KR20210056254A - Novel Human Epidermal Growth Factor-Trigger Factor Fusion Protein and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel human epidermal growth factor (hEGF)-trigger factor (TF) fusion protein and uses thereof. More particularly, the hEGF-TF fusion protein of the present invention is fused with a signal peptide of xylanase derived from Bacillus subtilis; hEGF; and Escherichia coli-derived TF, thereby not only improving the water solubility and expression rate of the target protein, but also remarkably enhancing useful effects such as skin cell growth increase and wound healing effects. The fusion protein will be widely used as an effective component of functional cosmetic compositions and pharmaceutical compositions in various industries.

Description

Novel Human Epidermal Growth Factor-Trigger Factor Fusion Protein and Uses Thereof}

The present invention relates to a novel human epithelial growth factor-TF fusion protein and use thereof.

Human epidermal growth factor (hEGF) has an excellent wound healing effect and has been used to treat general wounds, severe burns, bedsores, skin ulcers, and venous ulcers of the lower extremities. Recently, it is known to regenerate skin cells and promote the formation of collagen, and is also used as a raw material for functional cosmetics.

Although many studies have been conducted for the production of such human epithelial growth factors, problems that occur during the purification process, especially the reduction in recovery rate that occur during precipitation and frequent concentration processes, cause problems that are not suitable for mass production processes. I did. Thereafter, attempts to express the gene encoding the human epithelial growth factor in E. coli, Bacillus bacillus, yeast, etc. have been reported, but the expression rate in Bacillus bacillus and yeast was very low, and when expressed in E. coli, the protein was easily expressed in cells. It was confirmed that the expression rate and yield were low due to degradation by a degrading enzyme. After that, a process step such as a high-performance liquid chromatography (HPLC) purification method using a C18 column is required as a process for obtaining high-purity human epithelial cell growth factors, which has a disadvantage of being very expensive.

The ultimate goal in the field of recombinant protein production is to develop a recombinant protein production technology with high specificity and excellent production efficiency. To achieve this goal, studies on recombinant vectors and host cell systems are being conducted, and as an example, to express a target protein in host cells, such as Bacillus subtilis and E. coli, etc. Various expression vectors for this have been developed. However, in the expression of the target protein, the recombined protein is converted into an insoluble inclusion body in the host cell or is degraded by proteases, and chemical modification fails in the expression process. Defect factors still exist.

As one of the methods to solve such a conventional problem, by introducing a factor that promotes the secretion of the recombinant protein into the vector system, the formation of insoluble inclusion bodies, etc. is reduced, thereby increasing the expression level of the recombinant protein, that is, the production efficiency. There is a way to improve it. In general, host cells distinguish between proteins to be present in the cytoplasm and proteins to be separated outside the cell by signal peptides present in precursor proteins. In this regard, precursor proteins do not contain signal peptides. In this case, it has been reported that the protein is not secreted and thus is not expressed externally, and the expression pattern of the protein may change depending on the nature of the amino acids constituting the signal peptide. Accordingly, the presence or absence of the signal peptide And their properties have an important influence on the production of recombinant proteins.

On the other hand, in order to obtain an active recombinant protein in high yield in E. coli, the method of expressing by fusing a fusion partner to the amino terminal of the target protein is most commonly used. When the fusion partner is fused and expressed, it has the advantage of inducing the target protein to be water-soluble, and the amino terminal methionine problem can be solved by removing the fusion partner using an enzyme. In fact, several fusion partners that induce water-soluble expression of foreign proteins in E. coli have been studied and reported over the past several years.

However, since these fusion partners are relatively larger than the target protein, there are disadvantages that the yield of the target protein is significantly lowered depending on the size of the fusion part, and that they do not work universally for both medical or industrially useful proteins. . In addition, there are cases where the target protein is also produced in the form of a dimer by forming a dimer, and even if the target protein is induce water-soluble expression, it fails to induce the expression into an active form that performs its own function. In many cases, there is a problem in that there is a process irrationality in which the removal process of the fusion partner must be added in order to be used for a suitable purpose.

Until now, domestic recombinant proteins produced industrially by biotechnology have been focused on the development of the production process of expressible recombinant proteins, so the development of the expression system, which is a core source technology, is developed as an additional technology at a level of imitation or improvement compared to foreign countries. have. In addition, proteins that are very important commercially and medically are secretory proteins and membrane proteins, which are difficult-to-express proteins that create insoluble aggregates when expressed in Escherichia coli. have.

Therefore, in order to overcome this, it is important to secure the underlying technology by developing an expression system incorporating a signal peptide and a fusion partner to increase the expression efficiency of the actual target protein while utilizing the E. coli system that has various advantages. Do.

Under such circumstances, the present inventors have made diligent efforts to solve the above-described problems and to construct a recombinant protein expression system capable of producing human epithelial cell growth factor (hEGF) in a higher yield. As a result, when the present inventors use the gene construct for the production of the human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein of the present invention, the hEGF-TF fusion protein of the present invention is cultured. In addition to improving the function of excreting into the inside, it was confirmed that the produced hEGF-TF fusion protein of the present invention exerts superior cell growth and wound healing efficacy than commercial hEGF. In addition, when the optimal conditions for the production of the hEGF-TF fusion protein of the present invention were introduced, it was confirmed that hEGF-TF can be obtained in high yield. Was completed.

Accordingly, an object of the present invention is to provide a gene construct for the production of a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein.

In addition, another object of the present invention is to provide a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein.

In addition, another object of the present invention is to provide a recombinant expression vector for the production of a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein.

In addition, another object of the present invention is to provide a transformed recombinant microorganism transformed with the recombinant expression vector.

In addition, another object of the present invention is to provide a method for producing a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein.

In addition, another object of the present invention is to provide a pharmaceutical composition for treating or preventing skin diseases comprising a human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein as an active ingredient.

In addition, another object of the present invention is to provide a food composition for improving skin conditions comprising a human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein as an active ingredient.

In addition, another object of the present invention is to provide a cosmetic composition for improving skin conditions comprising a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein as an active ingredient.

The terms used in the present application are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate that a feature, step, structure, or a combination thereof described above exists in the name, and one or more other features, steps, structures, or It is to be understood that the presence or addition of any combination does not preclude the possibility.

Unless otherwise defined, all terms including technical or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present application. Does not.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention provides a gene construct for the production of a human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein, wherein the gene construct is Bacillus sub A gene encoding a Tilis-derived xylanase signal peptide; A gene encoding a protein of interest, human epithelial growth factor; And a gene encoding the fusion partner E. coli-derived TF (Trigger Factor); It characterized in that the linking by inserting a TA sequence between the gene encoding the hEGF and the gene encoding the TF.

The gene construct of the present invention is a gene encoding any Bacillus subtilis-derived xylanase signal peptide, as long as the object of the present invention can be achieved; Genes encoding any human epithelial growth factor; And any E. coli-derived TF (Trigger Factor) can be used, including the nucleotide sequence of the encoding gene.

According to a preferred embodiment of the present invention, the gene construct may be composed of a nucleotide sequence represented by SEQ ID NO: 7, but is not limited thereto. In addition, homologs of the nucleotide sequence are included within the scope of the present invention. Specifically, the gene is a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 7 It may include. The "% of sequence homology" for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

According to one embodiment of the present invention, the gene encoding the Bacillus subtilis-derived xylanase signal peptide may be composed of a nucleotide sequence represented by SEQ ID NO: 1, and preferably, an E. coli codon-optimized sequence. It consists of the nucleotide sequence represented by number 2; The gene encoding the human epithelial growth factor may be composed of a nucleotide sequence represented by SEQ ID NO: 3, and preferably consists of a nucleotide sequence represented by SEQ ID NO: 4 optimized for E. codon codon; The gene encoding the E. coli-derived TF (Trigger Factor) may consist of a nucleotide sequence represented by SEQ ID NO: 5, but is not limited thereto. In addition, homologs of the nucleotide sequence are included within the scope of the present invention. Specifically, the gene is a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 7 It may include.

In addition, in the present invention, the "codon optimization" refers to changing the codon of the polynucleotide encoding the protein as being preferentially used in a specific organism so that the encoded protein is more efficiently expressed in the organism. Although the genetic code is degenerate in that most amino acids are denoted by several codons, referred to as "synonyms" or "synonymous" codons, codon usage by certain organisms is not random and is biased towards certain codon triplets. Such codon usage bias may be higher with respect to a given gene, genes of common function or ancestral origin, highly expressed versus low copy number proteins, and the collective protein coding regions of the organism's genome. The nucleotide sequence of SEQ ID NO: 2 or 4 of the present invention is in the codon of E. coli so that the Bacillus subtilis-derived xylanase signal peptide-coding gene or the human-derived epithelial cell growth factor protein-coding gene can be expressed in E. coli. It is an optimized sequence.

