WO2012157945A2 - Method for expressing a target protein at the surface of a vacuole, and drug delivery carrier - Google Patents

Abstract

The present invention relates to a method for expressing a target protein at the surface of a vacuole of a microorganism, and to a drug delivery carrier. More particularly, the present invention relates to a method comprising the steps of: (a) creating a vector including a nucleotide sequence encoding a fusion protein containing a target protein and a vacuole-targeting transmembrane protein; (b) transforming a microorganism containing a vacuole using said vector; and (c) expressing the fusion protein from the transformed microorganism. According to the present invention, the expressed fusion protein targets the vacuole and the target protein of the fusion protein is expressed at the surface of the vacuole. The present invention relates to a method for preparing a drug delivery carrier which is safe and easily applied in a clinical setting by fusion expressing the target protein and vacuole targeting transmembrane protein on the surface of a vacuole of a yeast. Further, the present invention relates to a method for preparing a drug delivery carrier which is cost-efficient when the method of the present invention is industrialized, using yeast having more economically advantageous culturing conditions.

Description

 【Specification】

 [Name of invention]

 Method of expressing target protein on drug surface and drug delivery system

 The present invention relates to a method and a drug carrier for displaying a target protein on the surface of a vacuole of a microorganism.

Background Art

 Cancer, tumors, tumor-related diseases and neoplastic diseases are very serious and often cause life-threatening conditions. Numerous studies have been conducted to identify effective therapeutic agents for treating the disease characterized by the growth of fast-proliferating cells. The therapeutic agent prolongs the survival of the patient, inhibits rapid—proliferative cell growth associated with tumorigenicity and is effective in neoplastic degeneration. In general, surgical surgery and radiation therapy are primarily performed for the treatment of cancer, and chemotherapy for certain cancers is effective for the treatment of cancer, even if survival is low.

 Currently, most cancer therapies, including chemotherapy, radiotherapy and immunotherapy, function by indirectly inducing apoptosis in cancer cells. Because of the drawbacks of normal apoptotic mechanisms, the inability of cancer cells not to perform apoptosis is associated with increased resistance to chemotherapy, radiotherapy or immunotherapy-induced apoptosis. Due to apoptosis defects, the primary or acquired resistance of human cancers of different origins to recent treatment protocols is a major problem in cancer therapy (Lowe, et al., Carcinogenesis, 21: 485 (2000); Nicholson, Nature, 407: 810 (2000)).

In an attempt to improve more effective cancer treatments, therapies through specific targeting of monoclonal antibodies were developed in the 1970s. At least 15 monoclonal antibodies are currently approved for human use. Antibodies based on interactions with cell-specific markers may exhibit direct cytotoxicity according to a variety of mechanisms, for example, blocking growth factor receptors (Baselga, J. and Mendelsohn, J. Pharmacol. Ther., 64:

127154 (1994)), induction of apoptosis (Trauth, BC, et al., Science, 245: 301305 (1989)), formation of anti-idiotype antibodies (Fagerberg, J., et al., Cancer Immunol Immunother, 38: 149159 (1994)) or antibody-dependent cytotoxicity (ant i body-dependent eel hilar cytotoxicity, ADCC; Steplewski, Z., et al., Science, 221: 865867 (1983)) and complement- Indirect effects such as mediated cytotoxicity (complement-dependent cytotoxicity, CDC; Bal lare, C., et al., Cancer Inin nol. Immunother., 41: 1522 (1995)). On the other hand, the cytotoxicity induced by the antibody is often insufficient, and can be used together with various toxic substances (eg, radioisotopes and chemotherpeutic drugs) to increase the therapeutic efficacy. Recently, humanized monoclonal antibody trastuzumab (Herceptin) targeting human epidermal growth factor 2 (HER2 / neu), which is overexpressed in about 20-30% of breast cancers It is used for the treatment of this breast cancer (W0 2006/098978 Al; W0 2009/042618 A1). However, trastuzumab as a breast cancer drug has many side effects despite its approval. For example, fever, nausea, vomiting, infusion reaction diarrhea, infection, increased cold, headache, fatigue, shortness of breath, rash, neutropenia, anemia and myalgia are typical side effects.

 Although monoclonal antibodies offer very attractive properties for tumor targeting, monoclonal antibodies not only have high cost, various side effects and limited effects, but also due to their large size, are difficult to remove in the blood, liver influx and low tissue penetration rate. To overcome this, methods of using antibody fragments such as F (ab), diabody, scFv, Fv and various single domain antibodies have been developed. Approaches using such antibody fragments have some limitations but are very useful. To address stability issues such as hydrolysis and shelf-life, efforts have been made based on other protein scaffolds such as trinectins, ant ical ins and af ibody ligands. It is becoming.

On the other hand, a widely used vaccine delivery system is a method using viral liposomes. Viral liposomes direct the delivery of nucleic acids to specific targets while at the same time being present in body fluids and cytoplasmic organelles. ,

Pu / k M ^'' / ϋ 03811

Can protect against degrading enzymes. Tumor antigens are expressed in the background of semi-normal cells, as opposed to tumor cells. Nucleic acid vaccination offers the possibility of molecular immuno-physiological manipulation of antigen expression, which can be a useful means of cancer vaccine design. Examples of successfully delivering foreign DNA molecules into liposomes into cells include Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982) and Nicolau et al., Methods Enzymol. 149: 157—176 (1987). On the other hand, Lipofectamine (Gibco BRL) is the most used reagent for the transformation of animal cells using liposomes. In addition, a method of delivering an anticancer agent to a cancer cell by using an anticancer agent in liposomes has been used. However, the transport of an anticancer agent using liposomes has a high possibility of causing leakage of anticancer agents.

