Artificial hybrid promoter

Abstract

The present invention relates to an artificial hybrid promoter, and more particularly, to an artificial hybrid promoter including an upstream activation sequence of GAL10 gene and a core promoter sequence of PGK gene, a recombinant expression vector including same, a host cell, and a method of producing a target protein using the host cell.

Description

Artificial hybrid promoter

[0001] This application claims priority based on Korean No. 10-2024-0151409 filed on October 30, 2024, and all contents disclosed in the specification and drawings of said application are incorporated into this application.

[0002] The present invention relates to an artificial hybrid promoter, and more specifically, provides an artificial hybrid promoter comprising an upstream activation sequence of a GAL10 gene and a core promoter sequence of a PGK gene, a recombinant expression vector comprising the same, a host cell, and a method for producing a target protein using said host cell.

[0003] Yeast-based protein expression and secretion systems offer several advantages in the production of recombinant proteins. As unicellular eukaryotes, yeast can be easily mass-cultured at a low cost. Unlike prokaryotic expression systems such as E. coli, yeast is capable of post-translational modifications, such as glycosylation, making it particularly suitable for the production of proteins derived from eukaryotic cells. Furthermore, unlike expression systems in E. coli, yeast enables soluble expression in almost all cases without forming inclusion bodies. Additionally, its well-developed protein secretion pathways ensure that subsequent processes are not complex, giving it high industrial value.

[0004] To improve protein productivity in yeast, the use of high-expression promoters is indispensable. To date, various high-expression promoters have been developed to achieve high protein expression in yeast. Examples include constant high-expression promoters such as the promoter for the TDH3 gene (TDH3 promoter), the promoter for the PGK1 gene (PGK1 promoter), and the promoter for the ADH1 gene (ADH1 promoter), as well as inductive high-expression promoters such as the promoter for the GAL1 gene (GAL1 promoter) and the promoter for the GAL10 gene (GAL10 promoter). However, these natural promoters have limitations in transcriptional capacity, so hybrid promoters of a wider range of intensities are being developed to more precisely control gene expression by combining regulatory components and core components of various promoters. A traditional method for constructing a hybrid promoter involves combining the upstream activation sequence (UAS) of one promoter with the core sequence of another promoter.

[0005] Generally, the UAS of the GAL promoter is used, and in Non-Patent Literature 1, a UAS combining the UAS of the GAL10 promoter and the core sequence of the CYC1 promoter is used to express a heterologous protein in Saccharomyces cerevisiae. GAL10 -Core CYC1 Hybrid promoters have been reported. In addition, Non-patent Literature 2 describes the construction of various hybrid promoters with different induction strengths by combining the UAS of the GAL1 promoter with the core sequence of another promoter.

[0006] Against this background, the inventors completed the present invention by developing a novel artificial hybrid promoter capable of enhancing the expression of a target protein by at least 120% to a maximum of 330% compared to the existing GAL10 promoter by combining the UAS of the GAL10 promoter with the core sequence of the PGK promoter.

[0007] [Prior Art Literature]

[0008] [Non-patent literature]

[0009] (Non-patent Document 1) Proceedings of the National Academy of Sciences, 79(23), 7410-7414.

[0010] (Non-patent document 2) Biotechnology and Bioengineering, 109(11), 2884-2895.

[0011] The objective of the present invention is to provide an artificial hybrid promoter capable of enhancing the expression level of a target protein compared to the existing GAL10 promoter.

[0012] Another objective of the present invention is to provide a recombinant expression vector and a host cell comprising the artificial hybrid promoter.

[0013] Another objective of the present invention is to provide a method for producing a target protein using the host cell.

[0014] Another objective of the present invention is to provide a kit for producing a target protein comprising at least one of the artificial hybrid promoter, a recombinant expression vector, and a host cell, and a medium composition for inducing expression, and a method for producing a target protein using the same.

[0015] To solve the above-mentioned problem, the present invention provides an artificial hybrid promoter comprising the following (a) and (b):

[0016] (a) upstream activation sequence (UAS) of the GAL10 gene; and

[0017] (b) Core promoter sequence of the PGK gene.

[0018] In the present invention, the UAS of the GAL10 gene may be repeatedly located upstream of the core promoter.

[0019] In the present invention, the UAS of the GAL10 gene may include the nucleotide sequence of SEQ ID NO. 1, and the core promoter sequence of the PGK gene may include the nucleotide sequence of SEQ ID NO. 2.

[0020] In the present invention, the artificial hybrid promoter may include the nucleotide sequence of SEQ ID NO. 3, the nucleotide sequence of SEQ ID NO. 4, the nucleotide sequence of SEQ ID NO. 5, or the nucleotide sequence of SEQ ID NO. 6.

[0021] In addition, the present invention provides a recombinant expression vector comprising the aforementioned artificial hybrid promoter.

[0022] In the present invention, the recombinant expression vector may additionally include a gene encoding a target protein.

[0023] Additionally, the present invention provides a host cell comprising the aforementioned recombinant expression vector.

[0024] In the present invention, the host cell may be a yeast cell.

[0025] Additionally, the present invention provides a method for producing a target protein, comprising the step of culturing the aforementioned host cells to produce the target protein.

[0026] Furthermore, the present invention provides a kit for producing a target protein comprising at least one of (a) to (c) and (d):

[0027] (a) The aforementioned artificial hybrid promoter;

[0028] (b) a recombinant expression vector comprising the above promoter;

[0029] (c) a host cell comprising the above-mentioned recombinant expression vector; and

[0030] (d) A medium composition for inducing expression.