In particular, when a TA sequence is inserted and linked between the gene encoding hEGF and the gene encoding the TF in the construct of the present invention, the final product hEGF-TF protein production amount and secretion amount in the culture medium according to the insertion of the TA sequence It was the first to be discovered and demonstrated that this remarkable increase.

In addition, according to another aspect of the present invention, the present invention provides a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein.

The fusion protein of the present invention is artificially recombined so that Bacillus subtilis-derived xylanase signal peptide and E. coli-derived TF (Trigger Factor) are respectively bound to the N-terminus and C-terminus of human epithelial growth factor ( Fusion) means the hEGF-TF fusion protein, and is used interchangeably with "hEGF-TF protein" or "hEGF-TF" or "fusion protein" or "recombinant protein" herein.

In the present invention, the hEGF-TF fusion protein of the present invention is a novel protein consisting of an amino acid sequence represented by SEQ ID NO: 11 and exhibits increased cell growth and wound healing effects.

The range of the hEGF-TF fusion protein with increased cell growth and wound healing effect according to the present invention includes a protein having an amino acid sequence of SEQ ID NO: 11 and a functional equivalent of the protein. "Functional equivalent" is a result of the addition, substitution or deletion of amino acids, the amino acid sequence of SEQ ID NO: 11 at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95 It has% or more sequence homology, and refers to a protein that exhibits substantially the same activity as the protein represented by SEQ ID NO: 11. "Substantially homogenous activity" means cell proliferation, growth, increased survival and wound healing activity.

According to one embodiment of the present invention, the Bacillus subtilis-derived xylanase signal peptide consists of an amino acid sequence represented by SEQ ID NO: 8; The human epithelial cell growth factor consists of an amino acid sequence represented by SEQ ID NO: 9; The E. coli-derived TF (Trigger Factor) consists of an amino acid sequence represented by SEQ ID NO: 10.

In the present invention, in the fusion protein, the Bacillus subtilis-derived xylanase signal peptide and E. coli-derived TF (Trigger Factor) may be directly linked to the N-terminus and C-terminus of the EGF, respectively. , May be connected through a linker. The linker is not particularly limited as long as it exhibits an effect of improving the enzymatic activity of the fusion protein, but preferably glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, It can be linked using amino acids such as glutamine, glutamic acid, lysine, arginic acid, and the like, more preferably valine, leucine, aspartic acid, glycine, alanine, proline, etc. In consideration of the ease of genetic manipulation, one to five amino acids such as glycine, valine, leucine, and aspartic acid may be linked and used.

The fusion protein may include a polypeptide having a sequence different from the wild-type amino acid sequence of each domain contained therein and at least one amino acid residue. Amino acid exchanges in proteins and polypeptides that do not totally alter the activity of the molecule are known in the art. The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. In addition, it may include a protein whose structural stability to heat, pH, etc. of the protein is increased or protein activity is increased due to a mutation or modification in an amino acid sequence.

The fusion protein or the polypeptide constituting the fusion protein is prepared by a chemical peptide synthesis method known in the art, or a gene encoding the fusion protein is amplified by PCR (polymerase chain reaction) or synthesized by a known method. It can be produced by cloning into an expression vector and expressing it.

In addition, according to another aspect of the present invention, the present invention is a recombinant expression vector for the production of a human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein comprising the above-described gene construct and the recombinant It provides a host cell that is a transformed recombinant microorganism transformed with an expression vector.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells may express genes or gene segments that are not found in the natural form of the cell in either a sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in a natural state, but the gene is modified and reintroduced into the cell by artificial means.

In the present invention, the hEGF-TF coding gene with increased cell growth and wound healing effects may be inserted into a recombinant expression vector. The term “recombinant expression vector” refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid and vector can be used as long as they can replicate and stabilize in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene, and a translation control element.

An expression vector containing a gene sequence encoding an hEGF-TF fusion protein having an increased cell growth and wound healing effect and an appropriate transcription/translation control signal can be constructed by a method well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. In addition, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

Host cells capable of continuously cloning and expressing the vector of the present invention while being stable in prokaryotic cells may be any host cell known in the art, for example, E. coli BL21, E. coli Rosetta , E. coli JM109 , E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia Intestinal bacteria and strains such as Marsesons and various Pseudomonas species.

In addition, when transforming the vector of the present invention into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells, and the like can be used.

The host cell transformed with the recombinant vector according to an embodiment of the present invention may preferably be E. coli , more preferably E. coli BL21 (DE3), but is not limited thereto.

The method of transporting the vector of the present invention into a host cell includes the CaCl2 method, one method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580) and electroporation method when the host cell is a prokaryotic cell. It can be carried out by, for example. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. I can.

In addition, a polynucleotide encoding a tag for separation and purification may be operably linked to the fusion partner of the vector of the present invention or the gene of the target protein to facilitate separation and purification of the recombinant protein.

The target gene can be cloned through a restriction enzyme site, and if a polynucleotide encoding a protein cleavage enzyme recognition site is used, it is linked in frame with the polynucleotide, and the protein is cleaved after secretion of the target protein. When digested with enzymes, the foreign protein in its original form can be produced.

The term "operably linked" of the present invention means a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. . For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the coding sequence. The operative linkage with the expression vector may be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage may use enzymes generally known in the art.

In addition, according to another aspect of the present invention, the present invention provides a method for producing a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein comprising the following steps:

(a) The gene encoding the xylanase signal peptide derived from Bacillus subtilis, the gene encoding the human epithelial growth factor, which is a protein of interest, and the gene encoding the E. coli-derived TF (Trigger Factor), which is a fusion partner. As a step of preparing a recombinant expression vector comprising a fused construct,

The gene construct is a link between the gene encoding the hEGF and the gene encoding the TF by inserting a TA sequence;

(b) preparing a transformant by introducing the recombinant expression vector of step (a) into a microorganism; And

(c) culturing the transformant of step (b) to induce expression of a human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein and obtain the same.

Preferably, in the step (c), by additionally performing the step of culturing the transformant at a high concentration and replacing the medium before inducing the expression of the human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein. , can significantly increase the production yield of hEGF-TF fusion protein.

That is, the present invention provides conditions and methods for inducing protein expression for increasing the production of hEGF-TF fusion protein.

More specifically, a signal peptide and a fusion partner are linked to a gene of a protein of interest, and an expression vector into which a TA sequence is introduced is prepared, and the expression vector is introduced into a host cell to prepare a transformant, As a method for producing a recombinant protein comprising a step of inducing expression of the recombinant protein by culturing the transformant and obtaining the same, the method of producing a recombinant protein by introducing a Bacillus subtilis-derived xylanase signal peptide into a culture medium of the recombinant protein. By improving the excretion and expressing the E. coli-derived TF (Trigger Factor) gene as a fusion partner, the water solubility of the recombinant protein was improved.

The term "culture" of the present invention means a method of growing microorganisms in an appropriately artificially controlled environmental condition. In the present invention, the method of culturing the transformant may be performed using a method widely known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the fusion protein of the present invention, but may be continuously cultured in a batch process, injection batch, or repeated fed batch process. .

The medium used for cultivation must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. As a carbon source that can be used, a mixed sugar of glucose and xylose is used as the main carbon source. In addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut Oils and fats such as oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid are included. These materials can be used individually or as a mixture. Examples of nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. I can. These nitrogen sources may be used alone or in combination. The medium may contain first potassium phosphate, second potassium phosphate, and a corresponding sodium-containing salt as personnel. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used as the inorganic compound. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added in a batch, fed-batch, or continuous manner to the culture during the cultivation process, but are not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

In addition, the step of recovering the fusion protein from the culture may be performed by a method known in the art. Specifically, the recovery method is not particularly limited as long as the produced fusion protein of the present invention can be recovered, but preferably centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, Methods such as fractional dissolution (eg, ammonium sulfate precipitation), chromatography (eg, ion exchange, affinity, hydrophobicity, and size exclusion) can be used.

In addition, according to another aspect of the present invention, the present invention provides a pharmaceutical composition for treating or preventing skin diseases comprising a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein as an active ingredient.

In the present specification, the "prevention" refers to any action of inhibiting or delaying skin diseases by administration of the pharmaceutical composition. In addition, the "treatment" refers to any action in which the symptoms of skin diseases are improved or beneficially changed by the administration of the pharmaceutical composition.

In one embodiment of the present invention, the hEGF-TF fusion protein of the present invention exhibits more excellent cell growth and wound healing effects than commercially available hEGF.

