WO2020060202A1 - Expression cassette for preparing thymulin or argireline, and use thereof - Google Patents

Abstract

The present invention pertains to an expression cassette for preparing thymulin or argireline, and a use thereof, and specifically to: an expression cassette for preparing thymulin or argireline, the expression cassette containing a polynucleotide sequence encoding thymulin or argireline, the polynucleotide sequence being operably linked with a high-level secretory signal sequence and a promoter sequence that can be expressed in yeast; an expression vector comprising the expression cassette; a transformant comprising the expression vector; a method for preparing thymulin or argireline using the transformant; a hair-loss preventing or hair-growth promoting cosmetic composition and quasi-drug composition containing thymulin prepared by the method; and an anti-wrinkle cosmetic composition and quasi-drug composition containing argireline prepared by the method. High-purity thymulin or argireline can be prepared in large amounts at low cost by using the expression cassette for preparing thymulin or argireline, the expression vector, and the transformant comprising the expression vector of the present invention. Thymulin prepared according to the present invention has an excellent hair-loss preventing and hair-growth promoting effect, and can thus be widely used in many industries, such as the hair-loss preventing or hair-growth promoting cosmetic materials and quasi-drug industries. In addition, Argireline prepared according to the present invention has an excellent anti-wrinkle effect and very low toxicity, and can thus be widely used in many industries, such as the anti-wrinkle cosmetic materials and quasi-drug industries.

Description

Expression cassette for the production of thymulin or azirenine and use thereof

The present invention relates to an expression cassette for the production of thymulin or azirenine, and specifically, a polynucleotide sequence encoding thymulin or azirenine, wherein the polynucleotide sequence is a promoter sequence capable of being expressed in yeast and a high An expression cassette for thymulin or agireline production, an expression vector comprising the expression cassette, a transformant comprising the expression vector, and the transformant, which are operably linked to a secretion signal sequence. Method for producing thymulin or azirenine used, cosmetic composition for preventing hair loss or promoting hair growth, and medicinal product composition comprising thymulin prepared by the above manufacturing method, and wrinkles containing azirenin prepared by the above manufacturing method It relates to a cosmetic composition and a quasi-drug composition.

Thymulin is one of various hormones secreted by the human thymus, and is known to be involved in the differentiation of T cells and activation of T cells and NK cells. In addition to the paracrine and auto-organic effects on their thymus-dependent immune system, many studies have been conducted on neuroendocrine action, and in particular, effects on pro-inflammatory mediators / cytokines ( Timulin's role as an effector has been intensively studied.

In addition, various functional substances related to the prevention of hair loss are being developed, and shampoo products containing the same are being sold to the market, and the scale is constantly increasing. It has been found that various organic and inorganic materials, oils, natural extracts, antibacterial substances and peptides are involved in preventing hair loss and promoting hair growth. In particular, the Timullin of the present invention has been researched on the mechanism of action related to preventing hair loss and promoting hair growth, and specifically, Timullin attacks and kills hair follicle cells by immune cells by autoimmune reaction of the human body according to aging. It has been found to interfere with the process that occurs.

In this regard, Thymus skin of the United States has developed and marketed a functional shampoo that is effective in preventing hair loss and promoting hair growth using a young calf thymus extract. However, since the thymulin produced by such a chemical synthesis method contains chemical impurities and substances harmful to the human body, a high purity separation / purification process is required. In addition, the production cost is expensive, there is a limit in scale-up (scale-up), there is a problem that can not increase the price competitiveness with the above chemical synthesis method.

On the other hand, as a treatment material for improving the wrinkles of the skin, botulinum toxin (botox), which is currently most widely used, is a toxic substance extracted from a microorganism called Clostidium botulinum , a food poisoning bacteria. Botox breaks down the SNARE complex, a neurotransmitter complex that is involved in the repetitive contraction of muscles that cause wrinkles, thereby delaying the formation of wrinkles by preventing the transfer of acetylcholine, which is involved in muscle contraction, to muscle cells. The effect lasts 3-4 months until the SNARE complex is regenerated.

However, these botox is the most toxic substance among all the toxins and biological toxins developed by mankind, and the lethal dose is 0.5 g based on adult males when inhaling the mucosa. As such, Botox is very toxic, and can cause problems with human neurons, and upon continuous use, antibodies are generated in the body, resulting in resistance. In addition, various treatment side effects are occurring, and there is a 'samurai eyebrow', which is representative of eyebrow treatment. Due to the limitations of botox, there is an urgent need to develop a safety alternative material that can replace botox.