 Yeast vacuoles are the major organelles present in yeast, similar to mammalian lysosomes. That is, the vacuole is surrounded by a phospholipid dichroic membrane and contains a hydrolase inside it along with various membrane proteins. The advantage of research on vacuoles in yeast is that it is easier to access various gene molecules than higher eukaryotes. In particular, protein targeting studies are active.

 In the present invention, in order to effectively overcome the disadvantages of the delivery of the conventional liposome-based chemotherapy, the yeast-derived vacu-based drug delivery system for chemotherapy was developed. Numerous attempts at this study focus on treating a broader spectrum of neoplastic diseases.

As described above, since the conventional cancer treatment has toxicity and limitations, it is urgently required to develop more effective cancer treatment means. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained. / iu (mi. / υ υ

[Detailed Description of the Invention]

 [Technical problem]

 The present inventors have tried to develop a drug carrier that can be manufactured more safely and easily in the human body as a drug carrier of a protein or peptide. As a result, it has been found that vacuoles of microorganisms can be used as drug carriers of proteins or peptides, and that the vacuole drug carriers can be easily prepared, thereby completing the present invention.

 Accordingly, it is an object of the present invention to provide a method for displaying a protein of interest on the surface of a vacuole of a microorganism.

 Another object of the present invention is to provide a method for producing a drug delivery system. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

Technical Solution

 According to one aspect of the invention the invention provides a method for displaying a target protein on the vacuoles. Surface of a microorganism comprising the following steps:

 (a) constructing a vector comprising a nucleotide sequence encoding a fusion protein comprising a protein of interest and a vacuole targeting transmembrane protein;

 (b) transforming the microorganism comprising the vacuole using the vector; And

(c) expressing the fusion protein in the transformed microorganism, wherein the expressed fusion protein is targeted to a vacuole and the target protein of the fusion protein is displayed on the surface of the vacuole. The present inventors have tried to develop a drug carrier that can be manufactured more safely and easily in the human body as a drug carrier of a protein or peptide. As a result, it was found that vacuoles of microorganisms can be used as drug carriers of proteins or peptides, and that vacuole drug carriers can be easily prepared. It was clarified. The method for displaying the target protein on the surface of the vacuole of the microorganism of the present invention will be described in detail by dividing each step as follows:

Step (a): Construction of a vector encoding a fusion protein comprising a target protein and a vacuole targeting membrane transmembrane protein

 In the present invention the vector can typically be constructed as an expression vector.

The vector is a replicon, such as a plasmid, phage or cosmid, to which another DNA fragment is attached resulting in replication of the attached fragment. The term 'Replicon ¹ ' refers to all genetic factors (eg, plasmids, chromosomes and viruses, etc.) that act as autologous units of DNA replication in vivo.

 As used herein, an 'expression vector' refers to a DNA molecule containing a gene expressed in a host cell. Typically, expression of the gene is placed under the control of certain regulatory elements, including promoters and / or enhancers. The gene to be expressed is operatively linked to a regulatory element. The expression vector produced in the present invention is constructed to express a desired gene in the host cell. In general, expression vectors include expression constructs of promoter-gene-transcriptional sequences.

 For example, if the host cell is a yeast, the promoters available in the expression construct are GAL10 promoter, GAL1 promoter, ADH1 promoter, ADH2 promoter, PH05 promoter, GAL1-10 promoter, TDH3 promoter, TDH2 promoter, TDH1 promoter, PGK promoter, PYK promoter, EN0 promoter and TPI promoter, but are not limited to these.

 Transcription termination sequences available in the expression construct include, but are not limited to, ADH1 terminator, CYC1 terminator, GAL10 terminator, PGK terminator, PH05 terminator, EN0 terminator and TPI terminator.

The expression vector produced in the present invention includes an origin of replication that operates in the host cell. For example, if the host cell is yeast, the origin of replication included in the expression vector is a two plasmid replication system and an autonomous ARS. replicating sequences), but is not limited to such. remind

Examples of ARS include, but are not limited to, ARS1 and ARS3. In addition, the expression vector may further comprise centromeric sequences (CEN), which provide mitotic and mitotic stability, for example CEN3, CEN4 and CEN11.

 The expression vector produced in the present invention may include a selection marker to facilitate the screening operation. For example, when the host cell is yeast, the selection markers include auxotrophic selection markers such as UAR3, LEU2, HI S3, TRP1, ADE2 and LYS2 or drug resistance selection markers such as CAN1 and CYH2.

 In the preparation of the vector, the expression construct is constructed to express the fusion protein of the protein of interest and vacuole targeting transmembrane protein.

 The nucleotide sequence encoding the vacuole targeting membrane transmembrane protein used in the present invention is for various vacuole targeting membrane transmembrane proteins, and preferably includes PH08 protein, CPS1 protein, VAM3 protein, NYV1 protein, VPH1 protein and the like.

 Most preferably, the vacuole targeting membrane transmembrane protein is PH08 protein.

 According to a preferred embodiment of the present invention, the target protein or protein drug is bound to the N-terminus of the vacuole targeting membrane transmembrane protein. The protein of interest and the vacuole targeting membrane transmembrane protein may be linked directly or indirectly (eg, by linker linkage). Preferably, the target protein and vacuole targeting transmembrane protein of the fusion protein are linked by a linker.

As used herein, the term "linker" includes any peptide linker known in the art used as a linker. Preferred peptide linkers include Gly, Asn and Ser residues. Other neutral amino acids such as Thr and Ala can also be included in the linker sequence. Suitable amino acid sequences for linkers are described in Maratea et al. , Gene 40: 39-46 (1985); Murphy et al. , Proc. Natl. Acad Sci. USA 83: 8258-8562 (1986); US Patent Nos. 4, 935, 233, 4, 751, 180, and 5,990, 275. The linker sequence is 1-50 amino acid residues Can be configured. Exemplary linker sequences include "Gly Gly Gly Gly Gly Gly Gly Ser", "Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser", "Gly Ser" Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly "," Gly Ser Thr Ser Gly Lys Pro Ser Glu Gly Lys Gly "or" Gly Gly Gly Gly Gly Gly Ser ".

