JP6021116B2 - Proteolytic induction method in eukaryotic cells - Google Patents

Description

  The present invention relates to a method for efficiently inducing degradation of a target protein in eukaryotic cells.

  Controlling the expression level of a specific protein in the cell is very useful in analyzing the function of the protein in the cell and the life phenomenon in which the protein is involved. To this end, several methods have been developed so far based on separate principles.

For example, a method of performing transcriptional expression of a gene introduced into cultured cells in a tetracycline-dependent manner using a tetracycline-binding transcription repressing factor of Escherichia coli is known (Non-patent Documents 1 and 2). Transcription suppression systems based on this principle are commercially available, and many researchers have actually used them and have achieved results so far. However, since this method is a method of controlling transcription of the target gene, it often takes time for the actual protein to be removed from the cell even if expression is suppressed. In some cases, a decrease in the expression level may activate a secondary intracellular response.
Similarly, as a method for suppressing expression in a cultured cell, a method is known in which siRNA and shRNA are introduced into the cell and the mRNA of the target gene product is degraded using the intracellular RNA interference reaction. In fact, many companies have released products based on this method. However, since this method also suppresses expression at the mRNA level, it generally requires 24 to 48 hours to suppress expression. In addition, the degree of expression suppression depends on the target factor and target RNA sequence, and it is not very likely that expression suppression of 90% or more can be achieved.

  Recently, a method for controlling protein expression by introducing a degradation-inducing tag into a target protein and controlling the degradation of the tag has been put into practical use. A protein obtained by modifying the rapamycin-binding protein FKBP12 is stabilized when bound to Shield1, which is a rapamycin-like compound, and is destabilized and degraded in the cell if not bound. Utilizing this property, a method for fusing this FKBP12-derived protein to a target protein and controlling expression in a Shield1-dependent manner was disclosed (Non-patent Document 3). In this method, it is generally said that target protein is degraded in about 4 hours by Shield1 removal, and necessary plasmid sets and the like are on the market. In this method, it is necessary to always add Shield1 to the medium in order to stably express the protein. However, since Shield1 is a rapamycin-like compound, its cytotoxicity is questionable. In addition, since the degradation time required for expression suppression is about 4 hours, a system capable of faster degradation and removal has been desired for research on the cell cycle and the like.

  From this situation, the present inventor paid attention to plant-specific proteolysis induced by the plant hormone auxin, introduced this degradation pathway into budding yeast and mammalian cells, and added auxin to the medium for 15 to 30 minutes. A method of suppressing expression by degrading the target protein in a very short time was developed (Patent Documents 1 and 2).

JP 2008-187958 A International Publication No. 2010/125620 Pamphlet

Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89, 5547-5551, 1992. Gossen et al. Science, 268, 1766-1769, 1995. Banazynski et al. Cell, 126, 995-1004, 2006

However, the tag used in Patent Documents 1 and 2 is a plant Aux / IAA family protein. However, when a tag is added to a growth essential factor in budding yeast, the tag may be lethal. In some cases, the phenotype was not observed even when auxin was added because the degradation was not sufficient.
Accordingly, an object of the present invention is to provide a novel protein-inducible eukaryotic cell that does not let a host such as budding yeast kill, and has sufficient resolution, and a method for inducing proteolysis using the same.

  Therefore, the present inventor attempted to modify the Aux / IAA family protein used as a tag, and examined the use of a partial sequence of the IAA family protein in order to reduce the size of the tag. However, even when only the domain II region of the IAA family protein was used, the resolution was not sufficiently improved, and when the partial sequence containing domain I was used, the resolution was not improved. Then, when further examination was continued, a sequence consisting of a region containing at least two Lys residues on the N-terminal side and C-terminal side of the domain II region of the IAA family protein, or two or more of them were ligated unexpectedly. It was found that the use of the sequence as a tag dramatically improves the resolution and does not let the host be lethal, thereby completing the present invention.

  That is, in the present invention, degradation of a target protein is induced by auxins having a plant-derived TIR1 family protein gene and a chimeric gene that expresses the target protein labeled with a plant-derived Aux / IAA family protein partial sequence. A proteolytic inducible non-plant eukaryotic cell, wherein the partial sequence comprises at least two Lys residues on the N-terminal side and C-terminal side of the domain II region of the Aux / IAA family protein. Or a proteolytic-inducible non-plant eukaryotic cell, characterized in that it is a sequence formed by linking two or more such sequences.

  The present invention also provides an auxin that introduces a chimeric gene expressing a target protein labeled with a plant-derived Aux / IAA family protein partial sequence into a host non-plant eukaryotic cell. A proteolysis-inducing non-plant eukaryotic cell production method in which degradation of a target protein is induced by the method, wherein the partial sequence is at least on the N-terminal side and the C-terminal side of the domain II region of the Aux / IAA family protein Provided is a method for producing a proteolytic inducible non-plant eukaryotic cell, characterized in that it is a sequence comprising a region containing two Lys residues, or a sequence formed by linking two or more such sequences. It is.

