WO2007094391A1 - Method for suppressing gene expression utilizing interdomain interaction - Google Patents

Method for suppressing gene expression utilizing interdomain interaction Download PDF

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WO2007094391A1
WO2007094391A1 PCT/JP2007/052690 JP2007052690W WO2007094391A1 WO 2007094391 A1 WO2007094391 A1 WO 2007094391A1 JP 2007052690 W JP2007052690 W JP 2007052690W WO 2007094391 A1 WO2007094391 A1 WO 2007094391A1
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domain
protein
cell
expression
target
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PCT/JP2007/052690
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Japanese (ja)
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Shinsuke Fujiwara
Hiroki Higashibata
Hiroyuki Kitagawa
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Kwansei Gakuin Educational Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a method for suppressing gene expression using interaction between domains, and more specifically, a domain in protein folding that proceeds with translation from mRNA in a cell.
  • the present invention relates to a method for suppressing gene expression using a domain that interferes with the interaction between cells.
  • RNAi RNA interference
  • Gene expression is usually performed by (1) transcription of genetic information from genomic DNA to mRNA, (2) splicing (in the case of eukaryotic cells), (3) translation of mRNA into amino acid sequence in ribosomes, If necessary, it can be said to be a series of processes in which a protein having the original activity is finally produced in the cell through the processes of (4) post-translational modification such as glycosylation. But Theoretically, the expression of the target gene can be specifically suppressed by inhibiting the progress of any of the above processes.
  • the RNAi method described above is a method that inhibits the transcriptional translation process by specifically degrading the mRNA of the target gene and suppresses gene expression, but in order to realize various suppressions of gene expression. Therefore, development of a novel gene expression suppression method that inhibits a process different from RNAi method is desired.
  • Patent Document 1 proposes a method for inhibiting the folding of this protein, but no examples that actually inhibit the folding are disclosed in the same document.
  • Non-Patent Document 3 shows an example in which peptide refolding is inhibited in vitro using peptide fragments. This method does not allow protein folding in cells. It is essentially different from the method of inhibition.
  • Patent Documents 2 to 4 disclose methods for suppressing abnormal folding of amyloid monopeptide, prion protein, etc. using compounds such as peptides. It's not a way to suppress it.
  • Patent Document 1 International Publication No. WO 99/40435 Pamphlet
  • Patent Document 2 International Publication No. WO 96/39834 Pamphlet
  • Patent Document 3 International Publication No. WO 01/34631 Pamphlet
  • Patent Document 4 JP-A-2005-120002
  • Patent Document 5 JP-A-2005-278485
  • Non Patent Literature 1 Nature 391: 806-811 (1998)
  • Non-Patent Document 2 Nature 411: 494-498 (2001)
  • Non-Patent Document 3 PNAS, Vol.86, No.9, 3060-3064 (1989)
  • the present invention has been made in view of the above-mentioned problems, and the object thereof is to provide a novel gene expression suppression method that focuses on a process different from the conventional method, more specifically, To provide a novel gene expression suppression method by inhibiting protein folding in the target cell, and to provide a gene expression inhibitor and a gene therapeutic agent using this method. is there.
  • the RNAi method is known as a conventional gene expression suppression method.
  • the present inventor considered that this interference method cannot be applied at the protein level.
  • polypeptide chains translated from mRNA are folded sequentially from the N-terminal side to form a stable domain structure.
  • the protein After the interaction between multiple domains that are sequentially formed in this way, the protein finally acquires the tertiary structure (conformation) necessary for its activity.
  • the present inventor does not inhibit the folding of the protein by interfering with the interaction between the domains, or pays attention to the domain of the target protein itself as an interfering substance, and the domain If the protein is expressed in the cell and its folding is inhibited, the protein that has failed to take the correct structure is not degraded by proteases in the cell. I thought that it would be possible to obtain the effect of specifically suppressing the expression of.
  • the present invention is based on the above findings and includes the following inventions (1) to (: 16).
  • a method for suppressing the expression of a target gene in a cell by introducing a protein domain encoded by the target gene into the cell and inhibiting the folding of the protein.
  • a gene expression inhibitor comprising a vector constructed to express a protein domain encoded by the gene in a target cell in order to suppress the expression of the target gene in the cell.
  • a gene therapy drug comprising a vector constructed to express in a target cell a protein domain encoded by the gene in order to suppress the expression of a disease-related gene in the cell.
  • a method of promoting the activity or expression of a target protein in a cell by introducing a domain of the target protein into the cell.
  • FIG. 1 is a diagram showing the tertiary structure of CGTase derived from Thermococcus sp. B1001 modeled by SWISS-MODEL.
  • Domain A is the region from Metl to Tyr424
  • domain B is the region from Gly 425, et al., Pro512
  • domain C is the region from Thr513 to Thr604
  • domain D is the region from Asn605 to Arg712.
  • FIG. 2 is a diagram showing a strategy for constructing a co-expression vector.
  • Gene fragments encoding four globular domains of CGTase were introduced into pET vectors to prepare pET-A, pET-B, pET-C, and pET-D. Furthermore, the gene cgtA region encoding CGTase was inserted downstream.
  • Bm is BamH I
  • Nd is Nde I
  • Nc Nco I
  • Bp is Bpull02 I.
  • FIG. 3 is a diagram showing the results of confirming expression by SDS_PAGE. All of domains A, B, C, and D showed bands of the expected size, and both were confirmed to be expressed alone. When domains A and B were co-expressed, no CGTase band was observed. When domains C and D were co-expressed, CGTase bands were also confirmed.
  • M is a molecular mass marker
  • lane 1 is a cell before induction
  • lane 2 is a cell after induction
  • lane 3 is a cell-disrupted insoluble fraction
  • lane 4 is a cell-disrupted soluble fraction
  • lane 5 is soluble fraction at 80 ° C 2
  • the results of the supernatant fraction heat-treated at 0 minutes and centrifuged are shown.
  • PET-21a is a negative control for pET-A, C and D
  • pET_8c is a negative control for pET-B
  • CGTase is 83 kDa
  • domain A is 49 kDa
  • domain B is 9.7 kDa
  • domain C is 10.1 kDa
  • Domain D is 12.0 kDa.
  • FIG. 4 is a graph showing the effect of co-expression on activity.
  • the CD-synthesizing activity was measured using the soluble fraction of the cell disruption solution after induction of IPTG. When only CGTase was expressed (pC GTA), the activity value was defined as 100%, and the respective ratios were represented by graphs. Three measurements were taken for each sample. Error bars represent standard deviation.
  • FIG. 5 is a graph showing the effect of supplementation on activity.
  • the soluble fraction of the cell disruption fluid expressing each domain by IPTG induction was added to CGTase and the activity was measured.
  • a graph comparing the activity values of the E. coli lysates of empty vectors (pET-21a and pET-8c) with the activity values of cells containing lysate cells expressing domains A, B, and C. did. Each sample was measured three times. Error bars represent standard deviation.
  • the present invention provides (1) a method for suppressing the expression of a target gene in a cell by introducing a protein domain encoded by the target gene into the cell and inhibiting the folding of the protein.
  • the expression of a gene means that a protein encoded by the gene and having the original structure and activity is produced.
  • the protein expression has a protein having the original structure and activity. It means being produced.
  • Suppressing the expression of a target gene (or target protein) means reducing the production amount of this protein.
  • a method for introducing a domain into a cell a method for constructing a vector for expressing the domain in a target cell and introducing the expression vector into the cell as described later is preferable.
  • a method of introducing a domain protein directly into a cell is also conceivable.
  • a domain having the same structure as the domain of the target protein itself acts as an interference protein (interference peptide) and interferes with the interaction between domains during protein folding. Inhibits folding using domain interference (peptide interference).
  • the domain means a region having a unity in the three-dimensional structure of the protein. Proteins are often structurally divided into multiple regions, and these regions are usually called domains. In many cases, these regions are functional units.In addition, multiple domains are structurally and functionally linked to each other, such as domains close to each other to form a pocket, and this depression serves as a binding site for other molecules. There are many cases of dependence.
  • the folding interference domain of the present invention can be selected from domains determined by modeling the tertiary structure of the target protein. For example, as shown in the examples below, SWISS-MODEL (http: ⁇ swissmodel.expasy.org/) can be used to model the structure and determine the domain based on this structure. It is.
  • the interference domain of the present invention is preferably selected from globular domains of the target protein.
  • the globular domain means a region that is externally more structurally independent, and at least includes a domain that is currently recognized as a globular domain in various proteins.
  • the N-terminal domain for example, the N-terminal domain or the second domain from the N-terminal side
  • the interference domain of the present invention more effective expression suppression can be expected.
  • the length of the region selected as the interference domain of the present invention does not have to be very precise.
  • the length of the domain determined by structural modeling is closer to the N-terminal side or C-terminal side. Even a few residues longer (or shorter) can cause domain interferences of the present invention.
  • not all domains determined by structural modeling can be used as the interference domain of the present invention. Because domain interference occurred in the three domains, domain interference is considered to occur with a relatively high probability.
  • a vector is prepared so that the domain is expressed in the target cell, and the expression vector is introduced into the cell, thereby interfering. Domains can be expressed intracellularly.
  • a viral vector, a plasmid, a phage, or a cosmid can be used, and is not particularly limited.
  • a promoter to be used may be selected according to the type and purpose of the target cell and placed upstream of the DNA region on the vector encoding the interference domain.
  • the CGTase used in the examples described below is Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) derived from the hyperthermophilic archaeon, Thermococcus sp.
  • SEQ ID NO: 11 in the sequence listing shows the DNA base sequence and amino acid sequence of this thermostable CGTase, and SEQ ID NO: 12 shows the amino acid sequence thereof.
  • FIG. 1 shows the force showing the tertiary structure of this thermostable CGTase modeled by SWISS-MODEL.
  • this enzyme has four globular domains, domains A, B, C, and D. It consists of. Of these, domains A, B, and D showed suppression effects due to domain interference (Fig. 4).
  • CGTase is an enzyme belonging to the amylase family.
  • other members of the amylase family with similar structures such as isoamylase, pullulanase, amiguchi pullulanase, neopullulanase, and debranching enzyme, are also subject to domain interference. Therefore, it is considered that gene expression can be specifically suppressed.
  • thermostable protein having a very stable structure may more strongly interact between domains during folding. If so, it can be said that the method of the present invention for inhibiting folding is more effective in suppressing the expression of such thermostable proteins.
  • proteins and genes that can be suppressed in expression by applying the present invention are not particularly limited.
  • the present invention can be used for various studies in the life science field including functional analysis of genes.
  • the present invention that is, the vector constructed to express the domain of the protein encoded by the gene in the target cell in order to suppress the expression of the target gene in the cell is described in the present invention. And a method of using it as a gene expression inhibitor.
  • the present invention is not limited to research use, but also for the development of therapeutic drugs that use the activity regulation of the protein encoded by the gene by specifically suppressing the expression of the specific gene in the living body. Is also applicable.
  • the present invention can be applied to gene therapy for AIDS, cancer, various genetic diseases, etc. by using the vector as a gene therapy drug of the present invention and suppressing the expression of disease-related genes in the target cells. Is. It can also be applied to the treatment of conformational diseases such as Alzheimer's disease and prion disease that are caused by abnormal protein folding.
  • the vector is used as the gene therapy drug of the present invention
  • a known DDS drug delivery system
  • the vector is used as a carrier such as a ribosome. It may be encapsulated and administered in the body. At this time, if a carrier that specifically recognizes cells at the target site is used, the gene therapy drug of the present invention can be efficiently delivered to the target site.
  • the present invention may also be used for suppressing the expression of foreign genes.
  • a method of introducing the interference domain into the cell using a vector constructed to co-express the interference domain and the foreign gene in the target cell can be mentioned.
