JPH10234367A - Nuclear-transfer signal to introduce protein having local homology to human hepatic carcinoma cell-derived growth factor into nucleus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a nuclear-transfer-signal peptide of a protein having a local homology to human hepatic carcinoma cell-derived growth factor capable of elucidating nuclear transfer mechanism, biological function(s), and relation(s) of the mechanism with disease(s), by including a specified amino acid sequence. SOLUTION: This peptide has an amino acid sequence of formula I or II. The nuclear-transfer-signal peptide is obtained by culturing Escherichia coli transformed with a recombinant DNA molecule in which a DNA base sequence shown in formula III with the nuclear-transfer-signal peptides (HET-A-NLS1 and HET-A-NLS2) deleted so as not to work is integrated in a vector used in a host-vector system of E. coli.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an amino acid sequence for introducing a protein having local homology to a human hepatoma cell-derived growth factor (hereinafter abbreviated as "HET-A" throughout the present specification) into the nucleus. Abbreviated as "nuclear translocation signal" throughout the text).

[0002] HET-A, in which a DNA sequence encoding a nuclear transport signal has been disrupted, can be used as a reagent for analyzing the original physiological role of HET-A by analyzing cells into which HET-A has been introduced. In addition, this is achieved by introducing into the nucleus a protein that is physiologically expressed and functions in a place other than the nucleus or a microbial protein that is not originally involved in the physiological function of eukaryotes using the nuclear translocation signal. It is a reagent for analyzing changes in cells. The present invention contemplates providing such reagents.

[0003]

2. Description of the Related Art Human HDGF is a human liver cancer-derived cultured cell HuH-.
7 {Nakabayashi, H. et al (1982) Cancer Res. 42 3858
-3863.} Was roughly purified as a fraction having proliferative activity on Swiss3T3 cells {Nakamura, H. et al (1
989) Clinica Chimica Acta 183 273-284}. From this crude fraction, a protein with a molecular weight of about 25K was extracted as human HDGF and its gene was cloned {Nakamur
a, H. et al (1994) J. Biol. Chem. 269 25143-2514
9}. Furthermore, Northern hybridization of various organs of the mouse was performed using human HDGF as a probe, and it was found that mouse HDGF was expressed in a large amount in testis tissue.

[0004] In order to obtain a mouse homolog of human HDGF (ie, mouse HDGF), a cDNA library prepared from messenger RNA extracted from mouse testis tissue was used to obtain a human HDGF DNA (3-1-1 Takanodai, Tsukuba, Ibaraki, Japan). 1
Deposit of "RIKEN Genebank", Deposit No .: RDB
No. 1493) as a probe. As a result, not only mouse HDGF was obtained (JP-A-6-220094), but also two related proteins having high homology at the N-terminal about 100 amino acids of mouse HDGF (“HET-A
And "HET-B") were found (Japanese Patent Application No. 8-131788).

[0005] When the amino acid sequence of HET-A was homology-searched against a database, an amino acid sequence considered to be a nuclear localization signal was found. HET-A translocated to the nucleus and worked in the nucleus so far, if HET-A
Once it is clear that will move to the nucleus, it will be necessary to analyze new functions in that nucleus. It is significant to provide HET-A lacking a nuclear translocation signal as a reagent for this purpose.

At present, the following three types are known as nuclear transport signals. These are all said to have a common sequence called a motif.

(1) A type having almost no basic amino acids such as lysine and arginine. Very few examples of this motif. For example, influenza virus nucl
eoprotein nuclear translocation signal (AAFEDLRVLS) {Dav
y, J. et al (1985) Cell40 667-675}.

(2) A type containing a large amount of basic amino acids. There are numerous examples of this motif. For example, SV40 large
Has nuclear translocation signal (PPKKKRKV) for T antigen {Kaldero
n, D. et al (1984) Nature 311 33-38}.

(3) A type in which basic amino acids form clusters at intervals of about 10 amino acids. It is called a Bipartite-type nuclear translocation signal. There are many examples of this motif. For example, there is a nuclear translocation signal (KRPAATKKAGQAKKKK) of nucleoplasmin of Xenopus laevis {Robbins, J. e
tal (1991) Cell 64 615-623}.

However, amino acid sequences having these motifs are not necessarily translocated to the nucleus, and it is necessary to prove the ability of each sequence to translocate to the nucleus.

[0011] For this reason, at least one of the nuclear translocation signals is studied in many cases, and the mechanism of nuclear translocation is clarified.
In addition, elucidation of its biological function and elucidation of its relationship with disease are eagerly desired.

[0012]

The object of the present invention is to provide a HET-
It is to clarify that A is translocated to the nucleus, and to identify an amino acid sequence (nuclear translocation signal) having the ability to translocate the protein present in the amino acid sequence to the nucleus. It is possible to provide a reagent for discovering a new function of the protein by preparing HET-A lacking the nuclear localization signal identified in this way, introducing the HET-A into a cell, and analyzing a physiological change of the cell. it can. In addition, by introducing proteins having various physiological activities into the nucleus using the nuclear translocation signal, it becomes possible to analyze physiological changes of cells.

[0013]

SUMMARY OF THE INVENTION The present invention relates to HET-A and a protein which is never present in the nucleus, such as E. coli β-protein.
We succeeded in constructing an expression plasmid for a complex gene product with galactosidase, and found that HET-A was localized in the nucleus by retaining it in nucleated cells.

Therefore, the present invention provides (1) identification of a nuclear translocation signal on HET-A, and (2) HET-
A, and (3) a method of introducing into the nucleus a protein that is normally never present in the nucleus, using the translocation signal.

The invention of claim 1 provides a nuclear translocation signal peptide (HET-A-) of a protein (HET-A) having the amino acid sequence shown in FIG. 1A and having local homology to a human hepatoma cell-derived growth factor. NLS1).

The invention according to claim 2 provides a nuclear translocation signal peptide (HET-A-) of a protein (HET-A) having local homology to a human hepatoma cell-derived growth factor having the amino acid sequence shown in FIG. 1B. NLS2).

According to a third aspect of the present invention, there is provided a DNA encoding the nuclear localization signal peptide (HET-A-NLS1) according to the first aspect.
Is an array.

According to a fourth aspect of the present invention, there is provided a DNA encoding the nuclear localization signal peptide according to the second aspect (HET-A-NLS2).
Is an array.

The invention according to claim 5 is the invention according to claim 3 or 4.
A recombinant DNA molecule in which the described DNA base sequence is incorporated into a vector used in an E. coli host / vector system.

The invention according to claim 6 lacks the nuclear translocation signal peptide (HET-A-NLS1) according to claim 1 and the nuclear translocation signal peptide (HET-A-NLS2) according to claim 2 and does not function. A variant of the protein (HET-A) having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and having local homology to human hepatoma cell-derived growth factor.

[0021] The invention of claim 7 deletes the nuclear localization signal peptide (HET-A-NLS1) and the nuclear localization signal peptide (HET-A-NLS2) of claim 2 and does not function. As shown in SEQ ID NO: 1 in the sequence listing.
A variant of a protein (HET-A) having a DNA sequence and having local homology to human hepatoma cell-derived growth factor.