Recently, as a part of the amino acid structure of SNAP-25 protein, a component protein of the SNARE complex involved in muscle contraction, a peptide composed of amino acids 12 to 17 (Glu-Glu-Met-Gln-Arg-Arg) and Botox It showed the same effect, but the toxicity was analyzed to be about 100,000 times lower, and it was called 'argireline'. As described above, in the case of Botox, the secretion of acetylcholine, a neurotransmitter, and the SNARE complex, which is a neurotransmitter complex, are disrupted, but azirenine prevents nerve cells from contacting muscle cells, resulting in muscle cells of acetylcholine. Hinders the delivery of

Specifically, azirenine has a wrinkle improvement effect of 4000 times lower than that of Botox, but is 10 times lower in toxicity, and is a short peptide composed of 6 amino acid sequences, and has high skin permeability. Accordingly, instead of injections used in procedures such as Botox, it is highly likely to be used for cosmetic applications. In fact, cosmetics containing azirenine as botox to be applied are commercially available in China, and in Korea, products produced by Lipotec are imported and used.

However, currently azirenine is mainly produced by chemical synthesis, for example, it is produced by "Solid phase peptide synthesis" (International journal of cosmetic science. Volume 24, Issue 5, 3030- 310, October 2002). Since azirenine produced by such a chemical synthesis method contains chemical impurities and substances harmful to the human body, a high-purity separation / purification process is required. In addition, the production cost is expensive, there is a limit to the scale-up (scale-up), there is a problem that can not increase the price competitiveness with the above chemical synthesis method.

Accordingly, there is a continuous need for the development of a technology capable of mass-producing high-purity azirenine as a biological process using a yeast fermentation process with high safety instead of chemical synthesis. However, in the case of some peptides, it is sensitive to proteolytic enzymes located on the cell surface of yeast, so that peptides secreted and produced using yeast are decomposed by the degrading enzyme, and thus there is a possibility that the final product may be reduced ( Journal of Biotechnology 149 (2010) 1-7, 3p, right pannel).

The present inventors tried hard to produce thymulin or azirenine as a biological process using the fermentation process of yeast. As a result, Pichia pastoris, an expression vector containing a polynucleotide sequence encoding thymulin or azirenine, is a kind of yeast. Introduced in ( Pichia pastoris ), the present invention was completed by confirming that high-purity thymulin or azirenine can be produced at low cost by separating and purifying the expressed peptide by culturing the yeast.

One object of the present invention is timulin, which comprises a polynucleotide sequence encoding thymurine or azirenine, wherein the polynucleotide sequence is operably linked to a promoter sequence and a high secretion signal sequence that can be expressed in yeast. (thymulin) or azirenin (argireline) to provide an expression cassette for production.

Another object of the present invention is to provide an expression vector for producing thymulin or azirenine, which includes the expression cassette.

Another object of the present invention is to provide a transformant comprising the expression vector.

Another object of the present invention is culturing the transformant; And isolating and purifying thymulin or azirenine expressed from the cultured transformant.

Another object of the present invention is to provide a cosmetic composition for preventing hair loss or promoting hair growth, comprising thymeline prepared by the above manufacturing method.

Another object of the present invention is to provide a cosmetic composition for improving wrinkles, which includes azirenine prepared by the above-described manufacturing method.

Another object of the present invention is to provide a quasi-drug composition for preventing hair loss or promoting hair growth, comprising thymusin prepared by the above-described manufacturing method.

Another object of the present invention is to provide a quasi-drug composition for improving wrinkles, comprising azirenine prepared by the above-described manufacturing method.

By using the expression cassette for the production of thymulin or azirenine of the present invention, an expression vector, and a transformant containing the same, it is possible to manufacture high-purity thymulin or azirenine in large quantities at a low cost.

Timulin produced according to the present invention is excellent in preventing hair loss and promoting hair growth, and thus can be widely used in industries such as cosmetic materials, quasi-drugs, and medicines for preventing hair loss or promoting hair growth. In addition, since the azirenine produced according to the present invention has excellent wrinkle improvement effect and has very low toxicity compared to conventional botox, it can be widely used in industries such as cosmetic materials and pharmaceuticals.

Figure 1a is a schematic diagram schematically showing the gene synthesis sequence for thymusin production designed to high expression / high secretion of the thymulin of the present invention, Figure 1b is a high expression / high secretion of the azirenine of the present invention It is a schematic diagram schematically showing the designed expression cassette.

Figure 2a is a schematic diagram showing a vector map for the production of thymusin thymusin production synthetic sequence is introduced, Figure 2b is a schematic view showing a vector map for the production of azirenine synthesis sequence for the azirenine production is introduced.