  • the interference domain can be expressed at a high concentration around the ribosome, and effective suppression of expression can be expected.
  • the present invention suppresses the above-described gene expression using domain interference.
  • the present invention provides not only a method but also a gene expression promotion method using domain interference and a gene expression regulation method that is a concept encompassing these methods. Specifically, (1) a method for promoting (or regulating) expression of a target gene in a cell by introducing a domain of the protein encoded by the target gene into the cell, and (2) the target protein There is provided a method for promoting (or regulating) the activity or expression of a target protein in a cell by introducing the domain into the cell. Further, this promotion method and regulation method of the present invention can be used as a gene expression promoter or gene expression regulator as well as the above-described suppression method, and further a gene for treating a disease by regulating gene expression. It can be applied to therapeutic drugs.
  • the suppression method, the promotion method, and the regulation method of the present invention are the force that regulates gene expression by utilizing the domain interference effect on folding. Not only the unit but also the smaller structural unit motif (super-secondary structure), and even partial peptides with a secondary structure are considered to have similar interference effects. Accordingly, in each of the above methods of the present invention, a partial peptide having a motif or a secondary structure may be used instead of the domain.
  • the present inventor considered that the RNAi method of interference could be applied even at the protein level, and in this example, the possibility of inhibition of folding due to domain interference was examined.
  • CGTase has four globular domain forces, and finally converges to the correct conformation by the interaction between domains during folding.
  • CGTase will not be able to obtain the correct conformation. Proteins that are unable to take on the correct structure in the cell will soon be degraded by proteases.
  • specific gene expression suppression is expected to occur.
  • the possibility of specific gene expression suppression by domain interference was examined using CGTase derived from Thermococcus sp. B1001 as a model protein.
  • strains strains
  • plasmids Plasmids
  • Cloning was performed using E. coli TGI and vectors pET-21a (Novegen) and pET_8c. Expression vector construction was performed according to the strategy shown in FIG. pCGTA (J. Biosci. Bioeng. 89: 206-209 (2000)) was used as a saddle, and the gene fragment corresponding to each domain of CGTase of B1001 strain was amplified by PCR. A restriction enzyme recognition sequence was inserted on the primer for introduction into the pET vector. Af and Ar primers for domain A IJ, Bf and Br primers for domain B, Cf and Cr primers for domain C, and Df for domain D Dr primers were used (see Table 3 below for each primer sequence).
  • Domains A, C, and D were cleaved with restriction enzymes Nde I and BamH I and incorporated into pET_21a to prepare pET-A, pET-C, and pET-D, respectively.
  • Domain B was cleaved with restriction enzymes Nco I and BamH I and incorporated into pET-8c to prepare pET_B.
  • the CGTase gene was amplified by PCR using pCGTA as a saddle and CGT A-f and CGTA-r primers.
  • the transformant of E. coli BL2KDE3) by the prepared expression vector was cultured with shaking at 37 ° C in Luria-Bertani medium containing 50 ⁇ g / ml ampicillin.
  • IPTG was added to a final concentration of 0.1 mM to induce the expression of the target gene, and further cultured with shaking at 37 ° C for 4 hours.
  • the cells were collected by centrifugation (8000 X g, 10 minutes), and the resulting cells were suspended in a buffer solution (50 mM Tris-HCl, l mM CaCl, pH 7.8) and subjected to ultrasonic disruption.
  • the tertiary structure was modeled by SWISS-MODEL using the amino acid sequence of CGTase. More links include Bacillus stearothermophilus amylase, maltose, acarbose complex (lqho.pdb and maltose nemer (lqhp.pdb), Thermoanaerobactenum thermosulfu rigenes EMI derived CGTase and maltohexaose inhibitor complex (la47. pdb), CLrTase from T.
  • More links include Bacillus stearothermophilus amylase, maltose, acarbose complex (lqho.pdb and maltose nemer (lqhp.pdb), Thermoanaerobactenum thermosulfu rigenes EMI derived CGTase and maltohexaose inhibitor complex (la47. pdb),
  • tnermo sulfurigenes EMI (lciu.pdb)
  • deletion mutant ⁇ 145-150 (4cgt.pdb) of cyclodextrin glycosyl transferase from Bacillus circulans were used in the saddle type.
  • four domains were determined as shown in Figure 1. If the starting methionine of pCGTA is the first, domain A is from Metl to Tyr424, domain B is from Gly425 to Pro512, and domain C is from Thr513. Up to Thr604, domain D was changed from Asn605 to Arg712, and based on this, an expression vector was constructed.
  • CGTase band disappearance due to co-expression of domains A and B is thought to be because its expression was suppressed by a protein having the same amino acid sequence as the protein itself.
  • folding interference occurs when a protein having the same sequence and structure as CGTase domain A or B interacts with the CGTase during translation, causing intracellular interference.
  • CGTase which has lost its correct structure, is thought to have been degraded by proteases.
  • proteases For proteins with the same structure as domains C and D existing on the C-terminal side of CGTase, the correct structure is taken before most of the acting CGTase interferes.
  • domain C interference is thought to promote folding and, as a result, increase activity expression.
  • E. coli BL21 (DE3) strain transformed with each expression plasmid was induced with IPTG to co-express CGTase domains and CGTase.
  • Cyclodextrin (CD) synthesis activity was measured using the crude enzyme solution obtained by crushing the collected cells. CD-synthesizing activity was not found in the soluble fraction of bacterial cell disruption fluid in which only each domain was expressed alone, pET-21a, pET-8c (negative control) (Table 4).
  • pCGTA a-CD synthesis activity of the soluble fraction of E. coli BL2KDE3)
  • CGTase under translation sequentially follows the domain structure from the N-terminal side, and finally the domain It has cyclodextrin (CD) synthesis activity by taking correctness and conformation by the interaction.
  • CD cyclodextrin
  • the present invention introduces a protein domain encoded by a target gene, for example, by expressing it in a cell and inhibits the folding of the protein, thereby expressing the target gene in the cell. As described above, it can be used for various researches such as gene function analysis. It is.

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Abstract

The invention provides a method for suppressing the expression of a target gene in a cell by introducing a domain of a protein encoded by a target gene by expressing it in the cell or the like and inhibiting folding of the protein and the like. The introduced domain interferes an interdomain interaction during protein folding and inhibits the folding by the interferential action. The domain can be selected, for example, from domains determined by protein tertiary structure modeling.

Description

明 細 書  Specification
ドメイン間相互作用を利用した遺伝子発現抑制法  Gene expression suppression using interdomain interaction
技術分野  Technical field
[0001] 本発明は、ドメイン間相互作用を利用した遺伝子発現抑制法等に関し、より詳細に は、細胞内で mRNAからの翻訳に伴って進行するタンパク質のフォールデイング (fold ing:折りたたみ）におけるドメイン間相互作用に干渉するドメインを利用した遺伝子発 現抑制法等に関するものである。  [0001] The present invention relates to a method for suppressing gene expression using interaction between domains, and more specifically, a domain in protein folding that proceeds with translation from mRNA in a cell. The present invention relates to a method for suppressing gene expression using a domain that interferes with the interaction between cells.
背景技術  Background art
[0002] 細胞内での遺伝子の発現を抑制する技術、換言すれば、最終的な遺伝子産物で あるタンパク質の発現を抑制し、その細胞内での活性を調節する技術は、遺伝子の 機能解析をはじめとする生命科学分野の諸研究にとって不可欠のものといえる。さら に、こうした技術は、研究利用のみにとどまらず、生体での特定遺伝子の発現を特異 的に抑制することによって、その遺伝子にコードされるタンパク質の活性調節を利用 した治療薬の開発にも応用可能である。例えば、エイズ、がん、各種遺伝子疾患など 現状では治療困難な疾患に対して、疾患関連遺伝子の発現を抑制する遺伝子治療 への応用が期待される。  [0002] Technology that suppresses the expression of genes in cells, in other words, the technology that suppresses the expression of proteins, which are the final gene products, and regulates the activity in the cells, involves analysis of gene functions. It can be said that it is indispensable for various researches in the life science field. Furthermore, these technologies are not limited to research use, but can also be applied to the development of therapeutic drugs that specifically regulate the activity of proteins encoded by genes by specifically suppressing the expression of specific genes in the body. Is possible. For example, it is expected to be applied to gene therapy that suppresses the expression of disease-related genes for diseases that are currently difficult to treat, such as AIDS, cancer, and various genetic diseases.
[0003] 現在、遺伝子の発現を抑制する汎用的な方法として、 RNA干渉（RNAi： RNA interfe rence)を利用したものが知られている。 RNAiは、 siRNA (short interfering RNA)によつ てその配列特異的に mRNAが分解され、その結果遺伝子の発現が抑制される現象で ある（後記の非特許文献 1 · 2)。近年、この現象を利用した RNAi法による遺伝子機能 解析が盛んに行われるようになつている。さらに、この RNAi法による遺伝子サイレンシ ングの配列特異性と発現抑制効果の高さから、上述した遺伝子治療への期待が高ま つている。 [0003] Currently, a method using RNA interference (RNAi) is known as a general-purpose method for suppressing gene expression. RNAi is a phenomenon in which siRNA (short interfering RNA) degrades mRNA in a sequence-specific manner, resulting in suppression of gene expression (Non-patent Documents 1 and 2 below). In recent years, gene function analysis by the RNAi method using this phenomenon has been actively performed. Furthermore, expectations for gene therapy described above are increasing due to the sequence specificity of gene silencing by this RNAi method and the high expression suppression effect.
[0004] 遺伝子の発現は通常、（1)ゲノム DNAから mRNAへの遺伝情報の転写、（2)スプラ イシング (真核細胞の場合）、（3)リボソームでの mRNAからアミノ酸配列への翻訳、さ らに必要な場合は (4)糖鎖付加などの翻訳後修飾、の各プロセスを経て、最終的に 本来の活性をもつタンパク質が細胞内で生産される一連のプロセスといえる。したが つて、理論的には上記何れかのプロセスの進行を阻害することによって標的遺伝子 の発現を特異的に抑制することができる。上記 RNAi法は、標的遺伝子の mRNAを特 異的に分解することによって転写力 翻訳への過程を阻害し、遺伝子の発現を抑制 する方法といえるが、遺伝子発現の多様な抑制を実現するためには、 RNAi法とは異 なる過程を阻害する新規な遺伝子発現抑制法の開発が望まれる。 [0004] Gene expression is usually performed by (1) transcription of genetic information from genomic DNA to mRNA, (2) splicing (in the case of eukaryotic cells), (3) translation of mRNA into amino acid sequence in ribosomes, If necessary, it can be said to be a series of processes in which a protein having the original activity is finally produced in the cell through the processes of (4) post-translational modification such as glycosylation. But Theoretically, the expression of the target gene can be specifically suppressed by inhibiting the progress of any of the above processes. The RNAi method described above is a method that inhibits the transcriptional translation process by specifically degrading the mRNA of the target gene and suppresses gene expression, but in order to realize various suppressions of gene expression. Therefore, development of a novel gene expression suppression method that inhibits a process different from RNAi method is desired.
