JPH0797998B2 - Novel bioactive polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Field of Industrial Application The present invention relates to a novel bioactive polypeptide, a recombinant plasmid containing a DNA region encoding the polypeptide, a recombinant microbial cell transformed with the plasmid, and The present invention relates to a method for producing a novel bioactive polypeptide using the microbial cell. More specifically, a novel polypeptide having antitumor activity (hereinafter also referred to as a novel antitumor active polypeptide), a recombinant plasmid containing a DNA region encoding the polypeptide, and a set transformed by the plasmid The present invention relates to a modified microbial cell and a method for producing a novel antitumor active polypeptide using the microbial cell.

In the present specification, amino acids and polypeptides are IUPAC-I
It is abbreviated by the method adopted by the UB Biochemistry Committee (CBN), for example, the following abbreviations are used.

Ala L-alanine Arg L-arginine Asn L-asparagine Asp L-aspartic acid Cys L-cysteine Gln L-glutamine Glu L-glutamic acid Gly glycine His L-histidine Ile L-isoleucine Leu L-leucine Lys L-lysine Met L- Methionine Phe L-phenylalanine Pro L-proline Ser L-serine Thr L-threonine Trp L-tryptophan Tyr L-tyrosine Val L-valine The DNA sequence is abbreviated by the type of base contained in each deoxyribonucleotide constituting it. For example, the following abbreviations are used.

A adenine (representing deoxyadenylic acid) C cytosine (representing deoxycytidylic acid) G guanine (representing deoxyguanylic acid) T thymine (representing deoxythymidylate) Further, (H ₂ N)-and -(COOH) indicates the amine terminal side and the carboxy terminal side of the amine acid sequence, respectively, and (5 ')-and (3') indicate the 5'terminal side and the 3'terminal side of the DNA sequence, respectively. Is.

(2) Background of the Invention Carswell et al. Have found that a substance collected from mice after pre-stimulation with Bacillus Calmette-Guerin (BCG) or the like after administration of endotoxin contains a substance that causes hemorrhage and necrosis of cancer due to transplanted Meth A sarcoma. It was found that the substance is contained in the tumor necrosis factor (Tumor Necrosis Facto
r, sometimes abbreviated as TNF below) [EACar
Swell et al., Proc. Natl. Acad. Sci., USA, 72 , 3666 (197
Five)]. This TNF is found in many animals such as mice, rabbits, and humans, and since it works specifically in tumor cells and across species, it has been expected to be used as an anticancer agent.

Recently, Pennica et al. Performed cDNA cleaning of human TNF to elucidate the primary structure of human TNF protein and reported the expression of human TNF gene in Escherichia coli [D. Pennica et al., Nature, 312 , 724]. (1984)]. After that, Shirai et al. [T. Shirai et al., Nature, 313 , 803 (198
5)], Soumura et al. [Soumura et al. Cancer and Chemotherapy, 12 , 160 (198
5)], Wang et al. [AM Wang et al., Science, 228 , 149 (198
5)] and Marmenout et al. [A. Marmenout et al., Eur. J. Bioche
m., 152 , 515 (1985)] successively reported the expression of the human TNF gene in Escherichia coli.

As described above, the use of gene manipulation techniques has made it possible to obtain a large amount of pure human TNF protein, and the physiological activities other than the antitumor activity of TNF have been revealed. For example, cachectin, one of the causes of cachexia in terminal cancer and patients with severe infections, is very similar to TNF [B. Beulter et al., Nature, 316 ,
552 (1985)], because cachectin has lipoprotein lipase inhibitory activity, administration of TNF may increase the amount of triglyceride in blood, and as a result, may cause side effects such as hyperlipidemia. Was suggested. In addition, the effect on vascular endothelial cells [JR
Gamble et al., J. Exp. Med., 162 , 2163 (1985)], bone resorption [DR Beltolini et al., Neture, 319 , 516 (1986)] and the like have been reported.