[0031] In addition, the present invention provides a method for producing a target protein using the above kit.

[0032] The artificial hybrid promoter Pgal10-Ppgk according to the present invention can improve the expression level of a target protein by at least about 120% to a maximum of about 330% compared to the Pgal10 promoter, and thus can be used to efficiently produce various useful recombinant proteins on a yeast platform.

[0033] Figure 1 shows a gene construct in which a UAS (GAL10), a PGK core promoter, and a gene encoding a target protein are sequentially linked.

[0034] Figure 2 is a cleavage map of the UAS(GAL10)-PGK-MFα-TGFβ-GSlinker-RFP vector.

[0035] Figure 3 is a cleavage map of the GAL10-MFα-TGFβ-GSlinker-RFP vector.

[0036] Figure 4 compares the expression levels of the target protein in a strain transformed with the UAS(GAL10)-PGK-MFα-TGFβ-GSlinker-RFP vector and a strain transformed with the GAL10-MFα-TGFβ-GSlinker-RFP vector.

[0037] Figure 5 is a cleavage map of the UAS(GAL10)-TDH3-MFα-TGF-RFP vector.

[0038] Figure 6 is a cleavage map of the UAS(GAL10)-TEF1-MFα-TGF-RFP vector.

[0039] Figure 7 compares the expression levels of target proteins with a hybrid promoter in which Pgal10 is combined with a tef1 core promoter or a tdh3 core promoter as another homeostasis promoter, and a UAS(GAL10)-PGK hybrid promoter in which Pgal10 UAS is linked with various repeat numbers (UAS(GAL10)-PGK, UAS(GAL10)X2-PGK, UAS(GAL10)X3-PGK, and UAS(GAL10)X4-PGK, respectively).

[0040] Figure 8 is a cleavage map of the GAL10-MFα-RFP vector.

[0041] Figure 9 is a cleavage map of the UAS(GAL10)-PGK-MFα-RFP vector.

[0042] Figure 10 is a cleavage map of the UAS(GAL10)Х3-PGK-MFα-RFP vector.

[0043] The present invention will be described in more detail below.

[0044] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention.

[0045] All numbers expressing the size, quantity, and physical properties of a feature used in this specification and claims should be understood as being modified by the term "approximately" in all cases. Accordingly, unless otherwise indicated, the numerical parameters disclosed in this specification and claims are approximations that may vary depending on the desired properties to be obtained by a person skilled in the art using the teachings disclosed in this specification.

[0046] As described above, natural promoters have limited transcriptional capacity, so there is a need to develop artificial hybrid promoters to further enhance the expression of target proteins compared to existing natural promoters. Accordingly, in the present invention, a novel artificial hybrid promoter, 'Pgal10-Ppgk', was synthesized by combining the UAS of the GAL10 promoter with the core sequence of the PGK promoter, and a solution to the aforementioned problem was sought by confirming its strong promoter activity.

[0047] Accordingly, the first aspect of the present invention relates to an artificial hybrid promoter comprising the following (a) and (b):

[0048] (a) upstream activation sequence (UAS) of the GAL10 gene; and

[0049] (b) Core promoter sequence of the PGK gene.

[0050] In this specification, the term "promoter" refers to an untranscribed upstream nucleic acid sequence of an coding region that includes a binding site for polymerase and possesses transcription initiation activity for the mRNA (messenger RNA) of the gene downstream of the promoter. In the present invention, the term may be used interchangeably with artificial hybrid promoters for increasing the expression of a gene encoding a target protein. In eukaryotes, proteins called transcription factors are involved in inducing RNA polymerase by binding to the promoter region. A promoter can be said to refer to any DNA nucleic acid sequence region to which these transcription factors bind. In contrast, in prokaryotes, a promoter is generally defined as a binding site immediately near the transcription start site where RNA polymerase binds.

[0051] In the present invention, the GAL10 gene and the PGK gene may be derived from various biological species. For example, the GAL10 gene may be derived from various microbial species, specifically yeast, preferably Saccharomyces cerevisiae, but is not limited thereto. The PGK gene may be derived from humans or mice, or from microorganisms of the genus Saccharomyces, Kazachstania, Kluyveromyces, Aspergillus, Pichia, or Candida, but is not limited thereto. Alternatively, the core promoter sequences of the UAS of the GAL10 gene and the PGK gene may be synthesized de novo rather than being of natural origin.

[0052] In the present invention, the GAL10 gene is one of the genes involved in galactose utilization and refers to a gene encoding a gene that simultaneously possesses UDP (uracil-diphosphate)-glucose-4-epimerase and aldose 1-epimerase activities. The dual-functional UDP (uracil-diphosphate)-glucose-4-epimerase and aldose 1-epimerase are enzymes that catalyze the interconversion of UDP-galactose and UDP-D-glucose in galactose metabolism and catalyze the conversion of alpha-D-glucose or alpha-D-galactose into beta-anomers. The nucleotide sequence of the gene is disclosed in a known database such as NCBI. For example, the GAL10 gene derived from Saccharomyces cerevisiae may include or consist of the nucleotide sequence disclosed in NCBI Reference Sequence: NC_001134.8 (genomic) or NM_001178367.1 (mRNA), or said nucleotide sequence or said nucleotide sequence. Additionally, it may include or consist of a nucleotide sequence encoding an amino acid sequence disclosed in NCBI Reference Sequence: NP_009575.1 (protein), or said amino acid sequence.