According to the invention of claim 8, the nuclear localization signal peptide (HET-A-NLS1) according to claim 1 and the nuclear localization signal peptide (HET-A-NLS2) according to claim 2 are deleted and do not function. As shown in SEQ ID NO: 1 in the sequence listing.
It is a recombinant DNA molecule in which the DNA base sequence has been incorporated into a vector used in an E. coli host / vector system.

The ninth aspect of the present invention is the fifth aspect of the present invention.
E. coli transformed with the described recombinant DNA molecules.

According to a tenth aspect of the present invention, there is provided an animal cell transformed with the recombinant DNA molecule of the fifth or eighth aspect.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The details of the present invention will be described below.

(1) Proof that HET-A translocates to the nucleus In order to verify whether HET-A translocates to the nucleus, a plasmid is prepared in which the gene encoding the protein is linked downstream of an appropriate promoter. However, this is retained in nucleated cells, and nuclear translocation can be confirmed by an existing method such as an immunoantibody method using an antibody or an enzyme antibody method. Alternatively, as another method, a gene encoding the protein is constructed as a complex gene with an arbitrary protein which is never present in the nucleus, and a plasmid is prepared by binding the gene downstream of an appropriate promoter. Nuclear translocation can be confirmed by retaining the protein in a cell and using the activity of an arbitrary protein as an index, or an existing method such as an immunoantibody method or an enzyme antibody method using the antibody.

On the basis of the latter idea, a plasmid pRcβG-1 which can be easily fused with HET-A by modifying an arbitrary gene such as β-galactosidase which is never present in the nucleus can be prepared. . As a means for synthesizing a plasmid, for example, a Kozak sequence, a methionine codon, a restriction enzyme (eg, SpeI) sequence downstream of an arbitrary promoter such as SV40, and a β-galactosidase gene further downstream are used. By connecting, a plasmid can be constructed. Figure 2 shows the structure.

HET-A appropriately modified so that the translation frame matches SpeI of plasmid pRcβG-1 is prepared according to a conventional method such as PCR, and inserted into SpeI to express a fusion protein. If the plasmid thus prepared is transfected into cultured cells,
It becomes clear that the fusion protein is localized in the nucleus (Fig. 4
A). This means that HET-A translocates to the nucleus.

(2) Identification of nuclear translocation signal present on HET-A Next, in order to identify the nuclear translocation signal, HET-A was deleted by deleting a part of HET-A gene and -A variants can be created. By constructing an expression plasmid for a complex gene product of these and a protein that is never present in the nucleus, for example, β-galactosidase, and retaining this in a nucleated cell, the nuclear translocation signal is HET-.
You can identify where it is on A. That is, the mutant lacking the nuclear translocation signal does not translocate to the nucleus but remains in the cytoplasm.

In HET-A, for example, a codon encoding the first half amino acid sequence as shown in FIG. 3B is synthesized according to a conventional method such as PCR, and inserted so that the translation frame matches with SpeI of plasmid pRcβG-1. Can express the fusion protein. Transfection of the thus prepared plasmid into cultured cells reveals that the fusion protein is localized in the nucleus (FIG. 4B). This means that at least one nuclear localization signal exists in the first half amino acid sequence.

For example, a codon encoding the latter half of the amino acid sequence as shown in FIG. 3C is synthesized according to a conventional method such as the PCR method, and inserted into SpeI of plasmid pRcβG-1 so that the translation frame is in conformity with the fusion protein. Can be expressed. Transfection of the thus prepared plasmid into cultured cells reveals that the fusion protein is localized in the nucleus (FIG. 4C). This means that at least one nuclear localization signal exists in the latter amino acid sequence.

Then, when a homology search was performed on the amino acid sequence of HET-A against a database, for example, EMBL, Genbank, etc., two nuclear localization signals, HET-A-NLS1, were found in the amino acid sequence of the first half. HET-A-NLS2 can be found in the latter amino acid sequence. HET-A-NLS1 has the characteristics of a type I motif containing a large number of basic amino acids, and HET-A-NLS2 has the characteristics of a bipartite type, but their sequences are novel (FIG. 1).

It is necessary to confirm that HET-A-NLS1 in the amino acid sequence of the first half is the only nuclear translocation signal in the first half. For this purpose, for example, HET-A-NLS1 as shown in FIG. 3D
Codon encoding the first half amino acid sequence deleted
It is synthesized according to a standard method such as the PCR method, and the plasmid pRcβG-1
The fusion protein with β-galactosidase can be expressed by inserting the translation frame into SpeI. When the plasmid thus prepared is transfected into cultured cells, the nuclear translocation signal HET-
It is revealed that the amino acid sequence mutant in the first half where A-NLS1 is deleted does not translocate to the nucleus but remains in the cytoplasm (FIG. 4D). HET-
It can be said that A-NLS1 is the only nuclear translocation signal in the first half.

It is necessary to confirm that HET-A-NLS2 in the latter amino acid sequence is the only nuclear translocation signal in the latter half. For this purpose, for example, HET-A-NLS2 as shown in FIG.
Codon encoding the first half amino acid sequence deleted
It is synthesized according to a standard method such as the PCR method, and the plasmid pRcβG-1
And a fusion protein can be expressed by inserting the translation frame into SpeI. When the plasmid thus prepared is transfected into cultured cells, the nuclear translocation signal HET-A-
It becomes clear that the latter amino acid sequence mutant in which NLS2 was deleted does not translocate to the nucleus but remains in the cytoplasm (FIG. 4E). HET-A-
It can be said that NLS2 is the only nuclear translocation signal in the first half.

Further, both nuclear transport signals HET-A-NLS1 and
To confirm that HET-A-NLS2 is the only nuclear translocation signal overall, use HET-A-NLS1 and HET-
A codon encoding an amino acid sequence in which A-NLS2 has been deleted is synthesized according to a standard method such as PCR, and the plasmid is synthesized.
By inserting pRcβG-1 into SpeI so that the translation frame matches, the fusion protein with β-galactosidase can be expressed. When the plasmid thus prepared is transfected into cultured cells, the amino acid sequence mutant lacking both nuclear translocation signals HET-A-NLS1 and HET-A-NLS2 does not translocate to the nucleus but remains in the cytoplasm This becomes apparent (FIG. 4F). It can be said that HET-A-NLS1 and HET-A-NLS2 are the only nuclear translocation signals.

Analysis of changes in cells expressing a HET-A mutant having an amino acid sequence lacking both nuclear translocation signals can be expected to discover a new function of HET-A. Also this
The discovery of new functions of HET-A can be expected by preparing HET-A mutant proteins in Escherichia coli using standard DNA techniques and conducting various analyzes such as DNA binding experiments and phosphorylation experiments.

(3) Method of introducing into the nucleus a protein which is never present in the nucleus by using the nuclear localization signal The codon of the identified nuclear localization signal HET-A-NLS2, any transcription promoter portion, and the normal nuclear By preparing a plasmid having an arbitrary gene portion of a protein that is never present, and transfecting this into cultured cells, a gene product that is not originally present in the nucleus can be localized in the nucleus.