On the other hand, the recent progress in gene manipulation technology is to replace or add an arbitrary amino acid in a protein with another amino acid,
Or allowed to be deleted. In this way
Many studies have been carried out to modify naturally occurring proteins to create new proteins that serve specific purposes.

Some studies have been conducted on the modification of human TNF protein, and in the amino acid sequence of human TNF protein shown in FIG. 1, one or both of Cys ⁶⁹ and Cys ¹⁰¹ or other amino acid residues of other amino acid residues are used. Replacement (PCT application publication WO8
6/04606, Japanese Patent Application No. 61-106772), substitution of Gly ¹²² with another amino acid residue (Japanese Patent Application No. 61-106772, Japanese Patent Application No. 61-2380)
48), substitution of Ala ¹⁸ with another amino acid residue (Japanese Patent Application No. 61-
233337) has been reported. Also, regarding deletion of amino acid residues on the amino terminal side, TNF with 6 amino acids deleted has cell lifetime activity (JP-A-61-50923),
TNF with 7 amino acid deletion has cytotoxic activity (Japanese Patent Application No. 61-90087), TNF with 1-10 amino acid deletion has cytotoxic activity, and its specific activity is 6-8 amino acids. Maximize in deleted TNF (PCT application publication WO86 / 02381
No.), 10 amino acid-deleted TNF has cytotoxic activity (Japanese Patent Application No. 61-114754), and 11 amino acid-deleted TNF has cytotoxic activity (Japanese Patent Application No. 61-173822). issue),
And, based on TNF with 7 amino acids deleted, Pro ⁸ Ser ⁹ Asp ¹⁰
It has been reported that the substitution with ArgLysArg significantly increases the specific activity.

Therefore, the present inventors have conducted diligent research on modification of human TNF protein for the purpose of improving specific activity, stability, broadening reaction spectrum, and reducing side effects.
The present invention has been completed.

(3) Object of the Invention An object of the present invention is to provide a novel antitumor active polypeptide.

Another object of the present invention is to provide a recombinant plasmid containing a DNA region encoding a novel antitumor active polypeptide.

Still another object of the present invention is to provide a recombinant microorganism transformed with the above recombinant plasmid and a method for producing a novel antitumor active polypeptide using the recombinant microorganism cell.

Still other objects of the present invention will become more apparent from the following description.

(4) According to the configuration present inventors studies invention, the object of the present invention, the following amino acid sequence _{(H 2 N) -Arg-Lys} -Arg-Lys-Pro-Val-Ala-His-V
al-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-L
eu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-L
eu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-A
sn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-T
yr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-G
ly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-L
eu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-S
er-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-A
la-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-P
ro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-G
lu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-L
eu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-I
le-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-G
lu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-Ile-Ile-A
Disclosed is a novel physiologically active polypeptide represented by la-Trp- (COOH) or a polypeptide having Met linked to its amino terminus, and a recombinant plasmid containing a DNA region encoding the novel antitumor active polypeptide. And a recombinant microbial cell transformed by the recombinant plasmid thus obtained, a method for producing a novel antitumor active polypeptide of interest using the microbial cell, and the novel antitumor active polypeptide It has been found to be achieved by providing a pharmaceutical composition containing

The present invention will be described in more detail below.

(A) Cloning of human TNF gene; For the human TNF gene, an appropriate one is selected from several codons designating the amino acids [D. Pennica et al. It can be obtained by synthesizing. When designing the human TNF gene, it is desirable to select the most suitable codon for the host cell to be used, and to provide an appropriate restriction oxygen cleavage site at an appropriate position so that cloning and gene modification can be easily performed later. Is desirable. In addition, the DNA region encoding the human TNF protein preferably has a translation initiation codon (ATG) in a form in which the reading frame is aligned upstream, and a translation in a form in which the reading frame is aligned downstream. It is preferred to have a stop codon (TGA, TAG or TAA). It is particularly preferable to link two or more of the translation stop codons in tandem for the purpose of improving expression efficiency. Furthermore, this human TNF gene can be cloned into an appropriate vector by using the cleavage sites of restriction enzymes acting upstream and downstream thereof. An example of the nucleotide sequence of such human TNF gene is shown in FIG.