[0005] 上記（3)の翻訳過程においては、最終的に生産されるタンパク質がその活性に必 要な 3次構造を形成するため、リボソームでの翻訳と同時進行的に、既に翻訳された ポリペプチドの N末端側からタンパク質のフォールデイングが進行する。下記の特許 文献 1は、このタンパク質のフォールデイングを阻害する方法を提案しているが、実際 にフォールデイングを阻害した実施例は同文献に何ら開示されていない。一方、下 記の非特許文献 3には、ペプチド断片を用いて in vitroでタンパク質のリフォールディ ング (refolding)を阻害した実施例が示されている力 この方法は細胞内でタンパク質 のフォールデイングを阻害する方法とは本質的に相違する。さらに、下記の特許文献 2〜4には、ペプチド等の化合物を用いて、アミロイド 一ペプチド、プリオンタンパク 質などの異常なフォールデイングを抑制する方法が開示されているが、これらの方法 は遺伝子発現を抑制する方法ではなレ、。  [0005] In the translation process of (3) above, the finally produced protein forms a tertiary structure necessary for its activity, so that the already-translated poly- sylation is performed simultaneously with the translation in the ribosome. Protein folding proceeds from the N-terminal side of the peptide. The following Patent Document 1 proposes a method for inhibiting the folding of this protein, but no examples that actually inhibit the folding are disclosed in the same document. On the other hand, Non-Patent Document 3 below shows an example in which peptide refolding is inhibited in vitro using peptide fragments. This method does not allow protein folding in cells. It is essentially different from the method of inhibition. Furthermore, the following Patent Documents 2 to 4 disclose methods for suppressing abnormal folding of amyloid monopeptide, prion protein, etc. using compounds such as peptides. It's not a way to suppress it.
[0006] また、上記 RNAi法以外の遺伝子発現抑制法として、変異体のドミナントネガティブ 効果を利用した方法 (例えば、下記の特許文献 5)が挙げられるが、この方法を行うに はドミナントネガティブ効果を示す変異体の存在が必要である。  [0006] As a method for suppressing gene expression other than the RNAi method described above, there is a method using the dominant negative effect of a mutant (for example, Patent Document 5 below). In order to perform this method, a dominant negative effect is used. The presence of the indicated variant is necessary.
特許文献 1：国際公開第 WO 99/40435号パンフレット  Patent Document 1: International Publication No. WO 99/40435 Pamphlet
特許文献 2 :国際公開第 WO 96/39834号パンフレット  Patent Document 2: International Publication No. WO 96/39834 Pamphlet
特許文献 3 :国際公開第 WO 01/34631号パンフレット  Patent Document 3: International Publication No. WO 01/34631 Pamphlet
特許文献 4：特開 2005-120002号公報  Patent Document 4: JP-A-2005-120002
特許文献 5：特開 2005-278485号公報  Patent Document 5: JP-A-2005-278485
非特許文献 1 : Nature 391 :806-811 (1998)  Non Patent Literature 1: Nature 391: 806-811 (1998)
非特許文献 2： Nature 411 :494-498 (2001)  Non-Patent Document 2: Nature 411: 494-498 (2001)
非特許文献 3 : PNAS, Vol.86, No.9, 3060-3064 (1989)  Non-Patent Document 3: PNAS, Vol.86, No.9, 3060-3064 (1989)
発明の開示 発明が解決しょうとする課題 Disclosure of the invention Problems to be solved by the invention
[0007] 本発明は、上記の問題に鑑みなされたものであり、その課題'目的は、従来法とは 異なる過程に着目した新規な遺伝子発現抑制法を提供すること、より具体的には、 目的細胞内でのタンパク質のフォールデイングを阻害することによる新規な遺伝子発 現抑制法を提供すること、さらには、この方法を用いた遺伝子発現抑制剤および遺 伝子治療薬等を提供することにある。  [0007] The present invention has been made in view of the above-mentioned problems, and the object thereof is to provide a novel gene expression suppression method that focuses on a process different from the conventional method, more specifically, To provide a novel gene expression suppression method by inhibiting protein folding in the target cell, and to provide a gene expression inhibitor and a gene therapeutic agent using this method. is there.
課題を解決するための手段  Means for solving the problem
[0008] 従来の遺伝子発現抑制法として前述のように RNAi法が知られている力 本発明者 はこの干渉という手法をタンパク質レベルでも応用できない力と考えた。つまり、タン パク質のフォールデイングにおいて、 mRNAから翻訳されたポリペプチド鎖は N末端 側から順次折りたたまれ、安定な構造であるドメイン構造を形成していく。そして、この ように順次形成される複数のドメイン間の相互作用を通じて、最終的にタンパク質は その活性に必要な 3次構造 (コンフオメーシヨン）を獲得する。本発明者は、このドメィ ン間相互作用に干渉することでタンパク質のフォールデイングは阻害されるのではな レ、か、さらに、この干渉用物質として標的タンパク質自身のドメインに注目し、当該ドメ インを細胞内に発現させることによってフォールデイングが阻害され、正しい構造をと ることができな力 たタンパク質は、細胞内のプロテアーゼによって分解されるのでは ないか、すなわちこのようなドメイン干渉によって標的遺伝子の発現を特異的に抑制 する効果が得られるのではなレ、か、と考えた。  [0008] As described above, the RNAi method is known as a conventional gene expression suppression method. The present inventor considered that this interference method cannot be applied at the protein level. In other words, in protein folding, polypeptide chains translated from mRNA are folded sequentially from the N-terminal side to form a stable domain structure. Through the interaction between multiple domains that are sequentially formed in this way, the protein finally acquires the tertiary structure (conformation) necessary for its activity. The present inventor does not inhibit the folding of the protein by interfering with the interaction between the domains, or pays attention to the domain of the target protein itself as an interfering substance, and the domain If the protein is expressed in the cell and its folding is inhibited, the protein that has failed to take the correct structure is not degraded by proteases in the cell. I thought that it would be possible to obtain the effect of specifically suppressing the expression of.
[0009] そこで、シクロデキストリン合成酵素（CGTase)をモデルタンパク質として、このような ドメイン干渉による遺伝子発現抑制の可能性を検討した。その結果、後述するように CGTaseを構成する 4つのドメインのうち 3つのドメインを CGTaseと共発現させた場合 に当該酵素活性の顕著な抑制効果が認められ、 2つのドメインを発現させた場合に 実際に CGTaseの発現が抑制されていることが確認された。対照的に、これらの各ドメ インをフォールデイング後の CGTaseに添加しても活性の減少は認められなかったこと 力ら、これらドメインは干渉ドメイン（干渉ペプチド）として作用し、 CGTaseのフォール デイングを阻害したこと、さらに、解析を進めた結果ドメインによっては逆に当該ドメィ ンの発現により標的タンパク質の活性発現を促進する効果があることなどを明らかに し、本発明を完成するに至った。 [0009] Thus, the possibility of suppressing gene expression by such domain interference was examined using cyclodextrin synthase (CGTase) as a model protein. As a result, as described later, when 3 domains out of 4 domains constituting CGTase were co-expressed with CGTase, a remarkable inhibitory effect on the enzyme activity was observed, and when 2 domains were expressed, It was confirmed that the expression of CGTase was suppressed. In contrast, the addition of each of these domains to the CGTase after folding did not result in a decrease in activity, suggesting that these domains act as interfering domains (interfering peptides) and prevent CGTase folding. In addition, the results of the analysis revealed that, depending on the domain, the expression of the domain may have the effect of promoting the expression of the target protein. Thus, the present invention has been completed.
即ち、本発明は、上記知見に基づくものであり、以下の（1)〜（： 16)の発明を包含す る。  That is, the present invention is based on the above findings and includes the following inventions (1) to (: 16).
(1) 標的遺伝子がコードするタンパク質のドメインを細胞内に導入し、当該タンパク 質のフォールデイングを阻害することによって、細胞内の標的遺伝子の発現を抑制 する方法。  (1) A method for suppressing the expression of a target gene in a cell by introducing a protein domain encoded by the target gene into the cell and inhibiting the folding of the protein.
(2) 標的タンパク質のドメインを細胞内に導入し、当該タンパク質のフォールディン グを阻害することによって、細胞内の標的タンパク質の活性または発現を抑制する方 法。  (2) A method for suppressing the activity or expression of a target protein in a cell by introducing a target protein domain into the cell and inhibiting the folding of the protein.
(3) 上記ドメインは、タンパク質の 3次構造のモデリングによって決定されたドメイン の中から選択されることを特徴とする、上記（1)又は（2)記載の方法。  (3) The method according to (1) or (2) above, wherein the domain is selected from domains determined by modeling the tertiary structure of a protein.
(4) 上記ドメインは、タンパク質の球状ドメインの中から選択されることを特徴とする、 上記（1)又は（2)記載の方法。  (4) The method according to (1) or (2) above, wherein the domain is selected from globular domains of proteins.
(5) 上記ドメインを目的細胞内で発現するよう構築されたベクターを用いて、上記ド メインを細胞内に導入することを特徴とする、上記（1)又は（2)記載の方法。  (5) The method according to (1) or (2) above, wherein the domain is introduced into the cell using a vector constructed to express the domain in the target cell.
(6) 上記ドメインおよび標的遺伝子 (標的タンパク質)を目的細胞内で共発現するよ う構築されたベクターを用いて、上記ドメインを細胞内に導入することを特徴とする、 上記（1)又は（2)記載の方法。  (6) The above domain (1) or ( 2) The method described.
(7) 細胞内での標的遺伝子の発現を抑制するため、当該遺伝子がコードするタン パク質のドメインを目的細胞内で発現するよう構築されたベクターからなる遺伝子発 現抑制剤。  (7) A gene expression inhibitor comprising a vector constructed to express a protein domain encoded by the gene in a target cell in order to suppress the expression of the target gene in the cell.
(8) 細胞内での疾患関連遺伝子の発現を抑制するため、当該遺伝子がコードする タンパク質のドメインを目的細胞内で発現するよう構築されたベクターからなる遺伝子 治療薬。  (8) A gene therapy drug comprising a vector constructed to express in a target cell a protein domain encoded by the gene in order to suppress the expression of a disease-related gene in the cell.
(9) 標的遺伝子がコードするタンパク質、又は標的タンパク質が酵素である、上記（ 1)又は（2)記載の方法。  (9) The method according to (1) or (2) above, wherein the protein encoded by the target gene or the target protein is an enzyme.
(10) 標的遺伝子がコードするタンパク質、又は標的タンパク質がひ一アミラーゼフ アミリーに属する酵素である、上記（9)記載の方法。 (11) 標的遺伝子がコードするタンパク質、又は標的タンパク質がシクロデキストリン 合成酵素（CGTase)である、上記（10)記載の方法。 (10) The method according to (9) above, wherein the target gene encodes a protein, or the target protein is an enzyme belonging to the amylase family. (11) The method according to (10) above, wherein the protein encoded by the target gene or the target protein is cyclodextrin synthase (CGTase).
(12) 標的遺伝子がコードするタンパク質のドメインを細胞内に導入することによつ て、細胞内の標的遺伝子の発現を調節する方法。  (12) A method for regulating expression of a target gene in a cell by introducing a domain of a protein encoded by the target gene into the cell.
(13) 標的タンパク質のドメインを細胞内に導入することによって、細胞内の標的タ ンパク質の活性または発現を調節する方法。  (13) A method for regulating the activity or expression of a target protein in a cell by introducing a domain of the target protein into the cell.
(14) 標的遺伝子がコードするタンパク質のドメインを細胞内に導入することによつ て、細胞内の標的遺伝子の発現を促進する方法。  (14) A method of promoting expression of a target gene in a cell by introducing a protein domain encoded by the target gene into the cell.
(15) 標的タンパク質のドメインを細胞内に導入することによって、細胞内の標的タ ンパク質の活性または発現を促進する方法。  (15) A method of promoting the activity or expression of a target protein in a cell by introducing a domain of the target protein into the cell.
(16) 上記ドメインの代わりに、モチーフ（超二次構造）、又は二次構造をとる部分べ プチドを細胞内に導入することを特徴とする、上記（1)、（2)又は（12)〜（15)のいず れかに記載の方法。  (16) The above (1), (2) or (12), wherein a motif (super-secondary structure) or a partial peptide having a secondary structure is introduced into a cell instead of the domain. The method according to any one of to (15).
