JPH09322779A - Screening of dna-bonding protein, plasmid used therefor and dna-bonding protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening a DNA-bonding protein by which the protein capable of bonding to a specific target sequence, e.g. a cancer gene promoter sequence such as an HRE-I sequence can be selected at a high efficiency and obtain both a plasmid used therefor and a peptide capable of specifically bonding to the HRE-I sequence. SOLUTION: This plasmid is preferably used for screening a peptide having properties of bonding to a target gene and comprises a base sequence capable of coding a peptide to be tested for evaluating the degree of bonding to the target gene, a promoter containing the target gene and a reporter gene bonded to the promoter so as to enable the operation. The promoter contains the target gene so as to control the expression of the reporter gene according to the degree of properties of bonding the peptide to the target gene contained therein. Furthermore, the peptide comprises 7 residues having a specific sequence.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA binding protein screening method and a plasmid used therein. The present invention also relates to peptides that bind to the HRE-I sequence.

[0002] its expression by peptide binds to the promoter region or the like of the background art certain genes have been reported finding that is inhibited. The search for such peptides and the search method therefor have been the subject of research by many engineers. The Ras gene is known as a specific example.

One of the important factors in signal transduction related to cell proliferation is a GTP-binding protein called "Ras". Activating mutations in the Ras gene have been detected at a high rate of approximately 20% in various types of human spontaneous tumors (Bos. JL. (1989): Ras oncogenes in hum.
an cancer; a review, Cancer Res., vol.49, pp4682-468
9). In mammals such as humans, there are three types of Ras genes, N-, H-, and K-. The type and frequency of Ras activated depends on the type of tumor, especially pancreatic cancer,
Highly detected in biliary tract cancer, colon cancer, etc., and even in precancerous conditions such as myelodysplastic syndrome, colorectal adenoma, cutaneous papilloma, etc.
An activating mutation in the as gene has been recognized (Kataoka Tohru (1991) "Oncogene and suppressor gene", pp36-
57, edited by Masashi Shibuya, University of Tokyo Press). This means that R
It suggests the importance of the as gene in spontaneous cancer.

Binding sequences of various transcription factors have been found in the promoter of human H-Ras. From a comparative experiment of transcription activity using a series of deletion mutant promoters, 160 bases upstream from the transcription initiation point were found. It was suggested that there is a region in the vicinity that influences transcription intensity (Lee, W. & Keller, EB. (1
991): Regulation elements mediating transcription o
f the human Ha-ras gene, J. Mol. Biol., vol. 220, pp599-
661). Since the transcriptional activity was reduced to less than half when the deletion in the vicinity of -160, Lee et al. Found that the sequence "5'-CCGGAA-3 '" in the vicinity of -160 is important for the specific transcription of the H-Ras gene. HRE-I (H-Ras r
esposible element). Furthermore, when it is deleted up to -150, it is reduced to about 20%, suggesting that the GC-rich sequence (GC-II) present in this vicinity is also important. Therefore, it is considered that the peptide or protein that binds to HRE-I and efficiently inhibits the transcription of the H-Ras gene can effectively inhibit the development of spontaneous cancer.

As a method for screening a DNA-binding protein, there can be mentioned, for example, the method described in JP-A-4-504052 using a genetic engineering technique. This method comprises a DNA-binding peptide having two marker genes harmful to the growth of Escherichia coli and introducing a plasmid having a promoter gene operably linked to these into a host microorganism and observing its growth state. To screen. This method uses plasmid DNA containing a certain marker gene from a microorganism commonly used as a cloning host.
It is necessary to select a special microorganism that cannot stably maintain In addition, the introduced plasmid DNA may cause mutations at a high frequency, and screening is expected to be difficult.

[0006] By the way, as a DNA-binding protein, a protein having a zinc finger structure as found in the zinc finger domain encoded by the Drosophila ttk gene is known.

This zinc finger structure consists of an amino acid sequence of about 30 residues per unit, and 2
Two Cys residues and His residues have a loop-shaped structure, that is, a zinc finger structure, via a zinc ion. The C-terminal half of each zinc finger unit has an α-helix structure, and this part is D
It was presumed to be deeply involved in the binding to NA (Pabo, CO. & Sauer, RT. (1992): Transcription facters:
structural families and principles of DNArecogniti
on, Ann. Rev. Biochem., vol.61, pp1053-1095).

Mouse-derived zif268 (Pavletich, NP. &
Pabo, CO. (1991): Zinc finger DNA recognition: crystal
structure of a Zif268 ・ DNA complex at 2.1 A., Sci
ence, vol.252, pp809-817) and tt from Drosophila.
k (Fairall, L. et al. (1993): The crystal structure o
fa two zinc-finger peptide reveals an extensionto
the rules for zinc-finger / DNArecognition, Nature, v
According to the results of the three-dimensional structure analysis of the DNA-zinc finger peptide complex concerning ol.336, pp483-487) and the like, in most cases, 7 residues on the N-terminal side of the His residue in the α-helix region are converted to DNA. It is believed to be directly involved in binding.

[0009]

SUMMARY OF THE INVENTION The present inventors have now established a method by which DNA-binding peptides or proteins can be efficiently found. In particular, it was found that a DNA-binding peptide or protein can be obtained with extremely high efficiency by using a nucleotide sequence encoding a peptide having the above zinc finger structure, as compared with a conventionally known similar screening method. Obtained. The present invention is based on this finding.

The present invention is directed to specific target base sequences such as H
It is an object of the present invention to provide a screening method with high efficiency for a protein that binds to an oncogene promoter sequence such as RE-I sequence. Another object of the present invention is to provide a plasmid and a plasmid set preferably used in the above method. Another object of the present invention is to provide a peptide that specifically binds to the HRE-I sequence.

The plasmid according to the present invention is a plasmid preferably used for screening a peptide having a binding property to a target gene, and has a nucleotide sequence encoding a test peptide whose degree of binding to the target gene is evaluated. A promoter comprising the target gene and a reporter gene operably linked to the promoter, wherein the promoter comprises the reporter according to the degree of binding of the peptide to the target gene contained therein. What comprises the target gene so as to control the expression of the gene.

The plasmid set according to the present invention is a plasmid set comprising a first plasmid and a second plasmid which are preferably used for screening a peptide having a binding property to a target gene, wherein the first plasmid is the target. A second plasmid comprising a base sequence encoding a test peptide whose degree of binding to a gene is evaluated, wherein the second plasmid contains a promoter containing the target gene and a reporter operably linked to the promoter. A gene, and the promoter comprises the target gene so as to control the expression of the reporter gene depending on the degree of binding of the peptide to the target gene contained therein.

Further, the method for screening a peptide capable of binding to a target gene according to the present invention comprises transforming a host with the above-mentioned plasmid or plasmid set, culturing the host, and measuring the expression level of the reporter gene. And a step of performing. Further, the peptide according to the present invention is one described in any one of SEQ ID NOS: 1 to 64.

[0014]

DETAILED DESCRIPTION OF THE INVENTION Definitions In this specification, proteins and peptides are used interchangeably unless otherwise specified. In the present specification, the modified peptide means an amino acid sequence in which some bases or amino acids have been inserted, substituted or deleted, or one or both ends thereof have been added.

Screening method according to the invention and it
The plasmid used in the present invention is a method for efficiently searching a so-called DNA-binding protein, that is, a screening method for a DNA-binding protein. The basic principle of the screening method according to the present invention is as follows.

First, the target nucleotide sequence to which the protein binds is specified. In the present invention, this sequence is called "target gene". If this target gene controls the expression of the "specific gene", and the expression of the specific gene is affected by the binding of the protein to the target gene, for example, the expression is suppressed, then the protein Can control the expression of a specific gene. The present invention intends to provide a method for efficiently searching for such proteins.

In the present invention, the candidate protein having the binding property to the target gene is prepared as the base sequence encoding the candidate. In the present invention, this protein candidate is called a "test peptide". In the present invention,
On the other hand, a promoter containing a target gene and a reporter gene operably linked to this promoter are prepared. Here, the promoter comprises the target gene so that when the peptide binds to the target gene contained therein, the expression of the reporter gene is suppressed or enhanced. Since the promoter thus contains the target gene, such a promoter may be referred to herein as a “modified promoter”.

[0018] According to a preferred embodiment of the present invention, it is preferable that the expression of a reporter gene located downstream thereof is controlled according to the degree of binding. Then, the gene encoding the test peptide is expressed in the same system with the nucleotide sequence encoding the test peptide, the modified promoter and the reporter gene. The presence or absence of binding of this expression product to the target gene can be known as the presence or absence of expression of the reporter gene. Further, according to a preferred embodiment of the present invention, the degree of binding can be known as the degree of expression of the reporter gene. In the method according to the present invention, it goes without saying that the above-mentioned "system" must be formed for each gene encoding the test peptide.

According to a preferred embodiment of the present invention, it is preferable to use a peptide having a zinc finger (hereinafter sometimes simply referred to as "ZF") structure as a base sequence encoding this test peptide. Various nucleotide sequences have been reported as a nucleotide sequence encoding a peptide having this zinc finger structure. For example, Sp1 gene and WT1 gene derived from human, zif268 gene derived from mouse, WT1 gene and EGR1 gene, ttk gene derived from Drosophila, TFIII A gene derived from Xenopus laevis, APR1 gene derived from yeast (Akiyama Tohru (1995): Zn Transcription factors with finger structure, Experimental Medicine vol.13, pp745-750 ； Pabo, CO. & Sauer, RT. (1992) ，
Ann.Rev.Biochem., Vol.61, pp1053-1095, Pavletich, N
P. & Pabo, CO. (1991), Science, vol.252, pp809-817, F
airall, L. et al. (1993), Nature, vol.366, pp483-487)
And the like.

By modifying the base sequence encoding the peptide having this zinc finger structure to construct a base sequence group encoding the test peptide, and evaluating its binding property by the screening method of the present invention, the binding efficiency can be extremely improved. DNA binding proteins can be found. For example, as described in Examples below, the H-Ras promoter gene (for example, HRE) is used as a target gene.
-I sequence), a protein capable of binding to it could be found with a probability (efficiency) of about 4 × 10 ^-2 . On the other hand, for example, Proc.Natl.Acad.Sci.USA Vol.
91, pp. 3969-3973, April 1994, the probability (efficiency) was about 10 ⁻⁴ as a result of screening a protein having a binding property to a similar sequence.

According to a preferred embodiment of the present invention, for example, a gene derived from the Drosophila ttk gene (ttk gene) is used. The nucleotide sequence encoding the zinc finger structure of the ttk gene is as shown in SEQ ID NO: 65 of the sequence listing described later. This sequence forms a complex with zinc, for example, as shown in FIG.
It takes a structure like -1 and Unit-2. According to a preferred embodiment of the present invention, the amino acids 19 to 25 and 49 in SEQ ID NO: 65 constituting this Unit-1 and Unit-2 are
By substituting, adding, inserting, or deleting in the amino acid portion up to 55, a group of nucleotide sequences encoding various test peptides can be obtained. According to a particularly preferred embodiment of the present invention, the amino acid substitution, addition, insertion or deletion occurs at the amino acid number 19, 22, 25, 49, 52 or 55 of the amino acid sequence of SEQ ID NO: 65. Is preferred.

According to a preferred embodiment of the present invention, the nucleotide sequence encoding the above-mentioned test peptide and the modified promoter and the reporter gene bound thereto are placed in the same system. A configuration aspect and an aspect configured as separate plasmids (hereinafter sometimes referred to as “plasmid set”) are provided.