Through existing studies, epithelial cell growth factors are known to play a role in inhibiting the secretion of gastric acid, promoting epidermal proliferation, promoting angiogenesis and enhancing wound healing power, by stimulating the regeneration and differentiation of the intestinal mucosa, corneal epidermal tissue, and lung epidermal tissue. Since it was confirmed that the fusion protein provided by the present invention can improve the effect of the EGF, the hEGF-TF fusion protein of the present invention not only skin wounds and skin aging (reduction of elasticity), but also diseases in which EGF protein has been used previously, That is, it can be variously applied to the prevention or treatment of atopic dermatitis, contact dermatitis, corneal disease and gastric ulcer.

In the present invention, the term'skin disease' includes, but is not limited to, wounds, wounds, psoriasis, atopic dermatitis, contact dermatitis, foot ulcers, pressure ulcers, oral mucositis or burns.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention pertains. Or it can be prepared by incorporating it into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The pharmaceutical composition prepared as described above may be parenterally administered, that is, subcutaneous administration, intramuscular administration, or topical application, depending on the purpose, and the daily dose is 0.01 ng to 10000 mg/kg in an amount of 1 to It can be administered several times. The dosage level for a specific patient may vary depending on the patient's weight, age, sex, health status, administration time, administration method, disease severity, etc.

In addition, the present invention can provide a pharmaceutical composition for external application for skin comprising the fusion protein.

The term "external preparation for skin" of the present invention means a solid, semi-solid, or liquid external drug in which a drug is mixed with various bases such as oils and fats, petrolatum, lanolin, glycerol, and the like so that it can be easily applied to the skin. The external formulation is not particularly limited, but a powder, gel, ointment, cream, liquid or aerosol formulation is preferred.

For the purpose of the present invention, the external preparation for skin may be interpreted as a preparation containing the fusion protein of the present invention and containing a base that is an appropriate carrier for external preparation for skin, but is not particularly limited thereto.

In addition, since the composition of the present invention includes the fusion protein of the present invention described above as an active ingredient, overlapping contents are omitted in order to avoid excessive complexity of the present specification.

In addition, according to another aspect of the present invention, the present invention provides a food composition or a functional food composition for improving skin condition comprising the above-described human epidermal growth factor (hEGF)-TF (Trigger Factor) fusion protein as an active ingredient. do.

When the composition containing the hEGF-TF fusion protein of the present invention is prepared as a functional food composition or a food composition, it includes not only the hEGF-TF fusion protein as an active ingredient, but also an ingredient commonly added during the manufacture of a functional food or food, , For example, proteins, carbohydrates, fats, nutrients, flavoring and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glysirhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

When the functional food or food composition of the present invention is prepared as a drink, in addition to the active ingredients of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalothorax extract, jujube extract or licorice extract may be additionally included. I can.

In addition, since the composition of the present invention includes the fusion protein of the present invention described above as an active ingredient, overlapping contents are omitted in order to avoid excessive complexity of the present specification.

In addition, according to another aspect of the present invention, the present invention provides a cosmetic composition for improving skin conditions comprising the above-described human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein as an active ingredient.

The term "improving skin condition" of the present invention comprehensively refers to a process of treating, alleviating, or alleviating damage to the skin caused by an intrinsic factor or an exogenous factor of the skin, or an effect thereof, and more specifically, the present invention It refers to skin aging inhibition, skin elasticity improvement, skin regeneration, wound or wound recovery, cornea regeneration, skin irritation relief, etc. that can be induced by applying the fusion protein of

In the cosmetic composition according to an embodiment of the present invention, the content of the fusion protein of the present invention is preferably included in an amount of 0.000001 to 10% by weight based on the total weight of the cosmetic composition.

When the content of the protein is less than 0.000001% by weight, the effect of improving wrinkles and maintaining skin elasticity is insufficient, and when it is more than 10% by weight, the degree of increase in the effect of increasing the content is insignificant, and there may be problems with safety and stability of the formulation. have.

In addition to the active ingredients, the cosmetic composition of the present invention includes ingredients commonly used in cosmetic compositions, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents ( foaming agents), fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives , Conventional adjuvants such as lipid vesicles, and carriers.

The composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation , Wax foundation and spray may be formulated, but is not limited thereto. More specifically, skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisture cream, hand cream, essence, nutrition essence, pack, cleansing It can be prepared in cosmetic formulations of foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap and powder.

When the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. Can be used.

When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluoro Propellants such as hydrocarbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

In addition, the present invention can provide a functional cosmetic comprising the cosmetic composition for improving the skin condition.

The term "functional cosmetics (cosmedical, cosmeceutical)" of the present invention is a product that has a professional function that emphasizes physiologically active efficacy and effect unlike general cosmetics by introducing the professional treatment function of pharmaceuticals into cosmetics. It refers to cosmetics prescribed by the Ordinance of the Ministry of Health and Welfare among products that help and improve skin elasticity.

Functional cosmetics can be prepared by adding an appropriate carrier used in the manufacture of cosmetics for general skin to the cosmetic composition of the present invention. At this time, the carrier used is not particularly limited thereto, but preferably, oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be used alone or in an appropriate combination.

Functional cosmetics of the present invention exhibit skin condition improvement effects such as skin cell growth promotion and wound healing effect, and the formulation thereof is not particularly limited, but, for example, solutions, emulsions, suspensions, pastes, creams, lotions, gels , Powder, spray, surfactant-containing cleansing, oil, soap, liquid detergent, bath agent, foundation, makeup base, essence, lotion, foam, pack, softening water, sunscreen cream, sun oil, etc. , Preferably, it may be prepared in the form of an ointment for external use of the skin, a softening lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, a pack, an emulsion or an oil gel, wherein the carrier used is selectively used according to the formulation of the cosmetic product. I can.

For example, in the case of manufacturing cosmetics in the form of ointments, pastes, creams or gels, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier components. Can be used alone or in combination; In the case of manufacturing cosmetics in the form of powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, chlorofluorocarbon, propane/butane, dimethyl ether, etc. are used alone as carrier components. Can be used alone or in combination; When preparing cosmetics in the form of a solution or emulsion, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, cottonseed oil, peanuts as carrier components Oil, corn seed oil, olive oil, castor oil, sesame oil, glycerol fatty ester, polyethylene glycol or fatty acid ester of sorbitan, and the like may be used alone or in combination; When manufacturing cosmetics in the form of a suspension, water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide as carrier components , Bentonite, agar, tracanth, and the like may be used alone or in combination; When manufacturing cosmetics in the form of cosmetic soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzate, isethionate, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. are used as carrier components. They can be used alone or in combination.

Since the composition of the present invention contains the above-described fusion protein of the present invention as an active ingredient, overlapping contents are omitted in order to avoid excessive complexity of the present specification.

The human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein of the present invention includes a signal peptide of xylanase derived from Bacillus subtilis; Human epithelial growth factor (hEGF); And E. coli-derived TF (Trigger Factor) are combined to improve the water solubility and expression rate of the target protein, while remarkably enhancing useful effects such as increased skin cell growth and wound healing effect, and thus skin diseases such as refractory chronic skin ulcers and gastric ulcers It will be widely used in various industries as an active ingredient for not only therapeutic agents, but also functional cosmetic compositions and pharmaceutical compositions for improving skin conditions or for anti-aging for wrinkle improvement and skin regeneration promotion.

1 shows the gene construct used in this example.
hEGF-TF(A): a signal peptide of xylanase derived from Bacillus subtilis; Construct consisting of human epithelial growth factor (hEGF) and E. coli-derived TF (Trigger Factor),
hEGF-TF(B): a signal peptide of xylanase derived from Bacillus subtilis; Construct composed by connecting TA between human epithelial growth factor (hEGF) and E. coli-derived TF (Trigger Factor).
FIG. 2 shows the hEGF-TF (A) construct is inserted into E. coli BL21 (DE3) under various conditions in order to establish optimal conditions for increasing the production (emission) of the hEGF-TF fusion protein of the present invention. This is the result of confirming the amount of protein secreted into the cell and the culture medium over time by SDS-PAGE. Figure 2(A) shows the result of induction of expression after culturing to be 0.8 at OD600nm and addition of IPTG; Figure 2(B) shows the result of inducing expression by adding IPTG after culturing to be 2.5 at OD600nm and replacing the medium.
3 is an hEGF-TF (B) construct is inserted into E. coli BL21 (DE3) under various conditions in order to establish optimal conditions for increasing the production (emissions) of the hEGF-TF fusion protein of the present invention. This is the result of confirming the amount of protein secreted into the culture medium and intracellular protein over time by SDS-PAGE. Figure 3(A) shows the result of inducing expression after culturing to be 0.8 at OD600nm and adding IPTG; Figure 3(B) shows the result of inducing expression by adding IPTG after culturing to be 2.5 at OD600nm and replacing the medium.
4 is a result of confirming the protein purified by the osmotic pressure method by SDS-PAGE (A) and Western blot (B).
5 is a result showing the cell regeneration effect of commercially available hEGF (A) and hEGF-TF (B) on fibroblasts.
6 is a result showing the wound healing effect on the commercially available hEGF (A) and hEGF-TF (B) fibroblasts.