  Further, the present invention is directed to the degradation of the target protein by auxins having a plant-derived TIR1 family protein gene and a chimeric gene expressing the target protein labeled with a plant-derived Aux / IAA family protein partial sequence. A gene introduction vector for non-plant eukaryotic cells that is proteolytically induced, wherein the partial sequence includes at least two Lys at the N-terminal side and C-terminal side of the domain II region of the Aux / IAA family protein. Provided is a gene introduction vector for proteolytic inducible non-plant eukaryotic cells, characterized in that it is a sequence comprising a region containing residues, or a sequence formed by linking two or more such sequences. .

  Furthermore, the present invention provides a method for inducing degradation of a target protein in the non-plant eukaryotic cell, characterized in that auxins are added to the proteolysis-inducing non-plant eukaryotic cell.

  When the eukaryotic cell of the present invention is used, cell death is not caused, and degradation of the target protein can be induced reliably and efficiently in a short time by adding auxins. It is useful as a tool in the field of drug development targeting functions.

It is the schematic of an IAA family protein gene. A preferred partial sequence of an IAA family protein is shown. It is a figure which shows the resolution | decomposability of each recombinant. It is a figure which shows the resolution | decomposability of each recombinant. It is a figure which shows the resolution | decomposability of each recombinant.

  The proteolytic inducible non-plant eukaryotic cell of the present invention comprises (1) a plant-derived TIR1 family protein gene and (2) a chimeric gene that expresses a target protein labeled with a partial sequence of a plant-derived Aux / IAA family protein. And have. The combination of this TIR1 family protein gene and a chimeric gene that expresses the target protein labeled with a partial sequence of the Aux / IAA family protein causes a degradation pathway based on a plant ubiquitinase complex in a non-plant eukaryotic cell. It is formed. That is, ubiquitin / proteasome proteolysis is known in various eukaryotes such as animals, plants, and fungi. In this degradation system, ubiquitin is bound to a target protein by three enzymes, ubiquitin activating enzyme (E1) -ubiquitin-conjugating enzyme (E2) -ubiquitin ligase (E3), and the polyubiquitinated target protein is specific by the proteasome. Is a system that is recognized and disassembled. As this ubiquitin ligase, an E3 ubiquitinase complex (SCF complex) has been reported. This SCF complex is composed of four subunits: F-box protein, Skp1 protein, Cullin-1 protein, and Rbx1 protein. The plant SCF complex has a TIR1 family protein as an F-box protein, which is a receptor for the growth hormone auxin, and by receiving auxin, an inhibitor of the auxin signal transduction system Aux / IAA It has recently been elucidated to recognize family proteins and degrade them. Therefore, the present inventor uses a plant-derived TIR1 family protein to recognize a partial sequence of the Aux / IAA family protein using auxin as an inducer and decomposes the target protein labeled with the partial sequence of the Aux / IAA family protein. Successfully functioned in eukaryotic cells other than plants.

  With the proteolytic-inducible non-plant cells of the present invention, the expressed target protein can be degraded at any time by adding auxins. That is, according to the proteolysis-inducible non-plant eukaryotic cell of the present invention, for example, a target protein labeled with a partial sequence of an SCF complex containing a plant-derived TIR1 family protein and an Aux / IAA family protein is expressed. Therefore, under the coexistence of auxins, the plant-derived TIR1 family protein of the SCF complex accepts auxins, thereby recognizing the partial sequence of the Aux / IAA family protein by the TIR1 family protein, and Aux / Polyubiquitination to partial sequences of IAA family proteins occurs. The target protein labeled with the partial sequence of the polyubiquitinated Aux / IAA family protein is then degraded by the proteasome. For this reason, for example, it is possible to control the degradation of the expressed target protein by adding or not adding auxins.

  The TIR1 family protein gene used in the present invention is derived from a plant. Although the kind of plant is not restrict | limited here, For example, Arabidopsis thaliana, a rice, Zinnia, a pine, a fern, a tiger beetle, etc. are mentioned. In addition, rice is preferable when the host cell is a mammalian cell. Examples of the plant-derived TIR1 family protein genes include TIR1 gene, AFB1 gene, AFB2 gene, AFB3 gene, FBX14 gene, and AFB5 gene, and TIR1 gene is particularly preferable. More specifically, accession numbers registered in NCBI (National Center for Biotechnology Information) are registered. NM_001059194 (GeneID: 4335696), Os04g0395600 or the accession No. registered in the same database. The genes EAY93933 and OsI — 15707 are preferred.

  The plant-derived TIR1 family gene may be, for example, natural DNA extracted from the above-mentioned plant, for example, rice, Arabidopsis thaliana, or the like, or DNA synthesized by genetic engineering. The TIR1 family gene may be, for example, DNA containing exons and introns, or may be cDNA consisting of exons. The TIR1 family gene may be, for example, a full-length sequence in genomic DNA or a full-length sequence in cDNA. The TIR1 family gene may be a partial sequence in genomic DNA or a partial sequence in cDNA as long as the expressed protein functions as a TIR1 family protein. In the present invention, “functions as a TIR1 family protein” means, for example, recognition of a partial sequence of an Aux / IAA family protein in the presence of auxins. This is because if the plant-derived TIR1 family protein can recognize the partial sequence of the Aux / IAA family protein, the target protein labeled with the partial sequence of the Aux / IAA family protein can be degraded as described above. At this time, in the proteolytic-inducible non-plant eukaryotic cell of the present invention, the plant-derived TIR1 family protein, together with other subunits (Skp1 protein, Cullin protein and Rbx1 protein), is an SCF complex (E3 ubiquitinase complex). Body).

