JPH0851981A - Novel protein having cancer metastasis inhibitory activity and gene encoding the protein - Google Patents

Abstract

(57) [Summary] [Purpose] In order to be able to directly and highly efficiently produce a cancer metastasis-inhibiting protein, which was previously possible only by combining chemical synthesis and conjugation methods, by gene recombination techniques. The present invention provides a synthetic gene and a cancer metastasis inhibitory protein produced by using the synthetic gene. [Structure] A novel protein in which a peptide having cancer metastasis inhibitory activity is bound to the carboxyl terminal (C-terminal) of serum albumin protein, and in particular, it is frequently used in Schizosaccharomyces pombe cells of the fission yeast Schizosaccharomyces pombe. A gene encoding the above protein designed and synthesized using the codon, and a method for producing the above protein by the gene recombination technique using the gene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein having a novel cancer metastasis inhibitory activity (hereinafter simply referred to as “cancer metastasis inhibitory protein”), a gene encoding the same, and a recombinant vector containing the gene. The present invention relates to a transformant of a host cell transformed with the recombinant vector, and a method for producing a cancer metastasis inhibitory protein using the transformant.

[0002]

BACKGROUND ART Surgical therapy, radiation therapy and chemotherapy are mainly used for the treatment of cancer, but no satisfactory therapeutic effect has been mentioned in terms of preventing recurrence and metastasis of cancer.

Many of the anticancer agents currently used inhibit the biosynthesis system of nucleic acid or protein and cause cancer cells to die. However, since it is difficult to distinguish between cancer cells and normal cells with these anticancer agents, there is a major problem inherent in their effects, especially in terms of side effects. In addition, these anticancer agents are treated by shrinking the primary tumor, but what is always a problem in the treatment of cancer is that the cancer cells leave the primary tumor and metastasize to other organs where they proliferate and are fatal. The result is. Therefore, for the fundamental treatment of cancer, it is desired to develop an anti-tumor agent that suppresses the growth of cancer cells and exhibits an effective suppressive effect on metastasis.

Many studies have been conducted to elucidate the mechanism of cancer metastasis, and the search for substances relating to the inhibition of metastasis has been widely conducted. Cancer cells invade blood vessels after being released from the primary tumor. Then, after adhering to the blood vessel wall, it dives under the vascular endothelial cell layer to destroy the extracellular matrix and infiltrate into the parenchyma of the target organ. It is thought that cancer cells metastasize to other organs through these steps (LA Liotta et al .: Lab.
Invest., 49, 636-649 (1983)). Therefore, in order to develop a cancer metastasis inhibitor, it is considered necessary to develop a drug that suppresses any of the above steps. For example, a substance that inhibits adhesion of cancer cells to extracellular matrix (for example,
NJ Humphries et al .: Science, 223, 467-470 (198
6))), a substance that inhibits invasion into the lower layers such as the mesothelial cell layer and the vascular endothelial cell layer (for example, A. Isoai et al .: Jpn. J.
Cancer Res., 81, 909-914 (1990)), substances that inhibit the degradation of extracellular matrix (eg, RM Schultz et al .: Can).
cer Res., 48, 5539-5545 (1988)) and the like.

The present inventors have previously produced a complex (protein) of a peptide having a cancer metastasis inhibitory activity and a biopolymer by a chemical coupling method. That is, a peptide having a cancer inhibitory activity represented by the amino acid sequence of SEQ ID NO: 8 in the sequence listing (JP-A-3-34993, A. Isoai e
t al .: Jpn. J. Cancer Res., 81, 909-914 (1990) and A. Isoai et al .: Cancer Res., 52, 1422-1426 (19
92)) and a biopolymer such as serum albumin are bound with a water-soluble carbodiimide, it has been confirmed that they have excellent cancer cell invasion inhibitory activity and cancer metastasis inhibitory activity (Japanese Patent Application Laid-Open No. Heisei 4- No. 254000, 4-3
No. 00899, No. 4-300900 and A. Is
oai et al .: Biochem. Biophys. Res. Commun., 192, 7
-14 (1993)).

[0006] The protein having such useful cancer metastasis inhibitory activity is usually produced by a chemical protein binding method. However, that method has many steps,
In addition, separation of incomplete synthetic products, which are impurities, must be performed. Usually, these methods are complicated, and it is difficult to mass-produce them efficiently. Especially for the protein having 609 amino acid residues, the chemical protein binding method is always satisfactory in terms of cost and equipment. It wasn't something.

[0007]

The present invention has been made in view of the above circumstances, and is a complex of a peptide represented by the amino acid sequence of SEQ ID NO: 8 (cancer metastasis-inhibiting peptide) and a biopolymer such as serum albumin. It is an object of the present invention to provide a technique capable of achieving more efficient gene expression and production of a cancer metastasis inhibitory protein by using a gene recombination technique instead of the conventional chemical protein binding method.

[0008]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies in order to solve the above problems, and have created a novel system for producing a cancer metastasis inhibitory protein using gene recombination technology. Succeeded in producing. Specifically, a gene encoding a cancer metastasis inhibitory protein is prepared, the gene is integrated into an already established vector for heterologous protein production, and the obtained recombinant vector is introduced into a host cell to obtain a transformant. By making
It is possible to achieve production of a cancer metastasis inhibitory protein.

That is, according to the present invention, a novel cancer metastasis inhibitory protein represented by the amino acid sequence of SEQ ID NO: 1 is provided.

Further, according to the present invention, there is provided the gene represented by the nucleotide sequence of SEQ ID NO: 2 which codes for the above-mentioned cancer metastasis inhibitory protein.

