KR102553825B1 - Extracellular vesicle isolated from recombinant microorganism comprising polynucleotide encoding target protein and use thereof - Google Patents

Abstract

It relates to a recombinant microorganism comprising a polynucleotide encoding a target protein, an extracellular vesicle isolated therefrom, and uses thereof.

Description

Extracellular vesicle isolated from recombinant microorganism comprising polynucleotide encoding target protein and use thereof}

It relates to an extracellular vesicle derived from a recombinant microorganism comprising a polynucleotide encoding a target protein and uses thereof.

Most animal cells secrete extracellular vesicles of intracellular origin of varying size and composition. Both prokaryotes and eukaryotes are known to secrete extracellular vesicles. Conventionally, extracellular vesicles derived from Staphylococcus aureus have been known (Korean Patent Publication 10-2011-0025603).

Extracellular vesicles are membrane-structured vesicles ranging in diameter from about 20 nm to about 5 μm. Extracellular vesicles are heterogeneous in size and composition, and include exosomes (about 30 to 100 nm), ectosomes, microvesicles (about 100-1,000 nm), microparticles, It includes many different species, such as outer membrane vesicles. The characteristics of extracellular vesicles are influenced by the characteristics of the cells from which they originate.

On the other hand, intracellular substances (eg, DNA, RNA, proteins, etc.) are naturally loaded into extracellular vesicles and secreted out of the cells. Extracellular vesicles have high biocompatibility as they are the same as components of biological membranes, and have excellent mass transfer efficiency as they are small in size to nanometer size. Therefore, studies are underway to deliver drugs using extracellular vesicles instead of conventional delivery systems such as liposomes. However, when loaded into the extracellular vesicle, the loading efficiency of the target protein into the extracellular vesicle was low because intracellular materials such as other unnecessary intracellular proteins as well as genetic material in addition to the target protein were loaded together. Therefore, there is a need for a technology capable of stably loading a target protein into an extracellular vesicle with excellent efficiency. The present inventors have invented a technique for obtaining extracellular vesicles naturally loaded with a large amount of protein by stably loading a target protein into extracellular vesicles with excellent efficiency.

One aspect provides a recombinant microorganism comprising a polynucleotide encoding a target protein, wherein the microorganism is lactic acid bacteria or yeast.

Another aspect provides an extracellular vesicle derived from a recombinant microorganism described above.

Another aspect provides a composition for delivering a target protein comprising the recombinant microorganism-derived extracellular vesicle and a carrier as active ingredients to a subject.

Another aspect provides a method of treating a disease in a subject comprising administering the composition to the subject.

Another aspect provides a method of making up a subject comprising administering the composition to the subject.

Another aspect is culturing the microorganisms to obtain a culture; And it provides a method for producing extracellular follicles comprising the step of isolating the extracellular follicles from the culture.

A first aspect provides a recombinant microorganism comprising a polynucleotide encoding a target protein, wherein the microorganism is lactic acid bacteria or yeast.

The target protein may be a signal peptide linked to each other, that is, a fusion protein of the signal peptide and the target protein. The microorganism may load the target protein into the extracellular vesicle in an increased amount. In this case, the recombinant microorganism may have an increased extracellular vesicle (EV) loading capacity compared to a recombinant microorganism containing a polynucleotide encoding the target protein without a signal peptide. The extracellular vesicle loading capacity indicates the degree to which a target protein is included in EVs or the degree to which a target protein is expressed in EVs. Extracellular vesicle loading capacity may indicate loading capacity for parental microorganisms that do not contain a polynucleotide encoding a target protein.

The signal peptide may be any one of the amino acid sequences of SEQ ID NOs: 21 to 60 and a sequence including the same or a sequence similar thereto.

In the recombinant microorganism, the lactic acid bacteria may be a species belonging to a genus selected from the group consisting of Lactobacillus, Lactococcus, and Bifidobacterium. The lactic acid bacteria may be Lactobacillus paracasei , Lactobacillus brevis , or Lactobacillus plantarum .

In the recombinant microorganism, the yeast may be a species belonging to a genus selected from the group consisting of Saccharomyces, Pichia, and Hansenula. Saccharomyces may be S. cerevisiae. Pichia can be Pichia pastoris, Hansenula can be Hansenula polymorpha.

In the recombinant microorganism, the target protein may be a growth factor, a cytokine, an antibody, an enzyme, an inhibitory protein, or a fragment thereof. The growth factor may be a fibroblast growth factor. The target protein is fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (insulin-like growth factor) : IGF), placenta growth factor (PGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor factor: VEGF), thioredoxin (TRX), interleukin-1 (IL-1), interleukin-10, interleukin-22, interleukin-13, and tumor necrosis factor (TNF) It may be selected from the group consisting of. The target protein is, for example, IL-22, EGF, IGF1, FGF1 (hereinafter also referred to as aFGF (acidic fibroblast growth factor)), FGF2 (hereinafter referred to as bFGF (basic fibroblast growth factor)), FGF7 (hereinafter referred to as KGF (keratinocyte growth factor)), TGFa, and TRX.

In the recombinant microorganism, the signal peptide may be one encoded by SEQ ID NO: 4 or any one of the amino acid sequences of SEQ ID NOs: 21 to 60. The gene encoding the signal peptide may be linked such that the signal peptide is linked to the N-terminus of the target protein. The signal peptide may be natural or heterologous to the protein. The target protein may be a heterologous protein to the microorganism. The recombinant microorganism may express the target protein. The target protein may be loaded into the EV with the signal peptide cut off. The target protein may be loaded on the EV membrane or inside it.

In the recombinant microorganism, the polynucleotide encoding the target protein may be expressible. The polynucleotide may be operably linked to a transcriptional regulatory sequence. The transcription control sequence may be a promoter, operator, enhancer, or terminator. The polynucleotide may be operably linked to a translation control sequence. The translation control sequence may be a ribosome binding site or a ribosome entry site sequence. The polynucleotide may be integrated into the genome of the microorganism or may exist independently. The polynucleotide may be included in a vector. The vector may be an expression vector. The vector may be a plasmid or a viral vector.

A second aspect provides an extracellular vesicle isolated from the recombinant microorganism described above.

A third aspect provides a composition for delivering a target protein comprising the recombinant microorganism-derived extracellular vesicle and a carrier as active ingredients to a subject.

In the second or third aspect, the extracellular vesicles may be isolated from a culture medium of the microorganism. That is, the extracellular vesicles may be secreted extracellularly. The extracellular vesicle may have an average diameter of 20 to 500 nm, for example, 20 to 200 nm or 100 to 200 nm. The extracellular vesicle may contain the target protein described above. The target protein may be located on or inside the membrane of the extracellular vesicle.

The extracellular vesicles may be isolated from the culture medium by any method capable of separating them. For example, the extracellular vesicles can be separated by centrifugation, ultracentrifugation, filtration by filters, ultrafiltration, gel filtration chromatography, ion exchange chromatography, precipitation, and immunoprecipitation, free-flow electrophoresis, capillary electrophoresis, Or it can be separated by a combination thereof. The separation method may include processes such as washing and concentration to remove impurities. The extracellular vesicle may be produced by a method for isolating the extracellular vesicle described below. The extracellular follicles may be produced by ultrafiltration of the microbial culture medium using an ultrafine filter with a cutoff of 100 kD or more, for example, 300 kD or more, or 500 kD or more. The extracellular vesicles may be precipitated by ultracentrifugation of 100,000 xg or more of the microbial culture medium. The separation may be produced by the method for producing extracellular follicles according to the seventh aspect to be described below.

In the third aspect, the carrier may be physiologically acceptable, for example pharmaceutically or cosmetically acceptable. The carrier may include commonly used saline, sterile water, Ringer's solution, buffer, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, or a combination thereof. In addition, the carrier may include an antioxidant, a diluent, a dispersing agent, a surfactant, a binder, a lubricant, or a combination thereof.

The composition may have a dosage form for oral or parenteral administration. The parenteral dosage form may be a topical dosage form. Topical dosage forms may be dermal or mucosal dosage forms. The parenteral dosage form may be in the form of a solution, suspension, emulsion, skin external preparation, spray, or puff.

The composition may be administered to a subject by, for example, skin application, mucosal application, or intranasal administration.

The dose may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. A daily dose means an amount of an active ingredient sufficient to treat a disease state alleviated by being administered to a subject in need of such treatment. The dosage is 0.01 to 1000 mg/day, 0.01 to 500 mg/day, based on an adult individual with a body weight of 70 kg, and may be administered in divided doses once or several times a day at regular time intervals.

The composition may be a cosmetic composition. The cosmetic composition may include components commonly used in cosmetic compositions. The cosmetic composition may include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

The cosmetic composition may be a solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, wax foundation or spray. The cosmetic composition may have a formulation of nutrient cream, astringent lotion, softening lotion, lotion, essence, nutrient gel or massage cream.

The composition may be for promoting the growth of fibroblasts or keratinocytes or collagen synthesis in a subject. The composition may be used for preventing skin aging or improving wrinkles. In this case, the target protein may be a growth factor.