Figure 3a is a schematic diagram and a picture showing the process of transforming thymulin expression vector into yeast, Figure 3b is a schematic diagram and a picture showing the process of transforming azirenin expression vector into yeast and a fermenter for culturing yeast.

Figure 4 is a diagram and a picture showing the PCR results after inserting the vector for azirenin production and colony of yeast strains for azirenin production.

5 is a schematic diagram and a photograph schematically showing a method for separating and purifying azirenine of the present invention with high efficiency from cultured yeast.

Figure 6a is a diagram showing the results of SDS-PAGE confirming the production of thymullin through electrophoresis, Figure 6b is a diagram showing the results of SDS-PAGE confirming the production of azirenine fermentation through electrophoresis.

7 is a diagram showing the construction of an azirenine purity analysis system through HPLC and GC-MS.

Figure 8a is a diagram showing the chromatography showing the thymulin fermentation production through HPLC analysis, Figure 8b is a diagram showing the chromatography results showing the azirenine fermentation production through HPLC analysis.

9 is a diagram showing a chromatographic result showing the production of Timullin fermentation through ESI-MS analysis.

In order to achieve the above object, one aspect of the present invention comprises a polynucleotide sequence encoding thymulin or azirenine, and the polynucleotide sequence is operable with a promoter sequence and a high secretion signal sequence that can be expressed in yeast. It provides an expression cassette for thymulin or agireline production, which is tightly linked.

The term "timulin" of the present invention is one of various hormones secreted by the human thymus, and may be used interchangeably as 'Thymulin' in the present specification. In the present invention, Timulin can prevent the hair loss process by preventing immune cells from attacking and killing hair follicle cells by the autoimmune reaction of the human body according to aging, thereby preventing hair loss or promoting hair growth of Timullin. Is to use.

The term of the present invention, "expression cassette for the production of Timulin" means an expression structure capable of expressing Timulin.

In the present invention, the expression cassette may include the polynucleotide sequence encoding the thymusin, specifically, the polynucleotide sequence encoding the thymusin contained in the expression cassette consists of the nucleotide sequence represented by SEQ ID NO: 1 It may be, but is not limited thereto.

The term "azirenine" of the present invention is a partial amino acid structure of SNAP-25 protein, a constituent protein of the SNARE complex involved in muscle contraction, and is a peptide composed of amino acids 12 to 17 (Glu-Glu-Met-Gln- Arg-Arg). Azirenine showed the same wrinkle improvement effect as Botox, but the toxicity was analyzed to be 100,000 times lower. In the present specification, 'azirelin' or 'argirelin' may be used interchangeably.

The term "expression cassette for the production of azirenine" of the present invention means an expression structure capable of expressing azirenine.

In the present invention, the expression cassette may include the polynucleotide sequence encoding the azirenine, specifically, the polynucleotide sequence encoding the azirenine contained in the expression cassette consists of the nucleotide sequence represented by SEQ ID NO: 2 It may be, but is not limited thereto.

The nucleotide sequence used in the present invention is interpreted to include a sequence showing substantial identity with the sequence listed in the sequence list, when considering a variation having biologically equivalent activity. The term, 'substantial identity', aligns the sequence of the present invention with any other sequence as much as possible, and when the aligned sequence is analyzed using an algorithm commonly used in the art, it is minimal. It means a sequence that exhibits 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology.

Accordingly, the base sequence having high homology with the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, for example, its homology is 70% or more, specifically 80% or more, more specifically 90% or more It should be construed that the nucleotide sequence having is also included in the scope of the present invention.

In addition, the expression cassette may be one in which the polynucleotide sequence is operably linked to a promoter sequence and a high secretion signal sequence that can be expressed in yeast.

As a specific example, the promoter is a promoter of glyceraldehyde 3-phosphate dehydrogenase (GPD), a promoter of Threonine Dehydrogenase (TDH), a promoter of alcohol dehydrogenase (ADH), a promoter of Cytochrome c (CYC1), an SFORM5 promoter, or a combination thereof. However, it is not particularly limited as long as it is a promoter suitable for expression in yeast. The sequence of the promoter exemplified above may use a sequence known in the art.

As another specific example, the high secretion signal sequence may be the α-mating factor pre-sequence of S. cerevisiae , but the thymusin to be expressed is well secreted If possible, it is not particularly limited. The high secretion signal sequence illustrated above may use sequences known in the art.

It is preferred that the promoter and high secretion signal sequence are operably linked to a polynucleotide encoding Timulin.