Further, according to the present invention, there is provided the gene represented by the nucleotide sequence of SEQ ID NO: 3, which encodes the above-mentioned cancer metastasis inhibitory protein.

The present invention also provides a recombinant vector containing the gene represented by the nucleotide sequence of SEQ ID NO: 2 or 3.

The present invention also provides a transformant capable of producing the above-mentioned cancer metastasis inhibitory protein, which is obtained by transforming a host cell with the above-mentioned recombinant vector.

Further, according to the present invention, there is provided a method for producing the above-mentioned cancer metastasis-inhibitory protein, which comprises culturing the above-mentioned transformant, isolating the cancer-metastasis-inhibiting protein produced in the culture, and purifying it if desired. Provided.

The present invention will be described in detail below.

As described above, the conjugate (complex) of the peptide represented by the amino acid sequence of SEQ ID NO: 8 (cancer metastasis inhibitory peptide) and a biopolymer such as serum albumin has excellent cancer metastasis inhibitory activity. It has already been confirmed by the present inventors. However, the complex is one produced by a chemical protein binding method, and the binding relationship between the cancer metastasis-inhibiting peptide and serum albumin is in the state of being bound by a water-soluble carbodiimide, Not clear.

The inventors of the present invention have conducted various studies on the genetically engineered fusion protein in which a cancer metastasis-inhibiting peptide and serum albumin are bound, and as a result, have shown that the cancer metastasis-inhibiting activity can be sufficiently exerted. Considering the above, it is preferable to bind the cancer metastasis inhibitory peptide to a position that is located on the surface of the serum albumin protein and does not destroy the three-dimensional structure. It has been found that it is most preferable to attach a cancer metastasis inhibiting peptide to the terminal (C terminal). Then, they succeeded in producing a fusion protein obtained by binding the above-mentioned cancer metastasis-inhibiting peptide to the carboxyl terminus (C terminus) of serum albumin using a gene recombination technique.

The novel fusion protein (cancer metastasis inhibitory protein) according to the present invention is represented by the amino acid sequence of SEQ ID NO: 1, in which amino-terminal (N-terminal)
Sequences 1 to 588 from the side are serum albumin, 5
The 89th to 609th sequences are derived from the cancer metastasis inhibiting peptide. The synthetic gene according to the present invention is a synthetic gene encoding the fusion protein represented by the amino acid sequence of SEQ ID NO: 1, and theoretically, SEQ ID NO: 2 in the sequence listing.
Can be represented by the base sequence of 2, 147,
483,648 different sequences can be considered. However, it is preferable to match the frequency of codon usage of the host cell used for gene recombination, and it is preferable to design using the codon most frequently used.

The host cell used here is not particularly limited, but it is desirable that the culture method be easy,
Microorganisms that can be cultivated at low cost are good, for example, E. coli (Es
cherichia coli), various yeasts, Bacillus subtilis, filamentous fungi, etc., and host cells commonly used in the art. The method using a prokaryote as a host cell is not always effective for all polypeptides, and it is not always easy to reproduce a complex post-translational modification of a protein derived from eukaryote or the same three-dimensional structure as a natural body. Further, the presence of a specific endotoxin is not preferable because it may become a contaminant of the final product. For this reason, it is preferable to use various yeasts that do not contain endotoxin, have established culture methods, have been used in the fermentation and food industries for a long time, and that have safety established for humans.
Among them, the fission yeast Schizosaccharomyces pombe, which is expected to be able to obtain a gene product closer to a natural body, is said to have characteristics similar to animal cells genetically and molecularly. Is most preferred. The strain of the Schizosaccharomyces pombe, for example accession number ATCC38399 (leu-32h ^-) and ATCC38436 (ura4-294h ^-) American as such
Those deposited with the Type Culture Collection (ATCC) are listed and available.

Therefore, in the present invention, SEQ ID NO: 1
The gene encoding the cancer metastasis inhibitory protein represented by the amino acid sequence of is most preferably designed and synthesized using a codon most suitable for high expression in Schizosaccharomyces pombe. The optimal codon usage frequency of Schizosaccharomyces pombe is, for example, A. Nasim et al: Molecular Biology.
of the Fission Yeast p263, Academic Press (1989)
It can be known from etc. As a result of various studies, the present inventors have concluded that the gene represented by the nucleotide sequence of SEQ ID NO: 3 is most suitable, and designed and synthesized it. In the sequence of SEQ ID NO: 3, the nucleotide sequence from the 1'to 1758th position from the 5'-terminal side encodes serum albumin, 1765 to 18
The 30th nucleotide sequence encodes a cancer metastasis inhibitory peptide, and a translation termination signal (TAA) is added at 1828-1830th. Gene production (synthesis) was performed by the triester method (Nuc. Acid. Res. 10, p.655).
3, (1982)) and the phosphoamidite method (Tetrahedron Letters
22, p.1859, (1981)) and various other methods have already been developed, and any method may be used. DNA
Since synthesizers (DNA synthesizers) and the like are commercially available, they may be used.

Next, the gene thus synthesized is incorporated into a vector to prepare a recombinant vector. The vector to be used is not particularly limited, but it is capable of autonomously replicating in the host cell and has an insertion site capable of incorporating a synthetic gene (foreign gene). It is necessary to have a region that allows expression. As such a vector, for example, the foreign gene expression vector pTL2M (Japanese Patent Application No. 5-249310), which uses the Schizosaccharomyces pombe as a host, which has been successfully created by the present inventors, is advantageously used. The above synthetic gene can be easily incorporated into these vectors.