By being constructed as a single plasmid, the steps of constructing the plasmid can be simplified. In addition, by constructing a set of plasmids, the size of the plasmid becomes smaller than that of a single plasmid, so that the efficiency of plasmid construction can be increased and the efficiency of transformation can be increased. Further, the use of a low copy number origin of replication in a plasmid containing a reporter gene increases the probability that the test peptide and the target gene will associate with each other, so that the expression control of the reporter gene can be detected with higher sensitivity. Furthermore, the first plasmid containing the once-constructed nucleotide sequence encoding the test peptide can be generally used for the second plasmid containing various target genes.

The "reporter gene" in the present invention is
There is no particular limitation as long as its expression can be confirmed. For example, the β-galactosidase gene,
Examples include the luciferase gene and the chloramphenicol acetyltransferase gene. In particular, it is preferable to employ as a reporter gene a gene whose presence or absence can be visually confirmed by an enzymatic reaction or the like.

As the "target gene" in the present invention,
Disease-related genes are included. Examples of diseases include cancer, inflammatory diseases, immune diseases and the like. The "disease-related gene" refers to a gene involved in the onset of a disease or a disease state, and includes, for example, H-Ras promoter gene (for example,
Oncogene promoters such as HRE-I sequence), myc promoter gene (eg Myb binding sequence), jun promoter gene (eg AP-1 sequence), bcl-3 promoter gene (eg NF-κB binding sequence). Examples include genes.

“Promoter Comprising a Target Gene”
It is preferable that the inducer of the present invention does not require an inducer for the expression of a reporter gene linked downstream. For example, a lac promoter gene or trp promoter gene lacking the repressor binding sequence may be mentioned.

The plasmid of the present invention may contain a promoter operably linked to the nucleotide sequence encoding the test peptide. This promoter is preferably one whose promoter gene is activated by an inducer. For example, tac promoter, trp promoter, lac promoter, λPL promoter, r
Examples include the ecA promoter.

The tac promoter is isopropyl-β
It is activated in the presence of a repressor inactivating agent such as -D-thiogalactopyranoside (IPTG). Therefore, IPTG must be added to the medium in order to express the nucleotide sequence encoding the test peptide.

The plasmid in the present invention preferably comprises a selectable marker gene. Specific examples thereof include an ampicillin resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene, and a tetracycline resistance gene.

In addition to the above, the plasmid according to the present invention may have a terminator downstream of the nucleotide sequence encoding the test peptide or the reporter gene. The type of terminator is not particularly limited, and examples thereof include rrnB terminator, lac terminator,
It can be selected from aspA terminator, lpp terminator, and T7 terminator.

In addition to the above, the plasmid according to the present invention may further have an origin of replication. The origin of replication may be naturally occurring in the plasmid or foreign. The plasmid according to the present invention may contain a lacI ^q gene for strictly controlling the expression of a test peptide or a T7 promoter or SP6 promoter for enabling transcription in vitro.

According to another aspect of the present invention, there is provided a screening method using the above-mentioned plasmid or plasmid set. That is, the screening method according to the present invention is a method for screening a peptide having a binding property to a target gene, which comprises transforming a host with the plasmid or plasmid set according to the present invention, culturing the host, and then using the reporter gene. Which comprises the step of measuring the degree of expression of

In the present specification, the term "screening (method)" refers to finding out from a plurality of candidates those having a binding property to a target gene, and confirming whether or not a peptide has a binding property to a target gene. (Law) or test (law) shall be used in the meaning.

In the method according to the present invention, a host is transformed with the above-mentioned plasmid or plasmid set according to the present invention to obtain a transformant. In the present invention, this transformation can be appropriately selected and used from commonly used methods in consideration of the type of host and the properties of the plasmid.

The transformant obtained is then cultured. This culture needs to be carried out under the conditions in which expression of the test peptide takes place. Here, if the expressed test peptide binds to the modified promoter in the second plasmid of the plasmid or plasmid set according to the present invention, the expression of the reporter gene is suppressed or enhanced.
In the method according to the present invention, the presence or absence of the binding of the test peptide to the target gene and its degree can be known by measuring the degree of expression of the reporter gene, that is, the degree of suppression or enhancement thereof. . In the present invention, a method corresponding to the adopted reporter gene is selected as the method of “measuring the expression level of the reporter gene”. For example, when the enzyme gene as described above is used as a reporter gene, by adding a substrate for the enzyme reaction to the medium, the expression level of the reporter gene can be known through the change in the color tone of the colonies due to the reaction. It is also possible to know the degree of expression by measuring the enzyme activity of the expressed enzyme.

The host preferably used in the present invention is, for example, JM109 strain derived from Escherichia coli K12 strain,
HB101 strain, DH5 strain, DH5α strain, XL1-blue
e stock etc. are mentioned.

Further, according to a preferred embodiment of the present invention, the screening method according to the present invention comprises one or more steps for measuring the degree of binding between the test peptide and the target gene from the viewpoint of clarifying the screening. Further, it may be included. The degree of binding between the test peptide and the target gene is
Gel shift assay, fluorescence polarization method, immunoassay (E
LISA method) or surface plasmon resonance method.

The test peptide can be expressed in a large amount as a fusion protein with an appropriate protein in Escherichia coli or the like according to a conventional method. Further, according to another aspect of the present invention, it is also possible to detect and quantify the test peptide with an appropriate labeled tag or antibody.

Peptides According to another aspect of the present invention, there are provided peptides described in any one of SEQ ID NOS: 1 to 64. These peptides were found by the above-mentioned method according to the present invention as described later, and have the property of specifically binding to the HRE-I sequence. According to the examples described later, particularly SEQ ID NOS: 1, 2, 19, 22, 23, 35, 47, 4
Peptides 8, 54 and 64 have high binding ability to the HRE-I sequence.

Furthermore, the present invention also includes modified peptides of the peptides described in any one of SEQ ID NOS: 1 to 64, which peptides retain the ability to bind to the HRE-I sequence. It

As described above, the HRE-I sequence has the HR
It is an important part of the activity of the as promoter. Therefore, it is considered that these peptides according to the present invention can be used for the inhibition of tumor formation involving H-Ras and the normal reversion of cancer cells.

According to the present invention, further, the above SEQ ID NOs: 1 to
64. The peptide according to any one of 64 or HRE-I
Peptides comprising one or more modified peptides that retain the ability to bind to sequences are provided. According to a preferred embodiment of the present invention, specific examples of such peptides include peptides having a sequence represented by the following general formula (I). This peptide comprises one or two or more of the peptides described in any one of SEQ ID NOS: 1 to 64 or modified peptides thereof that retain the ability to bind to the HRE-I sequence, and the peptide as a whole contains the HRE-I sequence. It is expected to retain the ability to bind to. Therefore, it is considered that this peptide can also be used for suppressing tumor formation involving H-Ras and returning normal cells to cancerous cells. _{Y 1 - [- X 1 -Cys} -X 2 -Cys-X 3 -Z-His-X 4 -His-X 5 -] m-Y 2 (I) ( In the formula, X ₁ and X ₅ are, It may be the same or different and represents a bond, any amino acid sequence of 1 to 10 residues, or a bridging substance, provided that X ₁ and X ₅ are at the end of the peptide, they represent a bond, X ₂ is 2
Represents any amino acid sequence of 7 residues, X ₃ represents any amino acid sequence of 5 residues, X ₄ represents any amino acid sequence of 2 to 4 residues, Y ₁ and Y ₂ are They may be the same or different and represent a tag sequence and / or a translocation signal sequence or are absent, Z is a peptide or HR according to any one of SEQ ID NOs: 1 to 64 above.
Represents a modified peptide thereof that retains the ability to bind to the E-I sequence, and m represents an integer of 1 to 5)

In the above formula, X ₁ and X ₅ may be cross-linking substances other than peptides. Such cross-linking substances include (1) not destroying the α-helix structure other than the DNA binding site, (2) not destroying the structure as a zinc finger, and (3) a plurality of zinc fingers and / or DNA binding Those that optimize the configuration of the site. Specifically, an alkyldiimidate, an acyl azide, an isocyanate, a glutaraldehyde that crosslinks with a covalent bond (for example, an ester bond, an ether bond, a carbon-carbon bond), a noncovalent bond (for example, an ionic bond via a metal ion). And a divalent chelating reagent (for example, aminobenzyl EDTA) that crosslinks with.

Among the peptides represented by the above general formula, preferred are those in which m is 2 and any amino acid sequence of 6 to 8 residues when X ₁ and X ₅ are not present at the terminal of the peptide. , And at least one of Y ₁ and Y ₂ represents a tag sequence and a translocation signal sequence.

The tag sequence includes a secretory transport signal sequence, a carrier protein sequence, a purification detection tag sequence,
And peptidase recognition sequences. The secretory transport signal sequence is for transport after protein production, the carrier protein sequence is for stability of the produced protein, the purification detection tag sequence is for purification or detection of the protein, and the peptidase recognition sequence is the ZF peptide. Is useful for separating the above-mentioned three types of tag sequences from each other.

As a secretory transport signal sequence, malE
Origin transport signal (KIKTGARILALSALTT
MMFSASALA), ompA-derived transport signal (M
KKTAIAVALAGFATVAQA). As the carrier protein sequence, GST (glutathione-S-transferase) (Schistos
oma japonicum), ttkZF (C)
(From Drosophila melanogaster), MBP (from maltose binding protein) (from E. coli), lacZ (from E. coli),
TRX (thioredoxin) (from E. coli), DHFR
(Dihydrofolate reductase) (mouse, human, bovine,
E. coli, S. aureus etc.), Protein
A (from Staphylococcus aureus) can be mentioned.

The purification detection tag sequences include His tag (H6 to H10), S tag (KETAAAKFER).
QHMDS), strep tag (SAWRHPEFG)
C), c-MYC epitope tag (RQKLISEEE
DL), HA epitope tag (YPYDVPDY
A), VAV epitope tag (YTDIEMNRLG
K). As a peptidase recognition sequence, F
actor Xa Cribage site (IEGR ▽), En
terokinase cribage site (DDDK ▽),
Thrombin Cribage site (LVPR ▽ GS),
Collagenase cribage site (PV ▽ G
P), Ala64-subtilisin cribage site (GAHR∇).

Further, examples of the above-mentioned transfer signal sequence include a nuclear transfer signal sequence, for example, SV40la.
The nuclear transfer signal (PKKKKRKV) derived from rgeT antigen. The “nuclear localization signal sequence” is useful for transporting the peptide into the cell nucleus when the peptide is taken up into the cell or translated in the cytoplasm. The peptide according to the present invention has HRE-I binding ability and
Although the expression of the as gene is inhibited, the expression of the H-Ras gene is carried out in the cell nucleus. Therefore, when used for the purpose of inhibiting the expression of the H-Ras gene, this nuclear localization signal sequence is added. It is preferable.

The order of the tag sequence and the translocation signal sequence is not particularly limited, but the translocation signal is preferably present on the N-terminal side of the peptide.
The peptide according to the present invention may have a metal ion in the peptide. The metal ions include Zn ²⁺ ,
Examples thereof include Mn ²⁺ , Ni ²⁺ , Cd ^{2+ and the} like. The peptide of the above general formula can be obtained by a method commonly used in the art, for example, secretory production by Escherichia coli.