[0053] The UAS of the above GAL10 gene may include a nucleotide sequence corresponding to the UAS in the nucleotide sequence disclosed in NCBI Reference Sequence: NC_001134.8 or NM_001178367.1, or may be composed of said nucleotide sequence or said part thereof. Additionally, the UAS of the above GAL10 gene may include a nucleotide sequence encoding an amino acid sequence or a part thereof disclosed in NCBI Reference Sequence: NP_009575.1, or may be composed thereof. According to a specific embodiment of the present invention, the UAS of the above Saccharomyces cerevisiae-derived GAL10 gene may include or be composed of the nucleotide sequence of SEQ ID NO. 1.

[0054] CGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAATCCGACATGCTAT GATGCCCACTGTGATCTCCAGAGCAAAGTTCGTTCGATCGTACTGTTACTCTCTCTCTTTCAAACAGAATTGTCCGAATCGGTGACAACAACAGCCTGTTCTCACACACTCTTTTCTTCTAACCAAG (SEQ ID NO: 1)

[0055] In the present invention, the PGK gene is also referred to as PGK1 and is a gene encoding 3-phosphoglycerate kinase, the nucleotide sequence of said gene is disclosed in a known database such as NCBI. For example, a PGK gene derived from Saccharomyces cerevisiae may include or be composed of the nucleotide sequence disclosed in NCBI Reference Sequence: NC_001135.5 (genomic) or NM_001178725.1 (mRNA), or said nucleotide sequence, or said nucleotide sequence, or said nucleotide sequence. Additionally, it may include or be composed of a nucleotide sequence encoding an amino acid sequence or said nucleotide sequence disclosed in NCBI Reference Sequence: NP_009938.2 (protein).

[0056] The core promoter sequence of the above PGK gene may include a nucleotide sequence corresponding to the core promoter in the nucleotide sequence disclosed in NCBI Reference Sequence: NC_001135.5 or NM_001178725.1, or may be composed of said nucleotide sequence or said nucleotide sequence or said nucleotide sequence. Additionally, the core promoter sequence of the above PGK gene may include a nucleotide sequence encoding an amino acid sequence or said nucleotide sequence disclosed in NCBI Reference Sequence: NP_009938.2, or may be composed of said nucleotide sequence. According to a specific embodiment of the present invention, the core promoter sequence of the PGK gene derived from Saccharomyces cerevisiae may include or be composed of the nucleotide sequence of SEQ ID NO. 2.

[0057] GGGGTGGTTTAGTTTAGTAGAACCTCGTGAAACTTACATTTACATATATATAAACTTGCATAAATTGGTCAATGCAAGAAATACATATTTGGTCTTTTCTAATTCGTAGTTTTTCAAGTTCTTAGATGCTTTCTTTTTCTCTTTTTTACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATATAAAACA (SEQ ID NO: 2)

[0058] In the present invention, the UAS of the GAL10 gene may be repeatedly located upstream of the core promoter of the PGK gene. The preferred number of repetitions may be 1 to 10 times, but is not limited thereto, and may be repeated for example, 1 to 9 times, 1 to 8 times, 1 to 7 times, 1 to 6 times, 1 to 5 times, 1 to 4 times, or 1 to 3 times.

[0059] According to a specific embodiment of the present invention, artificial hybrid promoters in which the UAS of the GAL10 gene is positioned upstream of the core promoter of the PGK gene one to four times are named, in order, 'UAS(GAL10)-PGK', 'UAS(GAL10)X2-PGK', 'UAS(GAL10)X3-PGK', and 'UAS(GAL10)X4-PGK', respectively, and their nucleotide sequences may include or be composed of the nucleotide sequences of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 5, respectively, as shown in Table 1 below. In each nucleotide sequence of Table 1 below, the underlined sequence represents the nucleotide sequence of the PGK core promoter.

[0060]

[0061] The UAS of the Saccharomyces cerevisiae-derived GAL10 gene of the present invention may include or be composed of a sequence having sequence homology of 90% or more, 92% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% with respect to the sequence of SEQ ID NO. 1.

[0062] The core promoter sequence of the Saccharomyces cerevisiae-derived PGK gene of the present invention may include or be composed of a nucleotide sequence exhibiting sequence homology of 90% or more, 92% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% with the nucleotide sequence of SEQ ID NO. 2.

[0063] If an artificial hybrid promoter is combined with a core promoter sequence having 90% or more homology with the UAS of the GAL10 gene of the present invention and 90% or more homology with the core promoter of the PGK gene of the present invention, and exhibits an expression efficiency equivalent to that of the artificial hybrid promoter according to the present invention in yeast, it can be said to be a substantially equivalent artificial hybrid promoter.

[0064] Preferably, it may include or be composed of nucleotide sequences exhibiting sequence homology of 90% or more, 92% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, respectively, with respect to the nucleotide sequences of SEQ ID NOs 3 to 6.

[0065] In this specification, the term “homology” refers to the percentage of identity between two polynucleotide or polypeptide moietys. Homology between sequences from one moiety to another can be determined by known techniques. For example, homology can be determined by aligning sequence information and using readily available computer programs to directly align parameters, such as score, identity, and similarity, between two polynucleotide molecules or two polypeptide molecules (e.g., BLAST 2.0). Additionally, homology between polynucleotides can be determined by hybridizing the polynucleotides under conditions of stable double-stranded homologous regions, then digesting them with a single-strand-specific nuclease to determine the size of the digested fragment.

[0066] A second aspect of the present invention relates to a recombinant expression vector comprising the aforementioned artificial hybrid promoter.