Next, the above recombinant vector is introduced into a host cell to obtain a transformant. The method of introducing the recombinant vector into the host cell can be carried out by a conventionally used method, such as the competent cell method, the protoplast method, the calcium phosphate coprecipitation method, the electroporation method, the microinjection method, There are various methods such as the liposome fusion method and the particle gun method, and any method can be adopted depending on the host to be used. When using Schizosaccharomyces pombe as a host,
For example, the lithium acetate method (K. Okazaki et al., Nucleic A
Cids Res., 18, 6485-6489 (1990)) and the like can be used to efficiently obtain a transformant.

By culturing the transformant thus obtained, a cancer metastasis inhibitory protein is produced in the culture. The desired cancer metastasis inhibitory protein can be obtained by isolating it by a known method and optionally purifying it.

A medium for culturing the transformant is known, and a nutrient medium such as YPD medium (MD Rose et al.,
"Methods In Yeast Genetics", Cold Spring Harbor L
abolatory Press (1990)) and minimal medium such as MB medium (K. Okazaki et al., Nucleic Acids Res., 18, 6485-6
489 (1990)) and the like can be used. Cultivation of the transformant is usually at 16 to 42 ° C, preferably 25 to 37 ° C,
It is carried out for 8 to 168 hours, preferably for 24 to 72 hours. Both shaking culture and static culture are possible, but agitation and aeration may be added if necessary.

As a method for isolating and purifying the protein produced in the culture, a method utilizing a difference in solubility such as a known salting out or solvent precipitation method, a molecular weight such as dialysis, ultrafiltration or gel electrophoresis Difference, a method using a charge difference such as ion exchange chromatography, a method using a specific affinity such as affinity chromatography, a difference in hydrophobicity such as reverse phase high performance liquid chromatography Examples of the method include a method utilizing a difference in isoelectric points such as a method and an isoelectric focusing method.

As a method for confirming the isolated / purified protein, known Western blotting method, activity measuring method and the like can be mentioned. The structure of the purified protein can be clarified by amino acid analysis, amino terminal (N terminal) analysis, primary structure analysis, and the like.

[0027]

The present invention will be described more specifically by the following examples. However, the technical scope of the present invention is not limited by these examples. In addition, for each operation in the examples, unless otherwise specified, methods commonly used in the art (eg, J. Sambrook et al .: Molecu
lar Cloning. A Laboratory Manual. 2nd ed. Cold Spr
ing Harbor Laboratory Press, Cold Spring Harbor, N
ew York, USA, 1989.).

[Example 1] Preparation of gene represented by the nucleotide sequence of SEQ ID NO: 3 encoding cancer metastasis inhibitory protein Based on the amino acid sequence of SEQ ID NO: 1, the codon usage of Schizosaccharomyces pombe (Nasim , A. et al: Molec
ular Biology of the Fission Yeast, Academic Press,
1989, p263.), Two single-stranded oligo DNAs represented by the nucleotide sequences of SEQ ID NOS: 4 and 5 were synthesized using an automatic DNA synthesizer (Applied Biosystems). The base sequence of SEQ ID NO: 4 has a restriction enzyme Ba at the 5'end.
This is the sense strand of the gene in which the insertion site into mHI and the initiation codon ATG were introduced into the 3'end of the termination codon TAA and the insertion site into the restriction enzyme HindIII, and the nucleotide sequence of SEQ ID NO: 5 is its antisense strand. After deprotection and purification,
These two were annealed at 70 ° C.

On the other hand, apart from this, the plasmid pUC19
(Takara Shuzo Co., Ltd.) is double-digested with restriction enzymes BamHI (Takara Shuzo Co., Ltd.) and HindIII (Takara Shuzo Co., Ltd.), purified by phenol extraction and ethanol precipitation, and then subjected to agarose gel electrophoresis. A band corresponding to about 2600 base pairs was cut out and purified by a glass bead method using DNA-PREP (manufactured by Asahi Glass Co., Ltd.).

Both fragments were ligated with a DNA ligation kit (Takara Shuzo Co., Ltd.). This was introduced into Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) and transformed with ampicillin resistance, and
Positive clones displaying white colonies were screened on X-gal plates to obtain pI2A, which shows a cleavage fragment of about 70 base pairs upon double digestion of the plasmid of interest, restriction enzymes BamHI and HindIII. A large amount of pI2A was prepared according to the alkali-SDS method, and SEQ ID NO.
It was confirmed that the plasmid had the nucleotide sequence of.

Example 2 Construction of Expression Vector pTL2BmI Containing Cancer Metastasis Inhibitory Protein Gene The plasmid pI2A constructed in Example 1 was used as a restriction enzyme Nc.
Double-digest with oI (Takara Shuzo Co., Ltd.) and HindIII to adjust the ends, and a band corresponding to about 70 base pairs is cut out by acrylamide gel electrophoresis and eluted from the gel to encode a cancer metastasis inhibitory protein. It was used as a gene insert.

Separately from this, a linker fragment for gene transfer was prepared. That is, using the human serum albumin cDNA cloned from the human liver cDNA library on the plasmid pUC19 as a template, the DNA represented by the nucleotide sequence of SEQ ID NO: 6 was used as the 5'-end side primer, and represented by the nucleotide sequence of SEQ ID NO: 7. PCR was carried out using the DNA as a 3'terminal primer, and then the restriction enzymes NcoI and AflIII (New England Biolab
s) was used for terminal regulation (partial digestion). After purification by phenol extraction and ethanol precipitation, agarose gel electrophoresis was performed, and a band corresponding to about 1800 base pairs was cut out and purified by a glass bead method using DNA-PREP to obtain a linker fragment.