Control of Gene Expression According to the method of the present invention, a peptide capable of binding to a target gene and controlling the expression of a specific gene can be obtained. Therefore, according to another aspect of the present invention, a peptide obtained by the method according to the present invention capable of binding to a target gene and controlling the expression of a specific gene is used, the expression of which is controlled by a specific regulatory sequence. A method for controlling the expression of an existing gene and an expression control agent therefor are provided. In the present invention, the above-mentioned “peptide capable of controlling the expression of a specific gene by binding to a target gene obtained by the method of the present invention” means that the expression of a specific gene can be controlled by binding to a target gene. It is meant to include the modified peptide that retains the property. The method of controlling the expression of a gene whose expression is regulated by a specific regulatory sequence according to the present invention comprises a step of binding a peptide having a binding property to a target gene obtained by the above-mentioned method of the present invention to the regulatory sequence. Is included. Specifically, the binding of both is performed in vivo or in vitro. More specifically, it is considered that the peptide can be administered to animals including humans, or the peptide can be expressed in the body of the animal to control the expression of a specific gene. For example, a nucleotide sequence encoding the peptide according to the present invention or a DNA construct comprising the nucleotide sequence, such as a vector, is introduced into cells in animals including humans, such as cancer cells, by a suitable method, and expressed. It is thought that gene therapy can be performed for tumors and the like. Examples of the method of introducing the base sequence into cells in vivo include a method of directly introducing the base sequence into animal cells (eg, liposome method, DEAE-dextran method, calcium phosphate method, electroporation method, etc.). Examples of the method for introducing the vector into cells in vivo include a method of using a viral vector as a vector and introducing the vector into cells by virus infection. Examples of the vector used for gene therapy include a retrovirus vector, an adenovirus vector, an amino-associated virus vector and the like.

Expression inhibitors that control the expression of genes whose expression is regulated by a specific regulatory sequence are:
It comprises a peptide capable of binding to a target gene obtained by the method according to the present invention, or a base sequence encoding the peptide. The expression inhibitor according to the present invention may be a so-called pharmaceutical composition containing a pharmaceutically acceptable carrier and excipient. In addition, the expression inhibitor according to the present invention includes those used for expressing the target peptide in the body of animals including humans by the above-mentioned method.

As mentioned above, SEQ ID NOs.
64. The peptide according to any one of 64 and HER
The modified peptide which retains the ability to bind to the -I sequence, the peptide containing one or more of these peptides, and the peptide represented by the above general formula (I) include HRE-I as described above. It is a peptide that specifically binds to. Furthermore, these peptides according to the present invention are
It seems that it can be used for suppression of tumor formation involving H-Ras and restoration of cancerous cells to normal. Therefore, in a method for controlling the expression of a gene whose expression is controlled by a specific control sequence and an expression control agent therefor, as a peptide that controls the expression of a gene whose expression is controlled by a specific control sequence, Preference is given to using the peptides according to the invention described above. As is clear from the above, according to the present invention, when the expression of a specific gene appears as a certain disease, a method for treating the disease and a therapeutic agent therefor are provided. According to another aspect of the present invention, the use of the peptide according to the present invention or the nucleotide sequence encoding the peptide for the treatment of a disease involving the expression of a gene whose expression is regulated by a specific regulatory sequence. , Or its use for the manufacture of a therapeutic agent.

[0053]

EXAMPLES The present invention will be described with reference to the following examples, which should not be construed as limiting the invention thereto. The underlined numbers (for example, 1 ) in the examples correspond to the numbers (for example, 1 ) described in the drawings.

Common experimental procedure <Restriction enzyme treatment> Takara Shuzo, Toyobo, Nippon Gene, or New Eng
A restriction enzyme purchased from landBioLabs was used. The digestion was carried out by reacting for 1 to 2 hours at the reaction temperature and the buffer solution designated for each enzyme.

<Ligation reaction> Takara Shuzo "DNA
Ligation Kit ", BoehringerM
The reaction was carried out at 16 ° C. for 16 hours in the designated reaction solution using anneheim “T4 DNA ligase” or Nippon Gene “Ligation Pack”.

<Transformation> For cloning, Toyobo “Competent High; JM109 strain” or Nippon Gene “Competent E. coli” was used.
(E.P.); JM109 strain "was used. The former is HAN.
AHAN method (Hanahan, D (1983) J. Mol. Biol., 166, 557-58
0) based chemical transformation, the latter of which is electroporation by electric pulse (BioRad “G”).
enePulser "is used.) For protein production," BL21 "obtained from Pharmacia.
The "strain" was prepared according to the method described in Current Protocol. Competents for electroporation were prepared. In any transformation method, after transformation, the SOC medium or "Hi-Competence Broth" manufactured by Nippon Gene Co., Ltd. was used in accordance with a conventional method. "5 to 1
After adding 0 volume, and culturing at 37 ° C for 1 hour, it was spread on LB agar medium (predetermined antibiotic or selective substrate was added) and cultured overnight at 37 ° C to obtain a transformant. It was

<Analysis of Clones> From the colonies formed on the agar medium, the cells were inoculated in 1 to 5 ml of LB liquid medium, cultured at 37 ° C. overnight, and then the cells were subjected to the alkaline denaturation method. Was extracted. The plasmid can be extracted by Kurabo Industries "Plasmid Automatic Separator" or QIAGE
N company "QIAprep-spin PLASMID K
IT "was used. The extracted plasmid was judged by analyzing the digested fragment pattern by agarose gel electrophoresis after making it into a restriction enzyme. In addition, the base sequence was analyzed if necessary." Taq Dye "manufactured by PerkinElmer. D
eoxy ^TM Terminator Cycle Se
Quenching kit ”or the company's“ Dye Pr ”
imer Cycle Sequencing Kit "
To perform a dideoxy reaction using PerkinElm
It was analyzed with a DNA sequencer model "373A" manufactured by er.

<Chemical synthesis of DNA> Pharmacia
It was synthesized by a DNA synthesizer "Gene Assembler" or outsourced to a DNA synthesis maker such as Takara Shuzo, Kurabo, Wako Pure Chemical, Graner Japan.

<Preparation of DNA Fragment> (1) Separation / Purification by Agarose Electrophoresis Agarose gel electrophoresis was performed in TEA buffer, DNA was stained with ethidium bromide, and agarose gel containing the desired DNA fragment was cut out. , QIAGEN "QIAquick Gel Extraction"
Kit "or BIO 101 company" GENECLEA
Using N ^™ II ”and“ MERMAID ^™ Kit ”
DNA was extracted and purified from the agarose gel.

(2) Amplification / Purification by PCR Thermostable polymerases are "Ampli-Taq" manufactured by PerkinElmer and "TaKaRa manufactured by Takara Shuzo".
Ex Taq "," Taq DNApol "manufactured by Wako Pure Chemical Industries, Ltd.
The reaction apparatus for performing the PCR method is “GeneAmp ^™ ” manufactured by PerkinElmer.
model 9600 ”or“ Thermal Circler ”manufactured by ASTEC,“ Rapid Cycler model 100 ”manufactured by Idaho Technology.
2 "was used. The amplified fragment obtained using the desired template DNA and primer DNA was separated and purified by the method described in (1) above, or" QIAqu "manufactured by QIAGEN.
ick ・ spin PCR Purification
It was purified using Kit ".

Example 1 Expression of DNA binding protein
Induction-dependent reporter enzyme activity Tests whether the plasmid according to the present invention effectively works for selection of a test peptide using a single plasmid having a nucleotide sequence encoding an existing DNA-binding protein and its target protein. did. Escherichia coli was transformed with a total of four types of plasmids each having or lacking the ttkZF gene and the modified lac promoter in the X and Y regions in the plasmid of FIG. IPT
After culturing in a liquid medium with or without addition of G, galactosidase activity was measured.

(1) Construction of DNA-binding protein expression cassette 1 To allow introduction of a random DNA sequence, pKK
233-3 (purchased from Pharmacia) tac
Multiple cloning site downstream of promoter (MC
S) was modified (Fig. 3). The triplet codon is "nn
When synthesized as "s", 4 or more consecutive "A" to "T" do not appear, so the restriction enzyme MunI (CAA
A multi-cloning site containing TTG) and AflII (CTTAAG) was selected. pKK233-3 to EcoR
Double digestion with I and HindIII, insertion and ligation of synthetic DNA multi-cloning site to obtain "pNS34-
1 "was obtained. The expression cassette was inserted into a plasmid containing 2 reporter gene cassettes (Fig. 3). The expression cassette [P-tac: MCS: T-rrnB] of pNS34-1 was amplified by anchor PCR and EcoO109I / AatII. A fragment was obtained, EcoUC109 of pUC19 (purchased from Takara Shuzo).
Substitution insertion into the I.AatII site gave pNS34-2.

(2) Improvement of reporter cassette The promoter of the reporter gene derived from pUC19 (P
lac), like the tac promoter used as a DNA-binding protein expression cassette, induces expression in an IPTG-dependent manner, so it is necessary to eliminate IPTG-dependence. Furthermore, the reporter gene (lacZ ') derived from pUC19 acts in concert with the lacZω gene in the host bacterial genome to express β-galactosidase activity, and the expression of the lacZω gene is also induced in an IPTG-dependent manner. , Needs to be improved.

[0064] 3 for the lac promoter improvement was creating a unique restriction enzyme sites in the promoter upstream (Fig. 4). Amplification fragments were amplified by PCR using a primer set in which the nucleotide sequence of the SpeI site was added to the upstream of the lac promoter of pNS34-2, and the amplified fragment was SpeI.
Digested and self-ligated to obtain pNS34-3.

4 The lacZ ′ gene of pNS34-3 was removed, and a NotI site-containing linker DNA was inserted so that the β-galactosidase gene could be inserted (FIG. 4). pNS34-3 as HindIII and N
Double-digested with deI and double-digested in the same manner as "HindII
I-NotI-NdeI "Linker was ligated and inserted to obtain pNS34-4.

5 A β-galactosidase gene derived from pNASSβ was introduced (FIG. 4). pNASSβ (CLO
(Purchased from NTECH) was digested with NotI to separate and purify the DNA fragment encoding the β-galactosidase protein, which was ligated and inserted into the NotI site of pNS34-4 to obtain pNS34-5.

6 The lac promoter was modified to
The operator sequence that regulates TG-dependent expression was removed, and the target sequence of ttkZF was replaced and inserted (Fig. 5). pNS34-5 as SpeI and HindII
Double-digest with I to transform the lac promoter Plac into Pl
Substitution / insertion into ac ^{ttkBS gave pNS4113} (Fig. 5). The nucleotide sequences of Plac and Plac ^ttkBS were as shown in FIG.

7 The β-galactosidase gene of ^pNS4113 was translated into the translation control sequence "S" in "Plac ^ttkBS ".
For translation under the control of D ", HindIII and β-
The DNA sequence between the translation initiation codon ATG of the galactosidase gene was removed (Fig. 5). A DNA fragment containing the Eco8II site from the β-galactosidase translation initiation codon was amplified by anchor PCR using pNS4113 as a template. This amplified fragment was double-digested with HindIII and Eco8II, and ligated and inserted into pNS4113 digested in the same manner to obtain pNS4124.

(3) Construction of test plasmid 8 A peptide having 82 residues at the C-terminal side added to the ZF motif of ttkZF was used as a positive control, and a 457-base DNA encoding 146 residues was used.
The fragment was synthesized (Figure 7). ttkZF DNA is Hh
Divide into 3 at aI and HpaII sites to make a DNA fragment of about 160 bases each, and a total of 6 types of DNA on both strands.
Are chemically synthesized, each is annealed, digested with a predetermined restriction enzyme, and then joined in sequence to obtain a full-length t
A tkZF DNA fragment was obtained. The synthesized ttkZF gene was double-digested with MunI and AflII, and ligated and inserted into pNS4124 digested in the same manner to obtain pNS4123 (FIG. 5).

A plasmid deficient in both the ttkZF gene and Plac ^ttkBS was constructed for 9 control studies (FIG. 8). That is, pNS4124 is replaced with SpeI and Hin.
After double digestion with dIII, blunt end treatment (DNA
Blunting Kit: manufactured by Takara Shuzo Co., Ltd., and self-ligated to obtain pNS4129.