The composition may be for delivering a target protein locally to the subject. The composition may be for delivery through the skin, oral cavity, or mucous membranes.

In the composition, the subject may be a mammal. The mammal may be a human, dog, cat, horse, or pig.

A fifth aspect provides a method of treating a disease in a subject comprising administering the composition to the subject. The subject may be a mammal. The mammal may be a human, dog, cat, horse, or pig. The disease may be an inflammatory disease, wound, atopic dermatitis, psoriasis, or acne.

A sixth aspect provides a method of making up a subject comprising administering the composition to the subject. The administration may be applied to the skin of a subject. The administration may be applied to aged skin or wrinkled skin. The makeup method may be for improving aged skin or wrinkled skin.

The seventh aspect is culturing the microorganisms to obtain a culture product; And it provides a method for producing extracellular follicles comprising the step of isolating the extracellular follicles from the culture.

The culture may be incubation in a medium useful for the growth of the microorganism. The culturing may be performed under conditions known to be suitable for lactic acid bacteria or yeast, for example, temperature and stirring conditions.

The step of separating the extracellular follicles from the culture includes any separation of the extracellular follicles from the culture.

The separating may include centrifuging the culture and obtaining a supernatant; filtering the supernatant; And it may be one comprising the step of obtaining a precipitate by ultracentrifuging the filtrate.

In the separation step, in the step of obtaining the supernatant, centrifugation may be performed at 1,000 to 20,000 xg. In the step of filtering the supernatant, the filtration may be filtration using an ultra-fine filter. The filtration may be ultrafiltration of the supernatant using an ultra-fine filter having a cutoff of 100 kD or more, for example, 300 kD or more, or 500 kD or more. In the step of obtaining a precipitate by ultracentrifugation of the filtrate, the ultracentrifugation may be performed at 100,000 xg or more, for example, 100,000 xg to 200,000 xg.

The method may further include suspending the precipitate.

According to the recombinant microorganism according to one aspect, it can be used to efficiently separate extracellular vesicles or target proteins therefrom.

According to the composition for delivering the above-described extracellular vesicle and target protein to a subject according to another aspect, it can be used to efficiently deliver a target protein to a subject.

According to a method for treating a disease of an individual according to another aspect, the disease can be efficiently treated.

According to a method of applying makeup to an individual according to another aspect, it is possible to efficiently apply makeup to an individual.

According to the method for producing extracellular vesicles according to another aspect, extracellular vesicles can be efficiently produced.

1 shows an expression vector for expressing a target protein in yeast cells.
2 is a diagram showing the expression level of target proteins in the supernatant and extracellular vesicles from S. cerevisiae transformed with p416G-MF-hEGF1 (IGF1, FGF1, FGF2, TGF alpha, and TRX).
3 is a diagram showing the expression level of target proteins in the supernatant and extracellular follicles from S. cerevisiae transformed with p416G-MF-hFGF1 or hTRX or p416G-hFGF1 or hTRX.
Figure 4 is a diagram showing the effect on cell proliferation of growth factor-containing extracellular follicles isolated from yeast.
5 is a diagram showing the production of IL-10 in cells treated with extracellular follicles containing IL-22 and extracellular follicles not containing IL-22, respectively.
Figure 6 shows the result of observing the degree of fusion of CFSE-labeled extracellular follicles with cells through cell flow analysis.
7 is a diagram measuring the toxicity of yeast-derived extracellular vesicles to the skin.
8 is a diagram showing the size and concentration distribution of EVs isolated from transformed lactic acid bacteria.
Figure 9 shows the results of Western blotting with respect to the EV solution.
Fig. 10 shows the results of Western blotting on EVs isolated from LMT1-21 transformed with pMT172 recombinant containing the aFGF-encoding gene either fused or not fused with the signal peptide gene.
11 is a view showing the effect on cell proliferation of growth factor-containing extracellular follicles isolated from lactic acid bacteria.
12 is a photograph of electrophoresis of cell-derived proteins contacted with LMT1-21-derived EVs.
13 shows the result of observing the degree of fusion of CFSE-labeled extracellular follicles with cells through cell flow analysis.
14 is a diagram measuring the toxicity of yeast-derived extracellular vesicles to the skin.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1: Extracellular vesicles derived from yeast cells

Recombinant yeast expressing the target protein was prepared, and extracellular vesicles were produced from the yeast. The specific process is as follows. Yeast cells were Saccharomyces cerevisiae.

1. Construction of expression vectors

1 shows an expression vector for expressing a target protein in yeast cells. This vector was constructed using the sequence of plasmid pRS416 GPD (SEQ ID NO: 1), and the target proteins were hEGF1, hIGF1, hFGF1, hFGF2, hTGF alpha, and hTRX. hEGF1, hIGF1, hFGF1, hFGF2, hTGF alpha, and hTRX have amino acid sequences of SEQ ID NOs: 14, 15, 12, 13, 17, or 18, respectively, and they are SEQ ID NOs: 5, 6, 7, 8, 10, and 11 It can be encoded by a nucleotide sequence. FGF7 may have a nucleotide sequence of SEQ ID NO: 9, and its amino acid sequence may be an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 9.

The vector of FIG. 1 was named p416G-MF-hEGF1 (IGF1, FGF1, FGF2, TGF alpha, and TRX) according to the target protein.

First, target protein genes codon-optimized according to the codon usage frequency of S. cerevisiae, that is, human EGF1, IGF1, FGF1, FGF2, TGF alpha, and TRX genes were commissioned and synthesized by Macrogen. The expression vector shown in FIG. 1 was constructed using the p416GPD vector (ATCC87360) (SEQ ID NO: 1) for these genes. The expression vector of FIG. 1 includes a sequence in which a polynucleotide (SEQ ID NO: 4) encoding the mating factor alpha-1 signal peptide (MF) of S. cerevisiae is linked upstream of a target protein gene. As a control, a gene to which a polynucleotide encoding a signal peptide (MF) (SEQ ID NO: 4) was not linked was used. In addition, a vector was similarly prepared using the p426GPD vector (ATCC 87361) (SEQ ID NO: 2) instead of the p416GPD (ATCC 87360) vector. The p416GPD vector is a low-copy vector present in cells, and the p426GPD is a high-copy vector present in cells. In p416GPD and p426GPD, GPD represents the nucleotide sequence of the promoter GPD (SEQ ID NO: 3).

In FIG. 1, the vector contains the CEN/Ars sequence, which is the origin of replication of S. cerevisiae, the Amp ^r sequence of the ampicillin resistance gene, the ColE1 ori sequence, which is the origin sequence of E. coli replication, the promoter GPD sequence, which is the promoter sequence of S. cerevisiae, and the S. cerevisiae origin sequence. It includes the CYC terminator sequence, the ScCYC term sequence, the F1 ori sequence, which is the origin of replication of Bacteriophage, and the S.cerevisiae promoter, ORF, and terminator sequence (ScURA3p-URA3).

2. Expression of target proteins in yeast

The p416G-MF-hEGF1 (IGF1, FGF1, FGF2, TGF alpha, and TRX) vectors were respectively transformed into S. cerevisiae CEN.PK2-1 strains according to the LiCl method. The resulting transformed strain was mixed with 2 mL of minimal ura-drop out medium (Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. No. Y0626) 6.7 g/L, and Yeast synthetic drop-out without uracil (Sigma-Aldrich: Cat. No. Y1501) 1.92 g/L, glucose 2 (w/v)%) for 1 day, and the cultured strain is 15 mL of minimal ura-drop containing 1% of Casamino acids The medium was inoculated so that the starting OD ₆₀₀ was 0.5, and the main culture was performed. This culture was stirred and cultured at 220 rpm for 2 days at 30 ° C., and a sample group was prepared using the supernatant from which the cells were removed. In addition, the supernatant was filtered using a 100 kDa cut-off membrane (Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane (100K), millipore: Cat. No. UFC910024) to obtain a concentrated filtrate, which was 50,000 Extracellular vesicles (EVs) were separated by ultracentrifugation for 2 hours at g and suspended in 1ml PBS. Here, western blot was performed on the supernatant and the obtained extracellular follicle sample to confirm the level of protein expression.

2 is a diagram showing the expression level of target proteins in the supernatant and extracellular vesicles from S. cerevisiae transformed with p416G-MF-hEGF1 (IGF1, FGF1, FGF2, TGF alpha, and TRX). Lanes 1 and 2 are Western blot pictures showing the expression levels of fusion proteins in which each target protein is linked to the signal peptide MF. Lane 1 contains all proteins expressed in yeast cells and isolated in culture, both target proteins loaded and unloaded in extracellular vesicles. Lane 2 shows only the target protein loaded in extracellular vesicles. As shown in FIG. 2, the target protein was significantly increased in extracellular vesicles in all six experimental groups.

FIG. 3 is a diagram showing the expression levels of target proteins in the supernatant and extracellular follicles from S. cerevisiae transformed with p416G-MF-hFGF1 or hTRX or p416G-hFGF1 or hTRX, respectively. That is, it indicates the degree of entrapment in extracellular vesicles according to the presence or absence of the signal peptide.