Hereinafter, the examples are only for describing the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention, those of ordinary skill in the art to which the present invention pertains. It will be self-evident to you.

Example 1. Construction of a recombinant expression vector and a transformed recombinant microorganism for the production of a human epithelial growth factor (hEGF)-TF (rigger factor) fusion protein

The present inventors constructed 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.

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

In addition, the gene encoding the xylanase signal peptide derived from Bacillus subtilis and the gene encoding hEGF used a codon-optimized sequence, and are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

At this time, the construct hEGF-TF(A) (sequence) in which the TA sequence was directly linked without inserting the TA sequence between the gene encoding hEGF and the gene encoding TF as a control to check the difference in effect depending on whether or not the TA sequence was inserted. Number 6) was used.

Table 1 lists the hEGF-TF construct sequence and hEGF-TF fusion protein sequence used in this example.

More specifically, the codon-optimized gene sequence encoding the xylanase signal peptide derived from Bacillus subtilis is SEQ ID NO: 2; The codon-optimized gene sequence encoding EGF is SEQ ID NO: 4; The gene sequence encoding TF is SEQ ID NO: 5; The construct containing the signal peptide-EGF-TF gene (hEGF-TF(A)) is SEQ ID NO: 6; And a construct (hEGF-TF(B)) linked by a TA sequence between the signal peptide-EGF and the TF gene is shown in SEQ ID NO: 7.

In addition, the gene sequence encoding the Bacillus subtilis-derived xylanase signal peptide in SEQ ID NOs: 6 and 7 is underlined; The gene sequence encoding EGF is in bold; And gene sequences encoding TF are shown in italics.

In addition, the amino acid sequence of the xylanase signal peptide derived from Bacillus subtilis is SEQ ID NO: 8; The amino acid sequence of EGF is SEQ ID NO: 9; The amino acid sequence of TF is SEQ ID NO: 10; And the amino acid sequence of the signal peptide-EGF-TF is shown in SEQ ID NO: 11.

In addition, the Bacillus subtilis-derived xylanase signal peptide sequence in SEQ ID NO: 11 is underlined; EGF sequence in bold; And TF sequences are shown in italics.

The synthesized gene constructs hEGF-TF(A) and hEGF-TF(B) were digested with Nde I (TaKaRa, Japan) and BamH I (TaKaRa, Japan) restriction enzymes and then into pET11a expression vector (Novagen, USA). Recombinant plasmid was constructed by cloning.

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

Sequence name order Bacillus subtilis xylanase-SP (signal peptide) nucleotide ATGTTTAAGTTTAAAAAGAATTTCTTAGTTGGATTATCGGCAGCTTTAATGAGTATTAGCTTGTTTTCGGCAACCGCCTCTGCT (SEQ ID NO: 1) codon-optimized SP nucleotide ATGTTCAAATTCAAAAAAAACTTCCTGGTCGGATTGTCAGCAGCCTTGATGTCTATCTCGCTTTTTAGTGCAACAGCTTCGGCA (SEQ ID NO: 2) human EGF nucleotide AATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAAGCATTGGACAAGTATGCATGCAACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGC (SEQ ID NO: 3) codon-optimized human EGF nucleotide GCCAATTCAGATTCGGAGTGTCCATTATCACACGATGGTTATTGTTTACATGATGGGGTGTGTATGTACATTGAGGCGTTAGACAAATACGCTTGTAATTGTGTGGTCGGCTACATTGGGGAGCGTTGCCAGTACCGTGACTTAAAGTGGTGGGAGCTTCGC (SEQ ID NO: 4) TF nucleotide ATGCAAGTTTCAGTTGAAACCACTCAAGGCCTTGGCCGCCGTGTAACGATTACTATCGCTGCTGACAGCATCGAGACCGCTGTTAAAAGCGAGCTGGTCAACGTTGCGAAAAAAGTACGTATTGACGGCTTCCGCAAAGGCAAAGTGCCAATGAATATCGTTGCTCAGCGTTATGGCGCGTCTGTACGCCAGGACGTTCTGGGTGACCTGATGAGCCGTAACTTCATTGACGCCATCATTAAAGAAAAAATCAATCCGGCTGGCGCACCGACTTATGTTCCGGGCGAATACAAGCTGGGTGAAGACTTCACTTACTCTGTAGAGTTTGAAGTTTATCCGGAAGTTGAACTGCAGGGTCTGGAAGCGATCGAAGTTGAAAAACCGATCGTTGAAGTGACCGACGCTGACGTTGACGGCATGCTGGATACTCTGCGTAAACAGCAGGCGACCTGGAAAGAAAAAGACGGCGCTGTTGAAGCAGAAGACCGCGTAACCATCGACTTCACCGGTTCTGTAGACGGCGAAGAGTTCGAAGGCGGTAAAGCGTCTGATTTCGTACTGGCGATGGGCCAGGGTCGTATGATCCCGGGCTTTGAAGACGGTATCAAAGGCCACAAAGCTGGCGAAGAGTTCACCATCGACGTGACCTTCCCGGAAGAATACCACGCAGAAAACCTGAAAGGTAAAGCAGCGAAATTCGCTATCAACCTGAAGAAAGTTGAAGAGCGTGAACTGCCGGAACTGACTGCAGAATTCATCAAACGTTTCGGCGTTGAAGATGGTTCCGTAGAAGGTCTGCGCGCTGAAGTGCGTAAAAACATGGAGCGCGAGCTGAAGAGCGCCATCCGTAACCGCGTTAAGTCTCAGGCGATCGAAGGTCTGGTAAAAGCTAACGACATCGACGTACCGGCTGCGCTGATCGACAGCGAAATCGACGTTCTGCGTCGCCAGGCTGCACAGCGTTTCGGTGGCAACGAAAAACAAGCTCTGGAACTGCCGC GCGAACTGTTCGAAGAACAGGCTAAACGCCGCGTAGTTGTTGGCCTGCTGCTGGGCGAAGTTATCCGCACCAACGAGCTGAAAGCTGACGAAGAGCGCGTGAAAGGCCTGATCGAAGAGATGGCTTCTGCGTACGAAGATCCGAAAGAAGTTATCGAGTTCTACAGCAAAAACAAAGAACTGATGGACAACATGCGCAATGTTGCTCTGGAAGAACAGGCTGTTGAAGCTGTACTGGCGAAAGCGAAAGTGACTGAAAAAGAAACCACTTTCAACGAGCTGATGAACCAGCAGGCGTAA (SEQ ID NO: 5) hEGF-TF(A)nucleotide ATGTTCAAATTCAAAAAAAACTTCCTGGTCGGATTGTCAGCAGCCTTGATGTCTATCTCGCTTTTTAGTGCAACAGCTTCGGCA GCC AATTCAGATTCGGAGTGTCCATTATCACACGATGGTTATTGTTTACATGATGGGGTGTGTATGTACATTGAGGCGTACATTCCGGGTAGTCGCTTGTACCGGGTGGTCTGAGTACCGGGT ATGCAAGTTTCAGTTGAAACCACTCAAGGCCTTGGCCGCCGTGTAACGATTACTATCGCTGCTGACAGCATCGAGACCGCTGTTAAAAGCGAGCTGGTCAACGTTGCGAAAAAAGTACGTATTGACGGCTTCCGCAAAGGCAAAGTGCCAATGAATATCGTTGCTCAGCGTTATGGCGCGTCTGTACGCCAGGACGTTCTGGGTGACCTGATGAGCCGTAACTTCATTGACGCCATCATTAAAGAAAAAATCAATCCGGCTGGCGCACCGACTTATGTTCCGGGCGAATACAAGCTGGGTGAAGACTTCACTTACTCTGTAGAGTTTGAAGTTTATCCGGAAGTTGAACTGCAGGGTCTGGAAGCGATCGAAGTTGAAAAACCGATCGTTGAAGTGACCGACGCTGACGTTGACGGCATGCTGGATACTCTGCGTAAACAGCAGGCGACCTGGAAAGAAAAAGACGGCGCTGTTGAAGCAGAAGACCGCGTAACCATCGACTTCACCGGTTCTGTAGACGGCGAAGAGTTCGAAGGCGGTAAAGCGTCTGATTTCGTACTGGCGATGGGCCAGGGTCGTATGATCCCGGGCTTTGAAGACGGTATCAAAGGCCACAAAGCTGGCGAAGAGTTCACCATCGACGTGACCTTCCCGGAAGAATACCACGCAGAAAACCTGAAAGGTAAAGCAGCGAAATTCGCTATCAACCTGAAGAAAGTTGAAGAGCGTGAACTGCCGGAACTGACTGCAGAATTCATCAAACGTTTCGGCGTTGAAGATGGTTCCGTAGAAGGTCTGCGCGCTGAAGTGCGTAAAAACATGGAGCGCGAGCTGAAGAGCGCCATCCGTAACCGCGTTAAGTCTCAGGCGATCGAAGGTCTGGTAAAAGCTAACGACATCGACGTACCGGCTGCGCTGATCGACAGCGAAATCGACGTTCTGCGTCGCCAGGCTGCACAGCGTTTCGGTGGCAACGAAAAACAAGCTCTGGAACTGCCGCGCGAACTGTTCGAAGAACAGGCTAAACGCCGCGTAGTTGTTGGCCTGCTGCTGGGCGAAGTTATCCGCACCAACGAGCTGAAAGCTGACGAAGAGCGCGTGAAAGGCCTGATCGAAGAGATGGCTTCTGCGTACGAAGATCCGAAAGAAGTTATCGAGTTCTACAGCAAAAACAAAGAACTGATGGACAACATGCGCAATGTTGCTCTGGAAGAACAGGCTGTTGAAGCTGTACTGGCGAAAGCGAAAGTGACTGAAAAAGAAACCACTTTCAACGAGCTGATGAACCAGCAGGCGTAA (SEQ ID NO: 6) hEGF-TF(B)nucleotide ATGTTCAAATTCAAAAAAAACTTCCTGGTCGGATTGTCAGCAGCCTTGATGTCTATCTCGCTTTTTAGTGCAACAGCTTCGGCA GCC AATTCAGATTCGGAGTGTCCATTATCACACGATGGTTATTGTTTACATGATGGGGTGTGTGTATGTACATTGAGGTCTGACTGTACCGGGTGGTCTGACTACCGGGT ATGCAAGTTTCAGTTGAAACCACTCAAGGCCTTGGCCGCCGTGTAACGATTACTATCGCTGCTGACAGCATCGAGACCGCTGTTAAAAGCGAGCTGGTCAACGTTGCGAAAAAAGTACGTATTGACGGCTTCCGCAAAGGCAAAGTGCCAATGAATATCGTTGCTCAGCGTTATGGCGCGTCTGTACGCCAGGACGTTCTGGGTGACCTGATGAGCCGTAACTTCATTGACGCCATCATTAAAGAAAAAATCAATCCGGCTGGCGCACCGACTTATGTTCCGGGCGAATACAAGCTGGGTGAAGACTTCACTTACTCTGTAGAGTTTGAAGTTTATCCGGAAGTTGAACTGCAGGGTCTGGAAGCGATCGAAGTTGAAAAACCGATCGTTGAAGTGACCGACGCTGACGTTGACGGCATGCTGGATACTCTGCGTAAACAGCAGGCGACCTGGAAAGAAAAAGACGGCGCTGTTGAAGCAGAAGACCGCGTAACCATCGACTTCACCGGTTCTGTAGACGGCGAAGAGTTCGAAGGCGGTAAAGCGTCTGATTTCGTACTGGCGATGGGCCAGGGTCGTATGATCCCGGGCTTTGAAGACGGTATCAAAGGCCACAAAGCTGGCGAAGAGTTCACCATCGACGTGACCTTCCCGGAAGAATACCACGCAGAAAACCTGAAAGGTAAAGCAGCGAAATTCGCTATCAACCTGAAGAAAGTTGAAGAGCGTGAACTGCCGGAACTGACTGCAGAATTCATCAAACGTTTCGGCGTTGAAGATGGTTCCGTAGAAGGTCTGCGCGCTGAAGTGCGTAAAAACATGGAGCGCGAGCTGAAGAGCGCCATCCGTAACCGCGTTAAGTCTCAGGCGATCGAAGGTCTGGTAAAAGCTAACGACATCGACGTACCGGCTGCGCTGATCGACAGCGAAATCGACGTTCTGCGTCGCCAGGCTGCACAGCGTTTCGGTGGCAACGAAAAACAAGCTCTGGAACTGCCGCGCGAACTGTTCGAAGAACAGGCTAAACGCCGCGTAGTTGTTGGCCTGCTGCTGGGCGAAGTTATCCGCACCAACGAGCTGAAAGCTGACGAAGAGCGCGTGAAAGGCCTGATCGAAGAGATGGCTTCTGCGTACGAAGATCCGAAAGAAGTTATCGAGTTCTACAGCAAAAACAAAGAACTGATGGACAACATGCGCAATGTTGCTCTGGAAGAACAGGCTGTTGAAGCTGTACTGGCGAAAGCGAAAGTGACTGAAAAAGAAACCACTTTCAACGAGCTGATGAACCAGCAGGCGTAA (SEQ ID NO: 7) SP protein MFKFKKNFLVGLSAALMSISLFSATASA (SEQ ID NO: 8) human EGFprotein NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR (SEQ ID NO: 9) TF protein MQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQA (SEQ ID NO: 10) hEGF-TFprotein A MFKFKKNFLVGLSAALMSISLFSATASA NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR MQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQA (SEQ ID NO: 11)