  The proteolytic-inducible non-plant eukaryotic cell of the present invention preferably further has a promoter sequence that controls transcription of the plant-derived TIR1 family gene. Thereby, plant-derived TIR1 family proteins can be expressed more reliably. The promoter is not limited and can be appropriately determined according to, for example, the type of cell.

  In the present invention, the chimeric gene is a chimeric gene that expresses a target protein labeled with a partial sequence of an Aux / IAA family protein. The expressed target protein may be labeled with the partial sequence of the Aux / IAA family protein, and the form thereof is not limited. For example, the expressed target protein is a fusion protein containing the target protein and the partial sequence of the Aux / IAA family protein. Preferably there is. The partial sequence of the Aux / IAA family protein may be added to either the N-terminal side or the C-terminal side of the target protein, for example.

  Further, the positional relationship between the target gene and the partial sequence of the Aux / IAA family gene is not limited, and both genes are functional so that the expressed target protein is labeled with the partial sequence of the Aux / IAA family protein. It suffices if they are arranged. As a specific example, the partial sequence of the Aux / IAA family gene is preferably arranged adjacent to the upstream (5 'side) or downstream (3' side) of the target gene. As long as the target protein is expressed and labeled with a partial sequence of the Aux / IAA family protein, for example, the partial sequence of the Aux / IAA family gene may intervene inside the target gene.

  The type of the Aux / IAA family gene is not limited as long as it is a plant-derived Aux / IAA family gene. Although the kind of the plant is not limited, the Arabidopsis thaliana IAA17 gene is preferable. Specific examples of Aux / IAA family genes include, for example, IAA1 gene, IAA2 gene, IAA3 gene, IAA4 gene, IAA5 gene, IAA6 gene, IAA7 gene, IAA8 gene, IAA9 gene, IAA10 gene, IAA11 gene, IAA12 gene, IAA13 Gene, IAA14 gene, IAA15 gene, IAA16 gene, IAA17 gene, IAA18 gene, IAA19 gene, IAA20 gene, IAA26 gene, IAA27 gene, IAA28 gene, IAA29 gene, IAA30 gene, IAA31 gene, IAA32 gene, IAA33 gene and IAA34 gene Is mentioned. The proteolytic-inducible non-plant eukaryotic cell may have any one kind of partial sequence of the Aux / IAA family gene, or may have two or more kinds. For example, the sequence of an Aux / IAA family gene derived from Arabidopsis thaliana is registered in TAIR (the Arabidopsis Information Resource), and the accession numbers of the respective genes are as follows. IAA1 gene (AT4G14560), IAA2 gene (AT3G23030), IAA3 gene (AT1G04240), IAA4 gene (AT5G43700), IAA5 gene (AT1G15580), IAA6 gene (AT1G52830), IAA7 gene (AT3G23050), A2G23050, A2G23050, A2G23050 (AT5G65670), IAA10 gene (AT1G04100), IAA11 gene (AT4G28640), IAA12 gene (AT1G04550), IAA13 gene (AT2G33310), IAA14 gene (AT4G14550), IAA15 gene (AT1G80390), IAA0G17AT ), IA 18 genes (AT1G51950), IAA19 gene (AT3G15540), IAA20 gene (AT2G46990), IAA26 gene (AT3G16500), IAA27 gene (AT4G29890), IAA28 gene (AT5G25890), IAA29 gene (AT4G32280), AA31A31A31A31A31) (AT3G17600), IAA32 gene (AT2G01200), IAA33 gene (AT5G57420) and IAA34 gene (AT1G15050).

  The present invention provides, as a partial sequence of the Aux / IAA family protein, a sequence comprising a region containing at least two Lys residues on the N-terminal side and the C-terminal side of the domain II region of the Aux / IAA family protein, or It is characterized in that an array formed by connecting two or more arrays is used. By using such a specific partial sequence, the ability to induce degradation of the target protein is improved and cell death is suppressed as compared to the case where the full length of the Aux / IAA family protein, the domain II region, or the like is used, Stable target protein degradation inducibility can be obtained.

  The Aux / IAA family protein is a protein having a total length of about 25 KDa as shown in FIG. 1, and has domain I, domain II, domain III, domain IV and the like from the N-terminal side. Among these, the domain II alone does not improve the ability to induce degradation of the target protein, and the sequence from the N-terminal to the domain II does not improve the ability to induce degradation of the target protein. On the other hand, a sequence comprising at least two Lys residues on the N-terminal side and C-terminal side of domain II, which does not include domain I and domain III may partially include Is used, the ability to induce degradation of the target protein is significantly improved. In addition, when a sequence in which two or more such sequences are linked is used, the ability to induce degradation of the target protein is further improved.

  As such a partial sequence, 32 to 80 amino acid residues are preferable, 50 to 80 amino acid residues are more preferable, 50 to 75 amino acid residues are further preferable, and 50 to 70 amino acid residues are further preferable.