[0067] In this specification, the term "vector" refers to an expression vector capable of expressing a target protein in a suitable host cell, comprising an essential regulatory element operably linked to allow the expression of a gene insert.

[0068] Generally, plasmid vectors are extrachromosomal circular double-stranded DNA that exist within cells and perform various functions. They act as inhibitors that kill similar strains or species by producing antibiotic-resistant substances and bacteriocins, and perform physiological functions such as pigment production, compound degradation, and nitrogen fixation. They possess restriction enzyme sites to insert foreign DNA fragments up to approximately 10 kb in length.

[0069] A significant limitation of plasmids and bacteriophages used as vectors is their inability to insert only relatively small DNA fragments; however, larger DNA fragments can be cloned using cosmids, which are engineered hybrids of plasmid and phage DNA. Cosmids contain a cos region that is packaged into the phage particle, as well as genes for the replication start point of the plasmid and for selecting the plasmid to replicate in the bacterial host. Like bacteriophage vectors, they are packaged in a protein envelope in vitro; however, after the packaged DNA infects an E. coli host cell, the DNA replicates in the form of a plasmid rather than bacteriophage DNA and is not lysed. The size is 2.5 kb, and after infecting the host cell and packaging, the cos region separates to 37 to 52 kb, allowing it to accommodate foreign DNA as an insert. Generally, 35 to 45 kb can be cloned into a cosmid vector.

[0070] In addition, the λ phage, a common form of bacteriophage vector, is derived from a 50kb double-stranded wild-type genome with cohesive termini or cos, which are single strands of 12 nucleotides capable of forming base pairs, and in the lytic pathway, the host cell lyses after new viruses are replicated and progeny viruses are released. This form of DNA can accommodate an additional 3kb on top of the total 52kb size or as little as 5% of the genome, and the vector that makes room for foreign DNA is a form from which non-essential DNA fragments have been removed.

[0071] When the host cell of the present invention is a eukaryotic cell, for example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. may be used as phage vectors or cosmid vectors, and pRS416, pRS426, pRS415, pRS425, YEp13, YEp24, YCp50, pBR system, pUC system, pBluescriptII system, pGEM system, pTZ system, pCL system, and pET system, etc. may be used as plasmid vectors, but are not limited thereto.

[0072] When the host cell of the present invention is a yeast cell, the yeast expression vector can be both an integrative yeast plasmid (YIp) and an extrachromosomal plasmid vector. The extrachromosomal plasmid vector may include an episomal yeast plasmid (YEp), a replicative yeast plasmid (YRp), and a yeast centromere plasmid (YCp). Additionally, artificial yeast chromosomes (YACs) can also be used as vectors of the present invention. As a specific example, available vectors are pYG, pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2 / CT, pYD1 Yeast Display Vector, pYES2, pYES2 / CT, pYES2 / NT A, pYES2 / NT B, pYES2 / NT C, pYES2 / CT, pYES2.1, pYES-DEST52, pTEF1 / Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, It may include, but is not limited to, pYJ405 and pYJ406.

[0073] In this specification, the term "operably linked" refers to a functional linkage between the aforementioned artificial hybrid promoter and the nucleotide sequence encoding the target protein to perform a general function. Operatory linkage with the recombinant vector can be prepared using gene recombination technology well known in the art, and site-specific DNA cleavage and linkage use enzymes, etc., generally known in the art.

[0074] In this specification, the term “regulatory element” means an untranscribed nucleic acid sequence that helps promote or influences the transcription, translation, or expression of a nucleic acid sequence encoding a protein. The recombinant expression vector of the present invention essentially includes the artificial hybrid promoter of the present invention as a regulatory element and may include expression regulatory sequences that can influence protein expression, such as a start codon, a stop codon, a polyadenylation signal, an enhancer, a signal sequence for membrane targeting or secretion, etc.

[0075] Polyadenylation signals increase transcript stability or facilitate cytoplasmic transport. Enhancer sequences are nucleic acid sequences located at various sites in the promoter that increase transcriptional activity compared to transcriptional activity by the promoter in the absence of the enhancer sequence. For signal sequences, PhoA signal sequences, OmpA signal sequences, etc., may be used when the host is a genus of Escherichia; α-amylase signal sequences, Subtilisin signal sequences, etc., when the host is a genus of Bacillus; and MFα (Mating factor α) signal sequences, SUC2 signal sequences, etc., when the host is a yeast, but are not limited thereto.

[0076] Additionally, the vector may include a selection marker. The selection marker is intended to select cells transformed by the vector, and markers that confer selectable phenotypes, such as drug resistance, nutrient requirements, resistance to cytotoxic agents, or the expression of surface proteins, may be used. Since only cells expressing the selection marker survive in an environment treated with a selective agent, it is possible to select the transformed cells. Representative examples of selection markers include auxotrophic markers such as ura4, leu1, and his3, but the types of markers that can be used in the present invention are not limited by the above examples.

[0077] In order to facilitate the purification of the target protein recovered in the present invention, additional sequences may be included as needed during the preparation of the vector. The additional sequences that may be included may be tag sequences for protein purification, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6xHis(hexahistidine; Quiagen, USA), and most preferably 6xHis(hexahistidine); however, the types of sequences required for the purification of the target protein are not limited by the above examples. In the case of a fusion protein expressed by a vector containing the fusion sequence, it may be purified by affinity chromatography. For example, when glutathione S-transferase is fused, glutathione, which is the substrate of this enzyme, can be used, and in the case of 6xHis, the desired target protein can be easily recovered using a Ni-NTA His-binding resin column (Novagen, USA).