As a means for synthesizing a plasmid, for example, a codon of a nuclear localization signal (HET-A-NLS2) as shown in FIG.
A fusion protein with β-galactosidase can be expressed by inserting pRcβG-1 so that the translation frame matches with SpeI. By transfecting the plasmid thus prepared into cultured cells, any gene product that is not originally present in the nucleus, such as β-galactosidase, can be localized in the nucleus (FIG. 4 HE
TA-NLS2).

The method for preserving the expression plasmid in a microorganism may be the same as the method for storing a general plasmid or vector in a microorganism. The microorganism to be used may be any microorganism, such as Escherichia coli or Pseudomonas aeruginosa, as long as it can retain the plasmid. The cultivation of the microorganism having the expression plasmid may be the same as a general culturing method. The culture is stopped when the plasmid reaches its maximum in the cells. Plasmid DNA is extracted from the obtained cells, and this DNA can be transfected into cultured eukaryotic cells.

The transfection method may be a conventional method such as a calcium phosphate method, an electroporation method, a liposome method and the like. The cells to be transfected may be any eukaryotic organisms, such as vertebrates including humans, having nuclei. Depending on the technique, it can be confirmed in several hours to several tens of hours that the target protein is localized in the nucleus.

The method of confirming nuclear translocation can be carried out by a method using the activity of the target protein, or by an existing method such as an immunoantibody method using an antibody or an enzyme antibody method. For example, in the case of β-galactosidase, its enzyme activity is determined by the method of Sarnes (Sanes, JR et al. (1986)
According to EMBO J. 53133), it can be detected by using X-gal as a coloring agent.

[0042]

According to the present invention, it was revealed that HET-A translocated to the nucleus, and its translocation signal was identified. Therefore, the function of the protein in cells can be analyzed by using HET-A in which the nuclear transport signal has been modified or deleted. In addition, by binding this nuclear translocation signal, proteins that are physiologically expressed and function in places other than the nucleus or proteins of microorganisms that are not intrinsically related to the physiological functions of eukaryotes are introduced into the nucleus to transform cells. The change can be analyzed.

EXAMPLES The present invention will be further described below by way of examples.

(A) Fusion protein with β-galactosidase
Preparation of Plasmid pRcβG-1 for Expression of Plasmid In order to prepare a fusion vector, plasmid pRcβG-1 was prepared by the method shown in FIG.

(Preparation of pβG-1) Sequence of HindIII restriction enzyme
(AAGCTT), Kozak sequence for expression (AGCCCCGCC),
The methionine codon (ATG), the sequence of the restriction enzyme SpeI for introducing the fusion partner gene (ACTAGT), and the β-galactosidase gene {Kalnins, A. et al (1983) EMBO
J. 2 (4) 593-597}, a sense primer (5′-CCGAAGCTTAGCCCCGCCATGACTAGTGTCGTTTTACAAC-) bound to a DNA sequence encoding the 9th to 15th amino acid sequence.
3 ') was designed. Next, 15-galactosidase 15
An antisense primer (5'-ATCGT) was used to amplify the restriction enzyme FspI site existing around the 0th base.
AACCGTGCATCTGCC-3 ′) was designed. These primers are used in an automated DNA synthesizer (Model 394, Applied Biosyste
ms) and β-galactosidase DNA (pMC1
871) was used as a template to perform a PCR (Polymeraze chain reaction) reaction to prepare an N-terminal fragment of β-galactosidase modified so that a fusion protein could be easily prepared.

First, plasmid pMC1871 containing β-galactosidase {Malcolm J. et al Methods of Enzymology
(1983) 21 293-308}.
oning1.33-1.34,1.42-1.43 "(Cold Spring Houbor Labor
atory Press 1989). 1 μg of the obtained plasmid pMC1871 DNA was dissolved in 50 μl of TE buffer. To 5 μl of this cDNA solution, 100 pmole of each of the previously synthesized sense primer and antisense primer was added, and 10 μl of a 10-fold concentration amplification solution (500 mM KCl, 100 mM Tris
_{HCl pH8.3, 15mMMgCl 2, 0.1%} (W / V) gelatin) and
16 μl of dNTPs mix (1.25 mM dATP, 1.25 mM dGTP, 1.25 mM d
CTP, 1.25 mM dTTP), add water and add 0.5 μl
Of Taq DNA polymerase (5 units / μl, manufactured by Perkin-Elmer Cetus) was added to make the volume of the reaction solution 100 μl. This reaction solution was placed at 94 ° C. for 3 minutes to denature the cDNA. Further denature at 94 ° C for 1 minute,
For 1 minute to perform annealing, and then at 72 ° C for 2 minutes.
The reaction of allowing the reaction to elongate after standing for 15 minutes was performed 15 times.
Then, it was left at 72 ° C. for 2 minutes to complete the extension reaction. A total of 10 such PCR reactions were performed, and the reaction solution, ie, 1 ml of the reaction product, was collected in a micro desalting tube "Suprec-
02 "(Takara Shuzo) and desalted and concentrated.

The PCR product remaining on the membrane of the tube was recovered with 20 μl of TE buffer. The recovered PCR product was mixed with 500 μl of restriction enzyme HindIII-FspI buffer (10 mM Tris-H
Cl, 10 mM MgCl ₂ , 50 mM NaCl, 1 mM DTT, pH 7.9)
The cells were treated with 100 units of HindIII-FspI at 37 ° C. for 2 hours. The reaction product was separated by 0.7% agarose electrophoresis in a 0.5 × TBE (meaning, 0.5-fold concentration of TBE, hereinafter the same) buffer solution to obtain a target band of about 200 bp. Therefore, the HindIII-FspI band containing the N-terminus of this β-galactosidase was cut out, and “Molecular Cloning
6.28-6.29 "(Cold Spring Houbor Laboratory Press 19
The DNA was recovered by electroelution in 0.5 × TBE buffer according to the method described in 89). This gives approximately 10 μg of DNA
Fragments were obtained and dissolved in 500 μl of TE buffer.

Next, 50 μg of the plasmid pMC1871 DNA was added to 500
μl of restriction enzyme FspI buffer (20 mM Tris-acetate, 10 mM
Mg-acetate, 50mM K-acetate, 1mM DTT, 100μg / ml BS
A, pH7.9) and treated with 100 units of restriction enzyme FspI at 37 ° C for 2 hours. The reaction solution was extracted by a phenol method, and DNA was precipitated by an ethanol precipitation method. This DNA was then added to 500 μl of restriction enzyme SalI buffer (150 mM NaCl, 10
mM Tris-HCl, 10 mM MgCl ₂ , 1 mM DTT, 100 μg / ml BSA, p
In H7.9), the cells were treated with 100 units of restriction enzyme SalI at 37 ° C. for 2 hours. The reaction product was added to 0.5% TBE buffer at 0.7%
Separated by agarose gel electrophoresis, the FspI-SalI band containing the C-terminus of β-galactosidase was cut out, and “Molec
ular Cloning 6.28-6.29 "(Cold Spring Houbor Laborat
ory Press 1989), and DNA was recovered by electroelution in 0.5 × TBE buffer. As a result, about 20 μg of a DNA fragment was obtained and dissolved in 200 μl of a TE buffer.