  The partial sequence includes 32 to 80 amino acid residues including 2 to 5, more preferably 2 to 4 Lys residues on the N-terminal side and C-terminal side of the domain II region of the Aux / IAA family protein. The sequence consisting of is more preferred.

  Among the IAA family proteins, examples of partial sequences of IAA17, IAA16, IAA14, IAA9, IAA27, IAA4, IAA1, IAA2, IAA3, IAA8, IAA5, and IAA6 are shown in FIG. 2 and SEQ ID NOs: 1-12. In the present invention, a sequence consisting of 50 to 80 amino acid residues including a sequence selected from these SEQ ID NOs is more preferred, and a sequence consisting of 50 to 75 amino acid residues is more preferred. The entire amino acid sequence of IAA17 is shown in SEQ ID NO: 23.

  In addition, the number of linkages of the sequence is preferably 2 to 5, more preferably 2 to 4, and even more preferably 2 or 3.

  The proteolytic inducible non-plant eukaryotic cell of the present invention preferably further has a promoter sequence that controls transcription of the chimeric gene. Thereby, the target protein labeled with the partial sequence of the Aux / IAA family protein can be expressed more reliably. The promoter is not limited and can be appropriately determined according to, for example, the type of cell.

  In the present invention, the gene of the target protein may be an endogenous gene present in the genome of a non-plant eukaryotic cell or a foreign gene introduced into a non-plant eukaryotic cell. Moreover, the said foreign gene may be integrated in genomic DNA, for example, and does not need to be integrated. In the former case, for example, it is in a state of being incorporated into genomic DNA by a gene transfer vector or the like in which a foreign gene is linked. In the latter case, for example, the gene introduction vector is present as a plasmid, and in the gene introduction vector, the target gene is functionally linked to the origin of replication. preferable.

  In the present invention, the host cell may be any eukaryotic cell other than a plant (non-plant), and examples thereof include cells such as animals, fungi, and protists. Examples of animals include mammals such as humans, mice, rats, and rabbits, fish and amphibians such as zebrafish and Xenopus, C.I. and invertebrates such as elegans and Drosophila. Moreover, the cell type is not limited, for example, cultured cells such as Hela cells, CHO cells, MCF, HEK293, HepG2, NIH3T3, COS cells, DT40; primary cultured cells; hematopoietic stem cells; B cells, T cells, leukocytes Monocytes / macrophages, red blood cells, blood cells such as platelets, blood cells; fertilized oocytes; ES cells and the like. Further, other various tissue cells may be used. Examples of fungi include budding yeast and fission yeast.

  In the present invention, when a mammalian cell is used, it preferably has a Skp1 gene, a Cullin gene, and an Rbx1 gene. Each of these genes is preferably an endogenous gene of a mammalian cell. In the proteolytic inducible mammalian cell of the present invention, an SCF complex is produced from proteins expressed from these genes (ie, Skp1 protein, Cullin protein and Rbx1 protein) and TIR1 family proteins expressed from rice-derived TIR1 family genes. Presumed to be composed.

  The proteolytic inducible non-plant eukaryotic cell production method of the present invention includes, for example, (1) a step of introducing a plant-derived TIR1 family gene into a host eukaryotic cell, and (2) a host eukaryotic cell. This can be performed by introducing a partial sequence of a plant-derived Aux / IAA family gene and forming a chimeric gene that expresses the target protein labeled with the partial sequence of the Aux / IAA family protein.

  In the present invention, the order of the step (1) and the step (2) is not limited and may be performed simultaneously. The introduction of the plant-derived family gene in the step (1) and the introduction of the partial sequence of the Aux / IAA family gene in the step (2) may be performed using, for example, separate vectors. It is preferable to use one vector into which the derived family gene and the partial sequence of the Aux / IAA family gene are inserted.

  In the present invention, the target gene may be an endogenous gene present in eukaryotic cell genomic DNA or a foreign gene, as described above. In the case of the endogenous gene, in the step (2), for example, a chimeric gene can be formed by introducing a partial sequence of an Aux / IAA family gene into a mammalian cell and operably linking to the endogenous target gene. Alternatively, a chimeric gene in which a partial sequence of the Aux / IAA family gene and the target gene are linked may be formed, introduced into a eukaryotic cell, and inserted into the genome by recombination with the genome.

  When the target gene is a foreign gene, for example, the foreign gene may be introduced into the eukaryotic cell prior to the introduction of the partial sequence of the Aux / IAA family gene. When the target gene is a foreign gene, for example, it may be introduced into a eukaryotic cell together with a partial sequence of the Aux / IAA family gene. Specifically, a chimeric gene in which a partial sequence of the Aux / IAA family gene and the gene of the target protein are functionally linked in advance can be prepared and introduced into eukaryotic cells.

  An example in which the target gene is a foreign gene and a TIR1 family gene and a chimeric gene are introduced by recombination using a vector will be described.

  First, a plant-derived TIR1 gene is incorporated into a vector to prepare a TIR1 gene introduction vector. The type of vector is not limited, and can be appropriately determined according to, for example, the type of eukaryotic cell. Specific examples include plasmid vectors and virus vectors. Examples of the plasmid vector include pCMV, pcDNA, and pACT. Examples of the viral vector include an adenovirus expression system.