Lane 1 shows the target protein in extracellular vesicles obtained from the culture solution of the strain expressed without the signal peptide, and Lane 2 shows the expression of the target protein in the extracellular vesicle obtained from the culture solution of the strain expressed together with the signal peptide sequence. indicate As shown in FIG. 3, the amount of target protein expressed in extracellular vesicles when the target protein is secreted and expressed outside the cell rather than when the target protein is expressed intracellularly, that is, FGF1: 0.667ng and TRX: 0.047ng per 100 million EVs, i.e. FGF1: 2.648ng and TRX: 35.518ng per 100 million EVs were remarkably high.

3. Confirmation of the effect of growth factor-containing extracellular vesicles on cell proliferation

After measuring the concentration of the target protein in the isolated extracellular follicle according to the method described in Section 2, 20 uL of the target protein was used as a starting concentration, and the protein concentration was sequentially diluted 4 times with PBS by 10 times. 20 uL of each dilution was added to a 96-well plate containing 5,000 cells of NIH3T3 cell line or HaCat cells per well, followed by incubation at 37°C for 48 hours. Then, 10 uL of Cell Counting Kit-8 (Dojindo) solution was added to each well. After 2 hours, absorbance was measured at 450 nm. NIH3T3 cells were used for aFGF, bFGF, and IGF, and HaCat cells were used for TGFa and EGF.

Figure 4 is a diagram showing the effect on cell proliferation of growth factor-containing extracellular follicles isolated from yeast. In FIG. 4, SC represents Saccharomyces cerevisiae.

As a result, the target protein-containing extracellular vesicle increased the number of cells in a dose-dependent manner. In FIG. 4 , Pichia EV-aFGF and Hansenula EV-aFGF were subjected to the same process as using S. cerevisiae, except that Pichia pastoris or Hansenula polymorpha transformed with aFGF was used. In Figure 4, the horizontal axis represents the target protein content concentration (w / v, ng / ml) in the extracellular vesicles in the medium. The vertical axis shows the degree of cell proliferation of the used extracellular vesicle-containing solution as a percentage compared to the control group (100%).

4. Identification of IL-22 Expression

Expression vector p426G-MF-IL-22 was constructed as described in Section 1 using IL-22 as the target protein, and transformed into S.cerevisiae CEN.PK2-1 strain as described in Section 2. As a control, the same p426G-MF vector was used except that it did not contain IL-22.

Specifically, the Colo205 cell line was cultured in RPMI medium in a 96-well plate for 48 hours, and then transformed with the p426G-MF-IL22 vector and the p426G-MF vector, respectively derived from yeast expressing IL-22 and yeast not expressing the same. Extracellular vesicles were purified. The extracellular follicles were suspended in PBS at a concentration of 0.5 mg/mL, added to each well of a 96-well plate in an amount of 20 uL, and further cultured for 6 hours. Thereafter, proteins were extracted from the cell lines and the expression levels of IL-10 were compared. IL-22 has the amino acid sequence of SEQ ID NO: 19. IL-22 is known to promote IL-10 production in cells.

5 is a diagram showing the amount of IL-10 protein production confirmed by extracting proteins from Colo205 cell lines treated with extracellular follicles containing IL-22 and extracellular follicles without IL-22, respectively. As shown in FIG. 5 , compared to extracellular follicles not containing IL-22, when the extracellular follicles containing IL-22 were cultured in contact with cells, the protein production of IL-10 was significantly increased. In FIG. 5, lane 1 shows the level of IL-10 protein production of the Colo205 cell line treated with extracellular follicles not containing IL-22, and lane 2 shows the IL-10 protein production level of the Colo205 cell line treated with extracellular follicles containing IL-22. -10 It represents the degree of protein production.

5. Fusion of yeast-derived extracellular vesicles and cells

Extracellular vesicles were isolated from the untransformed S. cerevisiae CEN.PK2-1 strain as described above. 1 ml of isolated extracellular vesicles (0.5 mg/ml PBS) was placed in a solution of 5-Carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) (5 uM) at room temperature for 30 minutes. Thereafter, the remaining CFSE was removed from the solution using a PD-10 desalting column (GE) to obtain CFSE-labeled extracellular follicles. After culturing the NIH3T3 cell line in 0.2 mL of RPMI medium for 48 hours in the wells of a 96-well plate, 10 uL (red) or 20 uL (green) of CFSE-labeled extracellular follicles in PBS was added and further cultured for 24 hours. Then, the cells were washed with PBS. The remaining cells were passed through a flow cytometer and their fluorescence was measured. As a control, 20 ul of BSA 0.5 ug/ml was labeled with the same CFSE.

Figure 6 shows the result of observing the degree of fusion of CFSE-labeled extracellular follicles with cells through cell flow analysis. In Figure 6, the negative control (left figure) is contacting cells with CFSE-labeled BSA, and the experimental group (right) is treated with 10 uL (red) and 20 uL (green) of CFSE-labeled extracellular follicles. Results observed after contacting the cells are shown. As a result, as shown on the right side of FIG. 6 , the cell was stained with CFSE, confirming that the extracellular vesicle was fused with the cell and the components of the extracellular vesicle were introduced into the cell. NIH3T3 cells are a standard fibroblast cell line.

6. Confirmation of skin toxicity of yeast-derived extracellular vesicles

The toxicity of yeast-derived extracellular vesicles to the skin was measured through a toxicity test on artificial skin according to the OECD guidelines. Neoderm ^TM -ED (manufactured by Taego Science) was used as the artificial skin.

Extracellular vesicles derived from S. cerevisiae, Pichia pastoris, or Hansenula polymorpha were isolated. S.cerevisiae-derived extracellular vesicles were isolated as described in Section 2. Extracellular follicles derived from Pichia pastoris or Hansenula polymorpha were isolated as described in Section 2, except that Pichia pastoris and Hansenula polymorpha were used.

Each of the isolated extracellular follicles and 30 uL of each of PBS as a negative control and 5% SDS as a positive control were applied to the Neoderm ^™ -ED artificial skin and incubated for 15 minutes. After washing the artificial skin with PBS, it was immersed in 2 ml assay medium (Taego Science Co., Ltd.) in a 12-well plate and further incubated for 42 hours.

The incubated artificial skin was taken out, transferred to a 0.3% MTT solution (0.3 mg/ml), and incubated for 3 hours. Thereafter, the artificial skin was picked up again, and the tissue was separated using an 8 mm biopsy punch, and then placed in 500 ul of 0.04N HCl-isopropanol and bleached for 4 hours. After measuring the absorbance at 570 nm, the viability (%) was obtained by comparing the absorbance of the control group in which the artificial skin was not reacted. As a result, if the measured viability was greater than the median value of the positive and negative control values, it was determined that there was no toxicity. Viability was calculated according to the following formula.

Viability = absorbance of test substance / absorbance of negative control X 100

7 is a diagram measuring the toxicity of yeast-derived extracellular vesicles to the skin. In FIG. 7 , 1: negative control (PBS), 2: positive control (5% SDS), 3: S. cerevisiae, 4: P. pastoris, and 5: extracellular vesicles derived from H. polymorpha are shown.

Example 2: Extracellular vesicles derived from lactic acid bacteria (LAB) cells

Recombinant lactic acid bacteria expressing the target protein were prepared, and extracellular vesicles were produced from the lactic acid bacteria. The specific process is as follows. Lactobacillus paracasei LMT1-21 (KCTC13422BP), Lactobacillus brevis LMT1-46 (KCTC13423BP) and/or Lactobacillus plantarum LMT1-9 (KCTC13421BP) were used as lactic acid bacteria cells.

1. Construction of gene expression vectors

The target gene derives a nucleotide sequence having a codon optimized for the lactic acid bacteria used using the Codon optimization tool (http://sq.idtdna.com/CodonOpt) from the amino acid sequence of the protein, and BamHI and BamHI at both ends of the sequence. A sequence having the recognition sequence of the XhoI restriction enzyme was designed, and DNA having this sequence was synthesized (Macrogen, Korea). The synthesized gene was cut using BamHI and XhoI restriction enzymes. In addition, the parent vector pMT182-PR4 (SEQ ID NO: 20) was digested using the same restriction enzyme, purified using a gel purification kit, and then dephosphorylated using alkaline phosphatase (AP). This parental vector contains DNA corresponding to the promoter and signal peptide SP4 (SEQ ID NO: 21) so as to express the target protein.

1 uL of vector DNA thus prepared, 3 uL of insert DNA, 0.5 uL of T4 DNA ligase (Takara, Japan), and 1 uL of buffer solution were added to 5.5 uL of distilled water to make a total volume of 10 uL. The reaction solution was incubated at 16 ° C for 12 hours to perform a binding reaction, and the resulting conjugate was transformed into E. coli top10 strain according to the method of Sambrook et al. (Sambrook et al., Molecular Cloning: A laboratory manual, 2nd ed. 1989) did The sequence of the plasmid obtained from each colony was analyzed and confirmed. FGF1, FGF2, EGF, IGF, KGF, TGFa, TRX, and IL-22 were used as target proteins. They have the amino acid sequences of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, and 19, respectively.