To obtain human TNF designed as described above, for each of the upper strand and the lower strand, for example, divide into several oligonucleotides as shown in the second diagram, synthesize them, and synthesize each oligonucleotide. It is desirable to take a method of linking nucleotides. The diester method [HG Khorana, “Some Recent Develo
pments in Chemistry of Phosphate Esters of
Biological Interest ″, John Wiley and Sons, In
c., New York (1961)], triester method [RL Letsin
ger et al., J, Am. Chem. Soc., 89 , 4801 (1967)] and the phosphite method [MD Matteucci et al., Tetrahedron Lett., 21 , 719].
(1980)], but the synthesis by the phosphite method using a fully automatic DNA synthesizer is preferable from the viewpoints of synthesis time, yield, easiness of operation and the like. For purification of the synthesized oligonucleotide, gel filtration, ion exchange chromatography, gel electrophoresis, high performance liquid chromatography using a reverse phase column, etc. can be used alone or in combination as appropriate.

The 5'-terminal hydroxyl group of the thus-obtained synthetic oligonucleotide was phosphorylated with, for example, T4-polynucleotide kinase and then annealed to give, for example, T4.
-Ligate using DNA ligase. As a method for producing a human TNF gene by ligating synthetic oligonucleotides, synthetic oligonucleotides are divided into several blocks and ligated, for example, pBR322 [F. Bolivar et al., Gene, 2 , 95].
(1977)] once cloned into a vector such as
The method of ligating the DNA fragments of each of these blocks is preferable. Block DNA that constitutes such human TNF gene
The fragment-containing plasmid is preferably pTNF1BR or pTNF.
2N or pTNF3 is used.

After ligating the DNA fragments of each block constituting the human TNF gene cloned as described above, and connecting it to the downstream of an appropriate promoter and SD (Shine-Dalgarno) sequence, an expression type gene can be obtained. . Examples of promoters that can be used include tryptophan operon promoter (trp promoter), lactose operon promoter (lac promoter), tac promoter, PL promoter, lpp promoter, and the like, and trp promoter is particularly preferable. The plasmid having the trp promoter is preferably pYS31
N, or pAA41 is used. Furthermore, for the purpose of improving expression efficiency, a terminator that functions efficiently in Escherichia coli can be added to the downstream of the human TNF gene. Examples of such terminator include lpp terminator, trp terminator and the like, but trp A terminator is particularly preferable, and pAA41 is preferably used as a plasmid having trp A terminator. An expression type plasmid can be prepared by cloning this expression type human TNF gene into a vector derived from pBR322, for example. As the human TNF gene expression type plasmid, pTNF 401NN or pTNF 401A is preferably used.

(B) Cloning of a novel antitumor activity polypeptide gene; the human TNF gene-expressing plasmid thus obtained is cleaved with an appropriate restriction enzyme to eliminate a specific region in the human TNF gene, and then an appropriate nucleotide sequence. Gene repair using a synthetic oligonucleotide having By using such a method, an expression type plasmid containing a gene encoding a novel antitumor activity polypeptide in the form of substituting, adding or deleting any amino acid in human TNF protein with other amino acid Can be created. As such a novel antitumor activity polypeptide gene expression type plasmid, pTNF 617 is preferably used.

(C) Confirmation of expression and evaluation of activity; Examples of the microbial host for expressing the human TNF gene and the novel antitumor activity polypeptide gene include Escherichia coli, Bacillus subtilis, yeast and the like. Among them, Escherichia coli (Escherichia coli coli)] is preferred. The human TNF gene-expressing plasmid and the novel antitumor activity polypeptide gene-expressing plasmid can be prepared, for example, by a known method [MV Norgard et al., Gene, 3 , 279 (1978)] using a microbial host such as Escherichia coli C 600r. −
It can be introduced into the m-strain (ATCC 33525).