Protein or peptide displayed on the vacuole surface in the present invention includes any protein or peptide. For example, proteins or peptides displayed on the vacuole surface by the present invention are hormones, hormone analogs, enzymes, inhibitors, signaling proteins or parts thereof, antibodies or parts thereof, short chain antibodies, affibodies, peptide aptamers, aptides Binding proteins, or binding domains thereof, antigens, adhesion proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators and blood coagulation factors. More specifically, the protein or peptide carried by the drug carrier of the present invention is insulin, IGF-1 (.insul in-like growth factor 1), growth hormone, erythropoietin, G-CSFs (granulocyte-colony) stimulating factors), GM-CSFs (gr anu 1 ocyt e / macr ophage-co 1 ony stimulating factors), interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin — 3, interleukin-4, interleukin- 6, interleukin-2, epidermal growth factors (EGFs), calcitonin (calcitonin), adrenocort icotropic hormone (ACTH), tumor necrosis factor (TNF), atobisban, buserel in, buseroel in Cetrorelix, deslorelin, desmopressin, dynorphin A (1-13), elcatonin, eleidosin, epifiva Tide (eptif ibatide), GHRH-I I (growth hormone releasing hormone— II), gonadorerin (g onadorel in, goserelin, hystrelin, leuprorel in, lypressin, octreotide, oxytocin, pitressin, Secretin, sincal ide, terripressin, thymopentin, thymoosine α 1, triptorelin, bivaludine irudin) ， Carbetocin, cyclosporin, exedine, lanreotide, LHRH (luteinizing hormone-releasing hormone), nafarelin ᅳ parathyroid hormone, praml int ide, T-20 (enfuvirtide), thymal fasin, ziconotide, antibodies, antigens, aphibodies, peptide aptamers and aptides.

 The aptide means a peptide material developed and patented by the inventors, and means the BPB laptide disclosed in the inventor's patent application WO 2010/047515. The BPB has been renamed Aptide. The disclosure of WO 2010/047515 is incorporated herein by reference. Aptide is a peptide substance that binds to a specific target, similar to conventional peptide aptamers, and has structural features that are largely different from conventional peptide aptamers. Step (b): Microbial Transformation

As used herein, the term "microorganism" includes all microorganisms including vacuoles. Vacuoles are giant organelles wrapped in membranes, and, like cell walls and cell contents, belong to the posterior form. Thickness refers to a substance that is secondary to protoplasm. Vacuoles are present in bacteria, fungi, plant cells and animal cells belonging to the genus Thioploca, Beggiatoa and Thiomargari ta and serve as storage organelles. Yeast vacuoles serve to store amino acids and break down toxicity. Yeast vacuoles also have a dynamic structure that can change its form quickly. Vacuoles are involved in homeostasis of cell pH, concentration of ions, osmotic control, storage and digestion of polyphosphates and amino acids. Toxic ions such as strontium (Sr ²⁺ ), cobalt (Co ²⁺ ) and lead n (Pd ²⁺ ) are transported to the vacuoles and degrade.

 Preferably the microorganism comprising the vacuole is a fungus or bacterium (eg Thioploca, Beggiatoa and Thiomargari ta) ^, more preferably the microorganism comprising the vacuole is a fungus, most preferably the fungus is a yeast.

 The term “yeast” refers to Saccharomyces, Ski 2: City—Scaromyces, Schizosaccharomyces,

Sporobolo yces, Torulopsis, Tre i chosporon, Wickerhamia, Ashbya, Blastomyces, Candida, Citeromyces, Crebrothecium ), Cryptococcus, Debaryomyces, Endomycops is, Geotri ri chum, Hansemila, Kloeckera, Lipomyses (Lipomyces), Pichia, Rhodospor i di or Rhodotomla genus, and more preferably Saccharomyces and Schizocarcinaceae Belonging to the yeast, most preferably Saccharomyces cerevisiae, ski irradiated caromyces ^ / yi ᅳ The term "transformation" refers to a method of transporting the produced vector into the microorganism, Cells that If the nuclear cells, CaCl ₂ method (Cohen, SN et al, Proc Natl Acac Sci USA, 9:..... 2110- 2114 (1973))., A method (Cohen, SN et al, Proc . Natl. Acad. Sci. USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al. , Nucleic Acids Res., 16: 6127-6145 (1988)). If the cell to be transformed is a eukaryotic cell, micro-injection (Capecchi, MR, Cell, 22: 479 (1980)), calm phosphate precipitation (Graham, FL et al., Virology, 52: 456 (1973)) , Electroporation (Neumann, E. et al:, EMB0 J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE Textlan treatment (Gopal, Mol. Cell. Biol., 5: 1188-1190 (1985)), and Gene Bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 ( 1990), etc., but is not limited thereto. For transformation of fungi such as yeast, Lithium acetate (RD Gietz, Yeast 11, 355360 (1995)) and heat shock (Keisuke Matsuura, Journal of Bioscience and Bioengineer ing, Vol. 100, 5; 538 5 544) (2005)) and electroporation (Nina SkoluckaAsian, Pacific Journal of Tropical Biomedicine, 94-98 (2011)). Step (c): Expression of Fusion Proteins After transformation, the fusion protein is expressed in the transformed microorganism. The expressed fusion protein is targeted to the vacuole and the target protein of the fusion protein is displayed on the surface of the vacuole.