2. Lactic acid bacteria transformation

The obtained strain was transformed into three types of lactic acid bacteria. Each strain was cultured in 50 mL of MRS until the OD ₆₀₀ reached 0.5, centrifuged at 4 °C and 7,000 rpm for 10 minutes, and 25 mL ice-cold EPS (1 mM K ₂ HPO ₄ KH ₂ PO ₄ , pH 7.4, containing 1 mM MgCl ₂ and 0.5 M sucrose) twice. The cells were suspended in 1 mL ice-cold EPS to prepare competent cells to be used for electroporation, and stored in a -80 °C deep freezer. 40 uL of competent cells and 1 ug/uL DNA of the vector were transferred to a cuvette and left on ice for 5 minutes. After pulses were given at 25 uF, 8 kV/cm, and 400 ohms, 1 mL MRS liquid medium was immediately added and incubated at 37 °C for about 1 hour. The cells were plated in MRS medium containing 10 ug/ml chloramphenicol and then cultured at 37° C. for 49 hours to obtain transformed cells.

3. Extracellular Vesicle (EV) Isolation

After the KCTC13422BP strain of each of the transformed lactic acid bacteria strains obtained was statically cultured in MRS liquid medium at 37 ° C. for 16 hours, this was again inoculated into MRS liquid medium in a 2% volume and then statically cultured for 16 hours. The obtained culture was centrifuged at 5,000 xg for 15 minutes to obtain a supernatant from which lactic acid bacteria were removed, and then ultrafiltered using a molecular weight cut-off (MWCO) 100 kDa ultrafiltration membrane and concentrated 20 times. This concentrate was ultracentrifuged at 150,000 xg for 3 hours to obtain a precipitated pellet, which was then resuspended in PBS to obtain an EV solution. The size and number of EVs obtained were measured using a NanoSight NS300 (Malvern). The results are shown in FIG. 8 .

8 is a diagram showing the size and concentration distribution of EVs isolated from transformed lactic acid bacteria. In Fig. 8, the horizontal axis is the diameter, and the vertical axis is the concentration (particles/ml). In FIG. 8, the lactic acid bacteria used is KCTC13422BP strain, and the target protein is aFGF.

As shown in FIG. 8, 90% of EVs were distributed between 80 and 250 nm.

4. Confirmation of presence of target protein in EVs

Western blot was performed on the EV solution obtained in Section 3 to confirm whether the target protein was present in the EV. The EV was isolated from KCTC13422BP strain (hereinafter also referred to as LMT1-21) transformed with a vector in which genes encoding aFGF and TRX were cloned into pMT182-PR4. At this time, the signal peptide, that is, a sequence fused or not fused with the SP4 sequence was used as the gene.

Western blot was performed as follows. After adding 4x loading buffer (thermo) and 10x reducing agent (thermo) to 5 uL of the EV solution, electrophoresis was performed on a gel for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After transferring the protein of the gel to a nitrocellulose membrane, the membrane was blocked by incubation in a blocking solution 5% skim milk-containing TBST (Tris-buffered saline with tween 20) for 2 hours. After washing with TBST three times for 5 minutes, the membrane and the primary antibody were added to the blocking solution and incubated for 2 hours to induce antigen-antibody binding. After a TBST wash, secondary antibodies were added. After being allowed to stand for 1 hour, the amount and location of the target protein were confirmed using an enhanced electrochemical (ECL) system.

Figure 9 shows the results of Western blotting with respect to the EV solution. As shown in FIG. 9, aFGF and TRX were expressed in EVs isolated from LMT1-21 transformed with a vector obtained by cloning aFGF fused with a signal peptide and TRX into pMT182-PR4, and thus aFGF and TRX were expressed. can know that

FIG. 10 shows Western blotting performed on EVs isolated from LMT1-21 transformed with a recombinant vector, that is, a pMT182-PR4-aFGF vector containing a gene encoding pMT182-PR4 fused with a signal peptide gene or a gene encoding unfused aFGF. show the result. In FIG. 10, lane 1 shows EVs when the aFGF gene was expressed without a signal sequence, and lane 2 shows EVs when a gene encoding a protein fused with aFGF signal sequence was expressed.

5. derived from lactic acid bacteria Contains growth factors extracellular vesicle Confirmation of efficacy on cell proliferation

According to the method described in Section 3, extracellular vesicles isolated from 1 L lactic acid bacteria culture medium were suspended in 1 ml of PBS. 5,000 cells of the NIH3T3 cell line (or HaCat cells) in DMEM medium were seeded in each well of a 96-well plate, and then incubated at 37° C. for 48 hours. Thereafter, 20 uL of a solution containing extracellular follicles expressing the growth factor or control PBS was added. After culturing for 48 hours under the same conditions, 10 uL of Cell Counting Kit-8 (Dojindo) solution was added to each well. After 2 hours, absorbance was measured at 450 nm. NIH3T3 cells were used for aFGF, bFGF, and IGF, and HaCat cells were used for KGF, TGFa, and EGF.

11 is a view showing the effect on cell proliferation of growth factor-containing extracellular follicles isolated from lactic acid bacteria. In FIG. 11, LAB represents lactic acid bacterium (KCTC13422BP strain). As a result, the cell concentration of the target protein-containing extracellular vesicles increased in a dose-dependent manner. In FIG. 11, the horizontal axis represents the concentration (w/v) of the target protein contained in extracellular vesicles. The vertical axis shows the degree of cell proliferation of the used extracellular vesicle-containing solution as a percentage compared to the control group.

6. Efficacy of EVs containing growth factors: confirmation of IL-10 expression

A vector expressing IL-22 was constructed according to Sections 1 and 2, and the vector was transformed into LMT1-21. EVs were isolated from LMT1-21 transformed with the vector expressing IL-22 according to Section 3. By confirming whether these EVs promote IL-10 expression in cells, the presence or absence of IL-22 was indirectly estimated.

Specifically, after culturing the Colo205 cell line in a 96-well plate in RPMI medium for 48 hours, lactic acid bacteria expressing IL-22 and lactic acid bacteria not expressing the IL-22-derived extracellular follicles were isolated, and the extracellular follicles were 0.5 mg /mL were suspended in PBS, and they were added to the wells in 20 uL portions and further incubated for 6 hours. Thereafter, protein was extracted from the cell line, that is, cell lysis was obtained and lysate was obtained, and the expression level of IL-10 among them was compared.

12 is a photograph of Western blotting of cell-derived proteins contacted with LMT1-21-derived EVs. In FIG. 12 , lane 1 is PBS, lane 2 is LMT1-21-derived EV, and lane 3 is LMT1-21-derived EV expressing IL-22.

7. Fusion of lactic acid bacteria-derived extracellular vesicles and cells

Extracellular follicles were isolated from the untransformed lactic acid bacteria strain (KCTC13422BP) as described above. 1 ml of isolated extracellular follicles (0.5 mg/ml) was placed in CFSE (5 uM) solution at room temperature for 30 minutes. Thereafter, the remaining CFSE was removed from the solution using a PD-10 desalting column (GE) to obtain CFSE-labeled extracellular follicles. After culturing the NIH3T3 cell line in 0.25 mL of RPMI medium (Gibco) for 48 hours in the wells of a 96-well plate, 10 uL (red) or 20 uL (green) of CFSE-labeled extracellular follicles in PBS was added and further cultured for 24 hours. cultured. Then, the cells were washed with PBS. The remaining cells were passed through a flow cytometer and their fluorescence was measured. As a control, 20 ul of BSA 0.5 ug/ml was labeled with the same CFSE.

13 shows the result of observing the degree of fusion of CFSE-labeled extracellular follicles with cells through cell flow analysis. In FIG. 13, the negative control (left figure) was contacted with CFSE-labeled BSA and cells, and the experimental group (right) was treated with 10 uL (red) and 20 uL (green) of CFSE-labeled extracellular follicles. Results observed after contacting the cells are shown. As a result, as shown on the right side of FIG. 13 , the cells were stained with CFSE, confirming that the extracellular vesicles were fused with the cells and the components of the extracellular vesicles were introduced into the cells. NIH3T3 cells are a standard fibroblast cell line.

8. Confirmation of skin toxicity of lactic acid bacteria-derived extracellular vesicles

The toxicity of lactic acid bacteria-derived extracellular vesicles to the skin was measured through a toxicity test on artificial skin according to the OECD guidelines. Neoderm ^TM -ED (manufactured by Taego Science Co., Ltd.) was used as the artificial skin.

Extracellular follicles derived from LMT1-21, LMT1-9, or LMT1-46 were isolated. These extracellular vesicles were isolated as described in Section 2. Each of the isolated extracellular follicles and 30 uL of each of PBS as a control and 5% SDS as a positive control were applied to the Neoderm ^™ -ED artificial skin and incubated for 15 minutes. After washing the artificial skin with PBS, it was immersed in 2 ml assay medium (Taego Science) in a 12-well plate and further incubated for 42 hours.