Separately, 5 μg of the plasmid vector pUC18 was added to 50 μl of a restriction enzyme HindIII buffer (10 mM Trial).
s-HCl, 10mM MgCl ₂ , 50mM NaCl, 1mM DTT, pH7.9)
The cells were treated with 50 units of the restriction enzyme HindIII at 37 ° C for 2 hours.
The reaction solution was extracted by a phenol method, and DNA was precipitated by an ethanol precipitation method. This DNA is then
l of restriction enzyme SalI buffer (150 mM NaCl, 10 mM Tris-HCl,
10mM MgCl ₂ , 1mM DTT, 100μg / ml BSA, pH7.9), 100
The cells were treated with units of the restriction enzyme SalI at 37 ° C. for 2 hours. The reaction product was separated by 0.7% agarose electrophoresis in 0.5 × TBE buffer, and the band of the plasmid vector pUC18 digested with HindIII-SalI was cut out.
6.28-6.29 "(Cold Spring Houbor Laboratory Press 19
According to the method described in 89), the DNA was recovered by electroelution in 0.5 × TBE buffer. This results in approximately 4 μg of DNA
Fragments were obtained and dissolved in 40 μl of TE buffer.

It contains 2 μl of this restriction enzyme HindIII-SalI digested plasmid vector pUC18, and a HindIII-FspI band containing the N-terminal of the aforementioned β-galactosidase and a C-terminal.
2 μl of each FspI-SalI band was added to 20 μl of T4 ligase buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP,
By treating with 10 units of T4 DNA ligase at 25 ° C. for 15 hours in 25 μg / ml BSA, pH 7.5) for 15 hours at the vector and HindIII-FspI band containing the N-terminus of β-galactosidase and FspI-containing C-terminus. The SalI band was ligated.
The resulting recombinant plasmid was named pβG-1.

The base sequence of the double-stranded cDNA of the obtained plasmid was determined based on the dideoxy method using an automatic sequencer (ABI 377 type). As the primer, a primer specific to the adjacent pUC18 vector region (RV-M or M13-47 manufactured by Takara Shuzo) or a primer prepared based on a known β-galactosidase sequence was used. As a result, it was confirmed that there was no mutation in the β-galactosidase base sequence, and that the sequence of the modified portion was correct.

(Preparation of pRcβG-1) Escherichia coli having the plasmid pβG-1 was purified by using “Molecular Cloning 1.33-1.34, 1.42-
This plasmid was prepared in large quantities according to the method of 1.43 "(Cold Spring Houbor Laboratory Press 1989).
50 μg of βG-1 was added to 500 μl of a restriction enzyme HindIII-XbaI buffer (10 mM Tris-HCl, 10 mM MgCl ₂ , 50 mM NaCl, 1 mM DTT,
50 units of restriction enzyme H in 100 μg / ml BSA, pH7.9)
The cells were treated with indIII-XbaI at 37 ° C. for 2 hours. The reaction product was separated by 0.7% agarose electrophoresis in 0.5 × TBE buffer, and HindIII-Xb containing β-galactosidase was isolated.
Cut off the aI band and "Molecular Cloning 6.28-6.29"
According to the method described in (Cold Spring Houbor Laboratory Press 1989), the modified β-galactosidase-containing DNA was recovered by electroelution in 0.5 × TBE buffer. As a result, about 20 μg of a DNA fragment was obtained and dissolved in 200 μl of a TE buffer.

Separately, the plasmid vector pRc / RS
The E. coli harboring V {WC Lin et al (1991) BioTechniques 11 344} was isolated from "Molecular Cloning 1.33-1.34, 1.42-1.
43 "(Cold Spring Houbor Laboratory Press 1989). Large amounts were prepared of 5 µg of this plasmid vector in 50 µl of restriction enzyme HindIII-XbaI buffer with 50 units of HindIII-XbaI restriction enzyme at 37 ° C for 2 hours. The reaction product was placed in 0.5 × TBE buffer at 0.7
% Agarose electrophoresis, cut the band of plasmid vector pRc / RSV digested with HindIII-XbaI, and cut it out with "Molecular Cloning 6.28-6.29" (Cold Spring Houb
or Laboratory Press 1989).
DNA was recovered by electroelution in 5 × TBE buffer.
As a result, about 4 μg of a DNA fragment was obtained and dissolved in 40 μl of a TE buffer.

20 μl of 2 μl of this restriction enzyme HindIII-XbaI digested plasmid vector pRc / RSV and 2 μl of the HindIII-XbaI band containing the modified β-galactosidase
The vector and β were treated with 10 units of T4 DNA ligase at 15 ° C for 15 hours in T4 ligase buffer.
-The HindIII-SalI band containing galactosidase was bound. The resulting recombinant plasmid was named pRcβG-1. (Fig. 2) (B) Introduction of hHDGF gene into pRcβG-1 The full length of HET-A gene was expressed in S of plasmid pRcβG-1 for expression.
It was introduced into the peI site as follows.

(Preparation of pA) The restriction enzyme SpeI was added to the 5 'end of the 843 base pair DNA sequence corresponding to the 283rd leucine from the 3rd cysteine to the amino acid sequence of HET-A to be introduced.
(ACTAGT) and the XbaI sequence (TCTAGA) at the 3 'end. For this purpose, this region is designated by PCR (Polymeraze cha
sense primer (5'-G
CACTAGTTGCTTCAGCCGCTCTAAGTA-3 ′) and antisense primer (5′-GCTCTAGACAGGCCTTCTATTTCTCCAAC-3 ′) were designed.

These primers were synthesized using an automatic DNA synthesizer (Model 394, manufactured by Applied Biosystems).
A PCR reaction was performed using HET-A cDNA (Japanese Patent Application No. 8-131788) as a template. The plasmid containing 1 μg of HET-A cDNA was dissolved in 50 μl of TE buffer. To 5 μl of this cDNA solution, 100 pmole of each of the synthesized sense primer and antisense primer was added, and 10 μl of a 10-fold amplification solution (500 mM KCl, 100 mM Tris HCl) was added.
pH 8.3, 15mM MgCl ₂ , 0.1% (W / V) gelatin) and 16
μl of dNTPs mix (1.25 mM dATP, 1.25 mM dGTP, 1.25 mM d
CTP, 1.25 mM dTTP), add water and add 0.5 μl
Of Taq DNA polymerase (5 units / μl, manufactured by Perkin Elmer Cetus) was added to make the volume of the reaction solution 100 μl. This reaction solution was placed at 94 ° C. for 3 minutes to denature the cDNA. Furthermore, denaturation was carried out by leaving the mixture at 94 ° C. for 1 minute, annealing was carried out at 50 ° C. for 1 minute, and then extension reaction was carried out at 72 ° C. for 2 minutes to carry out 15 reactions.
Then, it was left at 72 ° C. for 2 minutes to complete the extension reaction.
This PCR reaction was performed 10 times in total, and the reaction solution, ie, 1 ml of the reaction product, was added to a micro desalting tube "Suprec-0".
Desalted and concentrated by 2 "(Takara Shuzo).