For 10 control studies, a plasmid ^{carrying the} ttkZF gene and lacking Plac ^ttkBS was constructed (FIG. 8). That is, pNS4123 is replaced with SpeI and Hi.
After double digestion with ndIII, blunt end treatment (DNA
Blunting Kit (manufactured by Takara Shuzo) and self-ligated to obtain pNS4125.

(4) Reporter Enzyme Assay Molecular Cloning (2nd ed
s. ) [Cold Spring Harbor La
The galactosidase enzyme activity was measured according to the method described in "Boratory Press". Pre-culture (agar medium) Escherichia coli JM109 strain was transformed with plasmids pNS4123, pN
Transformed with S4124, pNS4125 and pNS4129. Antibiotic ampicillin (100 μg / m
l) minimum glucose-containing agar medium (60 mM K)
₂ HPO ₄ ; 33 mM KH ₂ PO ₄ ; 7.5 mM (N
H _{4) 2} SO _4; 1.7mM sodium citrate;
0.4% glucose; 1 ppm thiamine; 1 mM
MgSO ₄ ; 1.5% agar) or LB agar medium (1%
Colonies were formed on the plate by culturing overnight in bactotryptone; 1% NaCl; 0.5% bacto yeast extract; 1.5%; agar).

Pre-Culture (Liquid Medium) The colonies formed on the plate were inoculated into a liquid medium of the same composition and pre-cultured overnight. Main culture (liquid medium) After that, transfer to a fresh liquid medium supplemented with 1 mM ZnCl ₂ (inoculation with 1/50 volume of the preculture liquid of the medium amount),
After culturing for a predetermined period of time, the culturing was stopped when the growth amount measured at OD600 nm reached about 0.4, and the cells were subjected to an enzyme assay. In the main culturing, in order to evaluate the effect of inducing ZF expression, culturing was carried out at a final concentration of 0.1 mM in a medium to which IPTG was added and a medium without any addition.

Measurement of enzyme activity The culture solution was lysed in a buffer containing Z buffer, chloroform and SDS, mixed for 10 seconds, and then allowed to stand at a reaction temperature of 28 ° C., and o-nitrophenyl galactopyrano was used as a substrate. Add Sid (ONPG),
The reaction was allowed to proceed at 28 ° C. until the color development was visible, and the reaction was stopped by adding a sodium carbonate solution. OD42 of reaction solution
OD550 for 0 nm and background correction
nm was measured. The activity value represents the enzyme activity per unit time / unit cell. The results were as shown in FIG. Galactosidase activity dependent on the modified Lac promoter was observed, and when ttkZF was expressed, the activity was markedly reduced.

Example 2 Targeting of DNA binding proteins
Sequence selectivity E. coli was transformed with a total of three types of plasmids in which the target sequence of the Y region in the plasmid of FIG. 10 was changed. IPT
After culturing in a liquid medium with or without addition of G, galactosidase activity was measured. (1) Construction of test plasmid 11 To verify the selectivity of the ttkZF peptide for the target sequence, the ttkZF gene was retained and Plac was used.
^{Plac ttkBS (mu)} with mutation in the target sequence in ^ttkBS
(FIGS. 11 and 12). Between -10 sequence from -35 sequences in Plac ^TtkBS as shown in FIGS. 11 and 12, ^ttkBS Plac ttkBS was changed to ^(mu) (mu) from SpeI site HindII
Chemical synthesis of about 150 bases forward chain up to I site
It was double-stranded with R, double digested with SpeI and HindIII, and ligated and replaced with pNS4123 digested in the same manner to obtain pNS4126.

[0076] In order to verify the selectivity for the target sequence of 12 TtkZF peptide retains TtkZF gene was modified to Plac ^WT was changed to the sequence in accordance with a target sequence within Plac ^TtkBS of wild-type Plac ( Figure 1
1 and FIG. 12). As shown in FIG. 11 and FIG. 12, Plac ^WT in which the region between −35 and −10 in Plac ^ttkBS was changed to ^WT was chemically synthesized with a forward chain of about 150 bases from SpeI site to HindIII site, Double-stranded by PCR, SpeI and HindIII
Was double-digested and ligated and replaced with pNS4123 which had been digested in the same manner to obtain pNS4127.

(2) Reporter Enzyme Assay Escherichia coli JM109 strain was prepared by using plasmids pNS4123 and pN.
Transformed with S4216 and pNS4127. The preculture and main culture of Escherichia coli and the measurement of the activity of the reporter enzyme were carried out according to the method described in Example 1 (3). The results are as shown in Table 1.

[0078]

[Table 1]

When the target sequence inserted into the promoter linked to the reporter gene was "ttkBS", a remarkable decrease in galactosidase activity was confirmed. The change in galactosidase activity reflects the selectivity of the DNA binding protein for the target sequence.

Example 3 HRE-I of modified ZF peptide
Effect on Sequence (1) A DNA-binding protein for a specific target protein is designed, and a single plasmid having both nucleotide sequences is used to test whether the plasmid according to the present invention effectively works for selection of a test peptide. did. Escherichia coli was transformed with the plasmid shown in FIG. 13 and the galactosidase activity was measured.

(1) Construction of test plasmid 13 In order to develop a H-Ras transcription inhibitory peptide,
Pl with HRE-I and GC-II as target sequences
A modification to ac ^HRE was made (Fig. 14). As shown in FIG. 15, Plac ^HRE in which the region between −35 and −10 in Plac ^ttkBS was changed to ^HRE chemically synthesizes a forward chain of about 150 bases from SpeI site to HindIII site and PCR Strand and SpeI and Hin
pNS412 double digested with dIII and similarly digested
4 was ligated and replaced to obtain pNS4130.

The region of ttkZF (C) required for protein stabilization in 14 E. coli cells was pre-inserted (FIG. 14). ttkZF (C) using pNS4123 as a template
The DNA fragment obtained by amplifying the region by anchor PCR was double-digested with MunI and AflII, ligated and inserted into pNS4130 double-digested in the same manner to obtain pNS4131.
I got This plasmid contains Ptac and ttkZF (C)
Has MunI and NheI sites between
It is a screening vector capable of inserting a ZF (N) fragment consisting of about 230 base pairs into this site.

A thZF designed to bind to the 15 HRE sequence was constructed (FIG. 14). The DNA binding sequence ZF peptide and its target sequence are set forth in FIG. A peptide that binds to the target sequence HRE-I sequence was designed with reference to these. Specifically, ttkZ
Ser in ZF unit 1 of F to Asn, and tt
The Asn in ZF unit 2 of kZF was changed to Asp to obtain a modified ttzZF. Modified ttkZF
Was designated as "thZF". The modified amino acid sequences of Unit 1 and Unit 2 are as set forth in SEQ ID NOs: 1 and 2. About 230 bases of the thZF (N) forward chain shown in FIG. 16 were chemically synthesized and double-stranded by PCR.
It was double-digested with MunI and NheI and ligated and replaced with pNS4131 which had been similarly digested to obtain pNS41th.

(2) Reporter enzyme assay Escherichia coli JM109 strain was transformed with the plasmid pNS41th. The preculture and main culture of Escherichia coli and the measurement of the activity of the reporter enzyme were carried out according to the method described in Example 1 (3). The experiment was performed 5 times (Experiments 1 to
5). The results are as shown in Table 2.

[0085]

[Table 2]

The galactosidase enzyme activity for inducing the expression of the modified ZF was remarkably reduced. In the above table, the relative activity is
(-) 100% galactosidase activity during IPTG culture
It means the galactosidase activity at the time of (+) IPTG culture when it was made into%.

Example 4 HRE-I of modified ZF peptide
Effect on Sequence (2) The modified ZF peptide obtained in Example 3 was used for Pharmac
Insert into IA "pGEX5-1" vector and
A fusion protein with T was obtained (Fig. 17). Purified GS
For T-fusion proteins, Boehringer M
Annheim "DIG Gel Shift Ki"
Gel shift assays were performed using t ".

The DNA binding ability of thZF was tested in vitro.
In order to evaluate at 0, it is necessary to obtain a large amount of thZF peptide. 1) To purify the thZF peptide produced in Escherichia coli, a fusion protein with GST [glutathione S-transferase] was used to enable affinity purification with glutathione sepharose.
2) It was possible to obtain a full-length thZF peptide by introducing a His tag [(His) 6] at the C-terminus. 3)
On the N-terminal side of thZF, a c-MYC tag [Glu-Gln-
Lys-Ile-Ser-Glu-Glu-Asp-L
eu] has made it possible to detect and quantify with an antibody.

(1) Construction of test plasmid Since the reporter gene cassette in pNS41th is not required for 16 protein production, a plasmid having only a DNA-binding protein expression cassette was constructed (FIG. 18). This plasmid can also be induced into a binary protein expression plasmid. pHSG3
To remove 96 lacZ gene cassette (purchased from Takara Shuzo) and create MunI site, anchor PCR was used to detect chloramphenicol resistance gene and pMB.
The 1 origin of replication was amplified, this fragment was digested with MunI, and the ends were dephosphorylated to prevent self-cyclization. Separately, the DNA-binding protein expression cassette of pNS4131 was amplified by anchor PCR and digested with EcoRI. These two DNA fragments were ligated to each other to form pNS4141.
I got

The 17 MYC tag was inserted (FIG. 18).
The DNA fragment containing the MYC tag is chemically synthesized from forward-strand DNA (66 bases), double-stranded by PCR, and EcoRI.
PNS4 double digested with AflII and the same digestion
141 ttkZF (C) is substituted / inserted into pNS41
51V was obtained.

[0091] was inserted into the His tag to 18 thZF (Figure 19). ThZF was amplified by anchor PCR using pNS41th as a template, double-digested with NcoI and BglII, and inserted and ligated into similarly digested pQE-60 (purchased from QIAGEN) to obtain pNS41th: His.

[0092] 19 ThZF: inserting the c-MYC tag His (Figure 19). ThZF: His is amplified by anchor PCR using pNS41th: His as a template, and Mu
pN double digested with nI and AflII
It was inserted into S4151V and ligated to obtain pNS4151th.

20 MYC: thZF: His GST
And a fusion protein with (FIG. 20). pNS4151
MYC: thZF: H by anchor PCR using th as a template
Amplify is, double digest with EcoRI and XhoI,
PGEX5X-1 (Pharmasi) digested in the same manner
(Purchased from a company) and ligated to obtain pNS42th.

MYC tag and His tag were inserted into 21 ttkZF to prepare a fusion protein with GST (FIG. 20). ttkZF (N) was amplified by anchor PCR using pNS4123 as a template, double digested with EcoRI and NheI, and inserted / ligated into pNS412th digested in the same manner to obtain pNS42ttk.

(2) Preparation of GST fusion protein 1) Preparation of host recipient bacterium : Escherichia coli BL21 strain obtained from Pharmacia was used as LB agar medium [1% bactotryptone; 1% NaCl; 0.5% bactost extraction. The product was inoculated into 2 to 5 ml of an LB liquid medium, and precultured overnight at 37 ° C. Pre-culture 1 fresh 1
Inoculate into 000 ml LB liquid medium, wavelength 600nm
The cells were cultured at 37 ° C. until the absorbance at about 0.6. After cooling the culture on ice, centrifuge (4000 rpm
(= 1500 × g); 2 ° C .; 15 minutes) to recover the proliferating cells. The cells were suspended in cold sterilized water, precipitated by centrifugation and washed (repeated twice). Further, the cells were suspended in a cold 10% glycerol aqueous solution, precipitated by centrifugation and washed (repeated twice). The washed bacterial cells were suspended in cold 10% glycerol having the same volume as the bacterial cell pellet, and the suspension was dispensed into microtubes little by little. The host recipient bacteria prepared as described above were snap frozen in liquid nitrogen and stored in a deep freezer at -80 ° C until use.