The incubated artificial skin was removed, transferred to a 0.3% MTT solution (0.3 mg/ml), and incubated for 3 hours. Thereafter, the artificial skin was picked up again, and the tissue was separated using an 8 mm biopsy punch, and then placed in 500 ul of 0.04 N HCl-isopropanol and bleached for 4 hours. After measuring the absorbance at 570 nm, the viability (%) was obtained by comparing the absorbance of the control group in which the artificial skin was not reacted. As a result, if the measured viability was greater than the median value of the positive and negative control values, it was determined that there was no toxicity. Viability was calculated according to the following formula.

Viability = absorbance of test substance / absorbance of negative control X 100

14 is a diagram measuring the toxicity of lactic acid bacteria-derived extracellular follicles to the skin. 14, 1: negative control (PBS), 2: positive control (5% SDS), 3: LMT1-46, 4: LMT1-9, 5: LMT1-21 derived extracellular follicles are shown.

<110> Medytox Co., Ltd. <120> Recombinant microorganism comprising polynucleotide encoding fusion protein between signal peptide and target protein and use thereof <130> PN126717KR <160> 60 <170> KopatentIn 2.0 <210> 1 <211> 5778 <212> DNA <213> Artificial Sequence < 220> <223> pRS416 GPD vector <400> 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata 120 atttgggaat ttactctgtg tttattatt tttatgtttt gtatttggat tttagaaagt 180 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa 240 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata 300 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct 360 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata 420 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact 480 attttttctt taatttctt a ataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 720 attcaacatt tccgtgtcgc cctattccc ttttttgcgg cattttgcct tcctgttttt 780 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 840 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 900 cgttttccaa tgatgag cac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 960 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1020 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1080 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1140 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1200 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 1260 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1320 caacaattaa tagactggat ggaggcggat aaagttgca g gaccacttct gcgctcggcc 1380 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 1440 atcattgcag cactggggcc agatggtaag cctccccgta tcgtagttat ctacacgacg 1500 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 1560 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 1620 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 1680 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 1740 tcttcttgag atcctttttt tctgcgcgta atctgct gct tgcaaacaaa aaaaccaccg 1800 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 1860 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 1920 cacttcaagactgtagc accg cctaca tacctcgctc tgctaatcct gttaccagtg 1980 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2040 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2100 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2160 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg aga gcgcacg 2220 aggggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2280 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2340 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 2400 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 2460 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 2520 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 2580 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacct cact 2640 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg 2700 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt 2760 aaccctcact aaagggaaca aaagctggag ctcagtttat cattatcaat actcgccatt 2820 tcaaagaata cgtaaataat taatagtagt gattttccta actttattta gtcaaaaaat 2880 tagcctttta attctgctgt aacccgtaca tgcccaaaat agggggcggg ttacacagaa 2940 tatataacat cgtaggtgtc tgggtgaaca gtttattcct ggcatccact aaatataatg 3000 gagcccgctt tttaagctgg catccagaaa aaaaaagaat cccagcacca aaatattgtt 3060 ttcttcacca acc atcagtt cataggtcca ttctcttagc gcaactacag agaacagggg 3120 cacaaacagg caaaaaacgg gcacaacctc aatggagtga tgcaacctgc ctggagtaaa 3180 tgatgacaca aggcaattga cccacgcatg tatctatctc atttcttac accttctatt 3240 accttctgct ctctctgatt tggaaaaagc tgaaaaaaaa ggttgaaacc agttccctga 3300 aattattccc ctacttgact aataagtata taaagacggt aggtattgat tgtaattctg 3360 taaatctatt tcttaaactt cttaaattct acttttatag ttagtctttt ttttagtttt 3420 aaaacaccag aacttagttt cgacggattc tagaactagt ggatcccccg ggctgcagga 3480 attcgatatc aagcttatcg at accgtcga cctcgagtca tgtaattagt tatgtcacgc 3540 ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct 3600 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata 3660 tttcaaattt ttctt ttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa 3720 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg gtacccaatt 3780 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact 3840 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 3900 ggcgtaatag cgaagaggcc cg caccgatc gcccttccca acagttgcgc agcctgaatg 3960 gcgaatggcg cgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 4020 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 4080 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 4140 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 4200 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 4260 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 4320 cttttgattt ataagggatt t tgccgattt cggcctattg gttaaaaaat gagctgattt 4380 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttcc tgatgcggta 4440 ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt 4500 aaattgaagc tc taatttgt gagtttagta tacatgcatt tacttataat acagtttttt 4560 agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac 4620 cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag 4680 atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt 4740 catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct cttccac cca 4800 tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag 4860 tacccttagt atattctcca gtagataggg agcccttgca tgacaattct gctaacatca 4920 aaaggcctct aggttccttt gttacttctt ctgccgcctg cttca aaccg ctaacaatac 4980 ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtataacac 5040 ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga 5100 gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa 5160 aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa 5220 ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct 5280 tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt 5340 ccttatatgt agctttcgac atgatttatc ttcgt ttcct gcaggttttt gttctgtgca 5400 gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata 5460 ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa 5520 aaaaatttca aagaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaatt 5580 gaaaagctgt ggtatggtgc actctcagta caatctgctc tgatgccgca tag ttaagcc 5640 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 5700 ccgcttacag acaagctgg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 5760 catcaccgaa acgcgcga 5778 <210> 2 <211> 6606 <212> DNA < 213> Artificial Sequence <220> <223> pRS426 GPD vector <400> 2 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg 120 aggagtggca gcata tagaa cagctaaagg gtagtgctga aggaagcata cgataccccg 180 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac 240 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata 300 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc 360 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt 420 cctattctct agaaagtata ggaacttcag ag cgcttttg aaaaccaaaa gcgctctgaa 480 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga 540 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat 600 ccctatataa cctacc catc cacctttcgc tccttgaact tgcatctaaa ctcgacctct 660 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta 720 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag 780 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc 840 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc gggg atgcct 900 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt 960 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg 1020 acctacagtg caaaaagtta tcaagagact gcattataga gcgcaca aag gagaaaaaaa 1080 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa 1140 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta 1200 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca ttttgtttt 1260 acaaaaatga agcacagatt cttcgttgg t aaaatagcgc tttcgcgttg catttctgtt 1320 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt 1380 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcg c 1440 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 