The term, 'operably linked' (operably linked) refers to a state in which a nucleic acid sequence encoding a desired protein or peptide and a nucleic acid expression control sequence to perform a general function are functionally linked (functional linkage). For example, a promoter and a nucleic acid sequence encoding a protein or peptide are operably linked to affect the expression of the coding sequence. Operational linkage with an expression vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can use enzymes and the like generally known in the art.

In addition, the expression cassette is a polynucleotide encoding His, GST or intein upstream or downstream of the polynucleotide sequence in the expression cassette to facilitate purification of the expressed thymulin or azirenine. It may further include 3 to 9, the polynucleotide encoding the His, GST or intein and the polynucleotide encoding the thymulin or azirenine K (Lysine) or DDDDK (Aspartate-Aspartate-Aspartate-) Aspartate-Lysine) may be linked through a linker. However, it is not particularly limited as long as it can facilitate the purification of Timulin.

In addition, the expression cassette may include a promoter, a highly secreted signal sequence, a sequence encoding Timulin and a sequence such as His, a transcription enhancer, a terminator, an initiator, and other genetic regulatory factors or antigenicity or binding of recombinant Timulin. It may further include components that regulate the expression of thymusin, such as factors that give affinity.

In a specific embodiment of the present invention, the aox promoter, the signal sequence for secretion (saccharomyces cerevisiae α-conjugation factor full sequence), the polynucleotide sequence encoding Timulin, His-tag sequence, and termination sequence This sequentially inserted high expression / high cost expression cassette was constructed (FIG. 1A).

In another specific embodiment of the present invention, the AOX1 promoter, a signal sequence for secretion (all of the α-conjugation factor to Saccharomyces cerevisiae), a His-tag sequence, a polynucleotide sequence encoding azirenine, And an azirenine high expression / high cost expression cassette in which a termination sequence was sequentially inserted (FIG. 1B).

For reference, as a result of applying various types of peptides to the expression cassette of the present invention, it was confirmed that not all peptides can be produced. Through this, when the desired peptide sequence was inserted into the expression cassette of the present invention, actual production It can be confirmed that this can not be easily inferred unless it is confirmed through experiments.

Another aspect of the present invention provides an expression vector for producing thymulin or azirenine, comprising the expression cassette.

At this time, the definition of the 'expression cassette', 'timimulin' and 'azirenine' is as described above.

The term “expression vector” of the present invention is a means for efficiently inducing the expression of a target gene by introducing DNA into a host cell, and specifically, an essential regulatory element operably linked to express the target peptide, thymulin or azirenine. It may mean a genetic construct comprising a.

In the present invention, the expression vector may include an expression cassette for the production of thymulin, comprising a polynucleotide sequence encoding the thymulin or azirenine of the invention.

Specific examples of the expression vector include a plasmid vector, a cosmid vector, a bacteriophage vector, or a viral vector. More specific examples, plasmids derived from E. coli (pBR322, pBR325, pUC118, pUC119, pET30a, pET30c or pGEX-GST), Bacillus subtilus-derived plasmids (pUB110 or pTP5), yeast-derived plasmids (YEp13, YEp24, YCp50, pPINKα-HC , pPink-HC or pPink-LC), Ti plasmid, etc. can be used, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus or plant viruses can be used, pPZP, pGA And binary vectors such as the pCAMBIA family.

As a more specific example, the vector may be pPINKα-HC, a yeast-derived plasmid, but is not limited thereto as long as the expression cassette of the present invention can be introduced into a host cell.

In addition, the expression vector can be functionally linked to the expression control sequence. As a specific example, the vector may include, but is not limited to, a signal sequence or a leader sequence for membrane targeting or secretion in addition to an expression control element such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer. It can be manufactured in various ways according to the purpose of the invention. In addition, it may include a selectable marker and may be self-replicating or integrated into host DNA. The vector of the present invention can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation may use enzymes and the like generally known in the art.

In a specific embodiment of the present invention, the expression vector for thymulin production was prepared by inserting the expression cassette for thymurin production into the expression vector pPinkα-HC through a conventional restriction enzyme treatment method and ligation (Example 1) ).

In another specific embodiment of the present invention, the expression cassette for azirenine production (pBJY-Ar vector) is inserted into the expression vector through a conventional restriction enzyme treatment method and ligation for the expression cassette for preparing azirenine. It was produced (Example 1).

Another aspect of the present invention provides a transformant comprising the expression vector.

At this time, the definition of the 'vector' is as described above.

The term "transformant" of the present invention refers to an organism whose genetic trait has been changed by introducing a foreign genetic material.

