan, 1% sodium chloride, and 0.5% yeast extract) containing ampicillin. The shaking culture was performed at 37° C. This was again cultured in 1 L of the same medium so that the absorbance became 0.8 at 600 nm, and then IPTG was added to be 0.01 mM, and protein expression was induced at 20° C. for 24 hours. The culture medium was centrifuged at 4° C. and 8,000 rpm for 10 minutes to collect only the precipitated cells. The collected cells were purified by osmotic pressure by adding FO solution (3% NaCl) after cryopreservation.

[0131] As a result, as shown in FIG. 4, the protein was identified using SDS-PAGE, and the hEGF-TF fusion protein was confirmed by performing western blotting using the trigger factor antibody protein.

Example 6. Cell Regeneration Effect of hEGF-TF Fusion Protein of the Present Invention

[0132] In order to evaluate the effect of the hEGF-TF fusion protein of the present invention on human dermal fibroblast (HDF) proliferation, the present inventors have measured cell growth and viability using an MTT assay.

[0133] First, DMEM/F12 (3:1) mixed medium containing 10% FBS was used for culturing HDF cells, and HDF cells were inoculated in a 96-well plate in 1×10.sup.3 cells/well, and at the same time, samples were treated with 0 ng/μl (control), 0.1 ng/μl, 1 ng/μl, and 10 ng/μl hEGF-TF fusion protein, respectively. Then, they were cultured at 37° C. and 5% CO.sub.2 for 1 day and 3 days, respectively. At this time, as an experimental comparison group, an experiment was performed under the same concentration and conditions using commercially available hEGF (Sigma-Aldrich). After the culture was completed, the MTT solution was added. They were cultured for 4 hours, and then the medium was removed. DMSO was added thereto, and the absorbance was measured at 450 nm.

[0134] Cell growth rates are calculated as follows:

Cell growth rate (%)=((absorbance of experimental group−absorbance of control group)/absorbance of control group)×100.

[0135] As a result, as shown in FIG. 5, in the experimental group in which HDF cells were treated with 0.1 ng/μl of the hEGF-TF fusion protein of the present invention, the cell growth rate was increased to 4.8% on the 1st day and 57.5% on the 3rd day compared to the control group; in the experimental group treated with 1 ng/μl, the cell growth rate was increased to 10.6% on the 1st day and 73% on the 3rd day compared to the control group. In addition, in the experimental group treated with 10 ng/μl, the cell growth rate was increased to 50.7% on the 1st day and 84.9% on the 3rd day compared to the control group.

[0136] This demonstrates that the hEGF-TF fusion protein of the present invention exhibits excellently increased cell growth efficacy compared to the commercially available hEGF as a control group.

Example 7. Wound Healing Effect of hEGF-TF Fusion Protein of the Present Invention

[0137] The present inventors have evaluated the wound healing efficacy of the hEGF-TF fusion protein of the present invention on human fibroblasts.

[0138] First, 70 μl of fibroblasts (5×10.sup.5 cells/ml) were inoculated into a culture-insert culture dish (ibidi), and 24 hours later, the culture-insert was removed. They were treated with hEGF-TF at a concentration of 0 ng/μl (control), 0.1 ng/μl, and 1 ng/μl, respectively. At this time, an experiment was performed under the same concentration and conditions using commercially available hEGF (Sigma-Aldrich) as an experimental comparative group. Cell images at 3, 6, 9, 12, and 24 hours of culture were analyzed using the imageJ program to confirm the wound healing efficacy.

[0139] As a result, as shown in FIG. 6, the experimental group treated with 1 ng/μl of the hEGF-TF fusion protein of the present invention showed a higher wound healing effect as compared to the control group and the comparative group (commercially available hEGF) in general.

[0140] This demonstrates that the hEGF-TF fusion protein of the present invention exhibits excellently increased wound healing efficacy compared to the commercially available hEGF as a control group.

[0141] Overall, the novel human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein of the present invention produced by a construct in which a signal peptide gene is bound to the hEGF gene according to the present invention and a TA sequence is inserted between the hEGF gene and the TF gene improves the function of secreting the hEGF-TF fusion protein into the culture medium, that is, the function of producing in E. coli and secreting excess extracellularly so that thus the production capacity thereof is improved, and cell growth and wound healing efficacy thereof is excellent compared to that of commercially available hEGF. Therefore, it is widely used as an active ingredient for a cosmedical composition and a pharmaceutical composition for external application to skin.

[0142] Further, in the method according to the present invention, optimal conditions have been established to improve the production efficiency of the human epidermal growth factor, and thus human epidermal growth factor may be obtained in high yield so that it may be usefully applied to mass production.

[0143] As described above in detail a specific part of the content of the present invention, it will be obvious for those of ordinary skill in the art that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.