Example 2. Induction of expression of the hEGF-TF fusion protein of the present invention and confirmation of excretion into the culture medium

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

First, the transformed E. coli BL21 (DE3) prepared in Example 1 was inoculated in 5 mL of LB medium (1% tryptophan, 1% sodium chloride, and 0.5% yeast extract) to which ampicillin was added. It was cultured with shaking at 37°C. After culturing this in 500 mL of the same medium at an absorbance of 600 nm to 0.8, 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, 96 hours), and cells and culture media were separated at 4°C and 8,000 rpm, respectively. After the cells were completely suspended in distilled water, the cells were destroyed using an ultrasonic disruptor, and a solution containing the intracellular protein was separated.

In order to confirm whether the expressed proteins were discharged into the culture medium for each time period, the proteins were identified by SDS-PAGE using solutions and culture solutions containing the separated intracellular protein as samples.

The hEGF-TF(A) construct was inserted into E. coli BL21(DE3), and the amount of the intracellular protein and the protein secreted into the culture medium according to time are shown in FIG. 2(A); The hEGF-TF(B) construct was inserted into E. coli BL21(DE3), and the amount of intracellular protein and the protein secreted into the culture medium over time are shown in FIG. 3(A).

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

Example 3. Confirmation of optimal conditions for increasing the production amount (emissions) of the hEGF-TF fusion protein of the present invention

In order to establish optimal conditions for increasing the production (emissions) of hEGF-TF fusion protein, the present inventors applied conditions according to high concentration culture of recombinant microorganisms and medium replacement before induction of expression to determine the level of hEGF-TF fusion protein emission. It was measured.

Transformed E. coli BL21 (DE3) prepared in Example 1 was inoculated in 5 mL of LB broth (1% tryptophan, 1% sodium chloride, and 0.5% yeast extract) to which ampicillin was added and cultured with shaking at 37°C. I did. This was cultured in 500 mL of the same medium at an absorbance of 600 nm to 2.5, and then centrifuged 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, the cells were cultured at 20° C. and the cells and the culture medium were separated at 4° C. and 8,000 rpm at 12 hours, 24 hours, 48 hours and 96 hours, respectively. After the cells were completely suspended in distilled water, the cells were destroyed using an ultrasonic disruptor, and a solution containing the intracellular protein was separated.

In order to check whether the expressed proteins are discharged into the culture medium, the proteins were identified by SDS-PAGE using solutions and cultures containing intracellular proteins separated at 12 hours, 24 hours, 48 hours, and 96 hours respectively. I did.

The hEGF-TF(A) construct was inserted into E. coli BL21(DE3), and the amount of intracellular protein and the protein secreted into the culture medium according to time are shown in FIG. 2(B); The hEGF-TF(B) construct was inserted into E. coli BL21(DE3), and the amount of intracellular protein and the protein secreted into the culture medium over time are shown in FIG. 3(B).

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

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

That is, by demonstrating that the present invention exerts a synergistic effect of increasing the amount of hEGF-TF fusion protein according to the conditions of high concentration culture and medium replacement of the recombinant microorganism before induction of expression in the present invention, the optimum conditions for increasing the amount of hEGF-TF fusion protein were established.

Therefore, it can be usefully used to mass-produce a high-quality human epidermal growth factor (EGF)-E. coli-derived TF (Trigger Factor) fusion protein.

Accordingly, in the following examples, hEGF-TF produced by introducing the hEGF-TF(B) construct having a TA sequence inserted between the hEGF gene and the TF gene was used.