[0078] In the present invention, the recombinant expression vector may additionally include a gene encoding a target protein. The target protein refers to any protein that is intended to be produced by utilizing a protein secretion mechanism or a protein that a person skilled in the art intends to produce in large quantities, and which can be expressed in a host cell by inserting a polynucleotide encoding said protein into the recombinant expression vector. For example, the target protein may be one or more selected from the group consisting of hormones, hormone analogs, enzymes, enzyme inhibitors, cytokines, coagulation factors, transport proteins, receptors, receptor fragments, regulatory proteins, structural proteins, toxin proteins, transcription factors, growth factors, antigens, antibodies, antibody fragments, and monoclonal antibodies, but is not limited thereto.

[0079] The nucleic acid sequence encoding the above-mentioned target protein can be inserted downstream of the promoter and expressed.

[0080] A third aspect of the present invention relates to a host cell in which the aforementioned artificial hybrid promoter is introduced into the genome or which has been transformed with the aforementioned recombinant expression vector.

[0081] In this specification, the term "host cell" refers to a cell that receives nutrition by parasitizing other microorganisms or genes, and signifies a cell that exerts various genetic or molecular effects within the host cell upon transformation by a vector. The host cell is in a competent state capable of accepting external DNA, through which external DNA, such as a vector, can be inserted; if the vector is successfully introduced into the host cell, it provides the genetic traits of the vector to the host cell.

[0082] In the present invention, the host cell may be a yeast-derived cell. For example, the yeast may be an acid-resistant yeast selected from the group consisting of the genus Saccharomyces, Kazachstania saccharomyces, and Candida, and more specifically, may be any one selected from the group consisting of Saccharomyces cerevisiae, Kazachstania exigua, Kazachstania bulderi, and Candida humilis, but is not limited thereto.

[0083] Transformation methods can be used to introduce a vector into a host cell. "Transformation" refers to the phenomenon of introducing external DNA into a cell to artificially induce genetic changes, in which DNA is introduced into a host so that it becomes capable of replication as a chromosomal factor or through the completion of chromosomal integration. Any transformation method may be used, and it can be easily performed according to conventional methods in the industry. Generally, transformation methods include the CaCl2 precipitation method, the Hanahan method (which increases efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in the CaCl2 method), electroporation, calcium phosphate precipitation, protoplasmic fusion, agitation using silicon carbide fibers, Agrobacteria-mediated transformation, polyethylene glycol (PEG) transformation, dextran sulfate, lipopectamine, and drying / inhibition-mediated transformation methods. The method for transforming the plasmid of the present invention is not limited to the above examples, and transformation methods commonly used in the art may be used without limitation.

[0084] The above host cell is typically a host cell that has a high efficiency of DNA introduction and a high efficiency of expression of the introduced DNA. In one embodiment of the present invention, a cell derived from Saccharomyces cerevisiae was used, but it is not limited thereto, and any type of yeast is acceptable as long as the target DNA can be sufficiently expressed.

[0085] In addition, any commonly known gene manipulation method may be used to insert a gene construct, in which the artificial hybrid promoter and the gene encoding the target protein are operably linked, onto the chromosome of a host cell in the present invention. Examples include using a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a poxvirus vector, a lentivirus vector, a non-viral vector, etc.

[0086] A fourth aspect of the present invention provides a method for producing a target protein, comprising the step of culturing the aforementioned host cell to produce a target protein by the expression of a target gene.

[0087] In the present invention, the target protein can be obtained from a culture of host cells into which the recombinant expression vector has been introduced, and the culture may include both the host cells themselves and the culture medium, and may include all substances generated during the process of culturing the host cells.

[0088] In this specification, the term "culture" means growing a host cell in an environment that is appropriately artificially controlled. The culture medium and other culture conditions used for the culture may be any medium used for the culture of microorganisms of the genus Saccharomyces without any particular limitations. Specifically, the host cell of the present invention may be cultured under aerobic or anaerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compound, amino acid, and / or vitamin.

[0089] In the present invention, the carbon source may include, but is not limited to, carbohydrates such as glucose, fructose, sucrose, maltose, galactose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice husk, cassava, sugarcane residue, and corn steeping liquid may be used, and carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used in various ways without limitation. These carbon sources may be used individually or in combination of two or more types.

[0090] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0091] The above ingredients may include potassium monophosphate, potassium diphosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc.

[0092] In addition, the above medium may contain amino acids, vitamins and / or suitable precursors, etc.

[0093] The above medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.

[0094] In the present invention, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., to the culture in an appropriate manner during the cultivation of the strain. Additionally, during cultivation, the generation of bubbles can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state of the culture, oxygen or an oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state.

[0095] The temperature of the culture may be 25°C to 40°C, more specifically 28°C to 37°C, but is not limited thereto. The culture period may continue until the desired amount of useful substance is obtained, specifically 1 hour to 50 hours, but is not limited thereto.

[0096] The target protein produced by the culture of the present invention may be secreted into the culture medium or remain within the cell.

[0097] In the present invention, the method for producing the target protein may further include the step of recovering the target protein from a culture of host cells.

[0098] The above recovery may involve collecting the desired recombinant protein using a suitable method known in the art according to the host cell culture method of the present invention, such as a batch, continuous, or fed-batch culture method. For example, various chromatographs such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the desired recombinant protein may be recovered from the culture medium or host cell using a suitable method known in the art.