 Expression of the fusion protein is carried out by culturing the transformed microorganism (eg, yeast). In addition, when the expression vector promoter is an inducible promoter, an inducer suitable for a medium is added to induce the expression of the fusion protein.

 According to a preferred embodiment of the present invention, the fusion protein formed by expression is targeted in microbial (yeast) cells so that the target protein is displayed on the vacuole surface of the microorganism, and the protein drug has a topology of the vacuole membrane and its structure directed toward the inside. Has

 According to a preferred embodiment of the present invention, after step (c) of the present invention, the method further comprises the step of separating the solution on which the fusion protein is exhibited. According to another aspect of the present invention, the present invention provides a method for preparing a drug delivery system comprising the following steps:

 (a) constructing a vector comprising a nucleotide sequence encoding a fusion protein comprising a protein drug and a vacuol targeting transmembrane protein;

 (b) transforming the microorganism (eg, yeast) containing the vacuole using the vector; And

 (c) expressing the fusion protein in the transformed microorganism (eg, yeast), wherein the expressed fusion protein is targeted to a vacuole and the protein drug of the fusion protein is displayed on the surface of the vacuole.

 The drug delivery method of the present invention is almost the same as the method for displaying the target protein on the surface of the vacuole described above. Therefore, the contents common between the two are omitted in order to avoid excessive complexity of the present specification. According to another aspect of the present invention, there is provided a drug delivery agent that is bound to a vacuole targeting transmembrane protein and comprises a protein drug displayed on the surface of the vacuole by the vacuole targeting transmembrane protein.

The drug carrier of the present invention may be prepared by the above-described method of the present invention. Therefore, the contents common between the two are omitted in order to avoid excessive complexity of the present specification.

 According to a preferred embodiment of the present invention, the vacuole targeting membrane transmembrane protein is PH08 protein or CPS1 protein, NYV1 protein, VAM3 protein, VPH1 protein and the like, most preferably PH08 protein.

 According to a preferred embodiment of the invention, the protein drug is bound to the N-terminus of the vacuole targeting membrane transmembrane protein.

 According to a preferred embodiment of the invention, the protein drug and vacuole targeting membrane transit protein are linked by a linker.

 According to a preferred embodiment of the present invention, the desired protein or protein drug displayed on the surface of the vacuole has a topology of the structure facing outward of the vacuole membrane.

 According to a preferred embodiment of the present invention, the vacuoles are vacuoles of fungi or bacteria, more preferably vacuoles of fungi, most preferably vacuoles of yeast.

Advantageous Effects

 The features and advantages of the present invention are summarized as follows:

 (a) The present invention can provide a drug carrier that is safe for clinical application by fusion-expressing a target protein and a vacuole targeting transmembrane protein on the vacuole surface of yeast.

 (b) The present invention can easily prepare a drug carrier that delivers a variety of proteins or temide drugs using commonly known yeast.

 (c) The present invention can provide a drug delivery agent cost-effectively when the present invention is industrialized by using yeast having more economical culture conditions.

[Brief Description of Drawings]

 FIG. 1 shows a schematic of a vector encoding a fusion protein comprising a target protein and a vacuole targeting transmembrane protein.

Figure 2 confirms the expression of Affibody _HER2- PH08 fusion protein in yeast Immunoblotting results are shown. Anti-PH08 (ALP, alkaline phosphatase) antibody and anti-myc antibody were used to express Affibody 瞧_2- PH08 fusion protein.

Confirmed.

Figure 3 shows the expression of Mfibody _HER2- PH08 fusion protein in isolated vacuoles

The results confirmed by immunoblotting using anti-PH08 antibody and anti-myc antibody

Shows.

Figure 4 shows the morphological size of the Affibody _HER 2 ᅳ vacuole (targeting vacuole)

With electrophoretic optical scattering spectrophotometer (ELS—8000, Otsuka Electronics)

The results showed. The distribution of the average 315.6 ^η πι is shown.

 Figure 5 shows the shape of Affibody 腿 2-vacuoles TEM

 Microscopy, Philips Electronic Instrument Corp., Mahwah, NJ)

It is the result observed using. As a result of the measurement, it has a spherical shape of 150-210rai

Shows that there is.

6 is Mfibody _HER2 - _HER2 binding of the affibody _HER2 expression in the vacuole

The results of the experiment (ELISA, enzyme-1 i nked immunosorbent assay) are shown.

Compared with the negative control group, Affibody _HE R2-vessels bind 19 times more well.

Confirmed.

Figure 7 is the result of measuring the affinity for _HER2 of Affibody _HER2 -vesicles.

Affibody 謹 一 vacuoles were detected using SPR (surface p 1 asmon resonance, BIAcore X).

The result is 3.57 times higher than the negative control group.

8 is a targeting experiment for HER2 expressing cells of Affibody 隱₂ -vessels

Show results. Af f ibody _HER2 -Valve 30 in SK0V-3 cells expressing _HER2

Treatment with / zg shows confocal microscopy.

9 is a targeting experiment for HER2-expressing cells of Affibody _HER2 -vessels

Show results. Negative control group 30 was applied to SK0V-3 cells expressing HER2.

The treatment shows confocal microscopy results. In comparison with FIG. 8

Affibody _HER2 — shows no fluorescence of vacuoles.

10 is a targeting experiment on HER2 non-expressing cells of Mfibody _HER2 -vessels.

Show results. HeLa cells that do not express _HER2 are treated with Af f ibody _HER2 -Valve 30 ¬ and show confocal microscopy results.