As specific examples, the transformant is Saccharomyces as MY access (Saccharomyces), Eisai Kosaka in my process (Zygosaccharomyces), blood teeth (Pichia), inclusive Vero My process (Kluyveromyces), Candida (Candida), ski irradiation Caro My process ( Schizosaccharomyces ), Issachenkia ( Yssachenkia ), Yarrowia ( Yarrowia ) or Hansenula ( Hansenula ) may be a strain belonging to, but is not particularly limited as long as it can express the Timulin .

In a more specific example, the transformant may be Saccharomyces cerevisiae , Schizosaccharomyces pombe , or Pichia pastoris , but expresses the thymusulin. If possible, it is not particularly limited.

In the present invention, the term ' Phichia pastoris ( Picchia pastoris )' refers to the yeast strain most widely used for the production of recombinant proteins with Saccharomyces cerevisiae . The yeast strain has the advantage of easy genetic manipulation, various expression systems have been developed, and mass culture is easy. In addition, when producing recombinant proteins derived from higher cells, such as human proteins, the secretion function to secrete proteins out of the cells, and the ability to perform post-translational modification functions of proteins such as sugar chain addition, etc. Gives The production of secretion of a recombinant protein enables the extracellular secretion by artificially fusion of the protein secretion signal and the target protein, and through the secretion process of the protein, folding or disulfide bond formation of the protein and sugar chain addition process As this progresses, there is an advantage that it is possible to produce a recombinant protein having biologically complete activity. In addition, since the protein having biological activity can be directly obtained from the medium, it is very economical because it does not require the step of crushing or refolding cells with low economic efficiency.

In a specific embodiment of the present invention, the expression vector for the production of thymusin is subjected to an electroporation transformation method, Pichia pastoris A transformant was prepared by introducing into the strain (transformation) (Example 2).

In another specific embodiment of the present invention, the expression vector (pBJY-Ar vector) for the production of azirenine is obtained by the method of electroporation transformation, Pichia pastoris. A transformant was prepared by introducing into the strain (transformation) (Example 2).

Another aspect of the present invention is culturing the transformant; And separating and purifying the thymulin or azirenine expressed from the cultured transformant.

At this time, the definitions of the 'transformants', 'timimulin' and 'azirenine' are as described above.

In the present invention, the step of culturing the transformant may be performed in consideration of nutritional requirements of the transformant. As a specific example, Pichia pastoris is a methylotrophic yeast cell, and a buffered complex methanol medium (BMMY), a buffered minimum methanol medium (BMM), etc. may be used for its culture, but is not limited thereto. No, it can be appropriately selected by those skilled in the art according to the purpose of the invention.

In addition, the recovery of thymulin or azirenine from the culture of the transformant can be performed by methods known in the art. Specifically, centrifugation, filtration, extraction, spraying, drying, distillation, precipitation, crystallization, electrophoresis, fractional lysis (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, affinity, hydrophobicity and size exclusion) ) Can be used, but is not particularly limited as long as the thymulin or azirenine of the present invention can be recovered.

In the present invention, the step of isolating and purifying the expressed thymulin or azirenine from the cultured transformant is 3 or more and 9 or less His, at the N-terminus or C-terminus of the expressed thymulin or azirenine, If there is GST or intein protein, it may further include the step of processing an enzyme that degrades the protein. In this case, Ni-column, Gel chromatograpy or Boiling may be used for the step of separating and purifying the thymulin or azirenine expressed from the cultured transformant, but the present invention is not limited thereto, and is performed by those skilled in the art according to the purpose of the present invention. It can be appropriately selected.

In a specific embodiment of the present invention, a Pichia pastoris transformant containing the expression vector for the production of thymuline is inoculated with a medium (inoculation media, yeast nitrogen-containing base (excluding amino acids) 6.7 g / L, synthetic drop of yeast) -Out medium supplement (adenine) 1.39 g / L, and glucose 40 g / L) and batch media (batch media, yeast extract 10 g / L, peptone 20 g / L, potassium phosphate 1.36 g / L, yeast nitrogen base 13.4 g / L, biotin 10 g / L, and glucose 40 g / L) were selected, and after incubation at a working volume of 5 L, pH 6.0, 500 rpm, and 30 ° C., this was collected and dissolved to separate Timulin. In addition, the thymulin was separated and purified from the fermentation broth by Ni-affinity chromatography, and the thymulin fermentation product was confirmed through SDS-PAGE, HPLC, and ESI-MS analysis (FIGS. 6A, 8A, and 9).

This suggests that the expression cassette for the production of timulin according to the present invention, the expression vector and the transformant containing the same can be usefully used for the production of timulin.