Separately from this, a Schizosaccharomyces pombe expression vector pTL2M was prepared. This vector pTL2M has been constructed by the present inventors (Japanese Patent Application No. 5-249310). The manufacturing method will be described below.

[Preparation of vector pRL2M] First, pcD4CAT prepared by a known method was cleaved with BamHI to remove the CAT gene and then ligated to obtain pcD4.
Was produced. pcD4 was partially cleaved with BamHI, blunt-ended, and ligated to prepare pcD4B (JP-A-5-15380).

This plasmid pcD4B was used as a restriction enzyme Sa
After digestion with cI, the ends were blunted with T4 DNA polymerase, further digested with a restriction enzyme BamHI, and then purified by phenol extraction and ethanol precipitation. Further, after agarose gel electrophoresis, DNA corresponding to about 4500 base pairs was purified by the glass bead method.

Separately from this, a human fibroblast-derived Okayama-Berg cDNA library (pcD vector) was prepared by a known method. Furthermore, the already known gene sequence of human lipocortin I (Nature, 320,
77, (1986)), the gene of lipocortin I was obtained by the colony hybridization method from the above library using the gene sequence of 50 bases encoding the N-terminal amino acid sequence of the protein as a DNA probe, and the nucleotide sequence was determined. By doing so, it was confirmed that it encodes the full-length lipocortin I protein. The obtained clone was named pcDlipoI. (JP-A-5
-15380). And this human lipocortin I
The vector pcDlipoI containing the gene (cDNA) was digested with restriction enzymes XmnI and BamHI, and then purified by phenol extraction and ethanol precipitation. Further, after agarose gel electrophoresis, the DNA corresponding to about 1300 base pairs was purified by the glass bead method.

After ligating both DNAs, this was introduced into Escherichia coli DH5 strain (manufactured by Toyobo Co., Ltd.) for transformation. A vector was prepared from the obtained transformant, and a transformant having the target vector pRL2L (FIG. 5) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

This lipocortin I expression vector pRL2
L was digested with restriction enzymes EcoRI and HindIII, extracted with phenol and precipitated with ethanol, a band corresponding to about 5000 base pairs was cut out by agarose gel electrophoresis, and purified by a glass bead method. Separately from this, a known plasmid pUC19 was added to the restriction enzyme EcoRI.
After digestion with HindIII and phenol extraction and ethanol precipitation, a band corresponding to about 60 base pairs was cut out by polyacrylamide gel electrophoresis and extracted and purified from the gel.

After ligation of these two fragments,
Target vector pR obtained by transforming Escherichia coli strain DH5
L2M (Figure 6) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

[Preparation of vector pTL2M] The above pRL
Oligodeoxyribonucleotide 5'- with 2M as template
TTGACTAGTTATTAATAGTA-3 'and oligodeoxyribonucleotide 5'-CTAGAATTCACATGTTTGAAAAAGTGTCTTTATC-
The target fragment was amplified by PCR using Taq polymerase with 3'as a synthetic primer. Restriction enzyme Sp
end-adjusted with eI and EcoRI, phenol extracted,
After ethanol precipitation, a band corresponding to about 600 base pairs was cut out by agarose gel electrophoresis and purified by a glass bead method.

Separately from this, pRL2M was digested with restriction enzymes SpeI and EcoRI, extracted with phenol and precipitated with ethanol, and a band corresponding to about 4500 base pairs was cut out by agarose gel electrophoresis and purified by a glass bead method. did. After ligation of these two fragments, Escherichia coli DH5 strain was transformed to screen the target vector pTL2M (FIG. 7). From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

The thus prepared pTL2M was double-digested with the restriction enzymes AflIII and HindIII,
A band corresponding to about 5000 base pairs was cut out.

Then, a total of 3 double digests of the gene insert fragment, the linker fragment and the vector pTL2M were
Ligation was performed using a DNA ligation kit. This was introduced into Escherichia coli DH5 strain (manufactured by Toyobo Co., Ltd.) for transformation, and then a target plasmid pTL2BmI as shown in FIG. 1 was obtained. A large amount of pTL2BmI was prepared according to the alkali-SDS method, and it was confirmed that the plasmid had the base sequence of SEQ ID NO: 3 by constructing a restriction map and determining the base sequence.

[Example 3] Expression vector pTL2BmI
Leucine auxotrophic strain of Schizosaccharomyces transformation of Mrs. pombe Schizosaccharomyces pombe using, h ^-
leu 1-32 (ATCC 38399) was grown in leucine-containing minimal medium MB-leu to reach 10 ⁷ cells / ml. After centrifugation and washing with water, 10 ⁹ cells /
100 mM lithium acetate (pH 5.
0), and incubated at 30 ° C. for 60 minutes.
Thereafter, pAL7 (K. Okazaki et al .: Nucl. Acids R digested with the restriction enzyme PstI was added to 100 μl of the above suspension.
es. 18, 6485-6489 (1990)) 1 μg and 2 μg of the expression vector pTL2BmI dissolved in 10 μl of TE buffer were added, and 50% PEG4000 was added to 290 μm.
After adding 1 well and mixing well, the mixture was incubated at 30 ° C. for 60 minutes, 43 ° C. for 15 minutes, and room temperature for 10 minutes in this order.
Then, after removing PEG4000 by centrifugation, 1
It was suspended in ml of the culture medium 1 / 2YEL-Leu.

100 μl of this suspension was taken, diluted with 900 μl of culture medium 1 / 2YEL-Leu, incubated at 32 ° C. for 30 minutes, and then 300 μl.
Was spread on the minimal agar medium MMA. After incubating at 32 ° C. for 3 days, the obtained transformant was transferred to a YEA medium containing 25 μg / ml of G418 and further cultured at 32 ° C. for 5 days, and the obtained clone was used as each target transformant.