2) Transformation into host strain : The recipient strain stored in a deep freezer at -80 ° C was thawed on ice and the plasmid pNS42thDNA solution (1 to 50n) was added.
(about g) was mixed and allowed to stand on ice for 5 minutes. Recipient-DNA in an ice-cooled electroporation cuvette (purchased from BioRad; "Genepar cuvette")
The mixed solution was poured and an electric pulse was applied to introduce DNA into the cells. 5 to 10 volumes of SOC liquid medium [2% bactotryptone; 0.5% bacto yeast extract; 10 mM NaCl; 2.5 mM KCl; 10
mM MgCl ₂ ; 20 mM glucose] and added 3
After culturing at 7 ° C for 1 hour, the LB agar medium supplemented with ampicillin at a final concentration of 100 µg / ml was smeared and cultured overnight at 37 ° C.

3) Cultivation of transformant: A colony of the transformant was inoculated into 5 ml of LB liquid medium to which ampicillin was added (final concentration 50 μg / ml), and precultured at 37 ° C. overnight. The preculture liquid was inoculated into a fresh 200 ml to 1 liter ampicillin-containing LB liquid medium and cultured at 37 ° C to 30 ° C until the absorbance at a wavelength of 600 nm reached about 0.5. Zn (CH ₃ CO) was added at a final concentration of 0.3 mM to induce expression of the GST fusion protein with IPTG, and further at a final concentration of 0.1 mM to stabilize the ZF peptide.
O) ₂ is added, and the mixture is further added at 37 ° C to 30 ° C for 3 to 5 ° C.
The culture was continued for an hour.

4) Extraction of protein from cultured cells : Proliferated cells were centrifuged [7000 rpm (4500 xg);
4 ° C .; 10 minutes]. PBS / Zn buffer [9.57 mM phosphate, pH 7.5 with 0.1 mM Z
n (CH ₃ COO) ₂ was added], frozen at −80 ° C., and thawed at room temperature. Freeze-thaw cells on ice for 5
Ultrasonic treatment for 10 minutes (Mr. Inoue "ULTRASO"
NIC GENERATOR model GSD5
0 ”5φ chip; output 50W / 28kHz),
Triton X-100 was added so that the final concentration was 1%, and the mixture was shaken at room temperature for 1 hour to obtain a lysate. Centrifuge the lysate [9000 rpm (8000 xg); 4
C .; 10 minutes] was collected and subjected to affinity purification.

5) Affinity purification : Glutathione Sepharose 4B column (purchased from Pharmacia) was centrifuged [500 × g; 5 minutes] to remove the solvent, and the resin was washed twice with PBS buffer. It was suspended in PBS buffer to obtain a 50% slurry. In the centrifugal supernatant obtained in 4), 0.5 ml to 2 ml of 50%
The slurry was added and shaken at room temperature for 30 minutes to allow the resin to adsorb the GST fusion protein. Then, centrifuge [500 xg; 5 minutes], discard the supernatant, and remove the resin by 1
It was washed twice with 0 volume of PBS / Zn buffer. Further, the resin was suspended in 1 volume of the eluate [10 mM reduced glutathione; 50 mM Tris-HCl, pH 8.0] and shaken at room temperature for 15 minutes to release the GST fusion protein from the resin. The supernatant containing the GST fusion protein was recovered by centrifugation [500 × g; 5 minutes].

(3) Determination of ZF peptide concentration Current Protocols in Immunology [Greene Publishin
The concentration of ZF peptide was quantified according to the "competitive ELISA method" described in g Associates and Wiley-Interscinece]. 1) Immobilization of MYC peptide on a microplate : 3
2-residue human c-MYC peptide (OncogeneS
Science company # PP06 was added to carbonate buffer (PI
ERCE BupH ^™ Carbonate-Bic
It was dissolved in a arbonate Buffer # 28382) to a final concentration of 1 μg / ml. The MYC peptide solution was added to a 96-well microplate (Nunc MaxS
orp Immuno Plate) 5 per well
After pouring 0 μl and mixing, the mixture was allowed to stand overnight at 4 ° C. to immobilize the MYC peptide. It was washed 3 times with 150 μl of sterile water.

2) Blocking : 200 μl of blocking solution [PBS was added to the plate on which MYC peptide was immobilized.
5% (w / v) bovine serum albumin in buffer (Sig
Ma company # A3803)] was added thereto, the mixture was reacted at room temperature for 1 hour or more, and then washed with 150 μl of PBS buffer three times.

3) Preparation of antigen / antibody mixture : anti-MYC antibody (Oncog) diluted 1000 times with blocking solution
50 μl of ene Science # OP10)
And a MYC-tagged protein sample or standard sample (human c-MYC peptide; Oncogene Science, # PP0, which was diluted stepwise with a blocking solution)
50 μl of 6) was mixed and allowed to react for 30 minutes or more at room temperature to obtain an antigen / antibody mixed solution.

4) Treatment with antigen / antibody mixture : 50 μl of the antigen / antibody mixture from 3) was poured into 2) the blocked plate and reacted at room temperature for 2 hours or more, and then the wells were set to 1
It was washed 3 times with 50 μl of PBS buffer. further,
Pour 150 μl of blocking solution into the wells and apply at room temperature for 10
After blocking for minutes, the wells were washed 3 times with 150 μl of PBS buffer.

5) Treatment with secondary antibody : 50 μl of secondary antibody (peroxidase-modified anti-mouse IgG antibody; "# PP273" manufactured by Binding Site, Inc.) diluted 1000 times with blocking solution was poured, and the mixture was allowed to stand at room temperature for 1 hour. After the above reaction, 150 μl of the washing solution [0.05% (v / v) Tween 20 was added to PBS buffer] was added to 3
Washed twice.

6) Treatment and detection of substrate solution : 150 wells
μl of substrate buffer [Sigma's # P492
2] and then washed with 100 μl of substrate solution [0.4 mg / ml of substrate buffer with o-phenylenediamine (S
Dissolve # P8787 manufactured by igma Co., Ltd.] was allowed to develop color for about 30 minutes at room temperature, and then 50 μl of 2N sulfuric acid was added to stop the reaction. Micro Plate Reader (Bio
The degree of color development was quantified by measuring the absorbance at a wavelength of 450 nm with a Rad model 450).

7) MYC tag tamper in protein sample
Calculation of mass : Using the concentration-absorbance curve for the MYC peptide used as a standard sample as a calibration curve, the amount of protein in the measurement sample was calculated and evaluated by the MYC tag conversion value.

(4) DNA by gel shift assay
Qualitative evaluation of binding ability 1) Preparation of ZF protein solution : The GST-ZF fusion protein purified in (2) above was treated with the Bradford method using BSA as a standard sample (“Comom manufactured by PIERCE”).
assie ^TM Protein Assay Reag
ent ”) to determine the protein concentration, and 50 μg / 10
It was prepared to have a volume of μl.

2) Preparation of probe: Each double-stranded oligonucleotide to be used as a probe is chemically synthesized (synthesized by Greiner Japan Co., Ltd.), and each single-stranded oligonucleotide is equimolar in TEN buffer [10 mM.
Tris-HCl; 1 mM EDTA; 0.1M Na
Cl, pH 8.0], heated at 95 ° C. for 10 minutes, and then slowly cooled to room temperature for annealing. The annealed double-stranded DNA is labeled as “DIG Gel Shi.
DIG-11-ddUTP was added to the end by the terminal transferase reaction attached to the ft Kit ", and DIG (d
A probe DNA labeled with igoxigenine) was obtained.

3) Binding reaction : The binding reaction solution was Fair
ll et al. (Fairall.L., et al. (1992): Sequence.
specific DNA binding by a two zinc ・ finger peptide
fromthe Drosophila melanogaster trametrack protei
n, J. Mol. Biol., vol.226, pp349-366) 2
0 mM MES-KOH (pH 6, 5), 1 mM Mg
Cl ₂ , 50 μM Zn (CH ₃ COO) ₂ , 0.1 m
Based on M to 1 mM DTT (dithiothreitol), 0.1 μg of polylysine (Sigma # P0879) was added to suppress non-specific binding and enhance specific binding. 50 μg ZF protein and 0.4 to 2 ng DIG-labeled probe D were added to the binding reaction solution.
Add NA to bring the total volume to 20 μl, then add 1 at 37 ° C.
The reaction was performed for 5 minutes.

4) Polyacrylamide electrophoresis : Using a polyacrylamide gel (PAGE EL ^™ # NPG-310L manufactured by Atto Co.) having a concentration gradient of 3% to 10%, 0.25 × TB buffer [25 mM Tri].
s; 25 mM boric acid]. The gel was prerun at 80V for 1 hour before loading the binding reaction sample. Glycerol was added to the binding reaction sample at a final concentration of 8% to load the gel, and the gel was electrophoresed at a constant voltage of 80 V for 2 hours.

5) Blotting : The DNA in the polyacrylamide gel was loaded with nylon membrane (H from Amersham).
electrobonded to ybond ^™ -N +).
The 0.25 × TB buffer was used as a blotting buffer, and a current of 400 mA was applied for 30 minutes. The nylon membrane after blotting was heated in an oven at 120 ° C. for 30 minutes to immobilize the DNA on the nylon membrane.

6) Detection by chemiluminescence method : The DIG-labeled probe on the surface of the blot prepared in 5) was detected with an anti-DIG antibody labeled with alkaline phosphatase. Then wash buffer [0.1 M maleic acid; 0.15 M NaC
1: 0.3% (v / v) Tween 20, pH 7.5]
For 5 minutes. Simply blocking buffer [0.
1M maleic acid; 0.15M NaCl: 1% (w /
v) The sample was immersed in a blocking agent attached to the kit, pH 7.5] and shaken for 30 minutes. 1000 with blocking buffer
The plate was immersed in a 0-fold diluted alkaline phosphatase-labeled anti-DIG antibody solution and shaken for 30 minutes. After washing twice with the washing buffer, the alkaline buffer [0.1M Tris-
HCl; 0.1 M NaCl; 50 mM MgCl ₂ ,
The blot was equilibrated by dipping in pH 9.5]. The substrate solution [alkaline buffer added with CSPD ^™ at a final concentration of 0.1 mg / ml] was dipped and reacted at 37 ° C for 15 minutes. It was exposed to Polaroid instant film (Type 667 manufactured by Polaroid) at room temperature for 40 minutes. The results were as shown in FIG. 21 and Table 3.

[0113]

[Table 3]

The modified ZF peptide bound to the probe (32 base pairs containing the HRE sequence) (lanes 2 and 7) and was competed with the unlabeled probe (lanes 3 and 8).
It was confirmed that the binding was specific to the RE sequence. In addition,
In Lanes 4 and 9, competition with "HRE" is not possible because this DNA is too short, and in Lanes 6 and 10, competition with "ttkBS" is due to similarity to the HRE sequence. Conceivable.

Example 5 HRE-I of modified ZF peptide
Effect on sequence (3) Modified ZF peptide obtained in Example 3, and ttkZ
F peptide was used as "pGEX5X-" manufactured by Pharmacia.
A fusion protein with GST was obtained by inserting it into a 1 "vector (Fig. 22). The DNA binding ability of the purified GST fusion protein was evaluated by a fluorescence polarization method using a fluorescence polarization measuring device (manufactured by PanVera). The dissociation constant (K
d) was calculated.

(1) pNS42th as a plasmid
In addition to using pNS42ttk, in addition to constructing a test plasmid, preparing a GST fusion protein, and ZF
Peptide concentration quantification was performed according to the method described in Example 4.