図面の簡単な説明 Brief Description of Drawings
[図 l]SWISS-MODELによりモデリングした Thermococcus sp. B1001株由来 CGTaseの 3次構造を示す図である。ドメイン Aは、 Metlから Tyr424までの領域、ドメイン Bは、 Gly 425力、ら Pro512までの領域、ドメイン Cは、 Thr513から Thr604までの領域、ドメイン Dは 、 Asn605から Arg712までの領域である。 FIG. 1 is a diagram showing the tertiary structure of CGTase derived from Thermococcus sp. B1001 modeled by SWISS-MODEL. Domain A is the region from Metl to Tyr424, domain B is the region from Gly 425, et al., Pro512, domain C is the region from Thr513 to Thr604, and domain D is the region from Asn605 to Arg712.
[図 2]共発現用ベクター構築のストラテジーを示す図である。 CGTaseのそれぞれ 4つ の球状ドメインをコードする遺伝子断片を pETベクターに導入し、 pET-A, pET-B, pE T-C, pET-Dを作製した。さらにその下流に CGTaseをコードする遺伝子 cgtA領域を 揷入した。図中、 Bmは BamH I、 Ndは Nde I、 Ncは Nco I、 Bpは Bpull02 Iを示す。  FIG. 2 is a diagram showing a strategy for constructing a co-expression vector. Gene fragments encoding four globular domains of CGTase were introduced into pET vectors to prepare pET-A, pET-B, pET-C, and pET-D. Furthermore, the gene cgtA region encoding CGTase was inserted downstream. In the figure, Bm is BamH I, Nd is Nde I, Nc is Nco I, and Bp is Bpull02 I.
[図 3]SDS_PAGEによる発現の確認結果を示す図である。ドメイン A, B, C, Dのいずれ も予想されるサイズのバンドが見られ、ともに単独で発現していることが確認された。 また、ドメイン A， Bを共発現させたとき、 CGTaseのバンドはみられなかった。ドメイン C, Dを共発現させた場合では CGTaseのバンドも確認できた。図中いずれも、 Mは分子 質量マーカー、レーン 1は誘導前の菌体、レーン 2は誘導後の菌体、レーン 3は菌体 破砕不溶性画分、レーン 4は菌体破砕可溶性画分、レーン 5は可溶性画分を 80°C 2 0分で熱処理し、遠心分離した上清画分、の結果を示す。また、 pET-21aは pET-A， C ， Dに対するネガティブコントロール、 pET_8cは pET-Bに対するネガティブコントロー ノレ、 CGTaseは 83 kDa、ドメイン Aは 49 kDa、ドメイン Bは 9.7 kDa、ドメイン Cは 10.1 kDa 、ドメイン Dは 12.0 kDa、である。 FIG. 3 is a diagram showing the results of confirming expression by SDS_PAGE. All of domains A, B, C, and D showed bands of the expected size, and both were confirmed to be expressed alone. When domains A and B were co-expressed, no CGTase band was observed. When domains C and D were co-expressed, CGTase bands were also confirmed. In each figure, M is a molecular mass marker, lane 1 is a cell before induction, lane 2 is a cell after induction, lane 3 is a cell-disrupted insoluble fraction, lane 4 is a cell-disrupted soluble fraction, lane 5 is soluble fraction at 80 ° C 2 The results of the supernatant fraction heat-treated at 0 minutes and centrifuged are shown. PET-21a is a negative control for pET-A, C and D, pET_8c is a negative control for pET-B, CGTase is 83 kDa, domain A is 49 kDa, domain B is 9.7 kDa, domain C is 10.1 kDa, Domain D is 12.0 kDa.
[図 4]共発現による活性への影響を示すグラフである。 IPTG誘導後の菌体破砕液の 可溶性画分を用いてひ -CD合成活性を測定した。 CGTaseのみを発現させたとき (pC GTA)の活性値を 100%とし、それぞれの割合をグラフで表した。それぞれのサンプル について 3回測定した。エラーバーは標準偏差をあらわす。  FIG. 4 is a graph showing the effect of co-expression on activity. The CD-synthesizing activity was measured using the soluble fraction of the cell disruption solution after induction of IPTG. When only CGTase was expressed (pC GTA), the activity value was defined as 100%, and the respective ratios were represented by graphs. Three measurements were taken for each sample. Error bars represent standard deviation.
[図 5]添カ卩による活性への影響を示すグラフである。 IPTG誘導により各ドメインを発現 させた菌体破砕液の可溶性画分を CGTaseに添加し活性を測定した。空ベクター (pE T-21aおよび pET-8c)の大腸菌破砕液の活性値とドメイン A， B, Cを発現させた菌体 破砕液添カ卩の場合の活性値とを比較してグラフで表した。それぞれのサンプルにつ レ、て 3回測定した。エラーバーは標準偏差をあらわす。  FIG. 5 is a graph showing the effect of supplementation on activity. The soluble fraction of the cell disruption fluid expressing each domain by IPTG induction was added to CGTase and the activity was measured. A graph comparing the activity values of the E. coli lysates of empty vectors (pET-21a and pET-8c) with the activity values of cells containing lysate cells expressing domains A, B, and C. did. Each sample was measured three times. Error bars represent standard deviation.
[0012] 以下、本発明について更に詳しく説明する。  [0012] Hereinafter, the present invention will be described in more detail.
[1]ドメイン干渉による遺伝子発現抑制法  [1] Suppression of gene expression by domain interference
本発明は、上記のように、 （1)標的遺伝子がコードするタンパク質のドメインを細胞 内に導入し、当該タンパク質のフォールデイングを阻害することによって、細胞内の標 的遺伝子の発現を抑制する方法、表現を変えれば、 （2)標的タンパク質のドメインを 細胞内に導入し、当該タンパク質のフォールデイングを阻害することによって、細胞 内の標的タンパク質の活性または発現を抑制する方法、を包含する。ここで、遺伝子 の発現とは、当該遺伝子によってコードされ、本来の構造と活性をもつタンパク質が 生産されることを意味し、タンパク質の発現も同様に、本来の構造と活性をもつタンパ ク質が生産されることを意味する。標的遺伝子 (又は標的タンパク質）の発現を抑制 するとは、このタンパク質の生産量を減少させることを意味する。  As described above, the present invention provides (1) a method for suppressing the expression of a target gene in a cell by introducing a protein domain encoded by the target gene into the cell and inhibiting the folding of the protein. In other words, (2) a method for suppressing the activity or expression of a target protein in a cell by introducing a domain of the target protein into the cell and inhibiting the folding of the protein. Here, the expression of a gene means that a protein encoded by the gene and having the original structure and activity is produced. Similarly, the protein expression has a protein having the original structure and activity. It means being produced. Suppressing the expression of a target gene (or target protein) means reducing the production amount of this protein.
[0013] ドメインを細胞内に導入する方法としては、後述のように、当該ドメインを目的細胞 内で発現するベクターを構築し、この発現ベクターを細胞内に導入する方法が好適 であるが、このようにドメインをコードする DNAを導入する方法以外に、ドメインタンパ ク質を直接細胞内に導入する方法も考えられる。 [0014] 本発明は、標的タンパク質自身が有するドメインと同じ構造のドメインを干渉タンパ ク質 (干渉ペプチド）として作用させ、タンパク質フォールデイング時のドメイン間相互 作用に干渉させることにより、 V、わばドメイン干渉（ペプチド干渉）を利用してフォール デイングを阻害する。 [0013] As a method for introducing a domain into a cell, a method for constructing a vector for expressing the domain in a target cell and introducing the expression vector into the cell as described later is preferable. In addition to the method of introducing a DNA encoding a domain, a method of introducing a domain protein directly into a cell is also conceivable. [0014] In the present invention, a domain having the same structure as the domain of the target protein itself acts as an interference protein (interference peptide) and interferes with the interaction between domains during protein folding. Inhibits folding using domain interference (peptide interference).
[0015] ここで、ドメインとは、タンパク質の立体構造上、まとまりをもった領域を意味する。タ ンパク質は、多くの場合、構造上複数の領域に分けることができ、これらの領域を通 常ドメインと呼ぶ。これらの領域が機能単位である場合も多ぐまた、ドメイン同士が互 いに寄り合い、ポケットを形成し、この窪みが他分子との結合部位になるなど、複数の ドメインが構造上および機能上相互依存的な場合も多い。  Here, the domain means a region having a unity in the three-dimensional structure of the protein. Proteins are often structurally divided into multiple regions, and these regions are usually called domains. In many cases, these regions are functional units.In addition, multiple domains are structurally and functionally linked to each other, such as domains close to each other to form a pocket, and this depression serves as a binding site for other molecules. There are many cases of dependence.
[0016] 本発明のフォールデイング干渉用ドメインは、標的タンパク質の 3次構造のモデリン グによって決定されたドメインの中から選択することができる。例えば、後述の実施例 に示すように、 SWISS- MODEL (http：〃 swissmodel.expasy.org/)などを利用すること によって構造のモデリングを行い、この構造をもとにドメインを決定することが可能で ある。  [0016] The folding interference domain of the present invention can be selected from domains determined by modeling the tertiary structure of the target protein. For example, as shown in the examples below, SWISS-MODEL (http: 〃 swissmodel.expasy.org/) can be used to model the structure and determine the domain based on this structure. It is.
[0017] また、本発明の干渉用ドメインを、標的タンパク質の球状ドメインの中から選択する ことは好ましい。ここで、球状ドメインとは外形的に構造上の独立性がより高い領域を 意味し、少なくとも現在、種々のタンパク質で球状ドメインとして認められているドメィ ンを含む意味である。球状ドメインのような構造のより安定したドメインを使用すること により、干渉タンパク質自身の安定性を確保することもでき、発現抑制効果の確実性 を高めることが期待できる。  [0017] In addition, the interference domain of the present invention is preferably selected from globular domains of the target protein. Here, the globular domain means a region that is externally more structurally independent, and at least includes a domain that is currently recognized as a globular domain in various proteins. By using a more stable domain such as a globular domain, the stability of the interference protein itself can be ensured, and the certainty of the expression suppression effect can be expected.
[0018] また、タンパク質のフォールデイングは N末端側力 順次折りたたまれていくので、 [0018] In addition, protein folding is folded sequentially N-terminal side force,
N末端側のドメイン (例えば、最も N末端側又は N末端側から 2番目のドメイン）を本発 明の干渉用ドメインとして使用することで、より効果的な発現抑制が期待できる。 By using the N-terminal domain (for example, the N-terminal domain or the second domain from the N-terminal side) as the interference domain of the present invention, more effective expression suppression can be expected.
[0019] 本発明の干渉用ドメインとして選択される領域の長さは、さほど厳密でなくてもよい と考えられ、例えば、構造のモデリングによって決定したドメインの長さより N末端側 又は C末端側に数残基長くても（あるいは短くても）本発明のドメイン干渉は生じ得る 。また、構造のモデリングによって決定したすべてのドメインが、本発明の干渉用ドメ インとして利用できるわけではなレ、が、後述の実施例に示すように、 4つのドメインのう ち 3つのドメインでドメイン干渉が生じたことから、比較的高い確率でドメイン干渉が生 じると考えられる。 [0019] It is considered that the length of the region selected as the interference domain of the present invention does not have to be very precise. For example, the length of the domain determined by structural modeling is closer to the N-terminal side or C-terminal side. Even a few residues longer (or shorter) can cause domain interferences of the present invention. In addition, not all domains determined by structural modeling can be used as the interference domain of the present invention. Because domain interference occurred in the three domains, domain interference is considered to occur with a relatively high probability.