In another specific embodiment of the present invention, the Pichia pastoris transformant containing the expression vector for preparing azirenine is inoculated medium (inoculation media, yeast nitrogen-containing base (except amino acid) 6.7 g / L, yeast Synthetic drop-out medium supplement (adenine) of 1.39 g / L, and glucose 40 g / L) and batch media (batch media, yeast extract 10 g / L, peptone 20 g / L, potassium phosphate 1.36 g / L, yeast Nitrogen base 13.4 g / L, biotin 10 g / L, glucose 40 g / L) was selected, and after incubation at 5 L, pH 6.0, 500 rpm, and 30 ° C., the azirenine was separated by collection and dissolution. . In addition, the azirenine was separated and purified from the fermentation broth by Ni-affinity chromatography, and the azirenine fermentation product was confirmed through SDS-PAGE (FIG. 6B). In addition, it was confirmed that the purity of the finally obtained azirenine was 95% or more, and the production concentration of azirenine showed a yield of 100 mg or more per 1 L of the culture medium (FIG. 7).

This suggests that the expression cassette for producing azirenine according to the present invention, an expression vector and a transformant comprising the same can be usefully used for the production of azirenine.

Another aspect of the present invention provides a cosmetic composition for preventing hair loss or promoting hair growth, comprising thymeline prepared by the above manufacturing method.

Another aspect of the present invention provides a cosmetic composition for improving wrinkles, comprising azirenine prepared by the above manufacturing method.

Timulin produced by the method of the present invention is excellent in preventing hair loss and promoting hair growth through promoting scalp and hair follicle cell activity, and thus can be usefully used as a cosmetic composition for preventing hair loss or promoting hair growth.

The hair to which the cosmetic composition of the present invention is applied may include various hairs such as hair, eyebrows (beauty), eyelashes (needle), pubic hair, armpit hair, chest hair, nose hair, and leg hair.

The azirenine produced by the production method of the present invention may exhibit an effect of improving skin wrinkles, and in particular, since it has a toxicity of 100,000 times lower than that of the previously used botox, it can be usefully used as a cosmetic composition for improving wrinkles.

The cosmetic composition of the present invention is a solution, ointment for external use, cream, foam, nutrition lotion, soft lotion, pack, soft water, emulsion, makeup base, essence, soap, liquid detergent, bathing agent, sunscreen cream, sun oil, suspension, Emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays can be prepared in a formulation selected from the group consisting of, but not limited to It is not.

The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and for example, oil, water, surfactant, moisturizer, lower alcohol, and thickener as common ingredients. , Chelating agents, pigments, preservatives, fragrances, etc. may be appropriately blended, but is not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention may vary depending on the formulation of the cosmetic composition.

When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. as carrier components This may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in the case of a spray, additionally, chlorofluorohard Propellants such as locarbon, propane / butane or dimethyl ether, but are not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil, and the like can be used, and in particular, cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan May be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar or trakant may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a soap, alkali metal salt of fatty acid, fatty acid hemiester salt, fatty acid protein hydrolyzate, isethionate, lanolin derivative, aliphatic alcohol, vegetable oil, glycerol, sugar, etc. are used as carrier components. It can be, but is not limited to. These may be used alone or in combination of two or more.

When the formulation of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosate, fatty acid as carrier component Amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used, but is not limited thereto. These may be used alone or in combination of two or more.

Another aspect of the present invention provides a quasi-drug composition for improving wrinkles, comprising thymusin prepared by the above manufacturing method.

Another aspect of the present invention provides a quasi-drug composition for improving wrinkles, comprising azirenine prepared by the above-described manufacturing method.

Timulin produced by the production method of the present invention is excellent in preventing hair loss and promoting hair growth through promoting scalp and hair follicle cell activity, and thus can be usefully used as a quasi-drug composition for preventing hair loss or promoting hair growth.

The hair to which the quasi-drug composition of the present invention is applied may include various hairs, such as hair, eyebrows (beauty), eyelashes (needle), pubic hair, armpit hair, chest hair, nose hair, and leg hair.

The azirenine produced by the manufacturing method of the present invention may exhibit an effect of improving skin wrinkles, and in particular, since toxicity is 100,000 times lower than that of the previously used botox, it can be usefully used as a quasi-drug composition for wrinkle improvement.

The quasi-drug composition of the present invention may be prepared in a formulation selected from the group consisting of body cleanser, soap, hand wash, hair cleanser, hair softener and hair tonic, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1: Preparation of expression cassette for production of thymulin or azirenine fermentation

1-1. Gene synthesis and expression vector production for timulin production

As shown in Figure 1a, in order to express in the Pichia pastoris ( Pchia pastoris ), a yeast (GRAS strain) recognized as safety as a production strain, codon optimization was performed to produce a synthetic gene for thymulin production.