11 is a targeting experiment for HER2 non-expressing cells of Affibody _HER2 -vesicles Show results. HeLa cells not expressing HER2 were treated with negative control group 30 / zg to show confocal microscopy results. Compared with FIG. 10, there is no fluorescence signal of Affibody 隱₂ -vessel. [Specific contents for implementation of the invention]

 Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention more specifically, and the scope of the present invention according to the gist of the present invention-it is obvious to those skilled in the art that they are not limited by these examples. something to do. Example

 Throughout this specification, unless otherwise indicated, “%” used to indicate the concentration of a particular substance is solid / solid as (weight / weight), solid / liquid as (weight / volume)%, and liquid / Liquid is (volume / volume)%. Example 1 Production of Gamma Aminobutyl Acid from Glutamic Acid or Glutamate

 1-1. Production of Gamma Aminobutyl Acid from Glutamic Acid Using Various Whole Cell Catalysts Treated with Organic Solvents

 Cells containing glutamic acid decarboxylase, E. coJi BL2KDE3),

E. col i JM101, Lactobaci 1 his paracasei and Lactobaci 1 lus cwj¾7 / 7 ^' s (KCTC) were recovered by incubation and divided into organic solvent treatment groups and controls. In the case of the organic solvent treatment group, the recovered cells were treated with 0.5% (v / v) of toluene and then treated at 30 ^° C. and 200 rpm for 10 minutes. After stirring to destroy the selective permeability of the microorganisms, they were suspended in sterile water. The control group was immediately suspended in sterile water without any special treatment. To the microbial suspension prepared above, 40 μΜ of pyridoxal-5_phosphate (PLP, Sigma Aldrich) was added, and 33% glutamic acid (target company) was added to measure gamma aminobutyl acid production rate. To compare the experimental results, the production rate value of the experimental group without organic solvent was divided by the production rate value of the experimental group without organic solvent. Experimental results, as shown in Figure 1 organic solvent, toluene The treated group was found to have improved the production rate of gamma aminobutyl acid about 1.2-5.5 times than the untreated group.

1-2. Production of Gamma Aminobutyl Acid from Whole Cell Catalysts and Glutamate According to Organic Solvents

After cultivating the bacteria ^ ( _^ Escherichia coH) containing glutamic acid decarboxylase and dilution, the organic solvent was used to destroy the permeability of the microorganisms, and then the production rate of gamma aminobutyl acid from sodium glutamate (Sigma Aldrich) was increased. Analyzed. The organic solvents used were all hydrophobic organic solvents, such as toluene, chloroform, xylene and benzene. Treatment conditions were treated 0.5% (ν / ν) of each organic solvent in the microbial suspension and stirred for 10 minutes at 3 (rC, 200 rpm condition. PLP 40 μΜ was added to each prepared microbial suspension, sodium glutamate The production rate of gamma aminobutyric acid was compared by adding 1% (ν / ν), and the results showed that the production rate of gammaaminobutyl acid increased about 5-8 times after hydrophobic organic solvent treatment as shown in Fig. 2.

1-3. Increased GABA Production Activity of Cells by Organic Solvent Treatment

The cells were recovered after culturing the cells (Escherichia coii) containing glutamic acid decarboxylase. The recovered cells were washed once with distilled water and stirred with an organic solvent to destroy the selective permeability. The organic solvent used at this time used toluene showing the fastest gamma amino butyric acid production rate in Singye Example 1-2. The experimental group treated with toluene and the untreated control group were divided into two groups. The experimental group treated with toluene was added 0.5% (v / v) of toluene to the microbial suspension, followed by stirring at 37 ^° C and 150 rpm for 10 minutes. D-. After stirring, the cells were washed once with distilled water. The prepared cells were suspended in acetate buffer solution (pH 4.6, 200 mM), and then added to PLP 0.04 ηιΜ and 1% sodium glutamate (ν / ν) to measure the GABA production rate. As a result, the GABA production activity of the toluene-treated cells was 5.72 μιηοΐ GABA / mg dcw / min, and the activity of the untreated cells was 0.75 umol GABA / mg dcw / min. 1-4. Of buffer solution. Improving the production rate of gamma aminobutyl acid according to pH Cultivation of Escherichia coli containing glutamic acid decarboxylase and treatment of the organic solvent, toluene, using the same method as described in Example 3 The treated cells were then suspended in each complete solution and sterile water. The complete solution used here had a concentration of 100 mM and a pH of 4.5, 6.0, 7.0 and 8.0, respectively. The preparation of a complete solution having a pH of 4.5 was performed using acetic acid, and the preparation of a buffer solution having a pH of 6.0, 7.0, and 8.0 was performed using phosphoric acid. PLP 0.04 mM-added amount was added to the suspension solution of the whole cell catalyst prepared as described above, and reaction was initiated by adding 10 3/4 glutamic acid. Since the initial production rate of gamma aminobutyl acid was measured to analyze the change in gamma aminobutyl acid production rate according to pH-. As a result, as shown in Figure 3 using a buffer solution of pH 6, when the initial pH was 3.9 the reaction rate was the maximum, the value was 116 g GABA / L / h.

1-5. Production of GABA Using Cells Treated with Organic Solvents

The cells were recovered by culturing Escherichia coli containing glutamic acid decarboxylase. At this time, the amount of dry cells recovered was 3.6 g. The cells were suspended in sterile water, toluene 0.5¾ (v / v) was added and stirred at 37 ^° C for 10 minutes. Toluene processor ^ᅵ After centrifugation to recover the cells, and washed once more with distilled water. The recovered cells were suspended in 2 L of a phosphate complete solution (pH 6.0, 100 mM), and then added to the semi-acupuncture vessel. The reaction was started by adding 0.04 mM PLP, 1 kg of glutamic acid and 50 ppm of an antifoaming agent (Polyoxyalkylene Glycol). Reaction conditions were 30 ^° C, 200 rpm-. At the start of the reaction, the pH was 4.5 and the concentration of residual glutamic acid was reduced to less than 1 wt% after 8 hrs, at which time the pH was 5.8. The hydrochloric acid solution was added to reduce the pH to 5.5, and then further reacted for 1 hr to convert all remaining glutamic acid to GABA. The total weight of the resulting GABA was 690 g, the molar conversion was 98% and the time taken was 9 hr in total.