The recombinant microbial cells thus obtained are cultured by a method known per se. As the medium, for example, M9 medium containing glucose and casamino acid [edited by T. Maniatis et al.,
“Molecular Cloning”, P 440, Cold Spring Harbor
Laboratory, New York (1982)], and it is desirable to add, for example, ampicillin, etc., if necessary. Culturing is carried out under conditions suitable for the target recombinant microorganism, such as shaking aeration and stirring at 37 ° C for 2 to 2
Perform for 36 hours. In addition, a drug such as 3-β-indoleacrylic acid may be added for the purpose of efficiently functioning the promoter at the start of the culture or during the culture.

After culturing, the recombinant microbial cells are collected, for example, by centrifugation, suspended in, for example, a phosphate buffer, the recombinant microbial cells are disrupted by, for example, sonication, and the lysate of the recombinant microbial cells is obtained by centrifugation. The proteins in the obtained lysate are separated by electrophoresis using a polyacrylamide gel containing sodium lauryl sulfate (hereinafter, sometimes abbreviated as SDS), and the proteins in the gel are stained using an appropriate method. The expression of the human TNF gene or the novel antitumor activity polypeptide gene is confirmed by comparing the migration patterns with a lysate of microbial cells containing no expression type plasmid as a control.

The antitumor activity of the human TNF protein and the novel antitumor activity polypeptide thus obtained was evaluated by in vivo activity assay (Carswell et al., Supra) to see the effect of necrosis of Meth A sarcoma transplanted in mice. , In vitro activity assay for cytotoxicity against mouse L cells [Ruff, J. Immunol., 12
6 , 235 (1981)] etc.

Separation and purification of human TNF protein and novel antitumor polypeptide from E. coli lysate may be carried out according to known notification known protein separation and purification methods, but affinity using antibodies against human TNF protein, etc. column·
Chromatography is advantageous. Among them, human TNF
Affinity column chromatography using mouse monoclonal antibodies against proteins and the like is particularly preferable. The use of human TNF protein and novel tumor active polypeptide purified product thus obtained, in
In vivo anti-cancer activity (described above) and side effects can be examined.

The side effects of human TNF protein and novel antitumor polypeptide are evaluated in vitro tr
The method can be carried out by the in vivo method of measuring the lethal dose or the degree of decrease in blood pressure by administering it to experimental animals such as mice.

Thus, according to the present invention, it becomes possible to obtain a novel bioactive polypeptide different from the conventionally known human TNF protein, and by using this novel antitumor active polypeptide, an excellent pharmaceutical composition for antitumor can be obtained. It is now possible to provide.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 (Design of human TNF gene) A human TNF gene having the nucleotide sequence shown in FIG. 1 was designed.
When designing, Pennica et al. [D. Pennica et al., Nature, 312 ,
724 (1984)], based on the base sequence of the structural gene portion of the human TNF precursor cDNA, a cleavage site by an appropriate restriction enzyme is provided at an appropriate position, and a translation initiation codon (ATG) is provided at the 5'side, And 2 translation stop codons (TGA
And TAA) respectively. In addition, a cleavage site by the restriction enzyme Cla I was provided upstream of the 5'side translation initiation codon
It was possible to ligate with the promoter while keeping the D sequence and the translation initiation codon in an appropriate state. Further, a cleavage site by the restriction enzyme Hind III was provided downstream of the 3'side translation stop codon so that it could be easily ligated to the vector / plasmid.