As shown in Figure 2a, the synthetic gene for the production of thymusin 30 ℃ transformation buffer (0.67% Yeast Nitrogen Base without amino acid, 0.076% Yeast Synthetic) by conventional restriction enzyme treatment method and ligation, etc. The transformation vector pBJY_Thymulin was produced by inserting it into the expression vector pPinkα-HC (Invitrogen) in Drop-out Adenine (2% Glucose).

Specifically, a P. pastoris custom promoter and a signal sequence for secretion were selected for high expression / high secretion, AOX1 promoter as the promoter, and Saccharomyces cerevisiae as the signal sequence for secretion. The full sequence of α-conjugation factor was used. After that, a polynucleotide sequence encoding thymurin was placed, followed by placing a His-tag sequence for high-efficiency separation and purification, and finally a thymurin high-expression / high-cost expression vector into which a terminator sequence was inserted was constructed. Did.

1-2. Gene synthesis and expression vector production for azirenine production

As shown in FIG. 1B, an expression cassette for expressing azirenine was prepared in Pichia pastoris , a yeast (GRAS strain) recognized for safety as a production strain.

As shown in Fig. 2b, the produced expression cassette was transformed at 30 ° C with a conventional restriction enzyme treatment method and ligation, etc. (0.67% Yeast Nitrogen Base without amino acid, 0.076% Yeast Synthetic Drop- out Adenine, 2% Glucose) was inserted into the expression vector pPinkα-HC (Invitrogen) to produce a transformation vector pBJY-Ar.

For high expression / high secretion, P. pastoris custom promoters and signal sequences for secretion were selected, the promoter being the AOX1 promoter, and the signal sequence for secretion was α- of Saccharomyces cerevisiae . The entire conjugation sequence was used. After that, a polynucleotide sequence encoding azirenine (Glu-Glu-Met-Gln-Arg-Arg) was placed, followed by a His-tag sequence for high-efficiency separation and purification, and the polynucleotide sequence and His- The tag sequence was ligated through an DDDDK (Aspartate-Aspartate-Aspartate-Aspartate-Lysine) linker, and finally, an azirenine high expression / high cost expression cassette into which an enterokinase site, a cleavage sequence, was inserted was constructed. In addition, the polynucleotide sequence encoding the target peptide azirenine was obtained through codon optimization.

Example 2: Cultivation of yeast introduced with thymulin or azirenine expression vector

As shown in Figure 3a, the thymeline expression vector prepared in Example 1 was introduced into the P. pastoris strain by electroporation transformation (transformation) to prepare a transformant.

As shown in Figure 3b, the transformation vector pBJY-Ar was introduced into the P. pastoris strain by electroporation transformation (transformation) to prepare a yeast strain for azirenine production.

In addition, a medium suitable for a yeast strain for production of thymulin or azirenine fermentation, which contains the components of Table 1 below, was selected, and the thymulin or azirenine at a pH of 6.0, 500 rpm, and 30 ° C was selected from the medium at a working amount of 5 L. The yeast strain for production was cultured.

Inoculation media
Yeast Nitrogen Base Without Amino Acid	6.7 g / L
Yeast Synthetic Drop-Out Medium Supplements (adenine)	1.39 g / L
Glucose	40g / L
Batch media
Yeast extract	10g / L
peptone	20g / L
Potassium phosphate	1.36 g / L
Yeast Nitrogen Base	13.4g / L
Biotin	10g / L
Glucose	40g / L

Example 3: High efficiency / high purity separation and purification of target peptide expressed from cultured yeast

3-1. High-efficiency / high-purity separation purification of Timulin

After collecting the cultured yeast and lysing the cells in the usual manner, the target peptide, Timulin, was isolated from the cell lysate. The target peptide was isolated using a physical / chemical separation and purification technique. Since the expression cassette prepared in Example 1 included the His-tag sequence, the thymosin was purified by Ni-affinity chromatography capable of adsorbing His well. Finally, the target peptide can be treated with enterokinase to obtain thymulin.

Specifically, 1 mL of Ni-NTA agarose and 4 mL of the separated and purified peptide sample were mixed and reacted at 4 ° C. for 60 minutes. After placing 5 mL of sample-Ni-NTA agarose in a disposable column, the lower part of the column was opened to allow gravity to flow. After that, 4 mL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole) was flowed twice, and 0.5 mL of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole) was flowed 4 times, and then a sample was secured. . 0.0064 µg / mg of enterokinase was added to the obtained sample and reacted at room temperature for 16 hours.