1-6. Production of high concentration GABA using organic solvent treated cells After incubating Escherichia coli containing glutamic acid decarboxylase, 0.25% (v / v) of toluene was added to the culture tank and stirred at 30 ^° C. for 10 minutes. After stirring, the microorganisms were recovered by centrifugation, and the weight of the dried cells was about 50 g. The microorganisms were suspended in a buffer solution or distilled water and then added to the reactor, and the reaction was started by adding 0.04 mM PLP, 8 kg of glutamic acid and 500 ppm of an antifoaming agent (Polyoxyalkylene Glycol). The reaction conditions were 30 ^° C, 200 rpm, and no other elements were controlled. The pH at the start of the reaction was 4.0, and the residual glutamic acid concentration decreased to less than 1 wt% after 10 hr of the start of the reaction, at which time the _P H was 5.87. Then, the pH was reduced to 5.6 by adding hydrochloric acid solution, followed by further 2 hr reaction to convert all remaining glutamic acid to GABA (FIG. 4). The concentration of produced GABA was 34 wt%, the molar conversion was 98%, and the total time required was 12 hr. 1-7. GABA Production Using Post-Recovered Cells

 In the same manner as in Example 1 to 2, 3.6 g of microorganism was treated with 0.5% (v / v) of toluene, followed by GABA production reaction using 0.5 kg of glutamic acid as a substrate. 9 hours after the reaction was terminated, microorganisms were recovered by centrifugation at 4000 rpm and 10 min. The microorganisms were recovered by distilled water as in Example 1, resuspended in acetic acid buffer (pH 4.6, 200 mM), and then PLP 0.04. The addition of mM and sodium glutamate 1% (v / v) was used to measure GABA production. The value was 4.80 μηιοΐ GABA / mg dcw / min. After separating and recovering the cells used for the GABA conversion reaction, 2 g of the recovered cells were added to a reaction vessel containing freshly prepared cells, and then the conversion was performed when the same reaction as in Example 1-2 was performed. It was almost the same as the secondary reaction and the total reaction time was reduced to 6 hr. Example 2: Obtaining Gamma Aminobutyl Acid from the Medium

The cells of the culture medium of Example 1 were removed, followed by a decolorization process after heat treatment. The cells were separated by centrifugation and the heat treated culture was decolorized with activated carbon using a stirrer. Discoloration To the culture was added 1.0-15.0% (activated carbon weight / gammaaminobutyl acid heavy ring ^:) activated carbon. Denatured protein produced after heat treatment was filtered off with activated carbon, concentrated and used for 2-pyrrolidone synthesis. As a result, as the amount of activated carbon increased, the color of the culture tended to become transparent.Table 1 shows the activated carbon used when bleaching with activated carbon in the culture medium with an initial concentration of 30.0% (weight of gammaaminobutyl acid / culture volume). The recovery rate of pyridone synthesis was shown as the concentration of activated carbon was increased. As the concentration of activated carbon was increased, the recovery rate was decreased by increasing the loss rate of GABA. Pyrrolidone synthesis was carried out according to the method described in Example 3-1 below.

Table 1

 To minimize the loss of gamma aminobutyl acid (4-aminobutyl acid) and most effectively remove the impurity pigments present in the culture,

(Weight / weight) It turned out that it is preferable to use activated carbon. Example 3: Preparation of 2 Papyridone from Gamma Aminobutyl Acid

Experiment ^ 1: Analysis of conversion of gamma aminobutyric acid according to reaction conditions The inventors made various experiments from the fact that gammaaminobutyl acid (4-aminobutyl acid) was converted to 2-pyridone and water at a melting point of 202 ^° C. As a result, 4-aminobutyl acid began to dissolve at 118t 120 ^° C in the presence of 2-pyrrolidone, at which time the dissolved solution was converted to 2-pyrrolidone and water. In addition, if the water produced during the reaction was removed under reduced pressure (10-110 mmHg), the reaction time was shortened and the conversion rate of 4-aminobutyl acid to 2 으로 pyridone was also increased. We also found that time was shortened (Table 2–4). Table 2-4 below shows a 1: 1 mixing of 4-aminobutyl acid and 2—pyridone by weight ratio at 120 ^° C (Table 2), 130 ^° C (Table 3) and 140 ^° C (Table 4), respectively. This is the result of checking the residual amount (%) of 4-aminobutyl acid by time under atmospheric pressure and reduced pressure while reacting. 4- The residual amount of aminobutyl acid was analyzed by HPLC (Hewlett Packard 1050 series, Hewlett Packard). There was no side reaction in the reaction, and 4-aminobutyl acid decreased and 2-pyrrolidone increased in the reaction solution.

 Table 2

 Table 3

 Table 4

3-1. Preparation of 2 \ Pyrlidone from 4-Iaminobutyl Acid (Method 1)

Into a 2 L reactor equipped with each distillation apparatus, 500 g of 2 k Plyridone (subject) was added and stirred. 600 g of 4-aminobutyl acid (in phase) was added thereto. The temperature was increased to 135 ^° C.-145 ^° C. under reduced pressure (60-80 mmHg), followed by stirring. The water produced during the reaction was removed through a distillation apparatus under reduced pressure, and when the reaction solution became transparent, the reaction was terminated, so that the residual water remaining in the reaction liquid under reduced pressure (10-20 mmHg) was gradually increased while increasing the vacuum degree. Was generated after 2-pyrrolidone was collected by distillation under reduced pressure (1-10 Pa Hg) to obtain 980 g (yield 98.5%, purity 99.5%) of a high purity 2-pyrrolidone as a colorless liquid.