Example 4. Quantitative analysis of the hEGF-TF fusion protein of the present invention

In order to confirm the amount of the hEGF-TF fusion protein discharged into the culture medium, the present inventors loaded a standard protein at 1, 4, and 8 ug onto an SDS-PAGE gel and prepared a calibration curve using the ImageJ program. As a result, the amount of the protein corresponding to the epithelial cell growth factor-Trigger Factor fusion protein band among the proteins discharged into the culture medium in Example 3-3 was confirmed.

As a result, as shown in Table 2 below, when the hEGF-TF protein released for each time period of FIG. 3(B) was quantitatively analyzed, it can be seen that the amount of the secreted protein increased with the incubation time.

Incubation time 12h 24h 48h 96h Epithelial Cell Growth Factor-Trigger Factor Amount (mg/L) 0 139.2 1171.1 1300.6

Example 5. Purification and confirmation of the hEGF-TF fusion protein of the present invention

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

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

Example 6. Cell regeneration effect of the hEGF-TF fusion protein of the present invention

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 measured cell growth and viability using an MTT assay.

First, HDF cells were cultured using a DMEM/F12 (3:1) mixed medium containing 10% FBS, and HDF cells were dispensed into a 96-well plate at 1×10 ³ cells/well, and at the same time, the sample was 0 ng/well. After treatment with ul (control), 0.1 ng/ul, 1 ng/ul, and 10 ng/ul, respectively, they were cultured under conditions of _{37° C. and 5% CO 2 for 1 and 3 days.} At this time, the experiment was performed under the same concentration and conditions using a commercially available hEGF (Sigma-Aldrich) as an experimental comparison group. After the cultivation was completed, the MTT solution was added and incubated for 4 hours, the medium was removed, and DMSO was added thereto to measure the absorbance at 450 nm.

Cell growth rate was calculated as follows:

X 100.Cell growth rate (%) =

X 100.

As a result, as shown in FIG. 5, in the experimental group treated with the hEGF-TF fusion protein of the present invention at 0.1 ng/ul to HDF cells, the cell growth rate increased to 4.8% on the first day and 57.5% on the third day compared to the control group; The experimental group treated with 1 ng/ul showed an increase of 10.6% on the first day and 73% on the third day compared to the control group. In addition, the 10 ng/ul experimental group showed an increase of 50.7% on the first day and 84.9% on the third day compared to the control group.

This proves that the hEGF-TF fusion protein of the present invention exerts an excellent increase in cell growth efficacy compared to the commercially available hEGF, which is a comparative group.

Example 7. The wound healing effect of the hEGF-TF fusion protein of the present invention

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

First, 70 ul of fibroblasts (5x10 ⁵ cells/ml) were inoculated into a culture-insert culture dish (ibidi), and after 24 hours, the culture-insert was removed, and hEGF-TF was added to 0 ng/ul (control), 0.1 It was treated at a concentration of ng/ul and 1 ng/ul. At this time, the experiment was performed under the same concentration and conditions using hEGF (Sigma-Aldrich), which is commercially available as an experimental control group. Cell images for 3, 6, 9, 12, and 24 hours of culture were analyzed using the imageJ program to confirm the wound healing efficacy.

As a result, as shown in FIG. 6, in the experimental group treated with 1 ng/ul of the hEGF-TF fusion protein of the present invention, overall, compared to the control group and the control group (commercially available hEGF), a high wound healing effect was exhibited.

This proves that the hEGF-TF fusion protein of the present invention exerts an excellent increase in wound healing efficacy compared to the commercially available hEGF, which is a comparative group.

Overall, the novel human epithelial cell growth factor (hEGF)-TF of the present invention produced by a construct in which a signal peptide gene is coupled to the hEGF gene according to the present invention and a TA sequence is inserted between the hEGF gene and the TF gene. (Trigger Factor) The fusion protein has improved production capacity by improving the function of discharging the hEGF-TF fusion protein into the culture medium, that is, producing it in E. coli and secreting it excessively outside the cell, and has improved cell growth and wound healing efficacy than commercially available hEGF. Since this is excellent, it will be widely used as an active ingredient in functional cosmetic compositions and pharmaceutical compositions for external application for skin.

In addition, in the method according to the present invention, since optimal conditions are established to improve the production efficiency of human epithelial cell growth factors, human epithelial cell growth factors can be obtained in high yield, and thus can be usefully applied to mass production. have.