  The TIR1 gene introduction vector preferably has a promoter sequence that controls transcription of the TIR1 family gene. The promoter is not limited and can be appropriately determined according to, for example, the type of eukaryotic cell. Specific examples include CMV promoter, SV40 promoter, GAL4 binding sequence and the like. In general, the promoter is preferably operably linked upstream (5 ′ side) of the TIR1 family gene. Further, it may be a cell type-specific or organ-specific promoter. Further, a TIR1 family gene having no promoter sequence may be incorporated downstream of an endogenous promoter in a host cell or host animal individual, for example. In this case, the TIR1 family gene may be targeted to a specific gene, for example, or may be randomly incorporated.

  Moreover, since the presence or absence of introduction into eukaryotic cells can be confirmed, the TIR1 gene introduction vector may further have a selection marker coding sequence. The selection marker coding sequence is not limited, and includes a sequence encoding a marker such as a known drug resistance marker, fluorescent protein marker, cell surface receptor marker and the like. The drug resistance marker is not limited, and examples thereof include a neomycin resistance marker, a puromycin resistance marker, and a hygromycin resistance marker. Examples of the fluorescent protein marker include GFP (Green Fluorescent Protein) and EGFP (mutant GFP). Examples of enzyme markers include luciferase and β-galactosidase. These selectable marker coding sequences may be synthesized by PCR or the like according to the sequence, or prepared from a commercially available vector having the selectable marker coding sequence. When a TIR1 gene introduction vector and a later-described chimeric gene introduction vector are used in combination, the selection marker in the TIR1 gene introduction vector and the selection marker in the chimeric gene introduction vector are preferably different markers. Furthermore, the TIR1 family gene is functionally linked to, for example, another protein gene or a tag sequence, and is a TIR1 protein labeled with a tag as a protein (for example, a fusion protein) containing the protein and the TIR1 protein. As such, it may be expressed.

  On the other hand, a target gene and a partial sequence of a plant-derived Aux / IAA family gene are linked to a vector to prepare a vector for introducing a chimeric gene. As described above, the chimeric gene is a gene that expresses a target protein labeled with a partial sequence of an Aux / IAA family protein. In the vector, the position of the target gene and the partial sequence of the Aux / IAA family gene is not limited as long as the labeled protein can be expressed as described above. As a specific example, the partial sequence of the Aux / IAA family gene may be arranged adjacent to the upstream (5 ′ side) or downstream (3 ′ side) of the target gene. A partial sequence of an IAA family gene may intervene.

  The vector is not limited and includes the same ones as described above. Moreover, the chimeric gene introduction vector preferably has a promoter sequence that controls transcription of the chimeric gene. Examples of the promoter include those described above. In addition, since the presence or absence of introduction into a eukaryotic cell can be confirmed, the vector for introducing a chimeric gene may further have a selection marker coding sequence as described above.

  Then, the above-described TIR1 gene introduction vector and chimeric gene introduction vector are introduced into eukaryotic cells which are host cells (step (1) and step (2)). The order of introducing both vectors is not limited.

  The method for introducing the vector is not particularly limited, and can be appropriately determined depending on, for example, the type of vector to be used and the type of host cell. Examples of the introduction method include a calcium phosphate precipitation method, a DEAE dextran transfection method, and an electroporation method. In addition, methods using a retrovirus vector, an adenovirus vector, and the like can be given.

  In the present invention, it is efficient to use an expression vector into which a TIR1 gene and a chimeric gene are inserted. Specifically, it is preferable to use these genes inserted into a vector having a virus-derived promoter and IRES (ribosome binding site inside mRNA). Usually, such a pIRES vector is designed to be used by inserting a target gene to be expressed between a virus-derived promoter and the IRES. However, the present inventor efficiently produced an expression vector by linking a rice-derived TIR1 family gene between a virus-derived promoter and IRES, and linking the chimeric gene downstream thereof, and It was found that degradation of the target protein was rapidly induced (see WO 2010/125620 pamphlet). Examples of the promoter of this expression vector include CMV promoter, SV40 promoter and the like, and CMV promoter is preferable.

  In addition, when the target gene is present in the genomic DNA, for example, when the target gene is an endogenous gene of a eukaryotic cell, or the target gene is a foreign gene, it has already been incorporated into the genomic DNA of the eukaryotic cell. In this case, it can be manufactured as follows. In this case, instead of the chimeric gene introduction vector, an Aux / IAA family gene introduction vector into which a partial sequence of the Aux / IAA family gene is inserted is used. This vector for gene transfer is, for example, a partial sequence of an Aux / IAA family gene at a site where a target protein can be labeled with a partial sequence of an Aux / IAA family protein during protein expression of eukaryotic genomic DNA. Is preferably a structure in which is incorporated. That is, the gene introduction vector has a structure in which a target gene in genomic DNA and a partial sequence of the Aux / IAA family gene are functionally linked to form a chimeric gene by recombination with genomic DNA. That's fine. By incorporating the partial sequence of the Aux / IAA family in this way, the target protein labeled with the partial sequence of the Aux / IAA family can be expressed. A chimera gene introduction vector can be constructed by a person skilled in the art from, for example, the locus of a target gene in genomic DNA, its sequence, and the like.