1500 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 1560 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 1620 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 1680 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaac g ttttccaatg 1740 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 1800 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1860 acagaaaagc atcttacgga tggcatgaca gtaagagaat ta tgcagtgc tgccataacc 1920 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1980 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2040 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 2100 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 2160 gactggatgg agg 2340 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 2400 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 2460 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 2520 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 2580 ccttttt ttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 2640 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 2700 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttca agaac 2760 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 2820 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2880 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2940 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3000 gcggacaggt atccggtaag cggcagg gtc ggaacaggag agcgcacgag ggagcttcca 3060 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 3120 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 3180 tttttac ggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 3240 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3300 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 3360 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3420 tggaaagcgg gcagtgagcg a gctggagct cagtttatca ttatcaatac tcgccatttc aaagaatacg 3660 taaataatta atagtagtga ttttcctaac tttatttagt caaaaaatta gccttttaat 3720 tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt acacagaata tataacatcg 3780 taggtgtctg ggtgaacagt tattcctgg catccactaa atataatgga gcccgctttt 3840 taagctggca tccagaaaaa aaaagaatcc cagcaccaaa atattgtttt cttcaccaac 3900 catcagttca taggtccatt ctcttagcgc aactacagag aacaggggca caaacaggca 3960 aaaaacgggc acaacctcaa tggagtgatg caacctgcct ggagtaaatg atgacacaag 4020 gcaattgacc cacgcatgta tctatctcat tttcttacac cttct attac cttctgctct 4080 ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag ttccctgaaa ttattcccct 4140 acttgactaa taagtatata aagacggtag gtattgattg taattctgta aatctatttc 4200 ttaaacttct taaattctac ttttatagtt agtctttttt ttagttttaa aacaccagaa 4260 cttagtttcg acggattcta gaactagtgg atcccccggg ctgcaggaat tcgatatcaa 43 20 gcttatcgat accgtcgacc tcgagtcatg taattagtta tgtcacgctt acattcacgc 4380 cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc 4440 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt 450 0 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag 4560 aaggttttgg gacgctcgaa ggctttaatt tgcggccggt acccaattcg ccctatagtg 4620 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 4680 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtatagc g 4740 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcg 4800 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 4860 ctacacttgc cagcgcccta gcgcccgctc ct ttcgcttt cttcccttcc tttctcgcca 4920 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 4980 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 5040 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 5100 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 516 0 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 5220 acgcgaattt taacaaaata ttaacgttta caatttcctg atgcggtatt ttctccttac 5280 gcatctgtgc ggtatttcac accgcatagg gtaataactg atataattaa attgaagctc 534 0 taatttgtga gtttagtata catgcattta cttataatac agttttttag ttttgctggc 5400 cgcatcttct caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag 5460 catcccttcc ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga 5520 ccacatcatc cacggttcta tactgttgac ccaatgcgtc tcccttgtca tctaaaccca 5580 caccgggtgt cataatca ac caatcgtaac cttcatctct tcccacccatg tctctttgag 5640 caataaagcc gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat 5700 attctccagt agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag 5760 gt tcctttgt tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca 5820 caccgtgtgc attcgtaatg tctgcccatt ctgctattct gtatacccc gcagagtact 5880 gcaatttgac tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt 5940 acttggcgga taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga 6000 tatccacatg tgtttttagt aaacaa attt tgggacctaa tgcttcaact aactccagta 6060 attccttggt ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga 6120 tattaaatag cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag 6180 ctttcgacat gatt tatctt cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga 6240 atactgggca atttcatgtt tcttcaacac tacatatgcg tatatatacc aatctaagtc 6300 tgtgctcctt ccttcgttct tccttctgtt cggagattac cgaatcaaaa aaatttcaaa 6360 gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga attgaattga aaagctgtgg 6420 tatggtgcac tctcagtaca at ctgctctg atgccgcata gttaagccag ccccgacacc 6480 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 6540 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 6600 gcgcga 660 6 <210> 3 <211> 644 <212> DNA <213> Artificial Sequence <220> <223> Promoter GPD <400> 3 tcattatcaa tactcgccat ttcaaagaat acgtaaataa ttaatagtag tgattttcct 60 aactttattt agtcaaaaaa ttagcctttt aattctgctg taacccgtac atgcccaaaa 120 taggggg cgg gttacacaga atatataaca tcgtaggtgt ctgggtgaac agttattcc 180 tggcatccac taaatataat ggagcccgct ttttaagctg gcatccagaa aaaaaaagaa 240 tcccagcacc aaaatattgt tttcttcacc aaccatcagt tcataggtcc attctcttag 300 cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg ggcacaacct caatggagtg 360 atgcaacctg cctggagtaa atgatgacac aaggcaattg acccacgcat gtatctatct 420 cattttctta caccttctat taccttctgc tctctctgat ttggaaaaag ctgaaaaaaaa 480 aggttgaaac cagttccctg aaattattcc cctacttgac taataagtat ataaagacgg 540 taggtattga ttgtaattct gtaaatctat ttcttaaact tcttaaattc tacttttata 600 gttagtcttt tttttagttt taaaacacca gaacttagtt tcga 644 <210> 4 <211 > 266 <212> DNA <213> Artificial Sequence <220> <223> Signal sequence of mating factor alpha <400> 4 atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct 60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120 tacttagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240 tctttggata aaagagaggc tgaagc 266 <210> 5 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> codon optimized EFG1 <400> 5 aattccgatt ccgagtgccc tctgtcacat gatggatatt gtctacatga cggagtgtgc 60 atgtatatag aagctctgga caagtatgcc tgtaactgtg ttgt cgggta tatcggcgag 120 agatgccaat acagggacct aaaatggtgg gaactaagg 159 < 210> 6 <211> 210 <212> DNA <213> Artificial Sequence <220> <223> codon optimized IGF1 <400> 6 ggccctgaga cattatgtgg tgcggagctt gtcgatgcat tacagttcgt atgcggagat 60 agaggcttct atttcaacaa acctacaggc tacggttcca gtt ccagaag ggcacctcaa 120 actggtatag ttgacgaatg ttgcttcaga agctgcgacc tgagaagact agaaatgtac 180 tgtgcgcccc tgaaaccagc caagagtgca 210 <210> 7 <211> 420 <212> DNA <213> Artificial Sequence <220> <223> codon optimized FGF1 <400> 7 tttaacttgc ctccggggaa ttacaagaag ccgaaattac tgtattgctc aaacggaggc 60 cacttcctga gaatccttcc cgacggtact gttgacggca cgagagaccg tagtgatcaa 120 cacatccagt tacaattgag cgccgagagc gtgggagaag tttacataaa gagcacagaa 180 actggccaat accttgcgat ggatacggac gggcttcttt atgggagcca gaccccaaac 240 gaggaatgct tatttcttga aaggctggag gagaatcatt acaatacata tattagtaaa 300 aaacatgcgg aaaagaattg gtttgtcggt ctgaaaaaga acggtagctg caaaagaggt 360 cccaggaccc attacgggca gaaggcgata ctatttctgc cgctacccgt ctcctccgac 420 420 <210> 8 <211> 438 <212> DNA <213> Artificial Sequence <220> <223> codon optimized FGF2 <400> 8 CCCCGCTAC CAGAGGACGGGGGAGCGGA GCGCCCCCCCCCCA gattttccgta cgggaagggta 120 gatgggagaagaagaagaag CGACCGCAC AGCTTCTTC AGAACGT T AATAGTACT TGGCGATGAA GGAGACGT 240 AGATTGCTGTG CCTCTAAATG TGTGACCGAC GAATGTTTTTTTTTTTCGAGTCC 300 AACAACTATATA acacatacag aagtcgtaaa tacacctcat ggtacgtggc gttgaaacgt 360 actggtcagt acaagcttgg ttctaaaaca ggaccaggtc aaaaagccat actttttcta 420 ccaatgtctg ccaagtcc 438 <210> 9 <211> 489 <212> DNA <213> Artificial Sequence <220> <223> codon optimized FGF7 <400> 9 tgcaacgaca tgactccaga gcagatggca accaacgtaa actgttcaag cccggagcgt 60 cacacaaggt cctatgatta tatggagggg ggggatattc gtgttaggag actattctgc 120 agaacacaat ggtatctgcg tattgataaa aggggcaagg tcaaaggaac tcaagaaatg 180 aaaaataact ataacattat ggagataaga acggtcgcgg tcgg gattgt tgcgattaaa 240 ggcgtggagt ccgaatttta ccttgccatg aataaggaag gaaaactgta cgccaagaag 300 gagtgcaacg aggattgtaa ctttaaggag ttgattttgg aaaaccatta caatacttat 360 gccagtgcaa agtggacgca taacgggggg gagatgttc g tcgccctgaa tcagaaaggt 420 atacctgttc gtggcaagaa gactaaaaaa gaacaaaaaa cagcacactt tcttccaatg 480 gcgatcact 489 <210> 10 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> codon optimized TGFalpha <400> 