KR102124035B1 - A expression cassette for preparation of thymulin and use thereof - Google Patents

Abstract

The present invention relates to an expression cassette for the production of timulin and its use, specifically comprising a polynucleotide sequence encoding timulin, and the polynucleotide sequence is operable with a promoter sequence and a high secretion signal sequence that can be expressed in yeast. The expression cassette for thymulin production, the expression vector containing the expression cassette, the transformant containing the expression vector, the production method of thymulin using the transformant, and the production method It relates to a cosmetic composition and a quasi-drug composition for preventing hair loss or promoting hair growth comprising the prepared Timulin.
Using the expression cassette for the production of thymulin of the present invention, an expression vector and a transformant comprising the same, it is possible to manufacture thymulin of high purity in large quantities at a low cost. In addition, the Timullin 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 and quasi-drugs for preventing hair loss or promoting hair growth.

Description

A expression cassette for preparation of thymulin and use thereof

The present invention relates to an expression cassette for the production of timulin and its use, specifically comprising a polynucleotide sequence encoding timulin, and the polynucleotide sequence is operable with a promoter sequence and a high secretion signal sequence that can be expressed in yeast. The expression cassette for thymulin production, the expression vector containing the expression cassette, the transformant containing the expression vector, the production method of thymulin using the transformant, and the production method It relates to a cosmetic composition and a quasi-drug composition for preventing hair loss or promoting hair growth comprising the prepared Timulin.

Thymulin is one of various hormones secreted by the human thymus, which is known to be involved in T cell differentiation 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 studied for 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.

Accordingly, there is a continuous need for the development of a technology capable of mass-producing high-purity timulin as a biological process using a fermentation process of yeast having 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).

Under this background, the present inventors tried hard to prepare thymulin as a biological process using the fermentation process of yeast, and thymulin is an expression vector containing a polynucleotide sequence encoding a Pichia pastoris, which is a kind of yeast. Introduced in pastoris ), the present invention was confirmed by separating and purifying the expressed peptide by culturing the yeast, thereby making it possible to mass-produce high-purity thymulin at low cost.

One object of the present invention is a thymulin, comprising a polynucleotide sequence encoding thymuline, wherein the polynucleotide sequence is operably linked to a promoter sequence and a high secretion signal sequence that can be expressed in yeast. It is to provide an expression cassette for production.

Another object of the present invention is to provide an expression vector for producing thymullin, 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 the expressed Timulin 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 quasi-drug composition for preventing hair loss or promoting hair growth, comprising thymeline prepared by the above manufacturing method.

In order to achieve the above object, one aspect of the present invention includes a polynucleotide sequence encoding thymuline, and the polynucleotide sequence is operably linked to a promoter sequence and a high secretion signal sequence that can be expressed in yeast. An expression cassette for producing thymulin is provided.

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 attacking and killing immune cells by attacking the hair follicle cells by the autoimmune reaction of the human body according to aging, thereby preventing the hair loss or promoting hair growth of Timullin. Is to use

The term "expression cassette for the production of Timulin" of the present invention 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 thymeline 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 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 to the maximal correspondence and analyzes the aligned sequence using an algorithm commonly used in the art. It refers to a sequence showing 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology.

Accordingly, a base sequence having high homology with the base sequence represented by SEQ ID NO: 1, for example, a base sequence having high homology of 70% or more, specifically 80% or more, and more specifically 90% or more It should also be interpreted as being 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 isoform 1, STE5 promoter, or a combination thereof. However, it is not particularly limited as long as it is a promoter suitable for expression in yeast. As the sequence of the promoter exemplified above, a sequence known in the art can be used.

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.

Preferably, the promoter and high secretion signal sequence are operably linked to a polynucleotide encoding thymulin.

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 are functionally linked to perform a general function. 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, in order to facilitate purification of the expressed thymusin, polynucleotides 3 to 9 encoding His, GST or intein upstream or downstream of the polynucleotide sequence in the expression cassette. The dog may further include a polynucleotide encoding the His, GST, or intein, and the polynucleotide encoding the Timulin, a K(Lysine) or DDDDK (Aspartate-Aspartate-Aspartate-Aspartate-Lysine) linker. It may be connected through. 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 A stimulin high expression/high cost expression cassette inserted in this order was produced (FIG. 1).

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 provides an expression vector for producing timulin, comprising the expression cassette.

At this time, the definition of the'expression cassette' and'timimulin' 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, specifically a gene comprising an essential regulatory element operably linked to express the target peptide, thymulin It can mean a construct.

In the present invention, the expression vector may include an expression cassette for preparing timulin, comprising a polynucleotide sequence encoding the timulin of the present 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) or Ti plasmid, and the like, animal virus such as retrovirus, adenovirus or vaccinia virus, insect virus such as baculovirus or plant virus can be used, pPZP, pGA And binary vectors such as the pCAMBIA family.

As a more specific example, the vector may be a yeast-derived plasmid, pPINKα-HC, 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 may 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 2) ).

Another aspect 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 means 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 if the expression of the Timulin is not particularly limited.

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 ( Pichia pastoris )'refers to the yeast strain most widely used for the production of recombinant proteins along 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 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, the folding or disulfide bond of the protein is formed and the sugar chain is added. As this progresses, there is an advantage that it is possible to produce a recombinant protein having a biologically complete activity. In addition, since the protein having biological activity can be obtained directly 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 thymulin is subjected to an electroporation transformation method, Pichia pastoris A transformant was prepared by introducing into the strain (transformation) (Example 2).