  When auxins are allowed to act on the proteolytic-inducible eukaryotic cells of the present invention, degradation of the target protein is induced. Degradation of the target protein is reliable and extremely rapid. For example, it is almost completely decomposed in 15 to 30 minutes.

  In the induction of target protein degradation, the amount of auxin added is not limited, and can be appropriately determined according to, for example, the type of auxin. Specific examples include 1 μM to 1 mM, preferably 20 μM to 500 μM.

  Examples of auxin include 1-naphthalene acetic acid (NAA) and indole-3-acetic acid. In addition to this, there are compounds having the same physiological activity as NAA and the like, for example, 2,4-dichlorophenoxyacetic acid, 4-chlorophenoxyacetic acid, (2,4,5-trichlorophenoxy) acetic acid. 1-naphthaleneacetamide, 2,4-dichlorophenoxyacetic acid, 4-parachloroacetic acid and the like. For example, a precursor that has physiological activity of auxin by metabolism can also be used. For example, a substance that is converted into a substance having auxin activity by esterase or β-oxidase in the host cell is preferable. Specific examples include indole-3-acetic acid methyl ester and indole-3-butyric acid.

  The auxins may be added to the medium containing the cells.

  Thus, since the degradation of the target protein can be induced promptly by the addition of auxins, the influence of the target protein can be examined by comparing with the case where auxins are not added. More specifically, a method for suppressing degradation of a newly expressed target protein by adding auxins to the cell to induce degradation of the expressed target protein and then removing the auxin; After the auxins are added to induce the degradation of the expressed target protein, an auxin inhibitor can be added to check the degradation of the newly expressed target protein.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

Reference example 1
1. Production of Saccharomyces cerevisiae strain The budding yeast strain | stump | stock which decomposes | disassembles EGFP by adding NAA was made into protein for the objective decomposition | disassembly. The promoter sequence and gene sequence of the budding yeast used can be found on the SGD website http: // www. yeastgenome. org /.

(1) Arabidopsis TIR1-expressing yeast strain (YNK2)
The pRS306-GAL plasmid vector was cut with Sal1 and Xho1 and self-ligated. The pRS306-GAL plasmid vector is described in the literature (L. Dury, G. Perkins and J. Diffley “The Cdc4 / 34/53 pathway targets Cdc6p for proteases in binding yeast”, 1997, 59-59. Yes. Thereby, the SalI site in the multicloning site of the plasmid vector was removed. The TIR1 gene (TAIR accession No. AT1G04250.1) of Arabidopsis thaliana was amplified by PCR using the following primer set 1 (1785 bp), and this amplified product was cloned into the Spe1-Not1 site of the plasmid vector. The obtained vector is called pMK26.
<Primer set 1>
F primer 7 (SEQ ID NO: 13)
5′-AGCTAGACTAGTATGCAGAAGCGAATACCCTT-3 ′
R primer 8 (SEQ ID NO: 14)
5′-ATCGATGCCGCCCGCAGATCTGCTAGTCGAACTAATCCGTTAGTAGTAATGA-3 ′

  The SalI-BglII fragment was cleaved from the pYM18 plasmid vector to excise the 9Myc tag portion (435 bp) and cloned into the SalI-BglII site of the vector pMK26. The pYM18 plasmid vector is described in the literature (C. Janke, M. Magiera, N. Rathfelder, C. Taxis, S. Reber, H. Maekawa, A. Moreno-Borchart, G. Doenges, E. Schwobel, E. Schiebel, E. and Schiebel. M. Knop. "A versatile toolbox for PCR-based tagging of yeast genes: new fluorescens proteins, more marques and promoter subs. The obtained vector is called pMK27. This vector pMK27 was cleaved with Stu1 inside the URA3 marker, transformed into the budding yeast wild strain W303-1a, and the plasmid vector was inserted into the URA3 site on the budding yeast genome by homologous recombination. The obtained recombinant was designated as an Arabidopsis TIR1 expression strain “YNK2”. In YNK2, a vector-derived GAL1-10 promoter is arranged upstream of the TIR1 gene.

The genotypes of wild strains W303-1a and YNK2 are shown below.
W303-1a
MATa ade2-1 ura3-1 his3-11, 15 trp1-1 leu2-3, 112 can1-100
YNK2
MATa ade2-1 his3-11, 15 trp1-1 leu2-3, 112 can1-100
ura3-1: URA3-GAL1-10 promoter-Arabidopsis TIR1 (pMK27 integrated)

Rice TIR1-expressing yeast strain preparation Rice TIR1 gene (NCBI, Accession No. NM_001059194) was PCR amplified from a rice cDNA library with the following primer set 2. The amplified product was cloned into a portion where pMK26 was treated with SpeI and SalI to remove the Arabidopsis TIR1 gene.
<Primer set 2>
F primer 5′-GGGGATCCCATGACGTACTCTCCGGAGGAGGT-3 (SEQ ID NO: 15)
R primer 5′-CCCGTCGATAGTAGATTTAACAAAAATTTGGGTG-3 ′ (SEQ ID NO: 16)
Rice TIR1 was introduced into Saccharomyces cerevisiae in the same manner as when Arabidopsis TIR1 was introduced.