As described above, a specific part of the content of the present invention has been described in detail, and for those of ordinary skill in the art, this specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. It will be obvious. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Korea Institute of Ocean Science and Technology <120> Novel Human Epidermal Growth Factor-Trigger Factor Fusion Protein and Uses Thereof <130> KIOS-1-33p-1 <150> KR 10-2019-0142493 <151> 2019-11-08 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Bacillus subtilis xylanase signal peptide nucleotide <400> 1 atgtttaagt ttaaaaagaa tttcttagtt ggattatcgg cagctttaat gagtattagc 60 ttgttttcgg caaccgcctc tgct 84 <210> 2 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> codon-optimized signal peptide nucleotide <400> 2 atgttcaaat tcaaaaaaaa cttcctggtc ggattgtcag cagccttgat gtctatctcg 60 ctttttagtg caacagcttc ggca 84 <210> 3 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> human EGF nucleotide <400> 3 aatagtgact ctgaatgtcc cctgtcccac gatgggtact gcctccatga tggtgtgtgc 60 atgtatattg aagcattgga caagtatgca tgcaactgtg ttgttggcta catcggggag 120 cgatgtcagt accgagacct gaagtggtgg gaactgcgc 159 <210> 4 <211> 162 <212> DNA <213> Artificial Sequence <220> <223> codon-optimized human EGF nucleotide <400> 4 gccaattcag attcggagtg tccattatca cacgatggtt attgtttaca tgatggggtg 60 tgtatgtaca ttgaggcgtt agacaaatac gcttgtaatt gtgtggtcgg ctacattggg 120 gagcgttgcc agtaccgtga cttaaagtgg tgggagcttc gc 162 <210> 5 <211> 1299 <212> DNA <213> Artificial Sequence <220> <223> TF nucleotide <400> 5 atgcaagttt cagttgaaac cactcaaggc cttggccgcc gtgtaacgat tactatcgct 60 gctgacagca tcgagaccgc tgttaaaagc gagctggtca acgttgcgaa aaaagtacgt 120 attgacggct tccgcaaagg caaagtgcca atgaatatcg ttgctcagcg ttatggcgcg 180 tctgtacgcc aggacgttct gggtgacctg atgagccgta acttcattga cgccatcatt 240 aaagaaaaaa tcaatccggc tggcgcaccg acttatgttc cgggcgaata caagctgggt 300 gaagacttca cttactctgt agagtttgaa gtttatccgg aagttgaact gcagggtctg 360 gaagcgatcg aagttgaaaa accgatcgtt gaagtgaccg acgctgacgt tgacggcatg 420 ctggatactc tgcgtaaaca gcaggcgacc tggaaagaaa aagacggcgc tgttgaagca 480 gaagaccgcg taaccatcga cttcaccggt tctgtagacg gcgaagagtt cgaaggcggt 540 aaagcgtctg atttcgtact ggcgatgggc cagggtcgta tgatcccggg ctttgaagac 600 ggtatcaaag gccacaaagc tggcgaagag ttcaccatcg acgtgacctt cccggaagaa 660 taccacgcag aaaacctgaa aggtaaagca gcgaaattcg ctatcaacct gaagaaagtt 720 gaagagcgtg aactgccgga actgactgca gaattcatca aacgtttcgg cgttgaagat 780 ggttccgtag aaggtctgcg cgctgaagtg cgtaaaaaca tggagcgcga gctgaagagc 840 gccatccgta accgcgttaa gtctcaggcg atcgaaggtc tggtaaaagc taacgacatc 900 gacgtaccgg ctgcgctgat cgacagcgaa atcgacgttc tgcgtcgcca ggctgcacag 960 cgtttcggtg gcaacgaaaa acaagctctg gaactgccgc gcgaactgtt cgaagaacag 1020 gctaaacgcc gcgtagttgt tggcctgctg ctgggcgaag ttatccgcac caacgagctg 1080 aaagctgacg aagagcgcgt gaaaggcctg atcgaagaga tggcttctgc gtacgaagat 1140 ccgaaagaag ttatcgagtt ctacagcaaa aacaaagaac tgatggacaa catgcgcaat 1200 gttgctctgg aagaacaggc tgttgaagct gtactggcga aagcgaaagt gactgaaaaa 1260 gaaaccactt tcaacgagct gatgaaccag caggcgtaa 1299 <210> 6 <211> 1545 <212> DNA <213> Artificial Sequence <220> <223> hEGF-TF(A) nucleotide <400> 6 atgttcaaat tcaaaaaaaa cttcctggtc ggattgtcag cagccttgat gtctatctcg 60 ctttttagtg caacagcttc ggcagccaat tcagattcgg agtgtccatt atcacacgat 120 ggttattgtt tacatgatgg ggtgtgtatg tacattgagg cgttagacaa atacgcttgt 180 aattgtgtgg tcggctacat tggggagcgt tgccagtacc gtgacttaaa gtggtgggag 240 cttcgcatgc aagtttcagt tgaaaccact caaggccttg gccgccgtgt aacgattact 300 atcgctgctg acagcatcga gaccgctgtt aaaagcgagc tggtcaacgt tgcgaaaaaa 360 gtacgtattg acggcttccg caaaggcaaa gtgccaatga atatcgttgc tcagcgttat 420 ggcgcgtctg tacgccagga cgttctgggt gacctgatga gccgtaactt cattgacgcc 480 atcattaaag aaaaaatcaa tccggctggc gcaccgactt atgttccggg cgaatacaag 540 ctgggtgaag acttcactta ctctgtagag tttgaagttt atccggaagt tgaactgcag 600 ggtctggaag cgatcgaagt tgaaaaaccg atcgttgaag tgaccgacgc tgacgttgac 660 ggcatgctgg atactctgcg taaacagcag gcgacctgga aagaaaaaga cggcgctgtt 720 gaagcagaag accgcgtaac catcgacttc accggttctg tagacggcga agagttcgaa 780 ggcggtaaag cgtctgattt cgtactggcg atgggccagg gtcgtatgat cccgggcttt 840 gaagacggta tcaaaggcca caaagctggc gaagagttca ccatcgacgt gaccttcccg 900 gaagaatacc acgcagaaaa cctgaaaggt aaagcagcga aattcgctat caacctgaag 960 aaagttgaag agcgtgaact gccggaactg actgcagaat tcatcaaacg tttcggcgtt 1020 gaagatggtt ccgtagaagg tctgcgcgct gaagtgcgta aaaacatgga gcgcgagctg 1080 aagagcgcca tccgtaaccg cgttaagtct caggcgatcg aaggtctggt aaaagctaac 1140 gacatcgacg taccggctgc gctgatcgac agcgaaatcg acgttctgcg tcgccaggct 1200 gcacagcgtt tcggtggcaa cgaaaaacaa gctctggaac tgccgcgcga actgttcgaa 1260 gaacaggcta aacgccgcgt agttgttggc ctgctgctgg gcgaagttat ccgcaccaac 1320 gagctgaaag ctgacgaaga gcgcgtgaaa ggcctgatcg aagagatggc ttctgcgtac 1380 gaagatccga aagaagttat cgagttctac agcaaaaaca aagaactgat ggacaacatg 1440 cgcaatgttg ctctggaaga acaggctgtt gaagctgtac tggcgaaagc gaaagtgact 1500 gaaaaagaaa ccactttcaa cgagctgatg aaccagcagg cgtaa 1545 <210> 7 <211> 1547 <212> DNA <213> Artificial Sequence <220> <223> hEGF-TF(B) nucleotide <400> 7 atgttcaaat tcaaaaaaaa cttcctggtc ggattgtcag cagccttgat gtctatctcg 60 ctttttagtg caacagcttc ggcagccaat tcagattcgg agtgtccatt atcacacgat 120 ggttattgtt tacatgatgg ggtgtgtatg tacattgagg cgttagacaa atacgcttgt 180 aattgtgtgg tcggctacat tggggagcgt tgccagtacc gtgacttaaa gtggtgggag 240 cttcgctaat gcaagtttca gttgaaacca ctcaaggcct tggccgccgt gtaacgatta 300 ctatcgctgc tgacagcatc gagaccgctg ttaaaagcga gctggtcaac gttgcgaaaa 360 aagtacgtat tgacggcttc cgcaaaggca aagtgccaat gaatatcgtt gctcagcgtt 420 atggcgcgtc tgtacgccag gacgttctgg gtgacctgat gagccgtaac ttcattgacg 480 ccatcattaa agaaaaaatc aatccggctg gcgcaccgac ttatgttccg ggcgaataca 540 agctgggtga agacttcact tactctgtag agtttgaagt ttatccggaa gttgaactgc 600 agggtctgga agcgatcgaa gttgaaaaac cgatcgttga agtgaccgac gctgacgttg 660 acggcatgct ggatactctg cgtaaacagc aggcgacctg gaaagaaaaa gacggcgctg 720 ttgaagcaga agaccgcgta accatcgact tcaccggttc tgtagacggc gaagagttcg 780 aaggcggtaa agcgtctgat ttcgtactgg cgatgggcca gggtcgtatg atcccgggct 840 ttgaagacgg tatcaaaggc cacaaagctg gcgaagagtt caccatcgac gtgaccttcc 900 cggaagaata ccacgcagaa aacctgaaag gtaaagcagc gaaattcgct atcaacctga 960 agaaagttga agagcgtgaa ctgccggaac tgactgcaga attcatcaaa cgtttcggcg 1020 ttgaagatgg ttccgtagaa ggtctgcgcg ctgaagtgcg taaaaacatg gagcgcgagc 1080 tgaagagcgc catccgtaac cgcgttaagt ctcaggcgat cgaaggtctg gtaaaagcta 1140 acgacatcga cgtaccggct gcgctgatcg acagcgaaat cgacgttctg cgtcgccagg 1200 ctgcacagcg tttcggtggc aacgaaaaac aagctctgga actgccgcgc gaactgttcg 1260 aagaacaggc taaacgccgc gtagttgttg gcctgctgct gggcgaagtt atccgcacca 1320 acgagctgaa agctgacgaa gagcgcgtga aaggcctgat cgaagagatg gcttctgcgt 1380 acgaagatcc gaaagaagtt atcgagttct acagcaaaaa caaagaactg atggacaaca 1440 tgcgcaatgt tgctctggaa gaacaggctg ttgaagctgt actggcgaaa gcgaaagtga 1500 ctgaaaaaga aaccactttc aacgagctga tgaaccagca ggcgtaa 1547 <210> 8 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> SP protein <400> 8 Met Phe Lys Phe Lys Lys Asn Phe Leu Val Gly Leu Ser Ala Ala Leu 1 5 10 15 Met Ser Ile Ser Leu Phe Ser Ala Thr Ala Ser Ala 20 25 <210> 9 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> human EGF protein <400> 9 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> 10 <211> 432 <212> PRT <213> Artificial Sequence <220> <223> TF protein <400> 10 Met Gln Val Ser Val Glu Thr Thr Gln Gly Leu Gly Arg Arg Val Thr 1 5 10 15 Ile Thr Ile Ala Ala Asp Ser Ile Glu Thr Ala Val Lys Ser Glu Leu 20 25 30 Val Asn Val Ala Lys Lys Val Arg Ile Asp Gly Phe Arg Lys Gly Lys 35 40 45 Val Pro Met Asn Ile Val Ala Gln Arg Tyr Gly Ala Ser Val Arg Gln 50 55 60 Asp Val Leu Gly Asp Leu Met Ser Arg Asn Phe Ile Asp Ala Ile Ile 65 70 75 80 Lys Glu Lys Ile Asn Pro Ala Gly Ala Pro Thr Tyr Val Pro Gly Glu 85 90 95 Tyr Lys Leu Gly Glu Asp Phe Thr Tyr Ser Val Glu Phe Glu Val Tyr 100 105 110 Pro Glu Val Glu Leu Gln Gly Leu Glu Ala Ile Glu Val Glu Lys Pro 115 120 125 Ile Val Glu Val Thr Asp Ala Asp Val Asp Gly Met Leu Asp Thr Leu 130 135 140 Arg Lys Gln Gln Ala Thr Trp Lys Glu Lys Asp Gly Ala Val Glu Ala 145 150 155 160 Glu Asp Arg Val Thr Ile Asp Phe Thr Gly Ser Val Asp Gly Glu Glu 165 170 175 Phe Glu Gly Gly Lys Ala Ser Asp Phe Val Leu Ala Met Gly Gln Gly 180 185 190 Arg Met Ile Pro Gly Phe Glu Asp Gly Ile Lys Gly His Lys Ala Gly 195 200 205 Glu Glu Phe Thr Ile Asp Val Thr Phe Pro Glu Glu Tyr His Ala Glu 210 215 220 Asn Leu Lys Gly Lys Ala Ala Lys Phe Ala Ile Asn Leu Lys Lys Val 225 230 235 240 Glu Glu Arg Glu Leu Pro Glu Leu Thr Ala Glu Phe Ile Lys Arg Phe 245 250 255 Gly Val Glu Asp Gly Ser Val Glu Gly Leu Arg Ala Glu Val Arg Lys 260 265 270 Asn Met Glu Arg Glu Leu Lys Ser Ala Ile Arg Asn Arg Val Lys Ser 275 280 285 Gln Ala Ile Glu Gly Leu Val Lys Ala Asn Asp Ile Asp Val Pro Ala 290 295 300 Ala Leu Ile Asp Ser Glu Ile Asp Val Leu Arg Arg Gln Ala Ala Gln 305 310 315 320 Arg Phe Gly Gly Asn Glu Lys Gln Ala Leu Glu Leu Pro Arg Glu Leu 325 330 335 Phe Glu Glu Gln Ala Lys Arg Arg Val Val Val Gly Leu Leu Leu Gly 340 345 350 Glu Val Ile Arg Thr Asn Glu Leu Lys Ala Asp Glu Glu Arg Val Lys 355 360 365 Gly Leu Ile Glu Glu Met Ala Ser Ala Tyr Glu Asp Pro Lys Glu Val 370 375 380 Ile Glu Phe Tyr Ser Lys Asn Lys Glu Leu Met Asp Asn Met Arg Asn 385 390 395 400 Val Ala Leu Glu Glu Gln Ala Val Glu Ala Val Leu Ala Lys Ala Lys 405 410 415 Val Thr Glu Lys Glu Thr Thr Phe Asn Glu Leu Met Asn Gln Gln Ala 420 425 430 <210> 11 <211> 514 <212> PRT <213> Artificial Sequence <220> <223> hEGF-TF protein <400> 11 Met Phe Lys Phe Lys Lys Asn Phe Leu Val Gly Leu Ser Ala Ala Leu 1 5 10 15 Met Ser Ile Ser Leu Phe Ser Ala Thr Ala Ser Ala Ala Asn Ser Asp 20 25 30 Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val 35 40 45 Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val 50 55 60 Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu 65 70 75 80 Leu Arg Met Gln Val Ser Val Glu Thr Thr Gln Gly Leu Gly Arg Arg 85 90 95 Val Thr Ile Thr Ile Ala Ala Asp Ser Ile Glu Thr Ala Val Lys Ser 100 105 110 Glu Leu Val Asn Val Ala Lys Lys Val Arg Ile Asp Gly Phe Arg Lys 115 120 125 Gly Lys Val Pro Met Asn Ile Val Ala Gln Arg Tyr Gly Ala Ser Val 130 135 140 Arg Gln Asp Val Leu Gly Asp Leu Met Ser Arg Asn Phe Ile Asp Ala 145 150 155 160 Ile Ile Lys Glu Lys Ile Asn Pro Ala Gly Ala Pro Thr Tyr Val Pro 165 170 175 Gly Glu Tyr Lys Leu Gly Glu Asp Phe Thr Tyr Ser Val Glu Phe Glu 180 185 190 Val Tyr Pro Glu Val Glu Leu Gln Gly Leu Glu Ala Ile Glu Val Glu 195 200 205 Lys Pro Ile Val Glu Val Thr Asp Ala Asp Val Asp Gly Met Leu Asp 210 215 220 Thr Leu Arg Lys Gln Gln Ala Thr Trp Lys Glu Lys Asp Gly Ala Val 225 230 235 240 Glu Ala Glu Asp Arg Val Thr Ile Asp Phe Thr Gly Ser Val Asp Gly 245 250 255 Glu Glu Phe Glu Gly Gly Lys Ala Ser Asp Phe Val Leu Ala Met Gly 260 265 270 Gln Gly Arg Met Ile Pro Gly Phe Glu Asp Gly Ile Lys Gly His Lys 275 280 285 Ala Gly Glu Glu Phe Thr Ile Asp Val Thr Phe Pro Glu Glu Tyr His 290 295 300 Ala Glu Asn Leu Lys Gly Lys Ala Ala Lys Phe Ala Ile Asn Leu Lys 305 310 315 320 Lys Val Glu Glu Arg Glu Leu Pro Glu Leu Thr Ala Glu Phe Ile Lys 325 330 335 Arg Phe Gly Val Glu Asp Gly Ser Val Glu Gly Leu Arg Ala Glu Val 340 345 350 Arg Lys Asn Met Glu Arg Glu Leu Lys Ser Ala Ile Arg Asn Arg Val 355 360 365 Lys Ser Gln Ala Ile Glu Gly Leu Val Lys Ala Asn Asp Ile Asp Val 370 375 380 Pro Ala Ala Leu Ile Asp Ser Glu Ile Asp Val Leu Arg Arg Gln Ala 385 390 395 400 Ala Gln Arg Phe Gly Gly Asn Glu Lys Gln Ala Leu Glu Leu Pro Arg 405 410 415 Glu Leu Phe Glu Glu Gln Ala Lys Arg Arg Val Val Val Gly Leu Leu 420 425 430 Leu Gly Glu Val Ile Arg Thr Asn Glu Leu Lys Ala Asp Glu Glu Arg 435 440 445 Val Lys Gly Leu Ile Glu Glu Met Ala Ser Ala Tyr Glu Asp Pro Lys 450 455 460 Glu Val Ile Glu Phe Tyr Ser Lys Asn Lys Glu Leu Met Asp Asn Met 465 470 475 480 Arg Asn Val Ala Leu Glu Glu Gln Ala Val Glu Ala Val Leu Ala Lys 485 490 495 Ala Lys Val Thr Glu Lys Glu Thr Thr Phe Asn Glu Leu Met Asn Gln 500 505 510 Gln Ala