(2) Quantitative evaluation of DNA binding ability by fluorescence polarization measurement method 1) Preparation of ZF protein solution : GST-ZF fusion protein purified by the method shown in Example 4 (2) was used. The protein concentration was quantified by the method shown in (3) of Example 4 and adjusted to 2 μM. Diluent [50 mM Tris-HCl;
50 μM Zn (CH ₃ COO) ₂ ] and then diluted,
A dilution series was prepared.

2) Preparation of probe: Each double-stranded oligonucleotide to be used as a probe was chemically synthesized (synthesized by Greiner Japan). However, one oligonucleotide was end-labeled with FITC. Each single-stranded oligonucleotide is equimolar to TEN buffer [1
0 mM Tris-HCl; 1 mM EDTA; 0.1
M NaCl, pH 8.0] and mixed at 95 ° C for 10
After heating for a minute, it was gradually cooled to room temperature and annealed. The annealed double-stranded DNA was treated with TN buffer [10 mM Tris-HCl; 0.1 M NaCl,
pH 8.0] to obtain 1 μM FITC-labeled probe DNA.

3) Binding reaction : The binding reaction solution was Faira.
ll et al. (Fairall, L ,. et al. (1992): Sequence
specific DNA binding by a two zinc ・ finger peptide
fromthe Drosophila melanogaster trametrack protei
n, J. Mol. Biol., vol.226, pp349-366) 2
0 mM MES-KOH (pH 6.5), 1 mM Mg
Cl ₂ , 50 μM Zn (CH ₃ COO) ₂ , 0.1 m
Based on M to 1 mM DTT (dithiothreitol) for stabilization of protein-DNA complex 1
0 μg / ml polylysine (Sigma # P087
9) and 10% glycerol were added. ZF protein with a final concentration of 1 μM or less and a final concentration of 0.5 in the binding reaction solution.
Or 2nM FITC-labeled probe DNA was added,
After setting the total volume to 1 ml, the reaction was carried out at 37 ° C. for 3 to 5 minutes.

4) Measurement of fluorescence polarization degree : The sample after the binding reaction was transferred to a non-fluorescence test tube and the fluorescence polarization degree was measured. For the measurement, a fluorescence polarization degree measuring instrument model VP2000 manufactured by Pan Vera was used. A sample containing no ZF protein and FITC-labeled probe was used as a blank, and a sample containing no ZF protein was used as a control, based on the difference between the fluorescence polarization degree measured in the measurement sample and the measured value in the control, according to a conventional method. The dissociation constant was calculated by a tics analysis. The results are as shown in Table 4.

[0121]

[Table 4]

The modified ZF peptide selectively bound to the HRE sequence. The binding of ttkZF is HG-0 / 1.
This reflects the presence of a sequence similar to "ttkBS" in the / 2 probe.

Example 6 Selection of HRE-I binding peptides
(1) Screening of ZF peptides that bind to DNA was carried out by the binary method using the reporter gene cassette and the protein expression cassette as separate plasmids (FIG. 2).
3).

(1) Construction of test plasmid 22 A reporter plasmid used for binary screening was constructed (FIG. 24). The replication origins pA15 and Amp ^R of the low copy plasmid pACYC177 (purchased from Nippon Gene) were used as the backbone. pACYC177 was digested with AvaI and treated with blunt ends [Takara Shuzo "DNA Blunting Ki
t ”] and further digested with EcoO1091 to obtain a DNA fragment containing the replication origins pA15 and Amp ^R. On the other hand, pNS4131 was digested with SpeI and blunt-ended [Takara Shuzo“ DNA Blunting Kit ”].
Then, it was further digested with EcoO1091 to obtain a DNA fragment containing the reporter gene cassette. Both of these DNA
The fragments were ligated together to give pNS4140.

A protein expression plasmid used for 23 binary screening was constructed (FIG. 2).
5). Anchor PCR using pNS41th: His as template
Amplifies ttkZF (C): His at MunI and A
pNS415 double digested with flII and similarly digested
Insertion and ligation into 1V gave pNS4154V.

24 A plasmid expressing the random peptide used in the actual screening was constructed (FIG. 2).
6). As shown in FIG. 27, 230 bases of r7ZF (N) normal chain having 7 residues in each ZF unit were chemically synthesized, double-stranded by PCR, EcoRI and N
pNS415 double digested with heI and similarly digested
Ligation / insertion into 4V gave pNS4154-r7.

(2) Screening A target sequence HRE-I / GC was used in a binary system.
A ZF-type DNA binding protein that binds to -II was selected. The reporter plasmid is a low copy number plasmid that retains the replication origin p15A derived from the pACYC-based plasmid, retains the ampicillin resistance gene as a selection marker, and "HRE-I / GC-II" as the target sequence.
Β regulated by the modified lac promoter with inserted sequence
-Holds the galactosidase gene as a reporter gene. On the other hand, the protein expression plasmid is pU
It is a high copy number plasmid that retains the replication origin pMB1 derived from the C-series plasmid, retains the chloramphenicol resistance gene as a selectable marker, and retains the r7ZF gene controlled by the tac promoter that can be inducible by a drug. ing.

1) Preparation of reporter plasmid retaining recipient
Made : Escherichia coli JM109 strain (Compiled by Nippon Gene Co., Ltd.
etent E. E. coli JM109 (EP) reporter plasmid pN by electroporation
S4140 was introduced and a recipient bacterium for electroporation was prepared from a colony grown on LB agar medium containing ampicillin according to the method described in (4) of Example 4.

2) Introduction of protein expression plasmid : p
The NS4140 transformed JM109 strain was transformed with the protein expression plasmid pNS4154-r7 by electroporation according to the method described in (3) of Example 4 and spread on LB agar medium containing chloramphenicol. The cells were cultured overnight at 37 ° C. to select a strain having a protein expression plasmid. In Example 6, 10 ng pN
Approximately 5000 using S4154-r7 for transformation
Single colonies were obtained (transformation efficiency: 5 x 10
⁵ c. f. u / μg DNA).

3) Primary selection : Chloramphenicol (C
m) Addition of Cm-supplemented protein-expressing plasmid-bearing strain colonized on LB agar medium with addition of Cm and Xg
al added, Cm, Xgal and IPTG added 3
The seeds were subcultured on LB agar medium of various types and cultured overnight at 37 ° C. Chloramphenicol (60 μg / ml) is a substrate for the reporter enzyme β-galactosidase, Xgal, for the selection of strains carrying protein expression plasmids.
(5-bromo-4-chloro-3-indolyl-β-D-
Galactoside (250 μg / ml) was added to confirm expression of the reporter gene, and IPTG, which is an expression inducer of the tac promoter, was added to induce expression of the ZF protein. The strain which becomes a blue colony on the medium and is white, slightly blue or does not grow on the medium is a strain in which the expression of the reporter gene accompanying the induction of ZF protein expression is suppressed or the growth is inhibited. , Was selected as a primary selection strain and used as a target strain for subsequent selection. When 800 strains of protein expression plasmids were selected, 151 primary selection strains were obtained.

4) Nucleotide Sequence Analysis-Secondary Selection : Plasmid DNA was extracted from the cells of the first selected strain, and the nucleotide sequence of ZF gene was analyzed. 2 ml of LB plasmid DNA
From the cells cultured overnight in liquid medium, "QI"
Prepare-sipin PLASMID KIT "
Extracted using "Taq" manufactured by PerkinElmer
Dye Deoxy ^™ Terminator Cyc
The Le Sequencing kit "was used to perform a dideoxy reaction, and the nucleotide sequence was analyzed with a DNA sequencer model 373A manufactured by PerkinElmer. ZF
A strain in which no deletion or insertion mutation was found in the nucleotide sequence in the gene was selected as a secondary selection strain, and was used as a target strain for subsequent selection. When the primary selection strains of 151 strains were analyzed, 65 secondary selection strains were obtained.

5) Confirmation of protein expression-Third selection : The second selection strain was cultured in LB liquid medium supplemented with IPTG, the protein was extracted from the cells, and the ZF protein was detected by Western analysis using an anti-MYC antibody. confirmed. After freeze-thawing the cells, lysis buffer [8M urea-added P
Lysis in BS buffer] and centrifugation (1000x
g; 10 minutes), the supernatant was collected, and BSA was used as a standard sample by the Bradford method (“Coo manufactured by PIERCE”).
massie ^™ Protein AssayReag
ent "), the protein concentration was quantified, and a protein sample corresponding to 7 μg was electrophoresed on a 15% denaturing polyacrylamide gel (PAGE ^™ # SPG-15S manufactured by Ato Co.). Hybond ^™ manufactured by the company
-ECL) electrical blotting to Current Prtoco
ls in Immunology [Greene Pubiishing Associates and
Wiley-Intrescinece] was performed according to the method described in [1]. Anti-MYC antibody as a primary antibody (# OP10 manufactured by Oncogene Science)
Anti-mouse I modified with peroxidase as a secondary antibody
gG antibody ("# PP27" manufactured by Binding Site)
3 "), an ECL system (Amersham ECL western blotting det.
Polaroid instant film (Type manufactured by Polaroid)
667). A strain in which the expression of ZF protein was observed by Western analysis was selected as a third-selected strain and used as a target for subsequent selection.

When the 65 secondary selected strains were analyzed, 31 tertiary selected strains were obtained. 31 selected Z
The amino acid sequence of F peptide (62 species) was as shown in FIG. 28 and SEQ ID NOs: 3 to 64.

Amino acid database NBRF-PIR (R44.
Homology search using 0) revealed that SEQ ID NO: 32 was yeast-derived YKR042w (entry number S38
114) and SEQ ID NO: 33 is a virus-derived phosphoprot
ein MI gene (entry number B46104), SEQ ID NO: 42 is human-derived in vitro nectin precursor gene (entry number SGHU1V), and SEQ ID NO: 57 is bacterial Cu-ATPase gene (entry number A4).
5995), each of which was independent of the DNA binding protein. In addition, no amino acid sequence was found that was completely identical with other sequences.

Furthermore, STN (The Scientific & Technical
Homology search was performed using the registry file (Release 172486-98-5) of Information Network).
No residue peptide was found.

Selection of HRE-I binding peptide (2) (1) Construction of test plasmid 25 Candidate peptide “r” obtained by screening
7ZF- # "was used as a fusion protein with GST (Fig. 2).
6). Anchor PCR using pNS4154-r7 # as a template
R7ZF (N) was amplified in PCR, double digested with EcoRI and NheI, and th of pNS42th digested in the same manner.
Substitution and ligation with ZF (N) gave pNS42r7- #. Note that “#” indicates the ZF peptide numbers 1 to 31 in FIG. 28.

By the method shown in (2) of Example 4, GST
The selected ZF peptide was prepared as a fusion protein with. Of these, peptide numbers 01, 06, 07, 08, 1
The peptides of 5, 17, 18, 19, 21, and 31 were not used in the following tests because a fusion protein of the desired size was not obtained as a major protein. Since no mutation in the nucleotide sequence of DNA was observed in these ZF peptides, it is possible that the translation of most ZF peptide molecules is interrupted because the codons of the random sequence portion are not compatible with E. coli. However, on the agar medium, the specified reactivity (ZF by IPTG
Since the expression of the reporter gene is inhibited when the protein expression is induced), it is considered that a small amount of the expressed full-length ZF peptide acts in E. coli cells. Therefore, in this Example, the in vitro DNA binding ability could not be verified by gel shift, but it is considered that the DNA binding ability of these ZF peptides is not denied.

(2) Screening (DNA of selected strain
Qualitative evaluation of binding ability-quaternary selection) 1) By the method shown in (4) of Example 4, gel shift assay was carried out on 31 third-selected strains (Experiment 1). The probe was a 32 bp DNA having the HRE-I sequence and the GC sequence shown in Table 3 (hereinafter referred to as "H
G-0 ") was used. The results are as shown in Table 5.