<Genetic type of rice TIR1-expressing yeast strain>
MATa ade2-1 his3-11, 15 trp1-1 leu2-3, 112 can1-100 ura3-1: URA3-GAL1-10 promoter-rice TIR1

Reference example 2
1. Production of Saccharomyces cerevisiae strain (1) Auxin degron (mcm4-ad) strain of endogenous protein Mcm4 A strain was prepared by using endogenous protein Mcm4 as a target protein for degradation and adding NAA. The endogenous protein Mcm4 is an essential protein involved in DNA replication. Nature Methods, Vol. 6, no. 12, p917 (Dec. 2009) was amplified by PCR using the following primer set 3. Then, using the obtained amplification product, the TIR1 expression strain YNK2 obtained in Reference Example 1 (1) was directly transformed, and by homologous recombination, the MCM4 5 ′ portion on the budding yeast genome was transformed into CUP1 Promoter-IAA17 was introduced. This recombinant is referred to as “YNK14”. The insertion into the genome was confirmed by PCR.

<Primer set 3>
F primer 180 (SEQ ID NO: 17)
5'-TTGTCCTTGGCGAGGGTGTAAGGAGATCAGTTCGCCTGAATAACCGTGTCCGTACGCTGCAGGTCGAC-3 '
R primer 181 (SEQ ID NO: 18)
5'-TCAATCGAGCCTACATACAGTATTGAATAGTGTTACAAAGCATAAGGATGATCGATGAATTCGAGCTCG-3 '

The genotype of YNK14 is shown below.
YNK14
MATa ade2-1 his3-11, 15 trp1-1 leu2-3, 112 can1-100
ura3-1: URA3-GAL1-10 promoter-Arabidopsis TIR1 (pMK27 integrated)
mcm4: mcm4-ad (kanMX)

2. Growth Experiment of Saccharomyces cerevisiae strain YNK4 strain was added to YPDCu agar medium without addition of NAA (2% peptone, 1% yeast powder, 2% glucose, 0.1 mM CuSO ₄ , 2% agar) and YPGNAA agar medium with NAA ( 2% peptone, 1% yeast powder, 2% galactose, 0.1 mM NAA, 2% agar) and a predetermined number of cells per plate (5 × 10 ⁵ , 5 × 10 ⁴ , 5 × 10 ³ 5 × 10 ² , 5 × 10), and cultured at 24 ° C. for 2 days. Cell growth was observed.

Example 1
In Reference Example 2, instead of the full-length tag (1-229) (SEQ ID NO: 23) of IAA17, IAA17 (1-132), IAA17 (1-34), IAA17 (Domain II), IAA17 (128-132) and A recombinant into which IAA17 (65-132) (SEQ ID NO: 1) was introduced was prepared.
In the same manner as in Reference Example 2 (2), the obtained recombinant was observed for the ability to induce protein degradation (cell growth) when auxin was not added and auxin was added.

These results are shown in FIG. The figure shows the results of culturing at 24 ° C. The number of cells spotted from the left is 5 × 10 ⁵ , 5 × 10 ⁴ , 5 × 10 ³ , 5 × 10 ² , and 5 × 10 for each strain. . Moreover, the figure on the left side of the figure is a growth image of cells when auxin is not added and the center figure is when auxin is added. The right side is a schematic design of the chimeric gene.

  From FIG. 3, the resolution was clearly reduced in IAA17 (1-94) and IAA17 (domain II) compared to the case where the full length of IAA17 was used as the degradation tag. On the other hand, the resolution of IAA17 (1-132), IAA17 (28-132) and IAA17 (65-132) is equal to or higher than the full length of IAA17. Among them, IAA17 (65-132) Codes part of the short IAA 17.

Example 2
Full length IAA17 or IAA17 (65-132) was added to the C-terminus of Psf2 involved in budding yeast chromosome replication. To add, pMK43 has the same sequence as pMK43 for adding full-length IAA and pMK43 for adding IAA17 (65-132), but the full-length IAA17 portion is modified to IAA17 (65-132) using the following primer set: DNA amplification was carried out by PCR using pMK68 as a template.

5′-CAGCATCTCTTACCGCTGGTACTGAAAATGATGAAGAAGAATTCAATATTCGTACGCTGCAGGTCGAC-3 ′ (SEQ ID NO: 19)
5′-AAATACATTCTATGCCCATTAACTAGGATACCACAACAAGTACATATATAATCGATGAATTCGAGCTCG-3 ′ (SEQ ID NO: 20)

After the obtained DNA was purified, it was transformed into the YNK2 strain and the target DNA was inserted downstream of the PSF2 gene by homologous recombination. Insertion into the genome was confirmed by PCR.
However, a strain having the full length IAA17 could not be created. This indicates that tagging can cause lethality. On the other hand, IAA17 (65-132) could be added without any problem by the same method. Further, the obtained strain expressing Psd2-IAA17 (65-132) has a predetermined number of cells per plate (5 × 10 ⁵ , 5 × 10 ⁴ , 5 × 10 ³ , 5 × 10 ² , 5 × 10) and cultured at 24 ° C. for 2 days. Then, cell growth was observed. As a result, a strain expressing Psd2-IAA17 (65-132) could not grow on a medium supplemented with NAA (FIG. 4, + auxin). This indicates that Psf2-IAA17 (65-132) is degraded in the cells.