Claims (17)

As a gene construct for the production of a human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein,
The gene construct may include a gene encoding a Bacillus subtilis-derived xylanase signal peptide; A gene encoding a protein of interest, human epithelial growth factor (hEGF); And a gene encoding the fusion partner E. coli-derived TF (Trigger Factor);
The gene construct is a gene construct, characterized in that a TA sequence is inserted and linked between the gene encoding the hEGF and the gene encoding the TF.
The method of claim 1,
The gene encoding the Bacillus subtilis-derived xylanase signal peptide is characterized in that it consists of a nucleotide sequence represented by SEQ ID NO: 2 optimized for E. coli codon, a gene construct.
The method of claim 1,
The gene encoding the human epithelial cell growth factor is characterized in that consisting of a nucleotide sequence represented by SEQ ID NO: 4 optimized E. codon codon, a gene construct (construct).
The method of claim 1,
The gene encoding the E. coli-derived TF (Trigger Factor) is characterized in that consisting of a nucleotide sequence represented by SEQ ID NO: 5, a gene construct (construct).
The method of claim 1,
The gene construct is characterized in that consisting of a nucleotide sequence represented by SEQ ID NO: 7, a gene construct (construct).
As a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein,
Bacillus subtilis-derived xylanase signal peptide; Human epithelial growth factor; And E. coli-derived TF (Trigger Factor), characterized in that the fusion, hEGF-TF fusion protein.
The method of claim 6,
The Bacillus subtilis-derived xylanase (xylanase) signal peptide is characterized in that consisting of the amino acid sequence represented by SEQ ID NO: 8, hEGF-TF fusion protein.
The method of claim 6,
The human epithelial cell growth factor is characterized in that consisting of the amino acid sequence represented by SEQ ID NO: 9, hEGF-TF fusion protein.
The method of claim 6,
The E. coli-derived TF (Trigger Factor) is characterized in that consisting of an amino acid sequence represented by SEQ ID NO: 10, hEGF-TF fusion protein.
The method of claim 6,
The hEGF-TF fusion protein is characterized in that consisting of the amino acid sequence represented by SEQ ID NO: 11, hEGF-TF fusion protein.
A recombinant expression vector for the production of a human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein comprising the gene construct of any one of claims 1 to 5.
A transformed recombinant microorganism transformed with the recombinant expression vector of claim 11.
A method for producing a human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein comprising the following steps:
(a) The gene encoding the xylanase signal peptide derived from Bacillus subtilis, the gene encoding the human epithelial cell growth factor (hEGF), the protein of interest, and the fusion partner E. coli-derived TF (Trigger Factor) are encoded. As a step of preparing a recombinant expression vector comprising a construct in which the gene is fused,
The gene construct is a link between the gene encoding the hEGF and the gene encoding the TF by inserting a TA sequence;
(b) preparing a transformant by introducing the recombinant expression vector of step (a) into a microorganism; And
(c) culturing the transformant of step (b) to induce expression of a human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein and obtain the same.
The method of claim 13,
In the step (c), prior to inducing the expression of the human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein, the step of culturing the transformant at a high concentration and replacing the medium is additionally performed. , Human epithelial cell growth factor (hEGF)-TF (Trigger Factor) fusion protein production method.
A pharmaceutical composition for treating or preventing skin diseases comprising the human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein of any one of claims 6 to 10 as an active ingredient.
A food composition for improving skin conditions comprising the human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein of any one of claims 6 to 10 as an active ingredient.
A cosmetic composition for improving skin conditions comprising the human epithelial growth factor (hEGF)-TF (Trigger Factor) fusion protein of any one of claims 6 to 10 as an active ingredient.

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