The reaction product remaining on the membrane of the tube was recovered with 20 μl of TE buffer. 500 collected PCR products
μl of restriction enzyme SpeI-XbaI buffer (10 mM Tris-HCl, 10 mM
M MgCl ₂ , 50 mM NaCl, 1 mM DTT, 100 μg / ml BSA, pH 7.
9) Inside, 37 ° C with 100 units of restriction enzyme SpeI-XbaI
Time processed. This reaction product is placed in 0.5 × TBE buffer,
Separation by 0.7% agarose electrophoresis yielded a target band of about 843 bp. So this HET-A
Cut out the SpeI-XbaI band containing the full length of "Molecula
r Cloning 6.28-6.29 "(Cold Spring Houbor Laborator
y Press 1989), and HET-A DNA was recovered by electroelution in 0.5 × TBE buffer. As a result, about 10 μg of a DNA fragment was obtained and dissolved in 100 μl of a TE buffer.

Separately, 5 μg of the plasmid vector pBluescriptII SK (+) was replaced with 50 μl of the restriction enzyme SpeI-XbaI.
37 ° C, 50 units of restriction enzyme SpeI-XbaI in buffer
Time processed. The reaction product was separated by 0.7% agarose electrophoresis in 0.5 × TBE buffer, and SpeI-Xba
Cut out the band of the plasmid vector pBluescriptII SK (+) digested with I, and remove the "Molecular Cloning 6.28-6.29"
DNA was recovered by electroelution in 0.5 × TBE buffer according to the method described in (Cold Spring Houbor Laboratory Press 1989). As a result, about 4 μg of a DNA fragment was obtained and dissolved in 40 μl of a TE buffer.

2 μl of this restriction enzyme SpeI-XbaI digested plasmid vector pBluescriptII SK (+) was added to the SpeI-XbaI
2 μl of the full-length cDNA fragment of HET-A digested with
The vector and the HET-A full-length DNA were ligated by treating at 15 ° C. for 15 hours with 10 units of T4 DNA ligase in a DNA ligase buffer. The resulting recombinant plasmid was named pA (Figure 3A).
The base sequence of the double-stranded cDNA was determined based on the dideoxy method using an automatic sequencer (ABI type 377). As a primer, the adjacent pBluescript II SK
(+) Vector specific primer (SK or K
S Toyobo) or a primer prepared based on the sequence of HET-A known in advance. As a result, it was confirmed that there was no mutation in the nucleotide sequence of HET-A, and that the sequence of the modified portion was also correct.

(Preparation of pRcβG-A) Recombinant plasmid pA
E. coli containing "Molecular Cloning 1.33-1.34, 1.
42-1.43 "(Cold Spring Houbor Laboratory Press 198
It was prepared in large quantities according to the method of 9). 50 μg of this plasmid pA was treated with 50 units of each restriction enzyme SpeI-XbaII in 500 μl of restriction enzyme SpeI-XbaII buffer at 37 ° C. for 2 hours. This reaction product is placed in 0.5 × TBE buffer,
Separated by 0.7% agarose electrophoresis, cut out the SpeI-XbaI band containing the full length of HET-A,
ing 6.28-6.29 "(Cold Spring Houbor Laboratory Press
1989), and HET-A DNA was recovered by electroelution in 0.5 × TBE buffer. This makes about 8
μg of DNA fragment was obtained and dissolved in 200 μl of TE buffer.

Separately, 5 μg of the expression plasmid vector pRcβG-1 was added to 50 μl of the restriction enzyme SpeI buffer.
The mixture was treated with 50 units of restriction enzyme SpeI (Toyobo Co., Ltd.) at 37 ° C. for 2 hours, and phenol extraction, chloroform extraction and ethanol precipitation were performed in a conventional manner to recover DNA, which was then subjected to 40 μl.
dissolved in 1 TE buffer.

2 μl of the expression plasmid vector pRcβG-1 digested with SpeI and 2 μl of the SpeI-XbaI band containing the full length of HET-A were added to 20 μl of T4 DNA ligase buffer in 10 units of T4 DNA ligase buffer. Lighace, 15
The vector and the full length of HET-A were ligated by treating at 15 ° C. for 15 hours. The resulting recombinant plasmid was pRcβG
Named -A. pRcβG-A has a high-efficiency promoter called the RSV LTR as a drive unit for transcription, and converts the HET-A cDNA integrated downstream into COS-7
It can be expressed using a cell or the like as a host.

(C) Transfection into COS-7 Cells The method of Chen-Okayama (Chen, C. and
According to Okayama, H. (1987) Mol. Cell. Biol. 72745-2752), a transformant of COS-7 cells was prepared using the plasmid pRcβG-A as follows.

COS-7 cells in the logarithmic growth phase were trypsinized by a conventional method, and contained 10 ml of 10% calf serum (Flow Laboratories) and 60 μg / ml kanamycin (Meiji Seika) in a 10 cm diameter plate. DME (Dulbecco´s modified Eagl
e´s) In the medium, sow 5 × 10 ⁵ cells, 37 ℃, 5
The cells were cultured overnight in CO 2%. 28 μg of plasmid DNA
(In 15 μl TE buffer) with 0.5 ml of 0.25 M CaCl ₂ and 0.5 ml of 2 × BBS (50 mM BES (N, N-bis [2-hydroxye
thyl] -2-aminoethanesulfonic acid) / 280mM NaCl / 1.5mM
Na ₂ HPO ₄ , pH 6.95} and left at room temperature for 20 minutes.
This solution was added dropwise to the medium, and the plate was spread well, and then cultured at 37 ° C. for 16 hours in a 5% carbon dioxide gas concentration. Thereafter, the medium was removed, and the COS-7 cells were replaced with 10 ml of PBS.
Wash twice with buffer, in DME medium containing 10% calf serum, 60 μg / ml kanamycin, in 5% gas carbonate, at 37 ° C.
The culture was continued overnight under the following conditions. The cells were trypsinized in a conventional manner, diluted by a factor of 4 and diluted with 10% calf serum, 60%.
The cells were cultured in DME containing μg / ml of kanamycin at a concentration of 5% carbon dioxide at 37 ° C. for 48 hours. The transformant thus obtained was named COS (pRcβG-A).

(D) Measurement of β-galactosidase activity and conclusion
To determine fruit fusion protein where to find a method of Sanesu {Sanes, JRet al. (1986 ) EMBO J. 5 3133}
The cells were stained with X-gal as a coloring agent. In this method, the portion where the fusion protein exists has a dark blue color.

Transformant CO on Petri dish or flask
After washing S (pRcβG-A) cells with phosphate buffer (PBS), fixative (2% (V / V) formaldehyde, 0.2% (V / V)
glutaraldehyde, but dissolved in PBS) for 5 minutes at room temperature. After washing twice with PBS, the staining solution (5 mM K fe
rricyanide, 5mM ferrocyanide, 2mM MgCl ₂ , 1mg / mlX-
gal but dissolved in PBS) for at least 2 hours. After washing with PBS, 10% formalin solution (10% f
ormaldehyde (dissolved in PBS) for 10 minutes at room temperature, washed with PBS and observed with a microscope. Samples were stored at 4 ° C.