[0020] 本発明の干渉用ドメインとして用いるドメインの領域 ·長さを決定後、当該ドメインを 目的細胞内で発現するようベクターを作製し、この発現ベクターを細胞内に導入する ことによって、干渉用ドメインを細胞内に発現させることができる。発現ベクターは、ゥ ィルスベクター、プラスミド、ファージ、又はコスミドなどを使用することができ、特に限 定されるものではなレ、。また、 目的細胞の種類、用途等に応じて使用するプロモータ 一を選択し、干渉用ドメインをコードするベクター上の DNA領域の上流に配置すれば よい。  [0020] After determining the region length of the domain to be used as the interference domain of the present invention, a vector is prepared so that the domain is expressed in the target cell, and the expression vector is introduced into the cell, thereby interfering. Domains can be expressed intracellularly. As the expression vector, a viral vector, a plasmid, a phage, or a cosmid can be used, and is not particularly limited. In addition, a promoter to be used may be selected according to the type and purpose of the target cell and placed upstream of the DNA region on the vector encoding the interference domain.
[0021] 後述の実施例では、ドメイン干渉を利用して大腸菌での CGTaseのフォールディン グを阻害したが、リボソームでの翻訳に伴って進行するタンパク質のフォールデイング はすべての生物に共通にみられる基本的生命現象であり、また、生体内でフォール デイングを助けるシャペロンは古細菌から哺乳類まで極めて良く保存されており、共 通の仕組みでフォールデイングが行われると考えられることから、ドメイン干渉を利用 してフォールデイングを阻害する本発明の方法は、古細菌や細菌での発現抑制に有 効なだけではなぐ真核細胞、植物細胞、動物細胞および哺乳類細胞での発現抑制 にも有効と考えられる。  [0021] In the examples described later, domain interference was used to inhibit CGTase folding in Escherichia coli, but protein folding that proceeds with ribosome translation is common to all organisms. Chaperones that are fundamental life phenomena and assist in folding in vivo are well preserved from archaea to mammals and are thought to be folded by a common mechanism. Thus, the method of the present invention for inhibiting folding is considered to be effective not only for suppressing expression in archaea and bacteria but also for suppressing expression in eukaryotic cells, plant cells, animal cells and mammalian cells. .
[0022] 後述の実施例で使用した CGTaseは、超好熱性始原菌 Thermococcus sp. B1001株 由来の Cyclodextrin glucanotransferase (CGTase; EC 2·4· 1.19)であり、而ォ熱十生 CGTa seである。配列表の配列番号 11には、この耐熱性 CGTaseの DNA塩基配列およびァ ミノ酸配列が、配列番号 12には、そのアミノ酸配列がそれぞれ示される。これらの配 列は、 DDBJ/EMBL/GenBank databasesのァクセッション番号 AB025721に記載され る配列と基本的に同一である力 シグナルペプチドに相当する領域が除かれている 点、及びコドンを改変している点で相違する。すなわち、本 CGTaseは、同ァクセッショ ン番号記載のアミノ酸配列のうち、ァミノ末端側の 28残基がシグナル配列になってお り、成熟状態の酵素では 29番目のパリンからの配列構造となる。後述のプラスミド pC GTAは、発現する CGTaseにおいて 2〜28番目のシグナルペプチド領域が除かれ、 2 9番目のノ リンが 2番目のアミノ酸になるように設計されている。また大腸菌での発現 性を高めるために AGT[Ser3]→AGC， CCC[Pro4]→CCG, TCA[Ser5]→TCT, CCA[ Pro7]→CCG, GGG[Gly9]→GGCと変更されてレ、る。 [0022] The CGTase used in the examples described below is Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) derived from the hyperthermophilic archaeon, Thermococcus sp. SEQ ID NO: 11 in the sequence listing shows the DNA base sequence and amino acid sequence of this thermostable CGTase, and SEQ ID NO: 12 shows the amino acid sequence thereof. These sequences are based on the fact that the region corresponding to the force signal peptide, which is basically the same as the sequence described in the accession number AB025721 of DDBJ / EMBL / GenBank databases, is removed, and the codon is modified. Is different. That is, in this CGTase, 28 amino acids at the amino terminal side are signal sequences in the amino acid sequence described in the same accession number, and the mature enzyme has a sequence structure from the 29th palin. The plasmid pCGTA described below is designed so that the 2nd to 28th signal peptide regions are removed from the expressed CGTase, and the 29th amino acid becomes the 2nd amino acid. Also expressed in E. coli AGT [Ser3] → AGC, CCC [Pro4] → CCG, TCA [Ser5] → TCT, CCA [Pro7] → CCG, GGG [Gly9] → GGC to improve the performance.
[0023] 図 1には、 SWISS-MODELによりモデリングしたこの耐熱性 CGTaseの 3次構造が示 される力 同図に示すように、この酵素はドメイン A， B, C, Dの 4つの球状ドメインから なる。このうちドメイン A， B, Dの 3つのドメインで、ドメイン干渉による抑制効果が認め られた（図 4)。 CGTaseは、 ひ一アミラーゼファミリーに属する酵素であり、イソアミラー ゼ、プルラナーゼ、アミ口プルラナーゼ、ネオプルラナーゼ、枝切り酵素など、類似の 構造を有する他のひ一アミラーゼファミリーに属する酵素も同様に、ドメイン干渉によ つて遺伝子発現を特異的に抑制できると考えられる。  [0023] Figure 1 shows the force showing the tertiary structure of this thermostable CGTase modeled by SWISS-MODEL. As shown in this figure, this enzyme has four globular domains, domains A, B, C, and D. It consists of. Of these, domains A, B, and D showed suppression effects due to domain interference (Fig. 4). CGTase is an enzyme belonging to the amylase family. Similarly, other members of the amylase family with similar structures, such as isoamylase, pullulanase, amiguchi pullulanase, neopullulanase, and debranching enzyme, are also subject to domain interference. Therefore, it is considered that gene expression can be specifically suppressed.
[0024] また、上記耐熱性 CGTaseのように構造が極めて安定な（変性剤に対して耐性を示 す）耐熱性タンパク質は、フォールデイング時により強力にドメイン間相互作用する可 能性がある。そうだとすると、フォールデイングを阻害する本発明の方法は、このような 耐熱性タンパク質の発現抑制により効果的であるといえる。もっとも、本発明を適用し て発現抑制可能なタンパク質、遺伝子は特に制限されるものではない。  [0024] In addition, a thermostable protein having a very stable structure (showing resistance to a denaturing agent), such as the above thermostable CGTase, may more strongly interact between domains during folding. If so, it can be said that the method of the present invention for inhibiting folding is more effective in suppressing the expression of such thermostable proteins. However, proteins and genes that can be suppressed in expression by applying the present invention are not particularly limited.
[0025] 本発明は、遺伝子の機能解析をはじめとする生命科学分野の様々な研究に利用 可能である。その 1つの利用態様として、前記ベクター、即ち、細胞内での標的遺伝 子の発現を抑制するため、当該遺伝子がコードするタンパク質のドメインを目的細胞 内で発現するよう構築されたベクターを、本発明の遺伝子発現抑制剤として利用する 方法が挙げられる。  [0025] The present invention can be used for various studies in the life science field including functional analysis of genes. As one of its utilization modes, the present invention, that is, the vector constructed to express the domain of the protein encoded by the gene in the target cell in order to suppress the expression of the target gene in the cell is described in the present invention. And a method of using it as a gene expression inhibitor.
[0026] さらに、本発明は、研究利用のみにとどまらず、生体での特定遺伝子の発現を特異 的に抑制することによって、その遺伝子にコードされるタンパク質の活性調節を利用 した治療薬の開発にも応用可能である。例えば、前記ベクターを本発明の遺伝子治 療薬として用レ、、 目的細胞内での疾患関連遺伝子の発現を抑制することによって、 エイズ、がん、各種遺伝子疾患などに対する遺伝子治療に本発明を適用可能である 。また、アルツハイマー病、プリオン病などタンパク質の異常なフォールデイングが原 因とされているコンフオメーシヨン病の治療への応用も可能である。  [0026] Furthermore, the present invention is not limited to research use, but also for the development of therapeutic drugs that use the activity regulation of the protein encoded by the gene by specifically suppressing the expression of the specific gene in the living body. Is also applicable. For example, the present invention can be applied to gene therapy for AIDS, cancer, various genetic diseases, etc. by using the vector as a gene therapy drug of the present invention and suppressing the expression of disease-related genes in the target cells. Is. It can also be applied to the treatment of conformational diseases such as Alzheimer's disease and prion disease that are caused by abnormal protein folding.
[0027] 前記ベクターを本発明の遺伝子治療薬として用いる場合は、公知の DDS (ドラッグ •デリバリー ·システム）を利用し、例えば、前記ベクターをリボソームなどの運搬体に 封入して体内投与してもよい。このとき標的部位の細胞を特異的に認識する運搬体 などを利用すれば、標的部位に本発明の遺伝子治療薬を効率よく運ぶことができ効 果的である。 [0027] When the vector is used as the gene therapy drug of the present invention, a known DDS (drug delivery system) is used. For example, the vector is used as a carrier such as a ribosome. It may be encapsulated and administered in the body. At this time, if a carrier that specifically recognizes cells at the target site is used, the gene therapy drug of the present invention can be efficiently delivered to the target site.
[0028] また、本発明を外来遺伝子の発現抑制に利用してもよい。この場合、例えば、干渉 用ドメインおよび外来遺伝子を目的細胞内で共発現するよう構築されたベクターを用 いて、干渉用ドメインを細胞内に導入する方法が挙げられる。この方法によれば、リボ ソーム周辺に干渉用ドメインを高濃度に発現させることができ、効果的な発現抑制が 期待できる。  [0028] The present invention may also be used for suppressing the expression of foreign genes. In this case, for example, a method of introducing the interference domain into the cell using a vector constructed to co-express the interference domain and the foreign gene in the target cell can be mentioned. According to this method, the interference domain can be expressed at a high concentration around the ribosome, and effective suppression of expression can be expected.
[0029] [2]ドメイン導入による活性発現の促進効果とモチーフ等の利用  [0029] [2] Activity promotion effect by domain introduction and use of motifs, etc.
これまで、ドメイン導入による遺伝子発現抑制効果について説明したが、さらに、解 析を進めた結果ドメインによっては逆に標的タンパク質の活性発現を促進する効果 があることがわ力、つてきた。例えば、後述の CGTaseを用いた実施例では、ドメイン Cを CGTaseと共発現させることによって CGTaseの酵素活性値の上昇が認められた（図 4 )。このこと力 、細胞へのドメイン Cの導入は CGTaseのフォールデイングを促進し、 結果としてその活性発現を高めたものと考えられる。  So far, the gene expression suppression effect by introducing the domain has been explained. However, as a result of further analysis, it has been proved that depending on the domain, there is an effect of promoting the active expression of the target protein. For example, in the examples using CGTase described later, an increase in the enzyme activity value of CGTase was recognized by co-expressing domain C with CGTase (FIG. 4). For this reason, it is considered that introduction of domain C into the cell promoted the folding of CGTase, and as a result, increased its activity expression.
[0030] このように、ドメイン領域によってはむしろ標的タンパク質の活性発現を高め、ドメイ ン干渉には活性発現の促進効果も認められるので、本発明は、ドメイン干渉を利用し た前述の遺伝子発現抑制法のみならず、ドメイン干渉を利用した遺伝子発現促進法 およびこれらを包括した概念である遺伝子発現調節法をも提供するものである。具体 的には、 （1)標的遺伝子がコードするタンパク質のドメインを細胞内に導入することに よって、細胞内の標的遺伝子の発現を促進 (又は調節）する方法、および、（2)標的 タンパク質のドメインを細胞内に導入することによって、細胞内の標的タンパク質の活 性または発現を促進 (又は調節）する方法、を提供する。そして、本発明のこの促進 法および調節法についても前述した抑制法と同様に、遺伝子発現促進剤あるいは遺 伝子発現調節剤として利用可能であり、更に遺伝子発現の調節によって疾患の治療 を図る遺伝子治療薬へ応用可能である。  [0030] In this way, depending on the domain region, the target protein activity expression is rather enhanced, and domain interference also has an effect of promoting activity expression. Therefore, the present invention suppresses the above-described gene expression using domain interference. The present invention provides not only a method but also a gene expression promotion method using domain interference and a gene expression regulation method that is a concept encompassing these methods. Specifically, (1) a method for promoting (or regulating) expression of a target gene in a cell by introducing a domain of the protein encoded by the target gene into the cell, and (2) the target protein There is provided a method for promoting (or regulating) the activity or expression of a target protein in a cell by introducing the domain into the cell. Further, this promotion method and regulation method of the present invention can be used as a gene expression promoter or gene expression regulator as well as the above-described suppression method, and further a gene for treating a disease by regulating gene expression. It can be applied to therapeutic drugs.