10 gtggtatctc attttaacga ctgccctgat tcacatactc aattttgttt ccacgggact 60 t gcaggttct tggtccaaga agataagccc gcgtgcgttt gccattcagg ttatgttggt 120 gcgaggtgtg aacacgctga cctgcttgct 150 <210> 11 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> codon optimized TRX1 gene <400> 11 atggtcaagc aaattgagag caaaacggcg ttccaggaag cactggacgc ggcggggac 60 aaacttgtag tagtggactt ctccgccaca tggtgcggtc catgcaaaat gatcaaaccg 120 ttcttccatt ccttgagcga aaagtacagc aacgtgatat ttcttgaagt ggatgtcgat 180 gactgtcaag acgttgcgtc cgagtgtgaa gttaaatgta tgccaacatt tcagttcttt 240 aagaaaggac agaaggtggg agagttcagc ggcgcaaata aagagaaatt agaggcaact 300 attaatgagc tagtg 315 < 210> 12 <211> 50 <212> PRT <213> Homo sapiens <400> 12 Val Val Ser His Phe Asn Asp Cys Pro Asp Ser His Thr Gln Phe Cys 1 5 10 15 Phe His Gly Thr Cys Arg Phe Leu Val Gln Glu Asp Lys Pro Ala Cys 20 25 30 Val Cys His Ser Gly Tyr Val Gly Ala Arg Cys Glu His Ala Asp Leu 35 40 45 Leu Ala 50 <210> 13 <211> 146 <212> PRT <213> Homo sapiens <400> 13 Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His 1 5 10 15 Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25 30 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp 35 40 45 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser 50 55 60 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly 65 70 75 80 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu 85 90 95 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr 100 105 110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser 115 120 125 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala 130 135 140 Lys Ser 145 <210> 14 <211> 53 <212> PRT <213> Homo sapiens <400> 14 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> 15 <211> 70 <212> PRT <213> Homo sapiens <400> 15 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 1 5 10 15 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 20 25 30 Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 35 40 45 Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 50 55 60 Lys Pro Ala Lys Ser Ala 65 70 <210> 16 <211> 163 < 212> PRT <213> Homo sapiens <400> 16 Cys Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser 1 5 10 15 Ser Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp 20 25 30 Ile Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile 35 40 45 Asp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr 50 55 60 Asn Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys 65 70 75 80 Gly Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu 85 90 95 Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile 100 105 110 Leu Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn 115 120 125 Gly Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg 130 135 140 Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met 145 150 155 160 Ala Ile Thr <210> 17 <211> 50 <212> PRT <213> Homo sapiens <400> 17 Val Val Ser His Phe Asn Asp Cys Pro Asp Ser His Thr Gln Phe Cys 1 5 10 15 Phe His Gly Thr Cys Arg Phe Leu Val Gln Glu Asp Lys Pro Ala Cys 20 25 30 Val Cys His Ser Gly Tyr Val Gly Ala Arg Cys Glu His Ala Asp Leu 35 40 45 Leu Ala 50 <210> 18 <211> 105 <212 > PRT <213> Homo sapiens <400> 18 Met Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp 1 5 10 15 Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys 20 25 30 Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys 35 40 45 Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp 50 55 60 Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe 65 70 75 80 Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys 85 90 95 Leu Glu Ala Thr Ile Asn Glu Leu Val 100 105 <210> 19 <211> 146 <212> PRT <213> Homo sapiens <400> 19 Leu Pro Val Asn Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln Gln 1 5 10 15 Pro Tyr Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser Leu 20 25 30 Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe Arg 35 40 45 Gly Val Ser Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val Leu Asn 50 55 60 Phe Thr Leu Glu Asp Val Leu Leu Pro Gln Ser Asp Arg Phe Gln Pro 65 70 75 80 Tyr Met Gln Glu Val Val Pro Phe Leu Thr Lys Leu Ser Asn Gln Leu 85 90 95 Ser Ser Cys His Ile Ser Gly Asp Asp Gln Asn Ile Gln Lys Asn Val 100 105 110 Arg Arg Leu Lys Glu Thr Val Lys Lys Leu Gly Glu Ser Gly Glu Ile 115 120 125 Lys Ala Ile Gly Glu Leu Asp Leu Leu Leu Phe Met Ser Leu Arg Asn Ala 130 135 140 Cys Val 145 <210> 20 <211> 4271 <212> DNA <213> Artificial Sequence <220> <223> pMT182-PR4 <400> 20 ggtacctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcgg cgagcg 60 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 120 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 180 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 240 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 300 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 360 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 420 cgctccaagc tgggctgtgt gcac gaaccc cccgttcagc ccgaccgctg cgccttatcc 480 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 540 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 600 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 660 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtag c 720 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 780 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 840 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 900 tttaaatcaa tctaaagtat atatgagtaa acttggtgaa ttcgcaaggt atagcccaat 960 atactttaact tcgtaaagtt tgagctctgc gaggcaaaaa cgtcagatat tgaatctaaa 1020 caagagtata ataaacacag ttacacaaat aaaaacaaaa ggagtagatc ttaaaaatgg 1080 aagcaaatcc aaagtggcaa gaaacaggaa gacaacaaaa agtaaa acaa cttgactatt 1140 cagaaacatt agcagtaaga atagacaatg tggaattata caactcagaa aagaaattat 1200 attatctagt aattcaaacc cttcttggga atccccaaaa aaatgaatgg cgtatccagg 1260 gacaaagtca aatattaaaa gaatcaaaac aaaaaataat tgt gtcataa aattattgca 1320 cattaaaagt cctaaaaatt attttgcatt aaagatttca aaactcattt aatgtaaaat 1380 aattttttct ttgataatcc catttagcaa caatctcgcg taccacttgg tcaacctttt 1440 gatcatcact atccgtatta atcaaatcac cattctcaac gtcttctaat tgcaactgct 1500 tgcgaatttc tttcagcaaa ccgccatagc taatttgccg ggaaccagcc aaagctcgtt 1560 ccaaatcatc aattacttgt aggtcttgtt cttgattatt agtcaaaata tctttagatt 1620 ttacctgata tttagccgtt tcttgagcac tagccaataa cgaattttta tggcgattca 1680 tattcggttt aactgcctca acattcacca ttggtacata agtcaatttc attgc ccgtt 1740 gccaaaaacc agtccattct acttgtgaaa tatagttatc agtcccctta aaataatggt 1800 tcttcacaaa aagcaaaaca tgcatatgat ggtggtacat tggctgaccc gcttcatgat 1860 taacagtcac ttcagtcgaa cgtacatagc ccaataaatt tttggcaact tttgtatact 1920 gaaaaatttt tgcaacagct cgtcccattt gacgtaactc actcttcaat tgatcaccag 1980 ttgtatt ctc aaccgtcaac gttaaaaata agaaccggcc cgtctttcgt tgtttaacag 2040 cttccgtcaa aatctgtgtt aactgattgg attgcttcat tgcccgccgc caattacata 2100 acggacacaa acgagaatgg caaaaccaag tctgcgccaa tttcttgtga ccatttttat 2 160 cttccacaaa acgcaaaact tcgccacatt ctttaactcg atgagctttc ttataatgca 2220 atatttgtaa atagtcacca tactgcaagt tctccaactt gcgctcccgc cacggccgaa 2280 ctttccctga ctttgaccga tcaaccaaga ctttttcatt agccaaaata aaaactcccc 2340 tcaccaacca cgtgagaaga gttaatctgt tatgtacttg cttcacttaa atcagtcaga 2400 aggcttgacg gcaa agggtt caagctttaa actatgtcta gtaaatccaa atacgatttt 2460 tacaagatta actcacgcgt tcgaggtcgg caaactttcg aagctcacgt gggttttttt 2520 tatatttatt ttataccaca ataatacgcc taaacccagt tgtgtcaagg gtttacctca 2580 cttt ttgaaa atgacgttgt ttctaatagt atcaagataa gaagaaaccg tcgaaaaaac 2640 gacggtttca aaccccaaaa agcagagaat tcggtacctg gagctgtaat ataaaaacct 2700 tcttcaacta acggggcagg ttagtgacat tagaaaaccg actgtaaaaa gtacagtcgg 2760 cattatctca tattataaaa gccagtcatt aggcctatct gacaattcct gaatagagtt 2820 cataaacaat cctgcatgat aaccatcaca aacagaatga tgtacctgta aagatagcgg 2880 taaatatatt gaattacctt tattaatgaa ttttcctgct gtaataatgg gtagaaggta 2940 attactatta ttattgatat ttaagttaaa cccagtaaat gaagtccatg gaataataga 3000 aagagaaaaa gcattttcag gtataggtgt tttggga aac aatttccccg aaccattata 3060 tttctctaca tcagaaaggt ataaatcata aaactctttg aagtcattct ttacaggagt 3120 ccaaatacca gagaatgttt tagatacacc atcaaaaatt gtataaagtg gctctaactt 3180 atcccaataa cctaactctc cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt 3240 tatcaccctt gtcactaaga aaataaatgc agggtaaaat ttata tcctt cttgttttat 3300 gtttcggtat aaaacactaa tatcaatttc tgtggttata ctaaaagtcg tttgttggtt 3360 caaataatga ttaaatatct cttttctctt ccaattgtct aaatcaattt tattaaagtt 3420 catttgatat gcctcctaaa tt tttatcta aagtgaattt aggaggctta cttgtctgct 3480 ttcttcatta gaatcaatcc ttttttaaaa gtcaatatta ctgtaacata aatatatatt acat cagtcg tgagagcatt 3720 gtgttgacag tgatcggcat cgtgttcggc tatctgctcg gcaatttgct gacagcctac 3780 attttgtatc aagccgaaac tgaggccgtg gtttttccac tcacgatcag cattgtcggc 3840 tacctcacgg ccacgttact catgtt ggcc ttcaccggcg tcgtcacctg gctcacgcat 3900 cgtcgactcc aacgggtgga catggtcgaa gccctgaaat caaacgaata acctacaatt 3960 ttgtcaggca gcgtcgtcac ggcgctgctt ttttcataca aaattcatca aaaattggga 4020 ttaaaaacgt tcatgatcgc aattttgaag cgcaaatgaa gattgagacc aactcctaac 