3-2. Preparation of 2-pyrrolidone from 4-aminobutyl acid (method 2)

A 2 L reaction vessel with 4—aminobutyl acid 1200 and a cold distillation unit was prepared. Into the prepared 2 L reactor, 200 g of 4-aminobutyl acid was first introduced. The temperature of the reactor was raised to the melting point of 4—aminobutyl acid (202 ^° C.), resulting in 2-pyridone and water as 4-aminobutyl acid dissolved. 200 g of 4-aminobutyric acid was further added to and dissolved therein while the temperature of the reaction mixture was naturally cooled. The remaining 4 g of aminobutyric acid was also added to and dissolved at a temperature of 135 -145 ^° C of the reactor. The water produced in the reaction was removed through a distillation apparatus under atmospheric pressure or reduced pressure (40-60 mmHg). When the reaction solution became transparent, the reaction was terminated. Residual water was removed. The resulting 2-pyrrolidone was distilled under reduced pressure (1-10 mmHg) to obtain 951 g (yield 96%, purity 99.5%) of a high purity 2-pyrrolidone as a colorless liquid. ^"Even if removal of the water produced at atmospheric pressure in the reaction, it was possible to obtain the 2-pyrrolidone to approximately the same value as the yield and purity of the above two more times longer than the time banung reduced pressure. 3-3. Preparation of 2-pyrrolidone from 4-aminobutyl acid (method 3)

In the 2 L reaction vessel with each distillation apparatus, 1200 g of 4 'aminobutyric acid was added while the stirrer was stopped. Increasing the temperature of the reactor to 200 ^° C— 2 K C produced some 2-pyrrolidone and water as some of the 4-aminobutyl acid dissolved. Slowly start the stirrer to check the agitation condition and start stirring if possible. The temperature of the reactor was naturally cooled with stirring, and the remaining 4-aminobutyl acid was dissolved at a temperature of 135-145 ^° C. of the reaction vessel. The water produced in the reaction was removed through a distillation apparatus at atmospheric pressure or under 60 kPa Hg. When the reaction solution became transparent, the reaction was terminated. Thus, the residual water remaining in the reaction solution was removed under reduced pressure while gradually increasing the vacuum degree. Thereafter, the produced 2 mu pirlidone was distilled under reduced pressure (1-10 mmHg) to obtain 960 g (yield 96.9%, purity 99.5%) of a high purity 2-pyrrolidone as a colorless liquid. Water generated during reaction Even when removed at atmospheric pressure, 2-pyrrolidone was obtained with values almost similar to the above yield and purity, but the reaction time was 2 hours longer than the reduced pressure conditions. 3-4. Preparation of 2 \ Pyrlidone from 4 I-Aminobutyl Acid

500 g of 2—pyrrolidone was added to a 2 L reaction vessel equipped with a cold-distillation apparatus and stirred. 600 g of 4-aminobutyric acid was added thereto. The temperature was raised to 118 ^° C. 12 CTC and stirred at this temperature for about 10 hours. A sample of the reaction solution was taken and analyzed by HPLC to confirm the residual concentration of 4—aminobutyl acid in 0.9%, and the reaction was terminated. The water produced after the reaction was removed through a distillation apparatus under 20-30 mmHg. Then, 2-pyrrolidone remaining in the reactor was collected by distillation under reduced pressure (1-10 mmHg) to obtain 974 g (yield 97.9%, purity 99.4%) of a colorless liquid, high purity 2—pyrrolidone. 3-5. Preparation of 2-Pyridone from 4-Aminobutyl Acid

500 g of 2-pyridone was added to a 2 L reaction vessel equipped with a cold distillation apparatus and stirred. 600 g of 4-aminobutyl acid was added thereto. The temperature was reduced to 118 ^° C-120 ^° C under reduced pressure (20–30 mmHg) and stirred to produce 2-pyrrolidone and water as the 4-aminobutyl acid dissolved. The resulting water was removed through a distillation apparatus under reduced pressure. After reaction for about 8 hours, a sample of the reaction solution was taken and analyzed by HPLC to confirm the residual concentration of 4 0aminobutyl acid at 0.¾, and the reaction was finished. The residual moisture was completely removed while gradually increasing the vacuum of the reaction solution. The 2—pyridone remaining in the reactor was then distilled under reduced pressure (1-10 mmHg) to give 985 g (yield 99¾ », purity 99.8%) as a colorless liquid, high purity 2-pyridone.

3-6. Preparation of 2-Pyridone from 4-Aminobutyl Acid

500 g of 2-pyrrolidone was added to a 2 L reactor equipped with a cold distillation apparatus and stirred. 600 g of 4-aminobutyric acid was added thereto. When the temperature was raised to 128 ^° C. 132 ^° C. under reduced pressure (50 mmHg at 3 ^° C.) and stirred, 4 ᅳ aminobutyric acid was dissolved, resulting in 2 ᅳ pyridone and water. The water produced is distilled under reduced pressure Removed through the device. After about 8 hours of reaction, ¾ of the reaction mixture was taken and analyzed by HPLC to confirm the residual concentration of 4—aminobutyl acid at 0.4%, and the reaction was terminated. The residual moisture was completely removed while gradually increasing the vacuum of the reaction solution. The remaining 2—pyridone in the reactor was then distilled under reduced pressure (1-10 mmHg) to obtain 980 g (yield 98.5¾, purity 99.6%) of a high purity 2-pyrrolidone as a colorless liquid.