[0031] また、本発明の抑制法、促進法および調節法は、フォールデイングに対するドメイン の干渉作用を利用して遺伝子発現を調節するものである力 この干渉作用はドメイン 単位に限らず、より小さな構造単位であるモチーフ（超二次構造）、更には二次構造 をとる部分ペプチドにおいても同様の干渉作用があると考えられる。したがって、本発 明の上記各方法において、ドメインの代わりに、モチーフ、又は二次構造をとる部分 ペプチドを使用してもよい。 [0031] In addition, the suppression method, the promotion method, and the regulation method of the present invention are the force that regulates gene expression by utilizing the domain interference effect on folding. Not only the unit but also the smaller structural unit motif (super-secondary structure), and even partial peptides with a secondary structure are considered to have similar interference effects. Accordingly, in each of the above methods of the present invention, a partial peptide having a motif or a secondary structure may be used instead of the domain.
[0032] 上記ドメイン干渉（又はモチーフ等のペプチド干渉）による発現促進効果は、ドメイ ンゃ部分ペプチドがシャペロン様の効果を持っている可能性を示すと共に、これらの 干渉作用によって遺伝子発現が調節されることは、対立遺伝子からの部分ペプチド による干渉によって遺伝子の発現レベルが増減するという未知の生命現象の可能性 を示唆するものといえる。  [0032] The above-described expression promoting effect by domain interference (or peptide interference such as motif) indicates that the domain peptide may have a chaperone-like effect, and gene expression is regulated by these interference actions. This suggests the possibility of an unknown life phenomenon in which gene expression levels increase or decrease due to interference by partial peptides from alleles.
実施例  Example
[0033] 以下、実施例により本発明をより具体的に説明するが、本発明は下記の実施例に よって何ら限定されるものではない。  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.
[0034] [1]本実施例の目的 [0034] [1] Purpose of this example
前述のように、本発明者は、 RNAi法の干渉という手法をタンパク質レベルでも応用 できないかと考え、本実施例では、ドメイン干渉によるフォールデイング阻害の可能性 を検討した。  As described above, the present inventor considered that the RNAi method of interference could be applied even at the protein level, and in this example, the possibility of inhibition of folding due to domain interference was examined.
[0035] つまり、タンパク質の折りたたみを干渉するような部分ペプチドを発現させることで、 翻訳されたタンパク質は正しい構造をとることができず、フォールデイングが完了しな い分子は、細胞内のプロテアーゼによって分解されると予想される。この干渉タンパ ク質として、標的タンパク質自身と同じアミノ酸配列を持ち、ある程度安定な構造をと れる様なタンパク質、すなわちドメイン単位に注目した。力、なり以前からドメインはそれ ぞれ独立して折りたたまれることが知られている。例えば、免疫グロブリンの F ドメイン  [0035] That is, by expressing a partial peptide that interferes with protein folding, the translated protein cannot take the correct structure, and the molecules that cannot be folded are Expected to be broken down. As this interference protein, we focused on proteins that have the same amino acid sequence as the target protein itself and have a somewhat stable structure, that is, domain units. For a long time, it has been known that domains can be folded independently. For example, the F domain of an immunoglobulin
ab は、それだけで折りたたまれた構造をつくる。つまり、ドメイン単位で干渉タンパク質を 設計することは、それ自身の安定性を確保する意味でも望ましいと考えられる。翻訳 途中のタンパク質のフォールデイングにこのような干渉タンパク質がとり込まれることに よって、正しい構造がとれなくなると予想される。この方法でフォールデイングを阻害 する干渉ドメインは、まったく同じアミノ酸配列をもつ標的タンパク質にのみ作用する と考えられ、このフォールデイング干渉でも RNAi法と同様に高い特異性を持つ遺伝 子発現の抑制が可能であると期待される。 ab creates a folded structure by itself. In other words, designing an interference protein on a domain basis may be desirable in terms of ensuring its own stability. By incorporating such interfering proteins into protein folding during translation, it is expected that the correct structure will not be obtained. Interfering domains that inhibit folding in this way are thought to act only on target proteins that have the exact same amino acid sequence. It is expected that child expression can be suppressed.
[0036] 前述のように、 CGTaseは 4つの球状ドメイン力 なり、フォールデイング時のドメイン 間相互作用によって最終的に正しいコンフオメーシヨンに収束する。この 4つのドメィ ンと同じアミノ酸配列を持つポリペプチドを翻訳中の CGTaseのフォールデイングに干 渉させることで、 CGTaseは正しいコンフオメーシヨンがとれなくなると推測される。細胞 内で正しい構造をとれなくなったタンパク質はプロテアーゼによってすぐに分解され るだろう。結果として、特異的な遺伝子発現の抑制が起こると期待される。本実施例 では、 Thermococcus sp. B1001株由来の CGTaseをモデルタンパク質として、ドメイン 干渉による特異的な遺伝子発現抑制の可能性について検討した。  [0036] As described above, CGTase has four globular domain forces, and finally converges to the correct conformation by the interaction between domains during folding. By interfering with the folding of CGTase during translation of a polypeptide having the same amino acid sequence as these four domains, it is speculated that CGTase will not be able to obtain the correct conformation. Proteins that are unable to take on the correct structure in the cell will soon be degraded by proteases. As a result, specific gene expression suppression is expected to occur. In this example, the possibility of specific gene expression suppression by domain interference was examined using CGTase derived from Thermococcus sp. B1001 as a model protein.
[0037] [2]実験材料 '方法  [0037] [2] Experimental material 'Method
[2-1]使用菌株 ·プラスミド '培地  [2-1] Strain used · Plasmid 'Medium
本実施例で使用した菌株（Strains)およびプラスミド（Plasmids)は、表 1に掲げるとお りである。また、培養には、 Luria-Bertani培地を用いた。この培地の組成および調製 法は表 2に掲げるとおりである。  The strains (Strains) and plasmids (Plasmids) used in this example are listed in Table 1. In addition, Luria-Bertani medium was used for the culture. The composition and preparation method of this medium is as shown in Table 2.
[0038] [表 1] istrains/Plasmids Characteristics  [0038] [Table 1] istrains / Plasmids Characteristics
5 thi Δ (/ac~proAB)/F〔 traD3Q proAB* lac /。 lacZ
Figure imgf000013_0001
5 thi Δ (/ ac ~ proAB) / F [traD3Q proAB * lac /. lacZ
Figure imgf000013_0001
F" 0/77/7 Γ 75£/SB(rB" mB") gal{ λ cl 8b / ind\ Saml n/n5F "0/77/7 Γ 75 £ / S B (r B " m B ") gal {λ cl 8b / ind \ Saml n / n5
£. co// BL2KDE3) £ .co // BL2KDE3)
/ac〃レ 5—T7 ? el ) dc (DE3) pET-21 a Ampr, Tllac promoter pET-8c Ampr, T7 promoter pCGTA Ampr, ΤΊ/ac promoter, cgtA / ac〃 5—T7? el) dc (DE3) pET-21 a Amp r , Tllac promoter pET-8c Amp r , T7 promoter pCGTA Amp r , ΤΊ / ac promoter, cgtA
[0039] [表 2] Luria - Bertani培地 [0039] [Table 2] Luria-Bertani medium
Bacto-tryptone 10 g  Bacto-tryptone 10 g
Yeast Extract 5 g  Yeast Extract 5 g
NaCI 5 g  NaCI 5 g
NaOHで pHを 7.3に合わせ、 milliQ水で 1000ml にメスアップする。  Adjust pH to 7.3 with NaOH and make up to 1000 ml with milliQ water.
寒天培地のときは 1.5%(w/v) となるよう Agarを加える。調製後、 121°C 20分オート クレープ滅菌する。  Add Agar to 1.5% (w / v) when using agar. After preparation, sterilize by autoclaving at 121 ° C for 20 minutes.
[0040] [2-2] CGTaseの構造のモデリング、ドメイン A, B, C, Dの決定 [0040] [2-2] CGTase structure modeling, domain A, B, C, D determination
Thermococcus sp. B1001株の CGTaseのアミノ酸配列をもとに、 SWISS-MODEL (htt p://swissmodel. expasy.org/)を用レ、て構造のモデリングを行つた。この構造をもとにド メイン A， B, C, Dをそれぞれ決定した。  Based on the amino acid sequence of CGTase of Thermococcus sp. B1001, the structure was modeled using SWISS-MODEL (htt p: // swissmodel. Expasy.org/). Based on this structure, domains A, B, C, and D were determined.
[0041] [2-3]発現ベクターの構築  [0041] [2-3] Construction of expression vector
E.coli TGIと、ベクター pET- 21a (Novegen)、 pET_8cを用いてクローニングを行った 。発現ベクター構築は図 2に示すストラテジーのとおりに行った。 pCGTA (J. Biosci. B ioeng. 89:206-209 (2000))を铸型とし、 PCR法にて B1001株の CGTaseの各ドメインに 相当する遺伝子断片の増幅を行った。 pETベクターに導入するため、プライマー上に 制限酵素認識配列を挿入した。ドメイン Aの配歹 IJには Af, Arプライマーを、ドメイン Bの 配列には Bf, Brプライマーを、ドメイン Cの配歹 IJには Cf， Crプライマーを、ドメイン Dの 酉己歹 IJには Df， Drプライマーを使用した (各プライマー配列については、下記表 3参照） 。ドメイン A, C, Dについては制限酵素 Nde I, BamH Iで切断し、 pET_21aに組み込み 、それぞれ pET-A， pET-C, pET-Dを作製した。ドメイン Bについては制限酵素 Nco I， BamH Iで切断し、 pET- 8cに組み込み、 pET_Bを作製した。 pCGTAを铸型とし、 CGT A-f, CGTA-rプライマーを用いて PCR法により CGTase遺伝子を増幅した。これを制 限酵素 BamH I, Bpul l02 Iで切断し、 pET- A， pET- B, pET-C, pET- Dに組み込み、 共発現用プラスミド pET- A- CGTA, pET-B-CGTA, pET- C-CGTA, pET- D-CGTAを 作製した。  Cloning was performed using E. coli TGI and vectors pET-21a (Novegen) and pET_8c. Expression vector construction was performed according to the strategy shown in FIG. pCGTA (J. Biosci. Bioeng. 89: 206-209 (2000)) was used as a saddle, and the gene fragment corresponding to each domain of CGTase of B1001 strain was amplified by PCR. A restriction enzyme recognition sequence was inserted on the primer for introduction into the pET vector. Af and Ar primers for domain A IJ, Bf and Br primers for domain B, Cf and Cr primers for domain C, and Df for domain D Dr primers were used (see Table 3 below for each primer sequence). Domains A, C, and D were cleaved with restriction enzymes Nde I and BamH I and incorporated into pET_21a to prepare pET-A, pET-C, and pET-D, respectively. Domain B was cleaved with restriction enzymes Nco I and BamH I and incorporated into pET-8c to prepare pET_B. The CGTase gene was amplified by PCR using pCGTA as a saddle and CGT A-f and CGTA-r primers. This is cleaved with restriction enzymes BamH I, Bpul 02 I and incorporated into pET-A, pET-B, pET-C, pET-D, and co-expression plasmids pET-A-CGTA, pET-B-CGTA, pET -C-CGTA and pET-D-CGTA were prepared.