[0099] In addition, the recovery may involve recovering the target protein from one or more substances selected from the host cell, its dried product, extract, culture, and lysate after the culture step.

[0100] In this specification, the term "culture" may refer to a product obtained by culturing the host cells of the present invention. For example, it may be a medium containing byproducts generated through metabolism after culturing host cells in the medium to consume nutrients, and it includes all forms of cultures that can be formed using the medium, such as a diluted solution or concentrate of the medium, a dried product obtained by drying the medium, a modified or purified product of the medium, or a mixture thereof. The culture of the host cells of the present invention may or may not contain the host cells. The culture is as described above.

[0101] In the present invention, "crushed material" may be a product obtained by crushing or lysing the host cell or its culture, or a supernatant obtained by centrifuging the crushed material.

[0102] The above-mentioned cultures, lysates, supernatants thereof, and fractions thereof are also included within the scope of the present invention. The method may additionally include a step of lysing host cells prior to or simultaneously with the recovery step. Lysing of host cells may be carried out by methods commonly used in the art to which the present invention belongs, for example, by a lysis buffer solution, a sonicator, heat treatment, and a French press. Furthermore, the lysis step may include, but is not limited to, enzymatic reactions such as cell wall degrading enzymes, nucleases, nucleotransferases, and proteases.

[0103] For the purposes of the present invention, dry yeast, yeast extract, yeast extract mix powder, and pure purified target protein containing a high content of the target protein can be produced through the method for producing the target protein, but are not limited thereto, and can be appropriately prepared depending on the target substance.

[0104] In this specification, the dry yeast may be used interchangeably with terms such as "dried strain." The dry yeast may be prepared by drying yeast cells that have accumulated recombinant protein, and specifically may be included in feed compositions, food compositions, etc., but is not limited thereto.

[0105] In this specification, the term "yeast extract" may be used interchangeably with terms such as "strain extract." The strain extract may refer to the material remaining after separating the cell wall from the cell of the strain. Specifically, it may refer to the remaining components obtained by lysing the cell, excluding the cell wall. The strain extract contains a target protein, and other components may include one or more of other proteins, carbohydrates, nucleic acids, and fibers, but are not limited thereto.

[0106] The method of the present invention may further include a step of purifying the target protein. The purification may be performed using a suitable method known in the art. For example, if the method for producing the target protein of the present invention includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or simultaneously or integrated into a single step, but are not limited thereto.

[0107] A fifth aspect of the present invention relates to a kit for producing a target protein and a method for producing a target protein using the same.

[0108] According to a specific embodiment of the present invention, the kit according to the present invention may include at least one of the following (a) to (c) and (d):

[0109] (a) Artificial hybrid promoter according to the first aspect;

[0110] (b) a recombinant expression vector comprising the above promoter;

[0111] (c) a host cell comprising the above-mentioned recombinant expression vector; and

[0112] (d) A medium composition for inducing expression.

[0113] In the kit according to the present invention, the description of (a) an artificial hybrid promoter according to the first aspect, (b) a recombinant expression vector comprising the promoter; and (c) a host cell comprising the recombinant expression vector is the same as that described in the first, second, and third aspects, so the description is omitted.

[0114] The "composition of a medium for inducing expression" used in the present invention is a medium used to regulate or induce the expression of the artificial hybrid promoter of the present invention, and may include carbon sources, nitrogen sources, inorganic salts, amino acids, vitamins, etc. suitable for the growth of host cells, and may additionally include galactose or a derivative thereof capable of inducing the activation of GAL10-derived UAS. However, the present invention is not limited thereto and encompasses all media containing other functional equivalents capable of inducing the expression of the promoter.

[0115] According to a specific embodiment of the present invention, the medium composition for inducing expression may include the medium component described in the fourth aspect.

[0116] A specific embodiment of the method for producing a target protein using the kit of the present invention may include the following steps:

[0117] (a) a step of constructing a recombinant expression vector by operably linking an artificial hybrid promoter according to the first aspect to the upstream of a gene encoding a target protein;

[0118] (b) a step of introducing the above recombinant expression vector into a host cell;

[0119] (c) a step of culturing the above host cells in a medium composition for inducing expression; and

[0120] (d) a step of recovering the target protein produced by the expression of the gene encoding the target protein; may be included.

[0121] In the method for producing a target protein according to the present invention, the specific description of each step (a) to (d) is the same as that described above, so the description is omitted.

[0122] The present invention will be explained in more detail below through examples. However, as the present invention is susceptible to various modifications and may take various forms, the specific examples and descriptions provided below are intended only to aid in understanding the invention and are not intended to limit the invention to the specific disclosed forms. The scope of the present invention should be understood to include all modifications, equivalents, and substitutions that fall within the spirit and technical scope of the invention.

[0123] [Example 1]

[0124] Production of a GAL10 promoter-based hybrid promoter and a recombinant vector containing the same

[0125] 1-1. Production of Pgal10-Ppgk Hybrid Promoter

[0126] First, a hybrid promoter was designed based on the GAL10 promoter (PGAL10) of Saccharomyces cerevisiae. The hybrid promoter was constructed by combining the transcriptional activation region of the GAL10 promoter with a potent core promoter found in yeast. Specifically, the hybrid promoter was completed by combining the core region of the PGK1 promoter while retaining the upstream activating sequence (UAS) region of the GAL10 promoter. The sequences specified in Table 2 were used for the UAS of the GAL10 promoter and the core sequence of the PGK1 promoter.