As a result, as shown in FIG. 4A, it was revealed that the fusion protein was localized in the nucleus. Ie HE
TA has been proven to translocate to the nucleus.

(Example 2) Identification of nuclear translocation signal present on HET-A (A) Preparation of split HDGF mutant gene In order to identify the nuclear translocation signal, a part of the HET-A gene was analyzed. HE with various deleted amino acids as shown in Fig. 3
A variant of TA was created.

First, the entire HET-A was divided into the first half and the second half. That is, in FIG. 3B, the restriction enzyme SpeI sequence (ACTAG) was added to the 5 ′ end of the 516 base pair DNA sequence corresponding to the cysteine from the third cysteine to the 174th histidine in the amino acid sequence of HET-A.
T) was ligated with an XbaI sequence (TCTAGA) at the 3 ′ end.
For this purpose, this region is called PCR (Polymeraze chain reaction)
Sense primer for amplification with (5'-GCACTAGTTGCTT
CAGCCGCTCTAAGTA-3 ') and antisense primer (5'-G
CTCTAGAATGCTCGGCTTGCTGATCTC-3 ′) was designed.

FIG. 3C shows the restriction enzyme SpeI sequence (ACTAGT) at the 5 'end of the 318 base pair DNA sequence corresponding to the 175th valine to the 283rd leucine in the amino acid sequence of HET-A.
Was ligated to the 3 'end with an XbaI sequence (TCTAGA). Therefore, a sense primer (5′-GCACTAGTGTGCAA) for amplifying this region by PCR (Polymeraze chain reaction)
GAGAAGCACCCTGA-3 ') and antisense primer (5'-G
CACTAGTCAGGCCTTCTATTTCTCCAAC-3 ′) was designed.

The primers of each set were synthesized using an automatic DNA synthesizer (Model 394, manufactured by Applied Biosystems), and a PCR reaction was carried out in the same manner as in Example 1 using HET-A cDNA as a template. The recovered PCR product was treated with the plasmid vector pBluescript II S in the same manner as in Example 1.
It was introduced into the SpeI site of K (+), and the obtained recombinant plasmids were named pB and pC, respectively. After confirming that there was no mutation in the nucleotide sequence, this was introduced into the expression plasmid vector pRcβG-1 in the same manner as in Example 1.
The obtained recombinant plasmids were named pRcβG-B and pRcβG-C.

(B) Expression in COS-7 Cells and Results pRcβG-B and pRcβG-C were converted to CO2 in the same manner as in Example 1.
S-7 cells were expressed as a host. As a result, it became clear that both fusion proteins were localized in the nucleus as shown in FIGS. 4B and 4C. That is, it was proved that both the first half and the second half of HET-A had at least one nuclear translocation signal. A homology search of the amino acid sequence of HET-A against a database, for example, EMBL, Genbank, etc., revealed that two nuclear translocation signals, HET-A-NLS1
Can be found in the first half amino acid sequence and HET-A-NLS2 in the second half amino acid sequence (FIG. 1). HET-A-
NLS1 has the characteristics of a type motif containing a large number of basic amino acids, and HET-A-NLS2 has the characteristics of bipartite type I, but their sequences are novel.

(C) HDGF Mutation Deleting Nuclear Localization Signal
Creation Figure 3 D body gene, E, F but is mutant obtained by deletion of nuclear localization signal, to create these is little necessary to devise.

(Preparation of pG) In order to remove the nuclear localization signal HET-A-NTS1, which exists at the 68th to 73rd positions of the amino acid sequence of HET-A, the 3rd to 67th positions and the 75th to 283rd positions of the amino acid sequence were removed. PCR sequence of the DNA sequence corresponding to the restriction enzyme NarI (GGCGCC)
Was interposed. First, the sequence of the restriction enzyme SpeI at the 5 'end of the DNA sequence corresponding to the third to 67th amino acid sequence
(ACTAGT) was ligated with a NarI sequence (GGCGCC) at the 3 'end. For this purpose, this region is used for PCR (Polymeraze chain rea
ction) sense primer (5'-GCACTAG)
TTGCTTCAGCCGCTCTAAGTA-3 ') and antisense primer
(5'-AATGGCGCCGAACTTCTCTTTGGACT-3 ') was designed. Next corresponds to positions 75 to 283 of the amino acid sequence.
The restriction enzyme NarI sequence (GGCGCC) at the 5 'end of the DNA sequence
Was ligated to the 3 'end with an XbaI sequence (TCTAGA). Therefore, a sense primer (5'-AATGGCGCCTTCAGCGAGGGGCTGTGGGA-3 ') and an antisense primer (5'-GCTCTAGACAGGCCTTCTATTTCTCCAAC) for amplifying this region by PCR
-3 ').

The primers of each set described above were synthesized by an automatic DNA synthesizer (Model 394, manufactured by Applied Biosystems), and a PCR reaction was carried out in the same manner as in Example 1 using HET-A cDNA as a template. The two kinds of the collected PCR products were introduced into the SpeI site of the plasmid vector pBluescriptII SK (+) in the same manner as in the case where the plasmid pβG-1 was prepared in Example 1, and the recombinant plasmid pG was obtained.

(Preparation of pH) 211 of the amino acid sequence of HET-A
The nuclear transport signal HET-A-NTS2 from the 227th to the 227th
DNA sequences corresponding to amino acids 3 to 209 and 228 to 283 of the amino acid sequence
R Restriction enzyme XhoI (CTCGAG) to amplify and connect both sequences
Sequence was interposed. First from the third amino acid sequence to 21
The restriction enzyme SpeI is located at the 5 'end of the 0th DNA sequence.
(ACTAGT) and the XhoI sequence (CTCGAG) at the 3 'end. For this purpose, this region is designated by PCR (Polymeraze cha
sense primer (5'-
GCACTAGTTGCTTCAGCCGCTCTAAGTA-3 ') and antisense primer (5'-CCGCTCGAGACTCCCCGGCTCCTCCA-3') were designed. Next, at the 5 'end of the DNA sequence corresponding to positions 228 to 283 of the amino acid sequence, the sequence of the restriction enzyme XhoI was added.
(CTCGAG) was ligated with an XbaI sequence (TCTAGA) at the 3 ′ end. Therefore, a sense primer (5'-CCTCTCGAGGCGGCTCCTGGTGCGTT-3 ') and an antisense primer (5'-GCACTAGTCAGGCCTTCTATTTC) for amplifying this region by PCR
TCCAAC-3´).

The primers of each set described above were synthesized by an automatic DNA synthesizer (Model 394, manufactured by Applied Biosystems), and a PCR reaction was carried out in the same manner as in Example 1 using HET-A cDNA as a template. The two kinds of the collected PCR products were introduced into the SpeI site of the plasmid vector pBluescriptII SK (+) in the same manner as in the case where the plasmid pβG-1 was prepared in Example 1, and the pH of the recombinant plasmid was obtained.

(Preparation of pD and pRcβG-D) pD is the pG Eco
It was prepared by replacing the RI fragment with the EcoRI fragment of pB.