[0042] [表 3] PCRに使用したプライマ一一覧 [0042] [Table 3] List of primers used for PCR
名称 塩基配列及ぴその特徴  Name Base sequence and its characteristics
5' -aactgcagtaaggaggaatagcatatggtcagcccgtcttatccggc-3' ( l)  5 '-aactgcagtaaggaggaatagcatatggtcagcccgtcttatccggc-3' (l)
Af  Af
Pstl SD配歹 'J Ndel  Pstl SD 'J Ndel
5' -ttggatcctcaataagctaaagcaggattatagcg-3' (2)  5 '-ttggatcctcaataagctaaagcaggattatagcg-3' (2)
Ar  Ar
BamHI  BamHI
5' -aaaagctttaaggaggaatagccatgggcctgatcgcgacccgctatgtaagcg-3' (3) 5 '-aaaagctttaaggaggaatagccatgggcctgatcgcgacccgctatgtaagcg-3' (3)
Bf Bf
Hindlll SD配歹 lj Ncol  Hindlll SD distribution lj Ncol
5' -ttggatcctcacggcgttgctttgtactgcc-3' 4)  5 '-ttggatcctcacggcgttgctttgtactgcc-3' 4)
Br  Br
BamHI  BamHI
5' -aaaagctttaaggaggaatagcatatgaccgatccgtgggtgggcgcaatte-3' (5)  5 '-aaaagctttaaggaggaatagcatatgaccgatccgtgggtgggcgcaatte-3' (5)
Cf  Cf
Hindlll SD配歹 IJ Ndel  Hindlll SD IJ Ndel
5' -ttggatcctcacgttagatattcaaaggctattcc-3' (6)  5 '-ttggatcctcacgttagatattcaaaggctattcc-3' (6)
Cr  Cr
BamHI  BamHI
5' -aactgcagtaaggaggaatagcatatgaacaaacaaattccggcgatttttgaag-3' v f ) 5 '-aactgcagtaaggaggaatagcatatgaacaaacaaattccggcgatttttgaag-3' v f)
Df Df
Pstl SD配列 Ndel  Pstl SD array Ndel
5* -ttggatcctcaacggttggcttcaactgaaac-3' (8)  5 * -ttggatcctcaacggttggcttcaactgaaac-3 '(8)
Dr  Dr
BamHI  BamHI
5' -aaggatcctaaggaggaatagcatatggtcagcccgtcttatccggc-3' (9)  5 '-aaggatcctaaggaggaatagcatatggtcagcccgtcttatccggc-3' (9)
CGTA-f  CGTA-f
BamHI SD配列 Ndel  BamHI SD array Ndel
5' -ttgctcagcggtcaacggttggcttcaactgaaac-3 (10)  5 '-ttgctcagcggtcaacggttggcttcaactgaaac-3 (10)
CGTA-r  CGTA-r
Bpu1102 I  Bpu1102 I
※ 表中、かっこ內の数字は、配列表の該当する配列番号を示す。  * Numbers in parentheses in the table indicate the corresponding sequence number in the sequence listing.
[0043] [2-4]タンパク質発現 [0043] [2-4] Protein expression
作製した発現ベクターによる大腸菌 BL2KDE3)の形質転換体を 50 β g/mlのアンピ シリンを含む Luria - Bertani培地で 37°Cにて振盪培養した。 660 nmの OD値が 0.3に達 した際に終濃度 0.1 mMとなるよう IPTGを添加し、 目的遺伝子の発現を誘導し、更に 3 7°Cで 4時間振盪培養した。菌体を遠心分離 (8000 X g、 10分)にて回収し、得られた菌 体を緩衝液（50 mM Tris-HCl, l mM CaCl , pH 7.8)に懸濁して超音波破砕を行った The transformant of E. coli BL2KDE3) by the prepared expression vector was cultured with shaking at 37 ° C in Luria-Bertani medium containing 50 β g / ml ampicillin. When the OD value at 660 nm reached 0.3, IPTG was added to a final concentration of 0.1 mM to induce the expression of the target gene, and further cultured with shaking at 37 ° C for 4 hours. The cells were collected by centrifugation (8000 X g, 10 minutes), and the resulting cells were suspended in a buffer solution (50 mM Tris-HCl, l mM CaCl, pH 7.8) and subjected to ultrasonic disruption.
2  2
。再び遠心分離 (16，000 X g、 30分)して上清画分を回収し、これを粗酵素液として使 用レた。  . Centrifugation (16,000 X g, 30 minutes) was performed again, and the supernatant fraction was collected and used as a crude enzyme solution.
[0044] [2-5]活性測定  [0044] [2-5] Activity measurement
Lejeune, Α·らの報告するメチルオレンジ法（Anal Biochem. 181 :6-11 (1989))を改 良してシクロデキストリン（ひ -CD)合成活性を測定した。 1450 μ 1の反応溶液（1%デン プン、 0.035 mMメチルオレンジ、 50 mMリン酸緩衝液 ρΗ6·0)をそれぞれの CGTase の反応至適温度で 10分間保温した後、 50 μ ΐの酵素液を加えた。 10分後、 6 N HC1を 75 /i 1加え反応を停止した。これを 16°Cで 30分以上保温した後、 505 nmの吸光度を 測定した。シクロデキストリン合成活性 1Uは 1分当たり 1 μ molの CDを合成する酵素量 とした。 The methyl orange method (Anal Biochem. 181: 6-11 (1989)) reported by Lejeune, J. et al. Was modified to measure cyclodextrin (H-CD) synthesis activity. 1450 μ 1 reaction solution (1% dens Pung, 0.035 mM methyl orange, 50 mM phosphate buffer (ρΗ6 · 0) were incubated at the optimum reaction temperature for each CGTase for 10 minutes, and then 50 μΐ of enzyme solution was added. After 10 minutes, 6 N HC1 was added at 75 / i 1 to stop the reaction. This was kept at 16 ° C for 30 minutes or more, and then the absorbance at 505 nm was measured. Cyclodextrin synthesis activity 1U was defined as the amount of enzyme that synthesizes 1 μmol of CD per minute.
[0045] [3]実験結果  [0045] [3] Experimental results
[3-1] CGTaseの 3次構造のモデリング  [3-1] Modeling the tertiary structure of CGTase
CGTaseのアミノ酸配列を用いて、 SWISS-MODELにより 3次構造をモデリングした。 モアリンクには Bacillus stearothermophilusのひ— amylaseと maltose、 acarboseの複合 体 (lqho.pdb汲び maltoseのネ¾合体 (lqhp.pdb)、 Thermoanaerobactenum thermosulfu rigenes EMI由来の CGTaseと maltohexaose inhibitorの複合体 (la47.pdb)、 T. tnermo sulfurigenes EMIの CLrTase(lciu.pdb)、 Bacillus circulans由来の cyclodextrin glycosyl transferaseの欠失変異体 Δ 145-150(4cgt.pdb)を铸型に用いた。この 3次構造をもとに 、図 1に示すように、 4つのドメインを決定した。 pCGTAの開始メチォニンを 1番目とす ると、ドメイン Aは Metlから Tyr424まで、ドメイン Bは Gly425から Pro512まで、ドメイン C は Thr513から Thr604まで、ドメイン Dは Asn605から Arg712となった。これに基づいて 発現用ベクターを構築した。  The tertiary structure was modeled by SWISS-MODEL using the amino acid sequence of CGTase. More links include Bacillus stearothermophilus amylase, maltose, acarbose complex (lqho.pdb and maltose nemer (lqhp.pdb), Thermoanaerobactenum thermosulfu rigenes EMI derived CGTase and maltohexaose inhibitor complex (la47. pdb), CLrTase from T. tnermo sulfurigenes EMI (lciu.pdb), and the deletion mutant Δ 145-150 (4cgt.pdb) of cyclodextrin glycosyl transferase from Bacillus circulans were used in the saddle type. In addition, four domains were determined as shown in Figure 1. If the starting methionine of pCGTA is the first, domain A is from Metl to Tyr424, domain B is from Gly425 to Pro512, and domain C is from Thr513. Up to Thr604, domain D was changed from Asn605 to Arg712, and based on this, an expression vector was constructed.
[0046] [3-2]発現の確認  [0046] [3-2] Confirmation of expression
それぞれ構築した発現用プラスミドで形質転換された大腸菌 BL2KDE3)を用いて B 1001株の CGTaseと、その 4つの球状ドメインそれぞれの誘導発現を行レ、 SDS-PAGE によって発現の確認を行った（図 3)。 CGTase及びドメイン A-Dレ、ずれにっレ、ても IPT G誘導により単独で発現させたとき、予想サイズのバンドが確認された。しかし、ドメイ ン A， Bと CGTaseを共発現させたとき、ドメイン A, Bのバンドは確認された力 CGTase のバンドが現れなかった。一方、ドメイン C， Dについては CGTaseのバンドも現れた。 ドメイン A， Bの共発現による CGTaseのバンド消失の結果は、このタンパク質自身と同 じアミノ酸配列を有するタンパク質によってその発現が抑制されたためと考えられる。 すなわち、 CGTaseのドメイン Aまたは Bと同じ配列と構造を有するタンパク質が翻訳中 の CGTaseとドメイン間相互作用することでフォールデイング干渉が起こり、細胞内で 正しい構造をとれなくなった CGTaseはプロテアーゼによって分解されたと考えられる 。 CGTaseの C末端側に存在するドメイン C，Dと同じ構造をもつタンパク質については 、作用する CGTaseの大部分が干渉する前に正しい構造をとるため、ドメイン A, Bが作 用したときと比べてプロテアーゼによる分解を受けにくぐ CGTaseのバンドが残った のではないかと考えた。特にドメイン Cの干渉はフォールデイングを促進し、結果とし て活性発現を高めたものと考えられる。 Using the E. coli BL2KDE3) transformed with the constructed expression plasmids, B 1001 CGTase and its four globular domains were inducibly expressed and confirmed by SDS-PAGE (Figure 3). ). A band of the expected size was confirmed when expressed alone by induction of CGTase and domain AD, slippage, or IPTG. However, when domains A and B and CGTase were co-expressed, the bands of domains A and B did not show the confirmed force CGTase band. On the other hand, CGTase bands also appeared for domains C and D. The result of CGTase band disappearance due to co-expression of domains A and B is thought to be because its expression was suppressed by a protein having the same amino acid sequence as the protein itself. In other words, folding interference occurs when a protein having the same sequence and structure as CGTase domain A or B interacts with the CGTase during translation, causing intracellular interference. CGTase, which has lost its correct structure, is thought to have been degraded by proteases. For proteins with the same structure as domains C and D existing on the C-terminal side of CGTase, the correct structure is taken before most of the acting CGTase interferes. We thought that a CGTase band remained that was difficult to be degraded by proteases. In particular, domain C interference is thought to promote folding and, as a result, increase activity expression.