[0139]

[Table 5]

Ten fourth selected strains (peptide number: 0
2, 03, 04, 09, 10, 23, 26, 28, 2
9, 30) was obtained.

2) In order to evaluate the binding specificity to the target sequence, the above-mentioned "HG-0" was used as a probe, and (a) unlabeled "HG-0" or (b) unlabeled Sp1 (= G
A large excess (corresponding to C-II sequence) (30 for probe)
The gel shift assay was carried out in the coexistence (0-fold amount) (Experiment 2). The gel shift assay was carried out for the 6 types judged to be "++++" and "++" in Experiment 1. Sp1
Is as shown below.

[0142]

Embedded image

The results are shown in Table 5 above. # 03 was slightly competed in (a) and was not competed in (b) at all, suggesting that it strongly binds specifically to the HRE sequence. # 04 was completely competed in (a) and not so much in (b), so HR
It was suggested that the E sequence was weakly bound specifically. #
Since 09 and # 23 were completely competed with each other in (a) and (b), it was suggested that they are weakly bound to the GC sequence. # 10 and # 28 are completely competed in (a), and (b)
It was suggested that it was weakly bound across the HRE sequence and the GC sequence since it competed considerably with.

Example 8 Selection of HRE-I binding peptides
Extraction (3) The 6 types of ZF peptides selected in Example 7 were subjected to the fluorescence polarization measurement method according to the method described in Example 5 (2). The probe is the HG-0 and the fourth.
HG-1 and HG-2 shown in the table were used. These were fluorescently labeled (FITC) at the 5'end of one chain. Kinetic analysis was performed to calculate the dissociation constant Kd (nM). The results are as shown in Table 6.

[0145]

[Table 6]

The increase in the Kd value of # 03 was observed in HG-1 and HG-2, suggesting that # 03 is bound to the HRE sequence in a fairly specific manner. # 04 is HG-0,
Since the Kd values were the same in 1 and 2, it was suggested that the selectivity for the base was weak or that they were bound to additional sequences other than HRE-GC. # 09, # 23
Showed an equivalent Kd value in HG-0,1, and was competed with Sp1 in the gel shift assay of Example 7, suggesting that it was bound to the GC sequence. In # 10, since the Kd value increased in HG-1,
Based on the results of the gel shift assay of Example 7, HR
Weakly bound across the E and GC sequences. #
No. 28 had an increased Kd value in HG-1 and HG-2. Therefore, based on the results of the gel shift assay of Example 7, HRE
It was suggested that the sequence was linked to the GC sequence.

Example 9 Conversion of HRE-I binding peptide
Confirmation of Copy Inhibitory Ability The peptides selected in Examples 7 and 8 were tested for their ability to inhibit transcription specific to a target sequence in animal cultured cells.
It was confirmed by the following experiment. Briefly describing the method, HaLa cells transformed with a firefly-derived luciferase gene driven by a human-derived c-H-ras promoter are used as a host, and a plasmid vector expressing a # 03 peptide is introduced into this host. # 03 in the cell
The peptide was expressed and the change in reporter expression at that time was examined. In the following, for the measurement of luciferase activity, 20 μl of cell lysate was dispensed into a light-shielding 96-well multiplate, and a detection reagent (Toyo Ink; Piccagene)
Immediately after the addition of 00 μl, the luminescence intensity for 5 seconds was measured with a luminometer (Verthold; MicroLuma).
t model LB96P).

(1) Preparation of Host Cell [0148] The human c-H-rasl genomic clone (# CO-001) c obtained from the JCRB gene bank was cloned upstream of the luciferase gene in the plasmid vector PGV-B (Toyo Ink Co., Ltd.). A reporter plasmid pNS2015B was constructed by inserting a SacI-NaeI fragment of about 450 base pairs containing the -H-ras promoter region (the region from -235 to +220 including the transcription start point). 17.5 μg of this reporter plasmid and a selection marker plasmid pSVneo (a plasmid having a neomycin resistance gene expressed in eukaryotic cells. Clo.
3.5 μg (purchased from ntech) was mixed, and then 1 ml of a mixed DNA suspension was prepared by the calcium phosphate method.
330 μl of this suspension was added to 10% fetal bovine serum (Gi
(hco) was added to HeLa cells cultured in RPMI1640 medium (Nissui) containing hco) for transformation. Selection of transformed cells was performed as follows. First, this DN
Approximately 24 hours after the addition of the suspension A, the medium was removed, and the cells were cultured for 1 week in the above medium supplemented with 0.4 mg / ml of the antibiotic G-418 (Sigma), and the surviving cells were collected. Furthermore, the luciferase activity of the recovered cells was measured, and the clone with the highest activity value was designated as H1-27 strain and used for the subsequent experiments.

(2) Construction of test plasmid Expression of the desired peptide in animal culture cells was performed by using Strat.
The LacSwitch ^™ system from agene was used. This system is a system that modifies the Escherichia coli lactose operon for eukaryotic cells and can transiently induce protein expression in animal cells by IPTG as in E. coli. This system is specifically composed of a Lac repressor expression plasmid p3'SS and a CAT driven by a promoter transcriptionally regulated by this repressor.
Plasmid vector pOPI3cat expressing (chloramphenicol acetyltransferase gene)
It is a system composed of and, which is capable of inducing and expressing a desired protein by substituting the latter CAT gene.

In order to express the # 03 peptide, first, a translation control sequence Kozak, a translation initiation codon, a protein detection tag sequence [YPYDYPDYA; derived from influenza virus haemagglutinin protein], a cloning site [ClaI, XhoI, SpeI], nuclear translocation. Signal sequence [PKKKKRKV; SV40 large T
Antigen-derived] and a chemically synthesized DNA (about 110 base pairs) containing a translation stop codon were substituted and inserted into the CAT gene portion in pOPI3cat to construct pOPI3tag. Then MnuI of pNS42r7- # 03
Anchor PCR from the site to the NheI site
The DNA fragment [ClaI-XbaI fragment] encoding the ZF unit of the # 03 peptide, which was amplified by
P inserted by inserting into 3 sites of ClaI and SpeI sites
OPI3 # 03 was obtained.

(3) Experiment for Transformation into Cultured Cells H1-27 cells grown in RPMI1640 medium supplemented with 10% fetal bovine serum, 10 mM dextran and 0.1 mM DTT were detached with trypsin, and the cells were washed with phosphate buffer to give 2 cells. After washing twice, 2 × 10 ⁷ cells / ml
A solution suspended in serum-free RPMI1640 medium so as to obtain a cell suspension for electroporation. 10
A DNA solution prepared by dissolving 10 μg of p3′SS and 10 μg of pOPI3 # 03 in 10 μl of TE buffer was added,
It was mixed with 300 μl of the cell suspension and left standing on ice for 10 minutes. After that, electroporation (Bio
Rad GenePulser; 0.3kV, 500μ
FD). After the pulse, the plate was allowed to stand on ice for 10 minutes, added with an Eagle MEM medium (Nissui) containing 10% fetal bovine serum, transferred to a φ100 mm petri dish, and cultured for about 16 hours. The surviving cells after culturing were peeled off with trypsin and collected. Recovered cells are 5 × 10 ⁴ cells / m
Eagle ME containing 10% fetal bovine serum to give 1
The cells were suspended in M medium, dispensed at 1 ml per well into a 24-well multiplate, and further cultured for 8 hours. Then, in the test section, the medium was replaced with a serum-free Eagle MEM medium containing IPTG (isopropyl-β-D-thio-galactopyranoside) so that the final concentration was 3 mM, and the cells were placed in a serum-free state 16 After culturing for a period of time, protein expression was induced. In the control section, IPT was used to prevent inducible expression of protein.
The same culture was performed without adding G. By culturing the cells in a serum-free medium, the residual luciferase activity due to the degradation of the luciferase protein can be expected to be reduced, and the serum-stimulated luciferase activity de novo can be expected.
It was made possible to prominently detect such inducible expression.
The cells of the test group and the control group exposed to the serum-free state were
Serum-stimulated in Eagle's MEM medium containing% fetal bovine serum and cultured for 0, 1, 2, 3, 4, or 5 hours,
100 μl / well of lysing agent (Toyo Ink; PG
The cells were fixed by lysing with C-50). Each cell lysate was stored at -80 ° C until luciferase activity measurement.
The luciferase activity of the cell lysate prepared as described above was measured. The result was as shown in FIG. In the figure, the value obtained by performing the same measurement using the cell lysing agent itself as a sample, correcting the measured value of each sample, and calculating the value relative to the measured value of the culture before serum stimulation as a sample Is shown on the vertical axis. The error bar on the vertical axis shows the standard error of two independent measurement results.
In the control group, luciferase activity was rapidly induced after serum stimulation, and increased to about 2-fold after 3 hours. However, in the test group in which the expression of # 03 peptide was induced in advance prior to serum stimulation, the expression of luciferase activity was significantly lower than that in the control group at least until 5 hours after serum stimulation. It was induced 16 hours before serum stimulation #
03 peptide inhibits the subsequent transcriptional activation of the c-H-ras promoter, and this peptide binds to the target sequence of the c-H-ras promoter to cause transcriptional inhibition. Strongly suggests that

[0152]

[Sequence list]

SEQ ID NO: 1 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence feature Characteristic symbol: binding-site Method for determining feature: E

SEQ ID NO: 2 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 3 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 4 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 5 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 6 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 7 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 8 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 9 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 10 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 11 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence feature Characteristic symbol: binding-site Method for determining feature: E

SEQ ID NO: 12 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 13 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 14 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 15 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 16 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 17 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence feature Characteristic symbol: binding-site Method for determining feature: E

SEQ ID NO: 18 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 19 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 20 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 21 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 22 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 23 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 24 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 25 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 26 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 27 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 28 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 29 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 30 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 31 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 32 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 33 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 34 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence feature Characteristic symbol: binding-site Method for determining feature: E

SEQ ID NO: 35 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 36 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 37 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 38 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 39 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 40 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 41 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 42 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 43 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence feature Characteristic symbol: binding-site Method for determining feature: E

SEQ ID NO: 44 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 45 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 46 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 47 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 48 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 49 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 50 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 51 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 52 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 53 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 54 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 55 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 56 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 57 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 58 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 59 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 60 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 61 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 62 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 63 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 64 Sequence length: 7 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E

SEQ ID NO: 65 Sequence length: 63 Sequence type: Amino acid Sequence type: Peptide Sequence characteristic feature symbol: binding-site Method for determining feature: E sequence Thr Lys Glu Gly Glu His Thr Tyr Arg Cys Lys Val Cys Ser Arg Val 1 5 10 15 Tyr Thr His Ile Ser Asn Phe Cys Arg His Tyr Val Thr Ser His Lys 20 25 30 Arg Asn Val Lys Val Tyr Pro Cys Pro Phe Cys Phe Lys Glu Phe Thr 35 40 45 Arg Lys Asp Asn Met Thr Ala His Val Lys Ile Ile His Lys Ile 50 55 60

[Brief description of drawings]

FIG. 1 is a diagram showing a recognition mode of a DNA sequence of ttkZF.

FIG. 2 is a diagram showing an outline of a plasmid used in Example 1.

FIG. 3 is a diagram showing steps of constructing a plasmid used in Example 1.

FIG. 4 is a diagram showing steps of constructing a plasmid used in Example 1.

FIG. 5 is a diagram showing steps of constructing the plasmid used in Example 1.

FIG. 6 is a diagram specifically showing the modification of the lac promoter sequence.

FIG. 7 is a diagram showing the entire sequence of ttkZF.