Example 3
In order to test whether multiple linked IAA17 (65-132) functions more, various modified IAA17 shown in FIG. 5 were added to the C-terminal of Sld3 involved in chromosomal replication in Saccharomyces cerevisiae. The following primers are used for addition: pMK43 for full-length IAA addition, pMK68 for IAA17 (65-132) addition, and pMK43 for multiple IAA17 (65-132) addition, but in the full-length IAA17 part. PMK74 with 2 × IAA17 (65-132) and pMK77 with 3 × IAA17 (65-132) were used as templates.

5′-ATAGCTCAAAAAGGAGAGTAAGAAGACGTTTATTTGCTCCAGAATCCACACGTACGCTGCAGGTCGAC-3 ′ (SEQ ID NO: 21)
5′-TTTAATTGTATACTCAAAGGCCCCCGAAGTGCGAAATTGTTGTAGCTTAGATCGATGAATTCGAGCTCG-3 ′ (SEQ ID NO: 22)

  After the obtained DNA was purified, it was transformed into the YNK2 strain, and the target DNA was inserted downstream of the SLD2 gene by homologous recombination. Insertion into the genome was confirmed by PCR.

When the obtained strain is a medium containing NAA (+ auxin) or a medium not containing NAA (-auxin), the number of cells per plate is a predetermined number (5 × 10 ⁵ , 5 × 10 ⁴ , 5 × 10 ^3). 5 × 10 ² , 5 × 10), and cultured at 24 ° C. for 2 days. Then, cell growth was observed.

  As a result, as shown in FIG. 5, only weak growth inhibition was observed in the strains to which full-length IAA17 and IAA17 (65-132) were added. This indicates that degradation of Sld2-IAA17 and Sld2-IAA17 (65-132) is not sufficiently efficient in the cell. On the other hand, when 2 × IAA17 (65-132) and 3 × IAA17 (65-132) were added, it became clear that growth inhibition became stronger as the number increased. This indicates that the plurality of IAA 17 (65-132) has higher decomposition efficiency than the original full length IAA 17 and IAA 17 (65-132).

Claims (7)

Proteolytic-inducible protein comprising a plant-derived TIR1 family protein gene and a chimeric gene expressing a target protein labeled with a partial sequence of a plant-derived Aux / IAA family protein, wherein auxins induce degradation of the target protein IAA17 (65-132) , which is a non-plant eukaryotic cell, wherein the partial sequence contains 2 to 5 Lys residues at the N-terminal side and C-terminal side of the domain II region of the Aux / IAA family protein. partial sequence consisting to a region IAA17 (1-132) from a region of SEQ ID NO: 1), or non-plant true proteolytic inductive, characterized in that the partial sequence is 2-4 linked comprising sequences Nuclear cells. The partial sequence, IAA17 (1-132), IAA17 ( 28-132) or IAA17 (65-132) (SEQ ID NO: 1) a partial sequence consisting of, or the partial sequence 2-4 linked comprising sequences there claim 1 Symbol placement proteolytic inducible non-plant eukaryotes. A non-plant eukaryotic cell is introduced with a plant-derived TIR1 family protein gene and a chimeric gene expressing a target protein labeled with a plant-derived Aux / IAA family protein partial sequence. A method for producing proteolytically induced non-plant eukaryotic cells, wherein the partial sequence comprises 2 to 5 Lys at the N-terminal side and C-terminal side of the domain II region of the Aux / IAA family protein. IAA17 (65-132) (SEQ ID NO: 1) a partial sequence consisting of a region to a region IAA17 (1-132), including residues, or that the partial sequence is 2-4 linked comprising sequences A method for producing a proteolytic inducible non-plant eukaryotic cell. The partial sequence, IAA17 (1-132), IAA17 ( 28-132) or IAA17 (65-132) (SEQ ID NO: 1) consisting of a sequence, or the partial sequence is 2-4 linked comprising sequences A method for producing a proteolytic-inducible non-plant eukaryotic cell according to claim 3 . Proteolytic-inducible protein comprising a plant-derived TIR1 family protein gene and a chimeric gene expressing a target protein labeled with a partial sequence of a plant-derived Aux / IAA family protein, wherein auxins induce degradation of the target protein a gene transfer vector for non-plant eukaryotic cell, partial sequence comprises a Lys residue of each 2-5 N-terminal and C-terminal domain II region of Aux / IAA family protein IAA17 (65-132) (SEQ ID NO: 1) a partial sequence consisting of up to the region of IAA17 (1-132) from the region of, or proteolytic, characterized in that the partial sequence is 2-4 linked comprising sequences A gene transfer vector for inducible non-plant eukaryotic cells. The partial sequence, IAA17 (1-132), IAA17 ( 28-132) or IAA17 (65-132) (SEQ ID NO: 1) consisting of a sequence, or the partial sequence is 2-4 linked comprising sequences The gene introduction vector for proteolytic-inducible non-plant eukaryotic cells according to claim 5 . 3. A method for inducing degradation of a target protein in a non-plant eukaryotic cell, comprising adding an auxin to the non-plant eukaryotic cell according to claim 1 or 2 .