The Escherichia coli carrying the plasmid pG was called "Molecu
lar Cloning 1.33-1.34, 1.42-1.43 "in a large amount. 50 μg of the obtained plasmid pG
With 500 μl of restriction enzyme EcoRI buffer (50 mM NaCl, 100 mM
M Tris-HCl, 10mM MgCl ₂ , 0.025% TritonX-100, pH 7.
In 5), the cells were treated with 50 units of restriction enzyme EcoRI at 37 ° C for 2 hours. The reaction product was placed in 0.5 × TBE buffer at 0.7
Separation by% agarose electrophoresis and cutting out the EcoI band containing the first half of HET-A lacking the nuclear translocation signal,
Molecular Cloning 6.28-6.29 "(Cold Spring Houbor L
aboratory Press 1989).
DNA was recovered by electroelution in BE buffer. As a result, about 5 μg of a DNA fragment was obtained and dissolved in 100 μl of TE buffer.

Separately, 5 μg of plasmid pB was
50 units of restriction enzyme Ec in 0 μl EcoRI buffer
Treated with oRI at 37 ° C for 2 hours. 0.5 g of this reaction product
Separation was performed by 0.7% agarose electrophoresis in × TBE buffer, and the EcoI band in the vector portion was cut out.
r Cloning 6.28-6.29 "(Cold Spring Houbor Laborato
DNA was recovered by electroelution in 0.5 × TBE buffer according to the method described in ry Press 1989). This allows
3 μg of the DNA fragment was obtained and dissolved in 30 μl of TE buffer.

2 μl of this restriction enzyme EcoRI digested plasmid vector pB and E. coli containing a portion lacking a nuclear localization signal
Both were bound by treating 2 μl of the coRI band in 20 μl of T4 ligase buffer with 10 units of T4 ligase at 15 ° C. for 15 hours. The resulting recombinant plasmid was named pD.

After confirming that the DNA fragment cloned in pD had no mutation in the nucleotide sequence, it was introduced into the expression plasmid vector pRcβG-1 in the same manner as in Example 1. The resulting recombinant plasmid was named pRcβG-D. (Preparation of pE and pRcβG-E) pE was prepared by replacing the Bsu36I-XbaI fragment of pF with the Bsu36I-XbaI fragment of pC.

The method of construction is the same as for pD, ie
2μl of the Bsu36I-XbaI band containing the HET-A part lacking the nuclear translocation signal extracted from pF and the restriction enzyme Bsu36I-XbaI
2 μl of the plasmid pC digested with the above was treated with 10 units of T4 ligase in 20 μl of T4 ligase buffer at 15 ° C. for 15 hours to bind them. The resulting recombinant plasmid was named pE.

After confirming that the DNA fragment cloned in pE had no mutation in the nucleotide sequence, it was introduced into the expression plasmid vector pRcβG-1 in the same manner as in Example 1. The resulting recombinant plasmid was named pRcβG-E.

(Preparation of pF and pRcβG-F) pF is the pG Eco
It was prepared by replacing the RI fragment with the EcoRI fragment at pH.

The preparation method is the same as that for pD and pE, that is, 20 μl of 2 μl of the EcoRI band containing the portion lacking the nuclear translocation signal extracted from pG and 2 μl of the plasmid vector pH digested with the restriction enzyme EcoRI. 10 units of T4 DNA ligase in 15 T4 DNA ligase buffer
Both were bound by treating at 15 ° C. for 15 hours. The resulting recombinant plasmid was named pF.

After confirming that the DNA fragment cloned in pF had no mutation in the nucleotide sequence, it was introduced into the expression plasmid vector pRcβG-1 in the same manner as in Example 1. The resulting recombinant plasmid was named pRcβG-F.

(D) Expression in COS-7 cells The resulting recombinant plasmids were pRcβG-D, pRcβG-E, pRc
cβG-F was expressed using COS-7 cells as a host in the same manner as in Example 1. As a result, it was revealed that all the fusion proteins were localized in the cytoplasm as shown in FIGS. 4D, 4E, and 4F.

That is, the fact that the amino acid sequence variant in the first half in which the nuclear transport signal HET-A-NLS1 is deleted does not translocate to the nucleus but remains in the cytoplasm means that HET-A-NLS1 is the only nuclear transport signal in the first half. be able to. Similarly HET-A-NL
The fact that the latter amino acid sequence mutant lacking S2 remains in the cytoplasm without translocating to the nucleus indicates that HET-A-NLS2 is the only nuclear translocation signal in the latter half.

Further, nuclear transport signals HET-A-NLS1 and HE
That the amino acid sequence mutant lacking TA-NLS2 does not translocate to the nucleus but remains in the cytoplasm means that HET-A-NLS1 and HE
It can be said that TA-NLS2 is the only nuclear translocation signal.

(Example 3) Method for introducing a protein which is never present in the nucleus into the nucleus using the nuclear translocation signal HET-A-NLS2 (A) Expression in which HET-A-NLS2 and β-galactosidase are fused
Construction of plasmid pKI-5 51 bp DNA corresponding to the nuclear localization signal HET-A-NTS2, which exists at positions 211 to 227 of the amino acid sequence of HET-A
The sequence of the restriction enzyme SpeI (ACTAGT) was
An XbaI sequence (TCTAGA) was ligated to the terminal side. for that reason,
To amplify this region by PCR (Polymeraze chain reaction), use a sense primer (5'-GCACTAGTAAGAGGAGCGCGGAG).
GAT-3 ') and antisense primer (5'-GCTCTAGACCTGG
GCCGTTTGAGAGGACAA-3 ′) was designed.

The above primers were synthesized using an automatic DNA synthesizer (Model 394, manufactured by Applied Biosystems) and
PCR using TA cDNA as template in the same manner as in Example 1.
The reaction was performed. The recovered PCR product was introduced into the SpeI site of the plasmid vector pRcβG-1 in the same manner as in Example 1, and each of the obtained recombinant plasmids was named pKI-5.

(B) Expression in COS-7 Cells pKI-5 was expressed using COS-7 cells as a host in the same manner as in Example 1. As a result, it was revealed that the fusion protein was localized in the nucleus as shown in FIG. 4 HET-A-NLS2. This indicates that the nuclear transport signal HET-A-NTS2 was able to localize β-galactosidase, which is not originally present in the nucleus, to the nucleus.