Another aspect is culturing the transformant; And isolating and purifying the expressed Timulin from the cultured transformant.

At this time, the definition of the'transformants' and'timulin' 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 from the culture of the transformant can be carried out by methods known in the art. Specifically, centrifugation, filtration, extraction, spraying, drying, distillation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation), chromatography (for example, ion exchange, affinity, hydrophobicity and size exclusion) ) May be used, but is not particularly limited as long as the thymulin of the present invention can be recovered.

In the present invention, the step of isolating and purifying the expressed thymeline from the cultured transformant is 3 or more and 9 or less His, GST or intein proteins at the N-terminus or C-terminus of the expressed thymusin. If present, 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 expressed thymulin from the cultured transformant, but is not limited thereto, and appropriately selected by a person skilled in the art according to the purpose of the invention Can be.

In a specific embodiment of the present invention, the Pichia pastoris transformant containing the expression vector for the production of thymulin is inoculated 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, incubated at 5 L of work volume, pH 6.0, 500 rpm, and 30° C., and then 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. 4 to 6 ).

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.

Another aspect of the present invention provides a cosmetic composition for preventing hair loss or promoting hair growth, comprising thymusin prepared by the above-described 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 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, a solubilizing agent or an emulsifying agent 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 may be, 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 surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcositate, fatty acid as a 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 provides a quasi-drug composition for improving wrinkles, comprising thymusin 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 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.

Using the expression cassette for the production of timulin of the present invention, an expression vector, and a transformant comprising the same, it is possible to manufacture a large amount of high-purity timulin at a low cost.

In addition, the Timullin 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.

1 is a schematic diagram schematically showing a gene synthesis sequence for thymusin production designed to highly express/secrete thymusin of the present invention.
Figure 2 is a schematic diagram showing a vector map for the production of thymulin Timulin production synthetic sequence is introduced.
Figure 3 is a schematic diagram and a photograph showing the process of transforming the thymulin expression vector into yeast.
4 is a view showing the results of SDS-PAGE confirming the production of Timullin fermentation through electrophoresis.
5 is a diagram showing a chromatography showing the production of Timulin fermentation by HPLC analysis.
6 is a diagram showing a chromatographic result showing the production of thymulin through ESI-MS analysis.

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: Gene synthesis and expression vector production for Timulin production

As shown in FIG. 1, in order to express in Pichia pastoris , a yeast (GRAS strain) that has been recognized for safety as a production strain, codon optimization was performed to produce a synthetic gene for timulin production.

As shown in FIG. 2, the synthetic gene for thymusin production 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 The transformation vector pBJY_Thymulin was produced by inserting it into the expression vector pPinkα-HC (Invitrogen) in Drop-out Adenine (2% Glucose).

Specifically, P. pastoris custom promoters and signal sequences 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.

Example 2: Cultivation of yeast introduced with thymulin expression vector

As shown in FIG. 3, the thymulin expression vector produced in Example 1 was subjected to P. pastoris by an electroporation transformation method. Introduced into the strain (transformation) to prepare a transformant.

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

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: Purpose expressed from cultured yeast Peptide High efficiency/high purity separation purification

After collecting the cultured yeast to lyse 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 His-tag sequence was included in the expression cassette prepared in Example 1, thymusin 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 adding 5 mL of sample-Ni-NTA agarose to 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, 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 enterokinase was added to the obtained sample, and the mixture was reacted at room temperature for 16 hours.

As a result, as shown in Figure 4, through the SDS-PAGE analysis results using electrophoresis of the peptide sample 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 5, through the HPLC analysis results, when compared with the thymeline standard (Thymulin standard), it was confirmed that thymulin 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 877.4015, the weight of the fermented produced Timullin 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.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing its technical spirit or essential characteristics. 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.

<110> KOREA INSTITUTE OF INDUSTRIAL TECHNOLOGY <120> A expression cassette for preparation of thymulin and use thereof <130> KPA180535-KR <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Thymulin <400> 1 catcaccatc atcaccatga cgacgatgat aaagaagcta agtctcaagg tggatctaac 60 taa 63 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Thymulin <400> 2 Glu Ala Lys Ser Gln Gly Gly Ser Asn 1 5

Claims (12)

It comprises a polynucleotide sequence encoding thymeline consisting of the nucleotide sequence of SEQ ID NO: 2,
Expression for production of thymulin, wherein the polynucleotide sequence is operably linked to the AOX1 promoter sequence that can be expressed in yeast and the α-mating factor pre-sequence, a high secretion signal sequence. As a cassette,
The expression cassette, characterized in that it further comprises 3 to 9 polynucleotides encoding His in the upstream or downstream of the polynucleotide sequence.
delete The expression cassette of claim 1, wherein the high secretion signal sequence is a customized secretion signal sequence of Pichia pastoris .
delete The expression cassette according to claim 1, wherein the polynucleotide sequence encoding the His and the polynucleotide sequence encoding the Timulin is linked through a K or DDDDK linker.
An expression vector for producing timulin, comprising the expression cassette of any one of claims 1, 3, and 5.
A transformant comprising the expression vector of claim 6.
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.
The method of claim 7,
The transformant is Saccharomyces cerevisiae ), Schizosaccharomyces pombe ) and Pichia pastoris ( Pichia pastoris ).
(A) culturing the transformant of claim 7; And
(b) separating and purifying the thymulin expressed from the cultured transformant, the thymeline production method. delete delete

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