Separately from this, transformants were prepared by the same method for plasmids pTL2M (described above) and pTL2Bm (Japanese Patent Application No. 5-249310) which do not have a cancer metastasis inhibitory peptide gene. It was used as a negative control. The plasmid pTL2Bm was prepared as follows.

[Preparation of plasmid pTL2Bm] Oligodeoxyribonucleotide 5'-AGACCA using the vector pILMALB5 containing human serum albumin cDNA provided by the National Institute of Preventive Health gene bank as a template.
The target fragment was amplified by PCR using Taq polymerase with TGGATGCACACACAAGAGTGAGGT-3 ′ and oligodeoxyribonucleotide 5′-CAGGAAACAGCTATGACCAT-3 ′ as synthetic primers. Restriction enzymes NcoI and H
End-adjusted with indIII, extracted with phenol, precipitated with ethanol, and then subjected to agarose gel electrophoresis to obtain about 1800
The band corresponding to the base pair was cut out and purified by the glass bead method.

Separately from this, pTL2M was used as a restriction enzyme Af.
After digestion with lIII and HindIII, phenol extraction and ethanol precipitation, a band corresponding to about 5000 base pairs was cut out by agarose gel electrophoresis and purified by the glass bead method.

After ligation of these two fragments,
Target vector pT obtained by transforming Escherichia coli DH5 strain
L2Bm (Figure 8) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

[Example 4] Culture of transformants and preparation of cell-free extract 50 ml of YPD medium containing the antibiotic G418 (GIBCO BRL) at a concentration of 200 µg / ml [(2% glucose (Wako Pure Chemical Industries, Ltd. )) 1% Bacto yeast extract (Di
fco), 2% bactopeptone (Difco)] in Example 3
The transformant prepared in 1. was inoculated and cultured at 32 ° C. for 5 days. After collecting 10 ⁸ cells from the culture and washing the cells,
The cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) and sonicated. 10% S so that the final concentration is 1%
The DS solution was added and heated at 80 ° C. for 15 minutes. A cell-free extract (supernatant) was obtained by centrifugation.

Separately from this, a cell-free extract was prepared in the same manner as above for the transformants introduced with the above-mentioned pTL2M and pTL2Bm having no cancer metastasis inhibitor protein gene, and used as a negative control.

[Example 5] Expression analysis of cancer metastasis inhibitory protein by SDS-polyacrylamide gel electrophoresis The expression analysis was carried out on the cell-free extract derived from each transformant prepared in Example 4 by SDS-PAGE. . The results are shown in Figure 2. As is clear from the figure, pT
The L2mBI transformant had a molecular weight of 6 as compared with the control pTL2Bm transformant.
The 9000 band (indicated by * in the figure) has a molecular weight of 71 due to the production of the cancer metastasis inhibitory protein.
It was possible to detect the movement to the 000 position (indicated by ** in the figure). When measured by a densitometer, the production amount of the cancer metastasis inhibitory protein was about 30% of the total microbial cell protein.

Example 6 Confirmation of Cancer Metastasis Inhibitory Protein by Western Blotting SDS-PAGE was performed on the cell-free extract derived from each transformant prepared in Example 4 in the same manner as in Example 5. Transfer the obtained gel to a PVDF membrane (Bio-Rad),
Antibodies specific for cancer metastasis inhibitory proteins (A. Isoai et a
l .: Biochem. Biophys. Res. Commun. 192, 7-14 (199
Western blotting using 3)) and ECL
(Manufactured by Amersham Co., Ltd.). The result is shown in Figure 3.
Shown in As is clear from the figure, since only a clear band was obtained at a position near the molecular weight of 71,000 corresponding to the sequence containing the cancer metastasis inhibitory protein, the amino acid sequence specific to the protein was included. It was confirmed that the protein containing the protein was produced.

Example 7 Purification of Cancer Metastasis Inhibitory Protein The transformant transformed with pTL2BmI was transformed into G
50 ml of YPD containing 418 at a concentration of 25 μg / ml
After pre-culturing in the medium at 32 ° C for 1 day, G418 was added to 200
1 x 10 ⁸ / in 1 liter of YPD medium containing μg / ml
The cells were inoculated at a ratio of ml and further cultured for 4 days. 50 mM Tris-hydrochloric acid buffer solution (pH 7.
5) [12 μM APMSF (manufactured by Wako Pure Chemical Industries, Ltd.), 2
5 μM leupeptin (manufactured by Wako Pure Chemical Industries, Ltd.), 2 mM E
It was suspended in DTA] and crushed at 0 ° C. using an equal amount of glass beads (bead beater). 12,000 rpm
The precipitate was centrifuged for 20 minutes, washed with the same buffer, and then solubilized in 50 mM Tris-HCl buffer (pH 7.5) containing 6 M guanidine hydrochloride and 10 mM dithiothreitol at 50 ° C. for 1 hour. 12,000 rpm, 2
The supernatant obtained by centrifuging for 0 minutes was treated with 0.1 M NaCl and 1 mM.
50 mM Tris-HCl buffer containing DTA, 2 mM reduced glutathione and 0.2 mM oxidized glutathione (pH
7.5) was gradually diluted 100 times (v / v) at 4 ° C. After being left overnight at 4 ° C., it was concentrated with an ultrafiltration membrane (Amicon), gel-filtered with Superose 12 column, and each fraction was analyzed by SDS-PAGE to have a molecular weight of 71,000.
Fractions showing a unique band at position 0 were collected and used as a purified cancer metastasis inhibitory protein.