Example 2 (Chemical synthesis of oligonucleotide) The human TNF gene designed in Example 1 is divided into 17 oligonucleotides and synthesized as shown in FIG. Oligonucleotides were synthesized by the phosphite method using a fully automatic DNA synthesizer (Applied Biosystems, model 380A). Purification of the synthetic oligonucleotide was performed according to the manual of Applied Biosystems. That is, by keeping an aqueous ammonia solution containing synthetic oligonucleotides at 55 ° C. overnight,
The protective group of the DNA base is removed, and the high molecular weight synthetic oligonucleotide fraction is separated by gel filtration using Sephadex G-50 Fine Gel (Pharmacia). Then, after polyacrylamide gel electrophoresis containing 7 M urea (gel concentration 20%), the migration pattern is observed by the ultraviolet shadowing method. Cut out the band of the desired size and crush the polyacrylamide gel fragment finely, then add 2-5 ml of elution buffer [5
00 mM NH ₄ OAc-1 mM EDTA-0.1% SDS (pH 7.5)] was added, and the mixture was shaken at 37 ° C. overnight. Target DN by centrifugation
The aqueous phase containing A was recovered. Finally, the solution containing the synthetic oligonucleotide was applied to a gel filtration column (Sephadex G-50) to obtain a purified product of the synthetic oligonucleotide. If necessary, polyacrylamide gel electrophoresis was repeated to improve the purity of the synthetic oligonucleotide.

Example 3 (Cloning of chemically synthesized human TNF gene) 17 synthetic oligonucleotides (TN
The human TNF gene was divided into three blocks and cloned using F-1 to TNF-17).

0.1-1.0 μg of synthetic oligonucleotide TNF-2 to TNF-
The 5'end of 6 is separately phosphorylated with 5 to 15 units of T4-polynucleotide kinase (E. coli B type, Takara Shuzo). Phosphorylation reaction is 10-20μ
50 mM Tris-HCl (pH 9.5), 10 mM MgCl ₂ , 5 mM dithiothreitol, 10 mM ATP aqueous solution at 37 ° C. for 30 minutes. After completion of the reaction, all the synthetic oligonucleotide aqueous solutions are mixed, and the T4-polynucleotide kinase is inactivated and removed by phenol extraction and ether extraction.
Add 0.1-
1.0 μg of synthetic oligonucleotides TNF-1 and TNF-7
Is added, heated at 90 ° C. for 5 minutes, then gradually cooled to room temperature, and annealed. Next, after drying this under reduced pressure,
30 μm 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl ₂ , 10 m
M dithiothreitol, dissolved in 1 mM ATP aqueous solution, 300
Add the unit's T4-DNA ligase (Takara Shuzo) to 11 ℃
The ligation reaction was carried out for 15 hours. After completion of the reaction, polyacrylamide gel electrophoresis (gel concentration 5%) is performed, and the migration pattern is observed by the ethidium bromide staining method. A band having a desired size (about 220 bp) is cut out, and the DNA is recovered from the polyacrylamide gel according to the method of Example 2.

On the other hand, 3 μg of E. coli plasmid pBR322 (about 4.4 Kb
p) 30 μm of 10 mM Tris-HCl (pH 7.5), 60 mM NaCl, 7
Dissolve it in mM MgCl ₂ aqueous solution and add 10 units of restriction enzyme Cla.
Add I (New England Biolabs),
The cleavage reaction was carried out at 37 ° C for 1 hour.

After digestion with the restriction enzyme Cla I, phenol extraction and ether extraction are performed, and the DNA is recovered by ethanol precipitation. This DNA was added to 30 μm of 50 mM Tris-HCl (pH 7.4), 100
It was dissolved in mM NaCl, 10 mM MgSO ₄ aqueous solution, 10 units of restriction enzyme Sal I (Takara Shuzo) was added, and the cleavage reaction was carried out at 37 ° C. for 1 hour. After completion of the reaction, agarose gel electrophoresis (gel concentration 0.8%) is performed and the cleavage pattern is observed by ethidium bromide staining. Plasmid
A band corresponding to a portion of DNA of about 3.7 Kbp containing most of pBR322 was cut out, and the agarose gel fragment was cut in a 3-fold amount (vo.
It was dissolved in an aqueous solution of 8 M NaClO _{4 (} l / wt). Chen et al.'S glass filter method [CW Chen et al., Anal. Biochem. 101 , 339 (1
980)], and a DNA fragment (Cla ISal I) of about 3.7 Kbp was recovered from the agarose gel.