As a result, as shown in Figure 6a, through the SDS-PAGE analysis results using electrophoresis of the peptide samples isolated and purified through the above method, it was confirmed that the thymulin was fermented from the cultured yeast.

In addition, as shown in Figure 8a, through the HPLC analysis results, when compared to the thymeline standard (Thymulin standard), it was confirmed that thymusin produced 11.109 mg / L in the obtained sample. More specifically, as shown in Figure 6, through the ESI-MS analysis results performed to obtain more accurate analytical data, the weight of the Timullin standard is 8877.4015, the weight of the Timulin produced fermented in the obtained sample In addition, since it shows the same value as 877.4015, it was confirmed that thymulin was fermented and produced in the obtained sample solution.

3-2. High efficiency / high purity separation purification of azirenine

As shown in Fig. 5, the cultured yeast was collected to lyse the cells in a general manner, and then the desired peptide, azirenine, was separated from the cell lysate. The desired peptide was isolated using a physical / chemical separation and purification technique. Since the His-tag sequence was included in the expression cassette prepared in Example 1, the desired peptide was purified by Ni-affinity chromatography capable of adsorbing His well. , Finally, the target peptide was treated with enterokinase to obtain azirenine.

Specifically, as shown in Figure 6b, after separating and purifying azirenine through the above method, through the SDS-PAGE results using electrophoresis, it can be confirmed that azirenine was fermented from the cultured yeast. there was.

In addition, as shown in FIG. 7, a spectrum showing the purity of azirenine was shown using HPLC and GC-MS. Through the analysis, it was confirmed that the purity of the finally obtained azirenine was 95% or more. In addition, as a result of calculating the production concentration of azirenine, it was found that the yield of 100 mg or more per 1 L of the culture medium.

Specifically, as shown in FIG. 8B, it was confirmed that 14.7 g / L of azirenine was produced, and considering that the existing chemical synthesis method has a price per 1 g of about 400,000 won, it is for producing azirenine of the present invention. It can be seen that, through fermentation production of azirenine using yeast strains, azirenine can be produced with higher efficiency than conventional chemical production methods.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the above detailed description and equivalent concepts thereof.

Claims (14)

1. A polynucleotide sequence encoding thymurine or azirenine,

   The polynucleotide sequence is operably linked to a promoter sequence and a high secretion signal sequence that can be expressed in yeast,

   Expression cassette for the production of thymulin or agireline.
2. The expression cassette according to claim 1, wherein the promoter is a GPD, TDH, ADH, CYC1 or STE5 promoter.
3. The expression cassette according to claim 1, wherein the high secretion signal sequence is a customized secretion signal sequence of Pichia pastoris .
4. The expression cassette of claim 1, wherein the expression cassette further comprises 3 to 9 polynucleotides encoding His, GST or Intein upstream or downstream of the polynucleotide sequence in the expression cassette. The expression cassette.
5. The expression cassette according to claim 4, wherein the polynucleotide sequence encoding the His, GST or intein and the polynucleotide sequence encoding the Timulin are linked through a K or DDDDK linker.
6. An expression vector for producing thymulin or azirenine, comprising the expression cassette of claim 1.
7. A transformant comprising the expression vector of claim 6.
8. The method of claim 7,

   The transformants My process (Saccharomyces), my process to Xi Kosaka (Zygosaccharomyces) a saccharide, blood teeth (Pichia), inclusive Vero My process (Kluyveromyces), Candida (Candida), ski irradiation Caro My process (Schizosaccharomyces), Ken and Escherichia (Issachenkia), Yarrow's (Yarrowia) and a century nyulra, transformant is selected from the group consisting of (Hansenula) strain belongs.
9. The method of claim 7,

   The transformant is selected from the group consisting of Saccharomyces cerevisiae , Schizosaccharomyces pombe and Pichia pastoris .
10. (A) culturing the transformant of any one of claims 7 to 9; And

    (b) separating and purifying the thymulin or azirenine expressed from the cultured transformant, a method for producing thymulin or azirenine.
11. A cosmetic composition for preventing hair loss or promoting hair growth, comprising thymeline prepared by the method of claim 10.
12. A cosmetic composition for improving wrinkles, comprising azirenine prepared by the method of claim 10.
13. A pharmaceutical composition for preventing hair loss or promoting hair growth, comprising thymeline prepared by the method of claim 10.
14. A quasi-drug composition for improving wrinkles, comprising azirenine prepared by the method of claim 10.

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