3-7. Preparation of 2-Pyrrolidone from 4-Aminobutyl Acid

 500 g of 2-pyrrolidone was added to a 2 L reactor equipped with a cold distillation apparatus and stirred. 600 g of 4-aminobutyric acid was added thereto. Raising the temperature to 138 ° C—142 ° C under reduced pressure (60 ° 80 mmHg) and stirring produced 4-pyridonone and water as the 4-aminobutyl acid dissolved. The resulting water was removed through a distillation apparatus under reduced pressure. After the reaction for about 3 hours, a sample of the reaction solution was taken and analyzed by HPLC to confirm the residual concentration of 4—aminobutyl acid in 0.3%, and reaction was completed. The residual moisture was completely removed while gradually increasing the vacuum of the reaction solution. The 2-pyrrolidone remaining in the reactor was then distilled under reduced pressure (1-10 mmHg) to obtain 985 g (yield 99%, purity 99.1) of a colorless liquid, high purity 2—Pyrlidone. Preparation of 2-pyrrolidone from aminobutyl acid

500 g of 2-pyrrolidone was added to a 2 L reaction vessel equipped with a cold distillation apparatus and stirred. 600 g of 4-aminobutyl acid was added thereto. The temperature was increased to 145 ° C—148 ° C under reduced pressure (70-110 mmHg) and stirred to produce 4-aminobutyric acid, resulting in 2-pyrididone and water. It's done. The resulting water was removed through a distillation apparatus under reduced pressure. After the reaction for about 2 hours, a sample of the reaction solution was taken and analyzed by HPLC to confirm 0.3% residual concentration of 4-aminobutyl acid, and the reaction was terminated. The residual moisture was completely removed while gradually increasing the vacuum of the reaction solution. Thereafter, 2-pyrrolidone remaining in the reaction period was collected by distillation under reduced pressure (1-lOmmHg), thereby obtaining 978 g (yield 98., purity 99.3%) of a colorless liquid, high purity 2—pyrrolidone. The specific parts of the present invention have been described in detail, and it is apparent to those skilled in the art that these specific technologies are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

[Range of request]  [Claim 1]  How to display the desired protein on the surface of the vacuoles of the microorganism comprising the following steps:  (a) constructing a vector comprising a nucleotide sequence encoding a fusion protein comprising a target protein and a vacuole targeting transmembrane protein;  (b) transforming the microorganism comprising the vacuole using the vector; And  (c) expressing the fusion protein in the transformed microorganism, wherein the expressed fusion protein is targeted to a vacuole and the target protein of the fusion protein is displayed on the surface of the vacuole. [Claim 2]  How to prepare a drug carrier comprising the following steps:  (a) constructing a vector comprising a nucleotide sequence encoding a fusion protein comprising a protein drug and a vacuole targeting transmembrane protein;  (b) transforming the microorganism comprising the vacuole using the vector; And  (c) expressing the fusion protein in the transformed microorganism, wherein the expressed fusion protein is targeted to a vacuole and the protein drug of the fusion protein is displayed on the surface of the vacuole. [Claim 3]  The method of claim 1 or 2, wherein the vacuole targeting transmembrane protein is a PH08 protein, a CPS1 protein, a VAM3 protein, a NYV1 protein, or a VPH1 protein. [Claim 4] 4. The method of claim 3, wherein the vacuole targeting transmembrane protein is a PH08 protein Method characterized by [Claim 5]  The method according to claim 1 or 2, wherein the target protein or protein drug is bound to the N ᅳ terminus of the vasculature targeting transmembrane protein. [Claim 6]  The method of claim 1 or 2, wherein the target protein and vacuole targeting transmembrane protein of the fusion protein are linked by a linker. [Claim 7]  The method according to claim 1 or 2, wherein the target protein or protein drug displayed on the vacuole surface of the microorganism has a topology of the structure facing outward of the vacuole membrane. [Claim 8]  The method according to claim 1 or 2, wherein the microorganisms containing the vacuoles are fungi or bacteria. [Claim 9]  9. The method of claim 8, wherein the microorganism comprising the vacuole is a fungus. [Claim 10]  10. The method of claim 9, wherein the fungus is yeast. [Claim 11] The method of claim 1 or 2, wherein the method further comprises the step of separating the vacuole after step (c). [Claim 12]  A drug carrier comprising a protein drug coupled to a vacuole targeting transmembrane protein and exhibited on the surface of the vacuole by the vacuole targeting transmembrane protein. [Claim 13]  The drug delivery vehicle according to claim 12, wherein the vacuole targeting transmembrane protein is PH08 protein, CPS1 protein, VAM3 protein, NYV1 protein or VPH1 protein. [Claim 14]  The drug delivery carrier of claim 13, wherein the vacuole targeting transmembrane protein is a PH08 protein. [Claim 15]  13. The drug delivery system according to claim 12, wherein the protein drug is bound to the N-terminus of the vasculature targeting transmembrane protein. [Claim 16]  13. The drug delivery system of claim 12, wherein the protein drug and vacuole targeting transmembrane protein are linked by a linker. [Claim 17]  The drug delivery vehicle according to claim 12, wherein the target protein or protein drug displayed on the surface of the vacuole has a topology of the structure facing outward of the vacuole membrane. [Claim 18] 13. The drug delivery vehicle of claim 12, wherein the vacuole is a vacuole of a fungus or bacterium. PCT / KR 2012. / 0 0 3 8 1 1 [Claim 19]  19. The method of claim 18, wherein the vacuoles are vacuoles of fungi Drug delivery system. [Claim 20]  20. The vacuole of claim 19 wherein the vacuole is a vacuole of yeast. Drug carrier,