Example 8 Measurement of Cancer Cell Invasion Inhibitory Activity of Purified Cancer Metastasis Inhibitory Protein The cancer cell invasion inhibitory effect of the cancer metastasis inhibitory protein purified in Example 7 was examined. The evaluation method is that of Albini et al. (Albini et al .: Cancer Res. 47, 3239-3245 (19
87)). Using a polycarbonate filter with a pore size of 8 μm, 10 μg of Matrigel (made by Collaborative Co., Ltd.) is placed on the upper surface of the chemotaxel (made by Kurabo Co., Ltd.) which is divided into an upper layer and a lower layer.
Was applied and dried overnight at room temperature. Immediately before use, it was swollen with a culture solution and set on a 24-well culture plate.
Cancer cells are highly metastatic clone B1 derived from B16 melanoma
6FE7 was used.

The cells were treated with 1.85 kBq / ml of [ ¹²⁵ I].
The cells were cultured in the presence of IUdR (manufactured by Amersham Co., Ltd.) for 2 days. Just before use, after collecting cells with trypsin solution,
The cells were suspended in a culture medium containing 0.1% bovine serum albumin, and the number of cells and the radioactivity of [ ¹²⁵ I] IUdR incorporated therein were measured. 20 μg / ml of human fibronectin was placed in the lower layer of chemotaxel, and 5 × 10 ⁴ cells were placed in the upper layer together with various concentrations of cancer metastasis-inhibiting protein, and cultured in a carbon dioxide gas incubator for 20 hours.

After the culture was completed, the cells remaining on the upper surface of the filter were scraped off with a cotton swab, and the filter was lysed together with the cells that had moved to the lower surface using a tissue solubilizer (manufactured by Amersham Co.), and the radioactivity was measured. FIG. 4 shows the results. As is clear from the figure, it was shown that the cancer metastasis inhibitory protein significantly inhibits invasion of cancer cells.

[0058]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, a cancer metastasis inhibitory protein that can be produced only by combining a chemical synthesis method and a binding method has been used as a recombinant DNA.
Only when the technology is used is it possible to produce directly and with high efficiency.

Therefore, the large-scale culture using the transformant of the present invention makes it possible to obtain a high productivity of the target cancer metastasis inhibitory protein, and it can be sufficiently used for production on an industrial scale. That is, it can be said that it has become possible to stably supply the cancer metastasis inhibitory protein as a drug.

[0060]

[Sequence list]