[0127] Sequence (5'→3') SEQ ID NO: GAL10 promoter UASCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAATCCGACATGCTATG ATGCCCACTGTGATCTCCAGAGCAAAGTTCGTTCGATCGTACTGTTACTCTCTCTCTTTCAAACAGAATTGTCCGAATCGTGTGACAACAACAGCCTGTTCTCACACACTCTTTTCTTCTAACCAAG1PGK1 Core PromoterGGGGTGGTTTTAGTTTAGTAGAACCTCGTGAAACTTACATTTACATATATATAAACTTGCATAAATTGGTCAATGCAAGAAATACATATTTGGTCTTTTCTAATTCGTAGTTTTTCAAGTTCTTAGATGCTTTCTTTTTCTCTTTTTTACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATATAAAACA2

[0128] 1-2. Vector Construction for Target Protein Expression

[0129] An expression vector using a Pgal10-Ppgk hybrid promoter was constructed for the expression of the target protein. The components of the vector and their sequences are shown in Table 3.

[0130] 서열(5'→3')서열번호하이브리드프로모터Pgal10-PpgkCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAATCCGACATGCTATGATGCCCACTGTGATCTCCAGAGCAAAGTTCGTTCGATCGTACTGTTACTCTCTCTCTTTCAAACAGAATTGTCCGAATCGTGTGACAACAACAGCCTGTTCTCACACACTCTTTTCTTCTAACCAAGGGGGTGGTTTAGTTTAGTAGAACCTCGTGAAACTTACATTTACATATATATAAACTTGCATAAATTGGTCAATGCAAGAAATACATATTTGGTCTTTTCTAATTCGTAGTTTTTCAAGTTCTTAGATGCTTTCTTTTTCTCTTTTTTACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATATAAAACA3MFα 신호펩타이드MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA7TGFβ-1ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS8GS 링커GGGGSGGGGSGGGGS9RFPMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK10CYC1 종결신호tcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggcc11

[0131] The hybrid promoter Pgal10-Ppgk is a regulatory element that induces galactose-induced expression, the MFα signal peptide is a signal peptide intended to promote protein secretion in yeast, TGFβ-1 is the target protein, RFP (Red Fluorescence Protein) is a fluorescent protein used to verify the expression level of the target protein, and the CYC1 termination signal is a signal sequence for precise transcription termination. The UAS region of the GAL10 promoter, the PGK1 core promoter, the MFα signal peptide, and the CYC1 termination signal, which are the major components of the hybrid promoter, were amplified via PCR from the gDNA of Saccharomyces cerevisiae and used. The RFP gene and TGFβ-1 were synthesized, and an ORF (open reading frame) encoding the target protein, the growth factor TGFβ-1, was linked to the front of the RFP using a GS linker. All of these elements were cloned using the Gibson assembly method, and the vector was amplified using Escherichia coli. The cleavage map of the constructed UAS(GAL10)-PGK-MFα-TGFβ-GSlinker-RFP vector is shown in Fig. 2, and the cleavage map of the GAL10-MFα-TGFβ-GSlinker-RFP vector used as a control is shown in Fig. 3. The sequence of GAL10 used in the control vector is shown in Table 4.

[0132] Sequence (5'→3') SEQ ID NO:Pgal10CGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAA AGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAA12

[0133] [Example 2]

[0134] Expression of target protein using the Pgal10-Ppgk hybrid promoter

[0135] A lithium acetate-based transformation method was used to transform Saccharomyces cerevisiae with a recombinant vector containing the hybrid promoter constructed in Example 1. The parent strain used for transformation was Saccharomyces cerevisiae CEN.PK-C (ATCC MYA-1108). Transformed yeast cells were cultured in SC-Ura selective medium. Successful transformation was confirmed by the growth of the transformed yeast in the selective medium. To induce the expression of the target protein in the transformed yeast cells, galactose was added to the culture medium to induce expression.

[0136] Specifically, the induction culture conditions were applied by culturing in YP (Yeast Peptone) medium with a final concentration of 3% glucose and 2% galactose added to the culture medium at 30°C. To confirm the expression of the target protein RFP, activation was set at 530 nm and detected at 600 nm using a fluorescence spectrophotometer.

[0137] As a result of confirming the TGFβ expression levels after 48 and 72 hours of induction culture, it was confirmed that strain HSY-TGF-072, transformed with the UAS(GAL10)-PGK-MFα-TGFβ-GSlinker-RFP vector as shown in Figure 4, exhibited TGFβ expression levels improved by approximately 48% and 62%, respectively, compared to the control strain HSY-TGF-088, transformed with the GAL10-MFα-TGFβ-GSlinker-RFP vector. However, regarding the expression amount, it was confirmed that 48 hours of induction culture was more appropriate, so the induction culture time was set to 48 hours in the following experiments.

[0138] [Example 3]

[0139] Confirmation of the expression-enhancing effect of the Pgal10-Ppgk hybrid promoter compared to hybrid promoters combining Pgal10 and other homeostatic promoters

[0140] 3-1. Construction of Pgal10-Ptef1 and Pgal10-Ptdh3 Hybrid Promoters and Recombinant Vectors Containing Them

[0141] It was constructed by combining the transcriptional activation region of the GAL10 promoter of Saccharomyces cerevisiae with the tef1 or tdh3 core promoter, which are known as potent promoters in yeast. Specifically, a hybrid promoter was completed by combining the tef1 core promoter or the tdh3 core promoter while retaining the UAS region of the GAL10 promoter of SEQ ID NO. 1. The sequences specified in Table 5 were used for the tef1 core promoter and the tdh3 core promoter.