[0047] [3-3]活性測定  [0047] [3-3] Activity measurement
それぞれの発現プラスミドで形質転換された大腸菌 BL21(DE3)株を IPTG誘導し、 C GTaseの各ドメインと CGTaseを共発現させた。集めた菌体を破砕して得られた粗酵素 液を用いて、シクロデキストリン (CD)合成活性を測定した。 pET-21a、 pET- 8c (ネガ ティブコントロール）、各ドメインのみを単独で発現させた菌体破砕液可溶性画分に ついて、 CD合成活性は認められなかった (表 4)。 CGTaseを単独で発現させた大腸菌 BL2KDE3) (pCGTA)の菌体破砕液可溶性画分の a -CD合成活性を 100%として、そ れぞれのドメインを共発現させた菌体破碎液の活性と比較した (図 4)。  E. coli BL21 (DE3) strain transformed with each expression plasmid was induced with IPTG to co-express CGTase domains and CGTase. Cyclodextrin (CD) synthesis activity was measured using the crude enzyme solution obtained by crushing the collected cells. CD-synthesizing activity was not found in the soluble fraction of bacterial cell disruption fluid in which only each domain was expressed alone, pET-21a, pET-8c (negative control) (Table 4). With the a-CD synthesis activity of the soluble fraction of E. coli BL2KDE3) (pCGTA) expressing CGTase alone as 100%, the activity of the cell disruption fluid co-expressed with each domain Compared (Figure 4).
[0048] [表 4] 菌体破碎液の CGTase活性  [0048] [Table 4] CGTase activity of bacterial cell lysate
vector 活性 (U/ml)  vector activity (U / ml)
pCGTA 1.52 ±0.16  pCGTA 1.52 ± 0.16
pET-21 a 0.00  pET-21 a 0.00
pET-8c 0.00  pET-8c 0.00
pET-A 0.00  pET-A 0.00
pET-B 0.00  pET-B 0.00
pET-C 0.00  pET-C 0.00
pET-D 0.00  pET-D 0.00
pET-A-CGTA 0.45 ±0.13  pET-A-CGTA 0.45 ± 0.13
pET-B-CGTA 0.49 土 0.05  pET-B-CGTA 0.49 Sat 0.05
pET-C-CGTA 2.04 ±0.07  pET-C-CGTA 2.04 ± 0.07
pET-D-CGTA 0.46 ±0.07 [0049] SDS-PAGEで CGTaseのバンドの消失が見られたドメイン A， Bを共発現した菌体破 砕液は CGTaseを単独で発現させたコントロールに対して活性が約 30%に減少した。こ のことから、これらの干渉タンパク質が CGTaseの発現を抑制したとレ、うことが示された 。また、ドメイン Dは SDS-PAGEでは CGTaseのバンドの消失は認められなかった力 ド メイン A， Bを共発現させたものと同等の値まで CGTase活性の減少がみられた。ドメイ ン Cについては抑制効果がみられなかった。この活性の減少が実際にフォールディ ング段階で働いているのかを調べるため、単独で発現させたドメイン A, B, Cの菌体 破砕液可溶性画分を CGTase酵素液に添カ卩し、その活性を測定した。その結果を表 5、図 5に示す。得られた CGTaseに各ドメインを添加しても活性の減少は認められな 力、つた。このことから、これらの活性の減少あるいは増加は干渉タンパク質によるフォ 一ルディングの阻害あるいは促進によって起きたことが示された。 pET-D-CGTA 0.46 ± 0.07 [0049] The disruption of bacterial cells co-expressing domains A and B, in which the disappearance of the CGTase band was observed on SDS-PAGE, decreased the activity to about 30% compared to the control in which CGTase was expressed alone. This indicated that these interfering proteins suppressed the expression of CGTase. In addition, domain D showed a decrease in CGTase activity to a level equivalent to that of co-expressed force domains A and B, in which no disappearance of the CGTase band was observed by SDS-PAGE. There was no inhibitory effect on domain C. In order to investigate whether this decrease in activity actually works in the folding stage, the cell fraction soluble fraction of domains A, B and C expressed alone was added to the CGTase enzyme solution, and the Activity was measured. The results are shown in Table 5 and Fig. 5. Even when each domain was added to the obtained CGTase, no decrease in activity was observed. This indicates that the decrease or increase in these activities was caused by inhibition or promotion of folding by interfering proteins.
[0050] [表 5] 千渉タンパク質の添加による活性の影響  [0050] [Table 5] Effect of activity by adding Senkyo protein
vector 活性 (U/ml)  vector activity (U / ml)
pET-21a 0.96 ±0.04  pET-21a 0.96 ± 0.04
pET- 8c 0.93 ±0.05  pET-8c 0.93 ± 0.05
pET-A 0.92 ±0.06  pET-A 0.92 ± 0.06
pET- B 0.96 ±0.10  pET- B 0.96 ± 0.10
pET- 0.93 ±0.03  pET- 0.93 ± 0.03
[0051] 以上のように、 Thermococcus sp. B1001株の CGTaseをモデルタンパク質として新規 な遺伝子発現抑制法について検討し、 CGTaseのドメイン A, B, Dに相当するアミノ酸 配列からなるタンパク質を大腸菌 BL2 DE3)内で共発現させたところ、その菌体破砕 液の CGTase活性は約 30%にまで減少した。特にドメイン A， Bについては SDS-PAGE によってタンパク質レベルにおいても発現が抑制されていることが確認された。また、 これらの各ドメインタンパク質をフォールデイング後の CGTaseに添カ卩しても活性の減 少は認められな力 た。これらの結果から、これら干渉タンパク質は翻訳途中で CGT aseのフォールデイングにおいて作用したことが示された。 [0051] As described above, a novel method for suppressing gene expression was studied using CGTase from Thermococcus sp. B1001 as a model protein, and proteins consisting of amino acid sequences corresponding to domains A, B, and D of CGTase were expressed in E. coli BL2 DE3) CGTase activity of the cell disruption solution decreased to about 30%. In particular, for domains A and B, it was confirmed by SDS-PAGE that the expression was also suppressed at the protein level. In addition, when these domain proteins were added to the CGTase after folding, no decrease in activity was observed. These results indicated that these interfering proteins acted on CGTase folding during translation.
[0052] 翻訳中の CGTaseは N末端側から順次ドメイン構造をとつていき、最終的にドメイン 間相互作用により正しレ、コンフオメーシヨンをとることでシクロデキストリン（CD)合成活 性を持つ。このフォールデイング中にそれぞれのドメインと同じ構造をもったタンパク 質を共発現させ、フォールデイング時に干渉させた結果、 CGTaseは正しい構造をと れなくなり、プロテアーゼによって分解されたと考えられる。 [0052] The CGTase under translation sequentially follows the domain structure from the N-terminal side, and finally the domain It has cyclodextrin (CD) synthesis activity by taking correctness and conformation by the interaction. As a result of co-expression of proteins with the same structure as each domain during folding and interference during folding, CGTase could not take the correct structure and was thought to have been degraded by proteases.
産業上の利用可能性 Industrial applicability
以上のように、本発明は、標的遺伝子がコードするタンパク質のドメインを細胞内に 発現させるなどして導入し、当該タンパク質のフォールデイングを阻害することによつ て、細胞内の標的遺伝子の発現を抑制する方法等に関するものであり、前述したと おり、遺伝子の機能解析をはじめとする様々な研究に利用可能であるば力りでなぐ 遺伝子治療などに用いることで種々の疾患治療に応用可能である。  As described above, the present invention introduces a protein domain encoded by a target gene, for example, by expressing it in a cell and inhibits the folding of the protein, thereby expressing the target gene in the cell. As described above, it can be used for various researches such as gene function analysis. It is.

Claims

請求の範囲 The scope of the claims
[I] 標的遺伝子がコードするタンパク質のドメインを細胞内に導入し、当該タンパク質の フォールデイングを阻害することによって、細胞内の標的遺伝子の発現を抑制する方 法。  [I] A method for suppressing the expression of a target gene in a cell by introducing a protein domain encoded by the target gene into the cell and inhibiting the folding of the protein.
[2] 標的タンパク質のドメインを細胞内に導入し、当該タンパク質のフォールデイングを 阻害することによって、細胞内の標的タンパク質の活性または発現を抑制する方法。  [2] A method for suppressing the activity or expression of a target protein in a cell by introducing a domain of the target protein into the cell and inhibiting the folding of the protein.
[3] 上記ドメインは、タンパク質の 3次構造のモデリングによって決定されたドメインの中 から選択されることを特徴とする、請求項 1又は 2記載の方法。 [3] The method according to claim 1 or 2, wherein the domain is selected from domains determined by modeling the tertiary structure of a protein.
[4] 上記ドメインは、タンパク質の球状ドメインの中から選択されることを特徴とする、請 求項 1又は 2記載の方法。 [4] The method according to claim 1 or 2, wherein the domain is selected from globular domains of proteins.
[5] 上記ドメインを目的細胞内で発現するよう構築されたベクターを用いて、上記ドメイ ンを細胞内に導入することを特徴とする、請求項 1又は 2記載の方法。 [5] The method according to claim 1 or 2, wherein the domain is introduced into the cell using a vector constructed to express the domain in the target cell.
[6] 上記ドメインおよび標的遺伝子 (標的タンパク質)を目的細胞内で共発現するよう構 築されたベクターを用いて、上記ドメインを細胞内に導入することを特徴とする、請求 項 1又は 2記載の方法。 [6] The domain according to claim 1 or 2, wherein the domain and the target gene (target protein) are introduced into the cell using a vector constructed to co-express in the target cell. the method of.
[7] 細胞内での標的遺伝子の発現を抑制するため、当該遺伝子がコードするタンパク 質のドメインを目的細胞内で発現するよう構築されたベクターからなる遺伝子発現抑 制剤。  [7] A gene expression inhibitor comprising a vector constructed to express a domain of a protein encoded by the gene in a target cell in order to suppress the expression of the target gene in the cell.
[8] 細胞内での疾患関連遺伝子の発現を抑制するため、当該遺伝子がコードするタン パク質のドメインを目的細胞内で発現するよう構築されたベクターからなる遺伝子治 療薬。  [8] A gene therapy drug comprising a vector constructed to express in a target cell a protein domain encoded by the gene in order to suppress the expression of a disease-related gene in the cell.
[9] 標的遺伝子がコードするタンパク質、又は標的タンパク質が酵素である、請求項 1 又は 2記載の方法。  [9] The method according to claim 1 or 2, wherein the protein encoded by the target gene or the target protein is an enzyme.
[10] 標的遺伝子がコードするタンパク質、又は標的タンパク質がひ一アミラーゼファミリ 一に属する酵素である、請求項 9記載の方法。  10. The method according to claim 9, wherein the protein encoded by the target gene, or the target protein is an enzyme belonging to the first amylase family.
[II] 標的遺伝子がコードするタンパク質、又は標的タンパク質がシクロデキストリン合成 酵素（CGTase)である、請求項 10記載の方法。  [II] The method according to claim 10, wherein the protein encoded by the target gene or the target protein is a cyclodextrin synthase (CGTase).
[12] 標的遺伝子がコードするタンパク質のドメインを細胞内に導入することによって、細 胞内の標的遺伝子の発現を調節する方法。 [12] By introducing the domain of the protein encoded by the target gene into the cell, detailed A method for regulating the expression of a target gene in a cell.
[13] 標的タンパク質のドメインを細胞内に導入することによって、細胞内の標的タンパク 質の活性または発現を調節する方法。  [13] A method for regulating the activity or expression of a target protein in a cell by introducing a domain of the target protein into the cell.
[14] 標的遺伝子がコードするタンパク質のドメインを細胞内に導入することによって、細 胞内の標的遺伝子の発現を促進する方法。 [14] A method for promoting the expression of a target gene in a cell by introducing a protein domain encoded by the target gene into the cell.
[15] 標的タンパク質のドメインを細胞内に導入することによって、細胞内の標的タンパク 質の活性または発現を促進する方法。 [15] A method of promoting the activity or expression of a target protein in a cell by introducing a domain of the target protein into the cell.
[16] 上記ドメインの代わりに、モチーフ、又は二次構造をとる部分ペプチドを細胞内に 導入することを特徴とする、請求項 1、 2又は 12〜： 15のいずれ力、 1項に記載の方法。 [16] The force according to any one of claims 1, 2 and 12 to 15, wherein a partial peptide having a motif or a secondary structure is introduced into a cell instead of the domain. Method.
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