SEQ ID NO: 1 Sequence length: 609 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly 1 5 10 15 Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu 20 25 30 Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr 35 40 45 Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp 50 55 60 Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr 65 70 75 80 Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu 85 90 95 Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn 100 105 110 Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe 115 120 125 His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala 130 135 140 Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys 145 150 155 160 Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala 165 170 175 Ala Cys Leu Leu Pro Lys Leu As p Glu Leu Arg Asp Glu Gly Lys Ala 180 185 190 Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly 195 200 205 Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe 210 215 220 Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr 225 230 235 240 Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp 245 250 255 Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile 260 265 270 Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser 275 280 285 His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro 290 295 300 Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr 305 310 315 320 Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala 325 330 335 Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys 340 345 350 Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His 355 360 365 Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu 370 375 380 Pro Gln Asn Leu Ile Lys Gln Asn Cy s Glu Leu Phe Lys Gln Leu Gly 385 390 395 400 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val 405 410 415 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 420 425 430 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 435 440 445 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 450 455 460 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 465 470 475 480 Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 485 490 495 Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 500 505 510 Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 515 520 525 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 530 535 540 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 545 550 555 560 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 565 570 575 Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Tyr Met Ala Glu Asp Gly 580 585 590 Asp Ala Lys Thr Asp Gln Ala Glu Ly s Ala Glu Gly Ala Gly Asp Ala 595 600 605 Lys 609 SEQ ID NO: 2 Sequence length: 1830 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence characteristics Characteristic symbol: CDS Location: 1. ． 1830 Method of characterizing: E sequence ATG GAT GCA CAC AAG AGT GAG GTT GCT CAT CGG TTT AAA GAT TTG GGA 48 GAA GAA AAT TTC AAA GCC TTG GTG TTG ATT GCC TTT GCT CAG TAT CTT 96 CAG CAG TGT CCA TTT GAA GAT CAT GTA AAA TTA GTG AAT GAA GTA ACT 144 GAA TTT GCA AAA ACA TGT GTA GCT GAT GAG TCA GCT GAA AAT TGT GAC 192 AAA TCA CTT CAT ACC CTT TTT GGA GAC AAA TTA TGC ACA GTT GCA ACT 240 CTT CGT GAA ACC TAT GGT GAA ATG GCT GAC TGC TGT GCA AAA CAA GAA 288 CCT GAG AGA AAT GAA TGC TTC TTG CAA CAC AAA GAT GAC AAC CCA AAC 336 CTC CCC CGA TTG GTG AGA CCA GAG GTT GAT GTG ATG TGC ACT GCT TTT 384 CAT GAC AAT GAA GAG ACA TTT TTG AAA AAA TAC TTA TAT GAA ATT GCC 432 AGA AGA CAT CCT TAC TTT TAT GCC CCG GAA CTC CTT TTC TTT GCT AAA 480 AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 528 GCC TGC CTG TTG CCA AAG CTC GAT GAA CTT CGG GAT GAA GGG AAG GCT 576 TCG TCT GCC AAA CAG AGA CTC AAA TGT GCC AGT CTC CAA AAA TTT GGA 624 GAA AGA GCT TTC AAA GCA TGG GCA GTG GCT CGC CTG AGC CAG AGA TTT 672 CCC AAA GCT GAG TTT GCA GAA GTT TCC AAG TTA GTG ACA GAT CTT ACC 720 AAA GTC CAC ACG GAA TGC TGC CAT GGA GAT CTG CTT GAA TGT GCT GAT 768 GAC AGG GCG GAC CTT GCC AAG TAT ATC TGT GAA AAT CAG GAT TAT ATC 816 TCC AGT AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC 864 CAC TGC ATT GCC GAA GTG GAA AAT GAT GAG ATG CCT GCT GAC TTG CCT 912 TCA TTA GCT GCT GAT TTT GTT GAA AGT AAG GAT GTT TGC AAA AAC TAT 960 GCT GAG GCA AAG GAT GTC TTC CTG GGC ATG TTT TTG TAT GAA TAT GCA 1008 AGA AGG CAT CCT GAT TAC TCT GTC GTG CTG CTG CTG AGA CTT GCC AAG 1056 ACA TAT GAA ACC ACT CTA GAG AAG TGC GGT GCC GCT GAT CCT CAT 1104 GAA TGC TAT GCC AAA GTG TTC GAT GAA TTT AAA CCT CTT GTG GAA GAG 1152 CCT CAG AAT TTA ATC AAA CAA AAC TGT GAG CTT TTT AAG CAG CTT GGA 1200 GAG TAC AAA TTC CAG AAT GCG CTA TTA GTT CGT TAC ACC AAG AAA GTA 1248 CCC CAA GTG TCA ACT CCA ACT CTT GTA GAG GTC TCA AGA AAC CTA GGA 1296 AAA GTG GGC AGC AAA TGT TGT AAA CAT CCT GAA GCA AAA AGA ATG CCC 1344 TGT GCA GAA GAC TAT CTA TCC GTG GTC CTG AAC CAG TTA TGT GTG TTG 1392 CAT GAG AAA ACG CCA GTA AGT GAC AGA GTC ACA AAA TGC TGC ACA GAG 1440 TCC TTG GTG AAC AGG CGA CCA TGC TTT TCA GCT CTG GAA GTC GAT GAA 1488 ACA TAC GTT CCC AAA GAG TTT AAT GCT GAA ACA TTC ACC TTC CAT GCA 1536 GAT ATA TGC ACA CTT TCT GAG AAG GAG AGA CAA ATC AAG AAA CAA ACT 1584 GCA CTT GTT GAG CTT GTG AAA CAC AAG CCC AAG GCA ACA AAA GAG CAA 1632 CTG AAA GCT GTT ATG GAT GAT GAT TTC GCA GCT TTT GTA GAG AAG TGC TGC 1680 AAG GCT GAC GAT AAG GAG ACC TGC TTT GCC GAG GAG GGT AAA AAA CTT 1728 GTT GCT GCA AGT CAA GCT GCC TTA GGC TTA TAC ATG GCN GAR GAY GGN 1776 GAY GCN AAR ACN GAY CAR GCN GAR AAR GCN GAR GGN GCN GGN GAY GCN 1824 AAR TAA 1830 (where N is A, T, G or C; Y is T or C; R
Represents A or G) SEQ ID NO: 3 Sequence length: 1830 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence characteristics Characteristic symbol: CDS Location: 1. ． 1830 Method of characterizing: E sequence ATG GAT GCA CAC AAG AGT GAG GTT GCT CAT CGG TTT AAA GAT TTG GGA 48 GAA GAA AAT TTC AAA GCC TTG GTG TTG ATT GCC TTT GCT CAG TAT CTT 96 CAG CAG TGT CCA TTT GAA GAT CAT GTA AAA TTA GTG AAT GAA GTA ACT 144 GAA TTT GCA AAA ACA TGT GTA GCT GAT GAG TCA GCT GAA AAT TGT GAC 192 AAA TCA CTT CAT ACC CTT TTT GGA GAC AAA TTA TGC ACA GTT GCA ACT 240 CTT CGT GAA ACC TAT GGT GAA ATG GCT GAC TGC TGT GCA AAA CAA GAA 288 CCT GAG AGA AAT GAA TGC TTC TTG CAA CAC AAA GAT GAC AAC CCA AAC 336 CTC CCC CGA TTG GTG AGA CCA GAG GTT GAT GTG ATG TGC ACT GCT TTT 384 CAT GAC AAT GAA GAG ACA TTT TTG AAA AAA TAC TTA TAT GAA ATT GCC 432 AGA AGA CAT CCT TAC TTT TAT GCC CCG GAA CTC CTT TTC TTT GCT AAA 480 AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 528 GCC TGC CTG TTG CCA AAG CTC GAT GAA CTT CGG GAT GAA GGG AAG GCT 576 TCG TCT GCC AAA CAG AGA CTC AAA TGT GCC AGT CTC CAA AAA TTT GGA 624 GAA AGA GCT TTC AAA GCA TGG GCA GTG GCT CGC CTG AGC CAG AGA TTT 672 CCC AAA GCT GAG TTT GCA GAA GTT TCC AAG TTA GTG ACA GAT CTT ACC 720 AAA GTC CAC ACG GAA TGC TGC CAT GGA GAT CTG CTT GAA TGT GCT GAT 768 GAC AGG GCG GAC CTT GCC AAG TAT ATC TGT GAA AAT CAG GAT TAT ATC 816 TCC AGT AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC 864 CAC TGC ATT GCC GAA GTG GAA AAT GAT GAG ATG CCT GCT GAC TTG CCT 912 TCA TTA GCT GCT GAT TTT GTT GAA AGT AAG GAT GTT TGC AAA AAC TAT 960 GCT GAG GCA AAG GAT GTC TTC CTG GGC ATG TTT TTG TAT GAA TAT GCA 1008 AGA AGG CAT CCT GAT TAC TCT GTC GTG CTG CTG CTG AGA CTT GCC AAG 1056 ACA TAT GAA ACC ACT CTA GAG AAG TGC GGT GCC GCT GAT CCT CAT 1104 GAA TGC TAT GCC AAA GTG TTC GAT GAA TTT AAA CCT CTT GTG GAA GAG 1152 CCT CAG AAT TTA ATC AAA CAA AAC TGT GAG CTT TTT AAG CAG CTT GGA 1200 GAG TAC AAA TTC CAG AAT GCG CTA TTA GTT CGT TAC ACC AAG AAA GTA 1248 CCC CAA GTG TCA ACT CCA ACT CTT GTA GAG GTC TCA AGA AAC CTA GGA 1296 AAA GTG GGC AGC AAA TGT TGT AAA CAT CCT GAA GCA AAA AGA ATG CCC 1344 TGT GCA GAA GAC TAT CTA TCC GTG GTC CTG AAC CAG TTA TGT GTG TTG 1392 CAT GAG AAA ACG CCA GTA AGT GAC AGA GTC ACA AAA TGC TGC ACA GAG 1440 TCC TTG GTG AAC AGG CGA CCA TGC TTT TCA GCT CTG GAA GTC GAT GAA 1488 ACA TAC GTT CCC AAA GAG TTT AAT GCT GAA ACA TTC ACC TTC CAT GCA 1536 GAT ATA TGC ACA CTT TCT GAG AAG GAG AGA CAA ATC AAG AAA CAA ACT 1584 GCA CTT GTT GAG CTT GTG AAA CAC AAG CCC AAG GCA ACA AAA GAG CAA 1632 CTG AAA GCT GTT ATG GAT GAT GAT TTC GCA GCT TTT GTA GAG AAG TGC TGC 1680 AAG GCT GAC GAT AAG GAG ACC TGC TTT GCC GAG GAG GGT AAA AAA CTT 1728 GTT GCT GCA AGT CAA GCT GCC TTA GGC TTA TAC ATG GCC GAG GAC GGT 1776 GAC GCC AAG ACC GAC CAA GCT GAG AAG GCT GAG GGT GCC GGT GAC GCC 1824 AAG TAA 1830 SEQ ID NO: 4 Sequence length: 73 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GATCC ATG GCC GAG GAC GGT GAC GCC AAG ACC GAC CAA GCT GAG AAG GCT 50 GAG GGT GCC GGT GAC GCC AAG TA 73 SEQ ID NO: 5 Sequence length: 73 Sequence type: Number of acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence AGCTTA CTT GGC GTC ACC GGC ACC CTC AGC CTT CTC AGC TTG GTC GGT CTT 51 GGC GTC ACC GTC CTC GGC CAT G 73 SEQ ID NO: 6 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AGACCATGGA TGCACACAAG AGTGAGGT 28 SEQ ID NO: 7 Sequence Length: 28 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTCACATGTA TAAGCCTAAG GCAGCTTG 28 SEQ ID NO: 8 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Glu Asp Gly Asp Ala Lys Thr Asp Gln Ala Glu Lys Ala Glu Gly 1 5 10 15 Ala Gly Asp Ala Lys 20 21