FIG. 8 is a diagram showing steps of constructing the plasmid used in Example 1.

FIG. 9 is a diagram showing the results of reporter enzyme activity in Example 1. X indicates the type of assay gene, and Y indicates the type of promoter operably linked to the reporter gene.

FIG. 10 is a diagram showing an outline of plasmids used in Example 2.

FIG. 11 is a diagram showing steps of constructing the plasmid used in Example 2.

FIG. 12 is a diagram specifically showing modification of the lac promoter.

FIG. 13 is a diagram showing an outline of plasmids used in Example 3.

FIG. 14 is a diagram showing steps of constructing the plasmid used in Example 3.

FIG. 15 is a diagram specifically showing modification of the lac promoter.

FIG. 16 is a diagram showing modification of the N-terminus of ttkZF.

FIG. 17 is a diagram showing an outline of the plasmid used in Example 4.

FIG. 18 is a diagram showing steps of constructing the plasmid used in Example 4.

FIG. 19 is a diagram showing steps of constructing the plasmid used in Example 4.

FIG. 20 is a diagram showing the steps of constructing the plasmid used in Example 4.

FIG. 21 is a diagram showing the results of gel shift assay in Example 4.

22 is a diagram showing an outline of the plasmid used in Example 5. FIG.

FIG. 23 is a diagram showing an outline of the plasmid used in Example 6.

FIG. 24 is a diagram showing the steps of constructing the plasmid used in Example 6.

FIG. 25 is a diagram showing steps of constructing the plasmid used in Example 6.

FIG. 26 is a diagram showing steps of constructing the plasmid used in Example 6.

FIG. 27 is a view showing a specific sequence of r7ZF (N).

FIG. 28 is a diagram showing the amino acid sequences of ZF peptides of 31 strains selected. Only the part with optionality is shown.

FIG. 29 shows changes in the expression of the luciferase gene, which is a reporter gene, when the peptide # 03 peptide selected in Examples 7 and 8 was expressed in animal cultured cells and then cultured.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] January 16, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] FIG.

[Correction method] Change

[Correction contents]

FIG. 21 is an electrophoretic photograph showing the result of gel shift assay in Example 4.

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] FIG.

[Correction method] Change

[Correction contents]

FIG. 21

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 1/21 C12P 21/02 C C12P 21/02 7823-4B C12Q 1/68 Z C12Q 1/68 G01N 33/53 D G01N 33/53 A61K 9/127 L // A61K 9/127 37/02 ADU (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (54)

[Claims] 1. A plasmid preferably used for screening a peptide having a binding property to a target gene, comprising a nucleotide sequence encoding a test peptide whose degree of binding to the target gene is evaluated, and the target gene. And a reporter gene operably linked to the promoter, wherein the promoter controls the expression of the reporter gene according to the degree of binding of the test peptide to the target gene contained therein. A plasmid comprising the target gene as described above. 2. The plasmid according to claim 1, wherein the base sequence encoding the test peptide is a base sequence encoding a peptide having a zinc finger structure. 3. A nucleotide sequence encoding the peptide having a zinc finger structure is a gene derived from the Drosophila ttk gene (ttk gene) or a mouse-derived zif.
268 gene and TFIII derived from Xenopus laevis
The plasmid according to claim 2, which is selected from the group consisting of A gene and modified genes thereof. 4. The plasmid according to claim 3, wherein the ttk gene encodes the amino acid sequence of SEQ ID NO: 65. 5. The plasmid according to claim 3 or 4, wherein the modified gene is a ttk gene in which the portions encoding the amino acid sequences of units 1 and 2 of the zinc finger structure are modified. 6. The modified gene is the 19th SEQ ID NO: 65.
The plasmid according to claim 5, which is a ttk gene in which the portions encoding the 25th to 25th amino acid sequences and the 49th to 55th amino acid sequences are modified. 7. The plasmid according to claim 1, wherein the promoter is a lac promoter or a modified promoter thereof. 8. The plasmid according to any one of claims 1 to 7, wherein the target gene is a disease-related gene. 9. The plasmid according to claim 8, wherein the disease-related gene is an oncogene promoter gene. 10. A promoter further operably linked to a nucleotide sequence encoding a test peptide chain,
The plasmid according to any one of claims 1 to 9. 11. The plasmid according to any one of claims 1 to 10, which further comprises a selection marker. 12. The plasmid according to claim 11, wherein the selectable marker is selected from the group consisting of ampicillin resistance gene, chloramphenicol resistance gene, kanamycin resistance gene, and tetracycline resistance gene. 13. A plasmid set comprising a first plasmid and a second plasmid which are preferably used for screening a peptide having a binding property to a target gene, wherein the first plasmid has a degree of binding to the target gene. Wherein the second plasmid comprises a promoter containing the target gene and a reporter gene operably linked to the promoter. And the promoter is
A plasmid set comprising the target gene so as to control the expression of the reporter gene according to the degree of binding of the test peptide to the target gene contained therein. 14. A base sequence encoding a test peptide comprises:
The plasmid set according to claim 13, which is a nucleotide sequence encoding a peptide having a zinc finger structure. 15. The nucleotide sequence encoding the peptide having a zinc finger structure is a Drosophila ttk.
Gene derived from gene (ttk gene), mouse derived zi
f268 gene and TFII derived from Xenopus laevis
The plasmid set according to claim 14, which is selected from the group consisting of the IA gene and its modified gene. 16. The plasmid set according to claim 15, wherein the ttk gene encodes the amino acid sequence of SEQ ID NO: 65. 17. The modified gene is a ttk gene in which the portions encoding the amino acid sequences of units 1 and 2 of the zinc finger structure are modified.
The plasmid set according to 5 or 16. 18. The modified gene according to SEQ ID NO: 65.
The plasmid set according to claim 17, wherein the portions encoding the amino acid sequences 9 to 25 and the amino acid sequences 49 to 55 are modified ttk genes. 19. The method according to claim 13, wherein the promoter is a lac promoter or a modified promoter thereof.
The plasmid set according to any one of 18. 20. The plasmid set according to any one of claims 13 to 19, wherein the target gene is a disease-related gene. 21. The set of plasmids according to claim 20, wherein the disease-related gene is an oncogene promoter gene. 22. The plasmid set according to any one of claims 13 to 21, wherein the first plasmid further comprises a promoter operably linked to a nucleotide sequence encoding a test peptide chain. 23. The method of claims 13-22, wherein the first plasmid and the second plasmid further comprise a selectable marker.
The plasmid set according to any one of 1. 24. The plasmid set according to claim 23, wherein the selectable marker is selected from the group consisting of an ampicillin resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene, and a tetracycline resistance gene. 25. A method for screening a peptide having a binding property to a target gene, which comprises the plasmid according to any one of claims 1 to 12 or the plasmid according to any one of claims 13 to 24. A method comprising transforming a host with the plasmid set, culturing the host, and measuring the degree of expression of the reporter gene. 26. The degree of expression of the reporter gene is
The method according to claim 25, which is measured by observing the color tone of the colony or by measuring the enzyme activity. 27. The method further comprises one or more steps of measuring the degree of binding between the test peptide and the target gene.
27. The method according to claim 25 or 26. 28. The method according to claim 27, wherein the degree of the binding is measured by gel shift assay, fluorescence polarization method, immunoassay, or surface plasmon resonance method. 29. A peptide obtained by the method according to any one of claims 25 to 28. 30. A nucleotide sequence encoding the peptide according to claim 29. 31. An expression inhibitor of a gene, the expression of which is regulated by a specific regulatory sequence, which comprises the peptide of claim 29 or the nucleotide sequence of claim 30. 32. A therapeutic agent for a disease involving expression of a gene, the expression of which is regulated by a specific regulatory sequence, which comprises the peptide of claim 29 or the nucleotide sequence of claim 30. 33. Use of the peptide according to claim 29 for the treatment of a disease involving the expression of a gene the expression of which is regulated by a specific regulatory sequence. 34. Use of the peptide according to claim 29 for the manufacture of a therapeutic agent for a disease associated with the expression of a gene the expression of which is regulated by a specific regulatory sequence. 35. Use of the nucleotide sequence according to claim 30 for the treatment of a disease involving the expression of a gene whose expression is regulated by a specific regulatory sequence. 36. Use of the nucleotide sequence according to claim 30 for the manufacture of a therapeutic agent for a disease associated with the expression of a gene the expression of which is regulated by a specific regulatory sequence. 37. A method for suppressing the expression of a gene, the expression of which is regulated by a specific regulatory sequence, which is obtained by the method according to any one of claims 25 to 28. A method comprising the step of linking a peptide having a binding property to a control sequence to the control sequence. 38. A method for treating a disease involving the expression of a gene, the expression of which is regulated by a specific regulatory sequence, which is obtained by the method according to any one of claims 25 to 28. D comprising a nucleotide sequence encoding a peptide
A method comprising introducing an NA construct into an animal body and expressing the peptide. 39. The method according to claim 37 or 38, wherein the gene whose expression is regulated by a specific regulatory sequence is an oncogene. 40. The method of claim 39, wherein the particular regulatory sequence is the HRE-I sequence and the oncogene is the Ras gene. 41. A peptide according to any one of SEQ ID NOs: 1 to 64 or a modified peptide thereof which retains the ability to bind to a HER-I sequence. 42. A peptide, comprising one or more peptides according to claim 41. [43] A peptide represented by the following general formula (I): _{Y 1 - [- X 1 -Cys} -X 2 -Cys-X 3 -Z-His-X 4 -His-X 5 -] m-Y 2 (I) ( In the formula, X ₁ and X ₅ are, May be the same or different, bond, 1
-10 residues of any amino acid sequence, or crosslinkers, provided that X ₁ and X ₅ represent a bond when present at the end of the peptide, X ₂ is any of 2-7 residues X ₃ represents an arbitrary amino acid sequence of 5 residues, X ₄ represents an arbitrary amino acid sequence of 2 to 4 residues, Y ₁ and Y ₂ may be the same or different. Often represents a tag sequence and / or a translocation signal sequence or is absent, Z represents a peptide according to claim 41 or 42 and m represents an integer from 1 to 5) 44. If m is 2 and X ₁ and X ₅ are not present at the terminal of the peptide, then 6 to
Represents any amino acid sequence of eight residues, at least one of Y ₁ and Y ₂ represent the tag sequence and localization signal sequence, peptide of claim 43. 45. The peptide according to claim 43 or 44, wherein the tag sequence is selected from the group consisting of a secretory transport signal sequence, a carrier protein sequence, a purification detection tag sequence, and a peptidase recognition sequence. 46. The peptide according to claim 43 or 44, wherein the translocation signal sequence is a nuclear translocation signal sequence or an intracellular organelle trafficking signal sequence. 47. The peptide according to any one of claims 41 to 46, which has a metal ion in the peptide. 48. A metal ion is Zn ²⁺ , Mn ²⁺ , N
^48. The peptide of claim 47 selected from i2 ⁺ and Cd2 ⁺ . 49. A therapeutic agent for a disease associated with H-Ras, which comprises the peptide according to any one of claims 41 to 48. 50. An HR comprising a nucleotide sequence encoding the peptide according to any one of claims 41 to 48.
A therapeutic agent for a disease involving as. 51. Use of a peptide according to any one of claims 41 to 48 for the treatment of diseases involving H-Ras. 52. Use of the peptide according to any one of claims 41 to 48 for the manufacture of a therapeutic agent for a disease involving H-Ras. [53] Use of a nucleotide sequence encoding the peptide according to any of [41] to [48] for the treatment of a disease involving H-Ras. [54] Use of a nucleotide sequence encoding the peptide according to any one of [41] to [48] for the manufacture of a therapeutic agent for a disease involving H-Ras.