4080 agtcctgtaa cgctgacgta acattgacac agtaaagtag cctttagtta atcaaatt 21 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide PR4 <400> 21 Met Lys Phe Asn Lys Val Met Ile Thr Leu Val Ala Ala Val Thr Leu 1 5 10 15 Ala Gly Ser Ala Ser Ala Val Thr Pro Val Phe Ala Asp Thr Ser 20 25 30 <210> 22 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide1 <400> 22 Met Lys Lys Leu Leu Ser Thr Val Leu Phe Ser Ala Val Ala Leu Ser 1 5 10 15 Ala Val Ala Leu Ser Lys Pro Ser His Val Ser Ala Ala Thr 20 25 30 <210> 23 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide2 <400> 23 Met Lys Lys Phe Leu Val Ser Val Gly Leu Leu Gly Met Leu Val Leu 1 5 10 15 Ser Thr Gly Ala Val Thr Ala His Ala Ala Asp 20 25 <210> 24 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide3 <400> 24 Met Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile Leu 1 5 10 15 Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala 20 25 <210> 25 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide4 <400> 25 Met Lys Asn Lys Leu Leu Met Ser Leu Val Leu Val Cys Ala Phe Leu 1 5 10 15 Gly Ile Ala Gly Ala His Thr Val His Ala Ala Asp 20 25 <210> 26 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide5 <400> 26 Met Lys Lys Thr Thr Leu Leu Leu Ser Thr Leu Phe Leu Gly Gly Thr 1 5 10 15 Leu Leu Ala Thr Thr Leu Ala Thr Pro Val Val Ala Asp Thr 20 25 30 <210> 27 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide1 <400> 27 Met Ile Leu His Thr Tyr Ile Ile Leu Ser Leu Leu Thr Ile Phe Pro 1 5 10 15 Lys Ala Ile Gly 20 <210> 28 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide2 <400> 28 Met Phe Ser Pro Ile Leu Ser Leu Glu Ile Ile Leu Ala Leu Ala Thr 1 5 10 15 Leu Gln Ser Val Phe Ala 20 <210> 29 <211> 40 <212> PRT < 213> Artificial Sequence <220> <223> Yeast Signal peptide3 <400> 29 Met Lys Ile Leu Ser Ala Leu Leu Leu Leu Phe Thr Leu Ala Phe Ala 1 5 10 15 Glu Val Ile Glu Leu Thr Asn Lys Asn Phe Asp Asp Val Val Leu Lys 20 25 30 Ser Gly Lys Tyr Thr Leu Val Lys 35 40 <210> 30 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide4 <400> 30 Met Lys Phe Ala Tyr Ser Leu Leu Leu Pro Leu Ala Gly Val Ser Ala 1 5 10 15 Ser Val Ile Asn Tyr Lys Arg Asp Gly Asp Ser Lys Ala Ile Thr Asn 20 25 30 Thr Thr Phe Ser Leu Asn Arg Pro 35 40 <210> 31 < 211> 40 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide4 <400> 31 Met Lys Ala Phe Thr Ser Leu Leu Cys Gly Leu Gly Leu Ser Thr Thr Thr 1 5 10 15 Leu Ala Lys Ala Ile Ser Leu Gln Arg Pro Leu Gly Leu Asp Lys Asp 20 25 30 Val Leu Leu Gln Ala Ala Glu Lys 35 40 <210> 32 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide5 <400> 32 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala <210> 33 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide7 <400> 33 Met Phe Tyr Asn Arg Trp Leu Gly Thr Trp Leu Ala Met Ser Ala Leu 1 5 10 15 Ile Arg Ile Ser Val Ser 20 <210> 34 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide8 <400> 34 Met Thr Leu Ser Phe Ala His Phe Thr Tyr Leu Phe Thr Ile Leu Leu 1 5 10 15 Gly Leu Thr Asn Ile Ala 20 <210> 35 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide9 <400> 35 Met Gln Leu Leu Arg Cys Phe Ser Ile Phe Ser Val Ile Ala Ser Val 1 5 10 15 Leu Ala <210> 36 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide10 <400> 36 Met Leu Lys Ser Ala Val Tyr Ser Ile Leu Ala Ala Ser Leu Val Asn 1 5 10 15 Ala <210> 37 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide11 <400> 37 Met Phe Thr Phe Leu Lys Ile Ile Leu Trp Leu Phe Ser Leu Ala Leu 1 5 10 15 Ala Ser Ala <210> 38 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide12 <400> 38 Met Phe Ala Phe Tyr Phe Leu Thr Ala Cys Ile Ser Leu Lys Gly Val 1 5 10 15 Phe Gly <210> 39 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide13 <400> 39 Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala 1 5 10 15 Ser Gln Val Leu Gly 20 <210> 40 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide14 <400> 40 Met Lys Leu Ser Val Leu Thr Phe Val Val Asp Ala Leu Leu Val Cys 1 5 10 15 Ser Ser Ile Val Asp Ala 20 <210> 41 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide15 <400> 41 Met Lys Leu Gln Leu Ala Ala Val Ala Thr Leu Ala Val Leu Thr Ser 1 5 10 15 Pro Ala Phe Gly 20 <210> 42 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide16 <400> 42 Met Phe Tyr Asn Arg Trp Leu Gly Thr Trp Leu Ala Met Ser Ala Leu 1 5 10 15 Ile Arg Ile Ser Val Ser 20 <210> 43 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide17 <400> 43 Met Leu Ser Phe Thr Thr Lys Asn Ser Phe Arg Leu Leu Leu Leu Ile 1 5 10 15 Leu Ser Cys Ile Ser Thr Ile Arg Ala 20 25 <210> 44 <211 > 20 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide18 <400> 44 Met Lys Phe Ser Thr Ala Leu Ser Val Ala Leu Phe Ala Leu Ala Lys 1 5 10 15 Met Val Ile Ala 20 < 210> 45 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide19 <400> 45 Met Lys Ile Phe Asn Thr Ile Gln Ser Val Leu Phe Ala Ala Phe Phe 1 5 10 15 Leu Lys Gln Gly Asn Cys Leu Ala 20 <210> 46 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide20\ <400> 46 Met Ser Leu Leu Tyr Ile Ile Leu Leu Phe Thr Gln Phe Leu Leu Leu 1 5 10 15 Pro Thr Asp Ala 20 <210> 47 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide21 <400> 47 Met Arg Phe Ser Thr Thr Leu Ala Thr Ala Ala Thr Ala Leu Phe Phe 1 5 10 15 Thr Ala Ser Gln Val Ser Ala 20 <210> 48 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide22 <400 > 48 Met Gln Arg Pro Phe Leu Leu Ala Tyr Leu Val Leu Ser Leu Leu Phe 1 5 10 15 Asn Ser Ala Leu Gly 20 <210> 49 <211> 32 <212> PRT <213> Artificial Sequence <220> <223 > Yeast Signal peptide23 <400> 49 Met Asn Leu Lys Gln Phe Thr Cys Leu Ser Cys Ala Gln Leu Leu Ala 1 5 10 15 Ile Leu Leu Phe Ile Phe Ala Phe Phe Pro Arg Lys Ile Val Leu Thr 20 25 30 <210> 50 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide24 <400> 50 Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys 1 5 10 15 Ile Ser Ala <210> 51 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide25 <400> 51 Met Lys Val Arg Lys Tyr Ile Thr Leu Cys Phe Trp Trp Ala Phe Ser 1 5 10 15 Thr Ser Ala <210> 52 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Yeast Signal peptide26 <400> 52 Met Ile Leu Leu His Phe Val Tyr Ser Leu Trp Ala Leu Leu Leu Ile 1 5 10 15 Pro Leu Thr Asn Ala 20 <210> 53 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 53 Met Lys Lys Lys Met Arg Leu Lys Val Leu Leu Ala Ser Thr Ala Thr 1 5 10 15 Ala Leu Leu Leu Leu Ser Gly 20 <210> 54 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 54 Met Gln Arg Lys Lys Lys Gly Leu Ser Ile Leu Leu Ala Gly Thr Val 1 5 10 15 Ala Leu Gly Ala Leu Ala Val Leu Pro Val Gly Glu Ile Gln Ala Lys 20 25 30 Ala <210> 55 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 55 Met Lys Lys Trp Phe Ile Ala Leu Ala Gly Leu Leu Leu Leu Thr Val Thr 1 5 10 15 Leu Ala Gly <210> 56 <211> 22 <212> PRT < 213> Artificial Sequence <220> <223> signal peptide <400> 56 Met Lys Lys Tyr Arg Lys Ile Leu Ala Met Leu Ala Val Leu Ala Ile 1 5 10 15 Val Leu Val Leu Ser Gly 20 <210> 57 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 57 Met Asn Asn Ala Leu Ser Phe Glu Gln Gln Phe Thr Asp Phe Ser Thr 1 5 10 15 Leu Ser Asp Ser Glu Leu Glu Ser Val Glu Gly Gly 20 25 <210> 58 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 58 Met Lys Ser Lys Lys Gly Leu Thr Leu Thr Ile Thr Leu Gly Thr Leu 1 5 10 15 Ala Leu Phe Leu Ser Gly 20 <210> 59 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 59 Met Asp Lys Ile Ile Lys Phe Gln Gly Ile Ser Asp Asp Gln Leu Asn 1 5 10 15 Ala Val Ile Gly Gly 20 <210> 60 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide <400> 60 Met Gln Ser Ser Leu Lys Lys Ser Leu Tyr Leu Gly Leu Ala Ala Leu 1 5 10 15 Ser Phe Ala Gly Val Ala Ala Val Ser Thr Thr Ala Ser Ala 20 25 30

Claims (13)

Isolating extracellular vesicles from recombinant microorganisms containing a polynucleotide encoding a heterologous target protein,
The microorganism is a lactic acid bacterium,
The target protein is loaded into the extracellular vesicle in an increased amount because a signal peptide is linked to it,
A method for producing extracellular vesicles loaded with increased amounts of a target protein. The method according to claim 1, wherein the lactic acid bacteria is a species belonging to a genus selected from the group consisting of genus 　Lactobacillus, Lactococcus, and 　Bifidobacterium. The method according to claim 2, wherein the lactic acid bacteria belong to a species selected from the group consisting of Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus paracasei. An extracellular vesicle loaded with an increased amount of a target protein obtained by the method of any one of claims 1 to 3. A cosmetic composition comprising the extracellular vesicle of claim 4 as an active ingredient, and a carrier. A pharmaceutical composition for preventing or treating inflammatory diseases, wounds, atopic dermatitis, psoriasis, or acne, comprising the extracellular vesicle of claim 4 as an active ingredient and a carrier.
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