[Brief description of drawings]

FIG. 1 is a constitutional view of an expression vector pTL2BmI.

FIG. 2 is an SDS-PAGE observation view.

FIG. 3 is a Western blot observation view.

FIG. 4 is a graph showing the measurement results of cancer cell invasion inhibitory activity.

FIG. 5 is a structural diagram of expression vector pRL2L.

FIG. 6 is a structural diagram of the expression vector pRL2M.

FIG. 7 is a structural diagram of the expression vector pTL2M.

FIG. 8 is a structural diagram of expression vector pTL2Bm.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12N 1/19 8828-4B C12P 21/02 C 9282-4B // A61K 38/00 ADU (C12N 1 / 19 C12R 1: 645) (C12P 21/02 C12R 1: 645) (72) Inventor Yoko Tsukamoto 1150 Hazawa-machi, Kanagawa-ku, Yokohama, Kanagawa Prefecture Asahi Glass Co., Ltd. Central Research Laboratory (72) Inventor Atsushi Atsushi, Kanagawa, Yokohama Asahi Glass Co., Ltd. Central Research Institute, 1150 Hazawa-machi, Kanagawa-ku, Japan (72) Inventor Hiromichi Kumagai 1150 Hazawa-machi, Kanagawa-ku, Yokohama, Kanagawa Prefecture Asahi Glass Co., Ltd.

Claims (8)

[Claims] 1. A protein having a cancer metastasis inhibitory activity, which is represented by the amino acid sequence of SEQ ID NO: 1. 2. A gene represented by the nucleotide sequence of SEQ ID NO: 2, which encodes the protein of claim 1. 3. A gene represented by the nucleotide sequence of SEQ ID NO: 3, which encodes the protein of claim 1. 4. A recombinant vector containing the gene according to claim 2 or 3. 5. The recombinant vector is the plasmid pTL2.
The recombinant vector according to claim 4, which is BmI. 6. A transformant capable of producing the protein according to claim 1, which is obtained by transforming a host cell with the recombinant vector according to claim 4 or 5. 7. The host cell is fission yeast Schizosaccharomyces.
The transformant according to claim 6, which is Pombe (Schizosaccharomyces pombe). 8. A method comprising culturing the transformant according to claim 6 or 7, isolating the protein having a cancer metastasis inhibitory activity produced in the culture, and purifying the protein if desired.
The method for producing the protein according to claim 1.