[0142] Sequence (5'→3') SEQ ID NO: tef1 core PromoterACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTT TTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAA13tdh3 core PromoterAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCT GAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAA14

[0143] For the expression of the target protein, an expression vector was constructed using the Pgal10-Ptef1 / tdh3 hybrid promoter in the same manner as in Examples 1-2. The UAS region of the GAL10 promoter, the tef1 and tdh3 core promoters, and the MFα signal peptide and CYC1 termination signal, which are the major components of the hybrid promoter, were amplified via PCR from the gDNA of Saccharomyces cerevisiae, while the RFP gene and TGFβ-1 were synthesized. All these components were cloned using the Gibson assembly method, and the vector was amplified using Escherichia coli. The cleavage maps of the constructed UAS(GAL10)-TDH3-MFα-TGF-RFP vector and UAS(GAL10)-TEF1-MFα-TGF-RFP vector are shown in Figures 5 and 6, respectively.

[0144] 3-2. Confirmation of Expression Enhancement Effect of Pgal10-Ppgk Hybrid Promoter Compared to Pgal10-Ptef1 and Pgal10-Ptdh3 Hybrid Promoters

[0145] The two vectors prepared in Example 3-1 were transformed into Saccharomyces cerevisiae using the same method as in Example 2. Induction culture conditions were applied by culturing in YP medium containing a final concentration of 3% glucose and 2% galactose at 30°C. To confirm the expression of the target protein, activation was set at 530 nm and detected at 600 nm using a fluorescence spectrophotometer.

[0146] As a result, as shown in Figure 7, it was confirmed that the productivity of the target protein was significantly increased when the pgk core promoter was applied compared to the tef1 core promoter and tdh3 core promoter.

[0147] [Example 4]

[0148] Comparison of target protein expression levels according to the number of Pgal10 UAS repeats

[0149] 4-1. Comparison of TGFβ1 expression levels according to the number of Pgal10 UAS repeats

[0150] A vector was constructed to compare the expression levels of the target protein when Pgal10 UAS was repeatedly added to the PGK core promoter. A hybrid promoter was constructed by conjugating a gene construct containing 1, 2, 3, or 4 repetitions of Pgal10 UAS to the PGK core promoter, and after transforming Saccharomyces cerevisiae in the same manner as in Example 2, the expression of the target protein was confirmed by culturing under induction culture conditions.

[0151] As a result, as shown in Figure 7, it was confirmed that the expression of the target protein of the hybrid promoter conjugated with PGK significantly increased compared to the existing Pgal10, and in particular, the cases of conjugating one UAS and three UASs were found to be the most superior. This was an increase of about 50% in secretion compared to the existing Pgal10.

[0152] 4-2. Comparison of RFP Expression Levels According to the Number of Pgal10 UAS Repeats

[0153] Using the top two hybrid promoters with high expression levels of the target protein identified in Example 4-1 (i.e., UAS(GAL10)-PGK and UAS(GAL10)-PGKX3), fluorescence was measured by directly attaching RFP to exclude interference with the expression amount of TGF-β1. The GAL10-MFα-RFP vector was used as a control. The cleavage maps of the constructed GAL10-MFα-RFP vector, UAS(GAL10)-PGK-MFα-RFP vector, and UAS(GAL10)X3-PGK-MFα-RFP vector are shown in Figures 8 to 10, respectively.

[0154] As shown in Table 6, it was confirmed that the expression intensity was superior in the order of P(UAS-PGK) and P(UASХ3-PGK) compared to the existing Pgal10.

[0155] Parent strain introduction vector supernatant fluorescence Saccharomyces cerevisiaeCENPK.CP(GAL10)-MFa-RFP15722P(UAS-PGK)-MFa-RFP46562P(UASХ3-PGK)-MFa-RFP51402

Claims

1. An artificial hybrid promoter comprising the following (a) and (b): (a) upstream activation sequence (UAS) of the GAL10 gene; and (b) Core promoter sequence of the PGK gene.

2. In claim 1, the UAS of the GAL10 gene is an artificial hybrid promoter that is repeatedly located upstream of the core promoter.

3. An artificial hybrid promoter according to claim 1, wherein the UAS of the GAL10 gene comprises the nucleotide sequence of SEQ ID NO. 1, and the core promoter sequence of the PGK gene comprises the nucleotide sequence of SEQ ID NO.

2.

4. In claim 1, the artificial hybrid promoter comprises the nucleotide sequence of SEQ ID NO. 3, the nucleotide sequence of SEQ ID NO. 4, the nucleotide sequence of SEQ ID NO. 5, or the nucleotide sequence of SEQ ID NO.

6.

5. A recombinant expression vector comprising an artificial hybrid promoter of any one of claims 1 to 4.

6. A recombinant expression vector according to claim 5, further comprising a gene encoding a target protein.

7. A host cell comprising the recombinant expression vector of claim 5.

8. In paragraph 7, the host cell is a yeast cell.

9. A method for producing a target protein, comprising the step of culturing the host cells of claim 7 to produce the target protein.

10. A kit for producing a target protein comprising at least one of (a) to (c) and (d) below: (a) an artificial hybrid promoter according to any one of paragraphs 1 through 4; (b) a recombinant expression vector comprising the above promoter; (c) a host cell comprising the above-mentioned recombinant expression vector; and (d) A medium composition for inducing expression.

11. Method for producing a target protein using the kit of claim 10.

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