[0094]

[Sequence list]

 Sequence number (SEQ ID NO): 1 Sequence length (SEQUENCE LENGTH): 783 Sequence type (SEQUENCE TYPE): Nucleic acid (Nucleic acid) Number of chains (STRANDEDNESS): Double topology (TOPOLOGY): Linear antisense (ANTI-SENCE): No Origin (ORIGINAL SOURCE): Mouse BAL B / C Sequence characteristics (FEATURE) (A) Character code indicating characteristics: CDS (B) Location: 1 to 783 Sequence (SEQUENCE DESCRIPTION) Met Ser Cys Phe Ser Arg Ser Lys Tyr Lys Thr Gly Asp Leu Val ATG TCT TGC TTC AGC CGC TCT AAG TAC AAG ACC GGG GAC CTG GTG 5 10 15 Phe Ala Lys Leu Lys Gly Tyr Ala His Trp Pro Ala Arg Ile Glu TTT GCC AAG TTA AAG GGC TAT GCC CAC TGG CCA GCG AGG ATC GAA 20 25 30 His Val Ala Glu Ala Asn Arg Tyr Gln Val Phe Phe Phe Gly Thr CAT GTC GCT GAA GCC AAC CGC TAC CAG GTG TTC TTC TTC GGG ACC 35 40 45 His Glu Thr Ala Leu Leu Gly Pro Arg His Leu Phe Pro Tyr Glu CAT GAG ACC GCC CTG CTG GGT CCC AGG CAC CTG TTC CCT TAT GAG 50 55 60 Glu Ser Lys Glu Lys Phe Gly Ala Phe Ser Glu Gly Leu Trp Glu GAG TCC AAA G AG AAG TTC GGC GCC TTC AGC GAG GGG CTG TGG GAG 65 70 75 Ile Glu His Asp Pro Met Val Glu Ala Ser Ser Ser Leu Cys Ser ATG GTT GAG GCC TCC TCT AGC CTG TGC TCA GAA GAG GAT CAG AGC 80 85 90 Pro Glu Leu Gly Gln Glu Leu Val Gln Glu Leu Glu Pro Glu Phe TAC ACA GAG GAT CCT ATC GAG CAC GAC CCT GGG CTA GCA GAG GAG 95 100 105 Glu Glu Asp Gln Ser Tyr Thr Glu Asp Pro Gly Leu Ala Glu Glu CCG GAA CTA GGA CAG GAG CTG GTG CAG GAG CTG GAG CCC GAA TTC 110 115 120 Met Pro Glu Leu Glu Ala Glu Pro Glu Met Pro Glu Thr Glu Cys ATG CCT GAG CTG GAG GCA GAA CCT GAG ATG CCT GAG ACC GAG TGT 125 130 135 Glu Gln Glu Pro Glu Pro Gln Pro Ala Tyr Asp Leu Leu Asp Ala GAA CAG GAG CCC GAG CCC CAG CCT GCC TAT GAC CTG CTG GAC GCC 140 145 150 Val Glu Glu Pro Gly Leu Thr Lys Ala Glu Pro Gly Asp Gln Gln GTG GAG GAG CCG GGC CTC ACC AAG GCC GAG CCA GGA GAT CAG CAA 155 160 165 Ala Glu His Val Gln Glu Lys His Pro Glu Val Glu Ala Glu Ala GCC GAG CAT GTG CAA GAG AAG CAC CCT GAG GTG GAG GCA GAG GCT 170 175 180 Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala Lys GAG GCT GAG GCT GAG GCC GAG GCA GAG GCG GAG GCG GAG GCC AAG 185 190 950 Ala Glu Val Glu Glu Pro Gly Ser Leu Glu Ala Ala Pro Gly Ala GCT GAG GTG GAG GAG CCG GGG AGT CTC GAG GCG GCT CCT GGT GCG 200 205 210 Leu Glu Met Glu Pro Ala Glu Glu Arg Glu Ala Glu Ala Ser Pro TTG GAG ATG GAG CCT GCT GAA GAG CGC GAG GCT GAG GCC TCC CCC 215 220 225 Phe Val Glu Glu Pro Asp Gln Ala Gln Glu Gln Leu Pro Pro Leu TTC GTG GAG GAG CCT GAC CAA GCC CAG GAG CAG CTG CCT CCG TTG 230 235 240 Glu Glu Glu Ala Thr Glu Lys Ala Val Gln Gly Leu Ile Val Gly GAG GAA GAG GCC ACA GAA AAG GCG GTC CAG GGC CTG ATT GTT GGA 245 250 255 Glu Ile Glu Gly Leu *** GAA ATA GAA GGC CTG TAG 260

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of the nuclear translocation signal present in HET-A and the DNA sequence encoding it, ie, (A) nuclear translocation signal HET-A-NLS1, and (B) nuclear translocation signal HET-
Shows A-NLS2.

FIG. 2. Modification of β-galactosidase gene,
4 is a chart showing a plasmid pβG-1 which can easily fuse a gene such as TA.

FIG. 3 is a chart showing various HET-A mutants created by deleting a part of the HET-A gene in order to identify a nuclear localization signal. NT (Nuclear transfer) indicates that the sample is transferred to the nucleus in the case of ○, and indicates that it is not transferred in the case of ×.

FIG. 4 is a micrograph of cells. A: After transfecting COS-7 cells with an expression plasmid pRcβG-A obtained by fusing HET-A and β-galactosidase genes, the enzyme activity of β-galactosidase was determined by the method of Surness to develop X-gal. It was detected by using it as an agent. B, C, D, E, F: Expression plasmid pRc in which various mutants of HET-A are fused with β-galactosidase gene
After transfecting βG-B to pRcβG-F into COS-7 cells, the enzyme activity of β-galactosidase was
It was detected by using X-gal as a color former according to the method of Surness. HET-A-NLS2: nuclear localization signals HET-A-NLS2 and β
After transfection of the expression plasmid pKI-5 fused with the galactosidase gene into COS-7 cells, the enzyme activity of β-galactosidase is detected by the method of Surness using X-gal as a color former. It was done.

Claims (10)

[Claims] 1. It has the amino acid sequence shown in FIG. 1A,
Nuclear translocation signal peptide (HET-A-NLS) of protein (HET-A) with local homology to human hepatoma cell-derived growth factor
1). 2. It has the amino acid sequence shown in FIG. 1B,
Nuclear translocation signal peptide (HET-A-NLS) of protein (HET-A) with local homology to human hepatoma cell-derived growth factor
2). 3. A DNA sequence encoding the nuclear localization signal peptide according to claim 1 (HET-A-NLS1). 4. A DNA sequence encoding the nuclear localization signal peptide according to claim 2 (HET-A-NLS2). 5. A recombinant DNA molecule comprising the DNA base sequence according to claim 3 or 4 incorporated into a vector used in an Escherichia coli host / vector system. 6. A sequence listing wherein the nuclear localization signal peptide (HET-A-NLS1) according to claim 1 and the nuclear localization signal peptide (HET-A-NLS2) according to claim 2 are deleted so that they do not function. A variant of a protein (HET-A) having local homology to a human hepatoma cell-derived growth factor having the amino acid sequence shown in SEQ ID NO: 1. 7. A sequence listing wherein the nuclear localization signal peptide (HET-A-NLS1) according to claim 1 and the nuclear localization signal peptide (HET-A-NLS2) according to claim 2 have been deleted so as not to function. A variant of a protein (HET-A) having local homology to a human hepatoma cell-derived growth factor having the DNA sequence shown in SEQ ID NO: 1. 8. A sequence listing wherein the nuclear localization signal peptide (HET-A-NLS1) according to claim 1 and the nuclear localization signal peptide (HET-A-NLS2) according to claim 2 are deleted so that they do not function. A recombinant DNA molecule comprising the DNA base sequence of SEQ ID NO: 1 incorporated into a vector used in an E. coli host / vector system. 9. Escherichia coli transformed with the recombinant DNA molecule according to claim 5 or 8. 10. The recombinant DNA according to claim 5 or 8.
Animal cells transformed with the molecule.