Approximately 220 bp DNA containing part of the previously obtained human TNF gene
The fragment was subjected to terminal phosphorylation reaction according to the method described above, and then approximately 3.7K containing most of the plasmid pBR322.
Mix with bp DNA solution in water. After ethanol precipitation, both DNA fragments were ligated according to the method described above.

For transformation of the Escherichia coli C600r-m- strain, a modified CaCl ₂ method (method by MV Norgard et al.) Was used. That is, 5 ml of L medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.2) was added to Escherichia coli C 6
The 00r-m- strain is inoculated with a culture medium for 18 hours and grown until the turbidity (OD ₆₀₀ ) at ₆₀₀ nm of the culture medium containing the cells reaches 0.3. Add bacterial cells to cold magnesium buffer [0.
1M NaCl, 5 mM MgCl ₂ , 5 mM Tris-HCl (pH 7.6,0
℃)] and washed with 2 ml of cold calcium buffer [100 mM CaCl ₂ , 250 mM KCl, 5 mM MgCl ₂ , 5 mM Tris
-HCl (pH 7.6, 0 ° C)] and left at 0 ° C for 25 minutes. Next, the cells were concentrated to 1/10 of this volume in calcium buffer, and the ligated DNA solution and 2: 1 (vo
l.:vol.) Mix. This mixture was kept for 60 minutes at 0 ° C., then 1 ml of LBG medium (1% tryptone, 0.5% yeast extract, 1%
% NaCl, 0.08% glucose, pH 7.2) and add 1 at 37 ℃
Incubate with shaking for hours. 100 μl of the culture solution in a selective medium [L medium plate containing 30 μg / ml of ampicillin (Sigma)]
Inoculate / plate ratio. The plate is cultured overnight at 37 ° C. to grow the transformant. From the obtained ampicillin-resistant colonies, DNA was prepared by a known method, and the target plasmid pTNF1BR (about 4.0 Kbp) was confirmed to be obtained by agarose gel electrophoresis. In Figure 3,
The method for constructing the plasmid pTNF1BR is shown below.

Synthetic oligonucleotide TNF was prepared by the same method as above.
Plasmid pTNF2N (about 3.1 Kbp) using -8 to TNF-13
Using the synthetic oligonucleotides TNF-14 to TNF-17, plasmid pTNF3 (about 2.4 Kbp) was prepared.
4 and 5 show the method for constructing the plasmids pTNF2N and pTNF3, respectively. The plasmids pTNF1BR, pRNF2N and pTNF3 containing a part of the human TNF gene thus obtained
The fact that the nucleotide sequence of the synthetic oligonucleotide used part of the above is as set is that the Maxam Gilbert method [AMMaxa
M. et al., Methods Enzymol., 65 , 499 (1980)].

Example 4 (Preparation of human TNF gene expression type plasmid) 10 μg of the plasmid pTNF1BR obtained in Example 3 was cleaved with restriction enzymes Cla I and Sal I in the same manner as in Example 3, and polyacrylamide gel electrophoresis (gel concentration) 5%),
According to the method of Example 2, a DNA fragment of about 220 bp (Cla ISal I) containing a part of human TNF gene was recovered from polyacrylamide gel.

Next, 1 μg of 10 μg of the plasmid pTNF2 obtained in Example 3 was used.
00μ of 10mM Tris-HCl (pH 7.5), 60mM NaCl, 7mM
Dissolved in MgCl ₂ aqueous solution, 40 units of restriction enzyme Pvu II
(Takara Shuzo) was added, and the cleavage reaction was carried out at 37 ° C. for 1 hour. Then, according to the method of Example 3, after digestion with a restriction enzyme Sal I and polyacrylamide gel electrophoresis (gel concentration 5%), according to the method of Example 2, about 170 bp containing a part of human TNF gene. DNA fragment (Sal IPvu II) was recovered from polyacrylamide gel.

In addition, 10 μg of the plasmid pTNF3 obtained in Example 3 was also 1
00μ of 10mM Tris-HCl (pH 7.5), 60mM NaCl, 7mM
Dissolved in MgCl ₂ aqueous solution, 40 units of restriction enzyme Pvu II
And 40 units of restriction enzyme Hind III (Takara Shuzo) were added, and a cleavage reaction was carried out at 37 ° C for 1 hour. Then, after polyacrylamide gel electrophoresis (gel concentration 5%), according to the method of Example 2, about 110 containing a part of human TNF gene.
A bp DNA fragment (Pvu IIHind III) was recovered from polyacrylamide gel.

On the other hand, the plasmid pYS31 having the E. coli trp promoter
5 μg of N (about 4.7 Kbp) was cleaved with restriction enzymes Cla I and Hind III in the same manner as above, and after agarose gel electrophoresis (gel concentration 0.8%), the plasmid p was prepared according to the method of Example 3.
A DNA fragment of about 4.7 Kbp containing most of YS31N (Cla IHind
III) was recovered from the agarose gel.

Thus obtained. 220b containing part of human TNF gene
p, about 170 bp and about 110 bp DNA fragments and plasmid pYS
After mixing with a DNA fragment of about 4.7 Kbp containing most of 31N and precipitating with ethanol, a ligation reaction with T4-DNA ligase was performed according to the method of Example 3. After completion of the reaction, it was introduced into the Escherichia coli C 600r-m- strain according to the method of Example 3, and a clone having the desired human TNF gene expression type plasmid pTNF401NN (about 5.2 Kbp) was selected from the transformants. did. FIG. 6 shows the method for constructing the plasmid pTNF 401NN.

In addition, 5 μg of the above plasmid pYS31N was partially digested with the restriction enzyme Pvu II according to the method described above, and then further digested with the restriction enzyme.
Cleaved with Hind III and subjected to agarose gel electrophoresis (gel concentration
0.8%), and according to the method of Example 3, a DNA fragment of about 2.7 Kbp containing the trp promoter [Pvu II (2) Hind II
I] was recovered from the agarose gel.

Next, an oligonucleotide having the base sequence shown in FIG. 7 was synthesized and purified according to the method of Example 2. 0.5 μg of each of the two synthetic oligonucleotides obtained was subjected to terminal phosphorylation according to the method of Example 3. After annealing, the DNA fragment of about 2.7 Kbp [Pvu II (2) Hind III] obtained above was mixed and ethanol-precipitated, followed by ligation reaction with T4-DNA ligase according to the method of Example 3. I did. After completion of the reaction, it was introduced into Escherichia coli C600r-m- strain according to the method of Example 3,
Depending on the transformant, the desired plasmid pAA41 (about 2.7 Kb
The clone with p) was selected. Such a plasmid is a multi-copy, high-efficiency expression vector in which E. coli trp A terminator is added to the downstream of the cloning site existing in the trp promoter, which is removed from the plasmid pYS31N in the copy number control region. Fig. 7 shows the method of preparation.

2 μg of this plasmid pAA41 was cleaved with restriction enzymes Cla I and Hind III in the same manner as above, and after agarose gel electrophoresis (gel concentration 0.8%), according to the method of Example 3,
A DNA fragment of about 2.7 Kbp containing most of the plasmid pAA41 (Cl
a IHind III) was recovered from the agarose gel.

In addition, the previously obtained human TNF gene expression type plasmid pTN
F 401NN 5 μg was treated with the restriction enzymes Cla I and Hind in the same manner as above.
After cutting with III and polyacrylamide gel electrophoresis (gel concentration 5%), according to the method of Example 2, a DNA fragment of about 490 bp containing the entire human TNF gene (Cla IHind III).
Was recovered from the polyacrylamide gel.

Thus obtained, containing most of the plasmid pAA41,
Approximately 490 bp D including 2.7 Kbp DNA fragment and entire human TNF gene
After mixing with the NA fragment and ethanol precipitation, the ligation reaction with T4-DNA ligase was performed according to the method of Example 3. After the reaction was completed, it was introduced into Escherichia coli 600r-m-strain according to the method of Example 3, and a clone having the desired plasmid pTNF 401A (about 3.2 Kbp) was selected from the transformants. This plasmid has the ability to more efficiently express the human TNF gene, and its construction method is shown in FIG.

Example 5 (Preparation of novel anti-tumor activity polypeptide gene expression type plasmid) Human TNF gene expression type plasmid pTN obtained in Example 4
20 μg of F 401A was added to the restriction enzyme Cla according to the method of Example 4.
After cutting with I and Hind III, polyacrylamide gel electrophoresis (gel concentration 5%) and agarose gel electrophoresis (gel concentration 0.8%), two DNAs produced according to the methods of Examples 2 and 3, respectively. Fragment (about 490 bp and about 2.7 Kb
p, both Cla IHind III) were recovered from the gel.

Approximately 490 bp DNA containing the entire human TNF gene obtained here
50 μm of the fragment was added to 10 mM Tris-HCl (pH 7.4), 10 mM MgSO 4.
_It was dissolved in a 4.1 mM dithiothreitol aqueous solution, 10 units of restriction enzyme Hap II (Takara Shuzo) was added, and a cleavage reaction was carried out at 37 ° C for 1 hour. After completion of the reaction, polyacrylamide gel electrophoresis (gel concentration 5%) was performed, and according to the method of Example 2, about 390 bp D containing most of the human TF gene was obtained.
The NA fragment (Hap IIHind III) was recovered from polyacrylamide gel.

An oligonucleotide having the nucleotide sequence shown in FIG. 9 was synthesized and purified according to the method of Example 2. 0.5 μg of each of the four synthetic oligonucleotides obtained was subjected to terminal phosphorylation according to the method of Example 3, annealed, and then ligated with T4-DNA ligase.

After completion of the reaction, the obtained double-stranded oligonucleotide was converted into a DNA fragment of about 2.7 Kbp (Cla IHind III) obtained above and a DNA fragment of about 390 bp (Hap I containing most of human TNF gene).
IHind III) and mixed with ethanol, followed by ligation reaction with T4-DNA ligase according to the method of Example 3. After the reaction was completed, it was introduced into Escherichia coli C 600r-m-strain according to the method of Example 3, and a clone having the desired plasmid pTNF471 (about 3.2 Kbp) was selected from the transformants. This plasmid has the following amino acid sequence _{(H 2 N) -Arg-Lys} -Arg-Lys-Pro-Val-Ala-His-V
al-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-L
eu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-L
eu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-A
sn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-T
yr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-G
ly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-L
eu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-S
er-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-A
la-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-P
ro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-G
lu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-L
eu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-I
le-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-G
lu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-Ile-Ile-A
It is an expression-type plasmid encoding an antitumor polypeptide represented by la-Leu- (COOH) or a polypeptide having Met bound to its amino terminus, and its construction method is shown in FIG.

On the other hand, the human TNF gene expression type plasmid p obtained above was used.
20 μg of TNF 471 was added to the restriction enzyme H according to the method of Example 4.
After cutting with ind III, 50 mM Tris-HCl (pH 7.4), 100
The cleavage reaction with the restriction enzyme Nco I (Takara Shuzo) is performed at 37 ° C. for 1 hour in an aqueous solution of mM NaCl and 10 mM MgSO ₄ . After completion of the reaction, agarose gel electrophoresis (gel concentration 0.7%) and polyacrylamide gel electrophoresis (gel concentration 5%) were performed, and according to the method of Example 2, a DNA of about 140 bp containing a part of the human TNF gene. Fragments (Nco IHind III) from polyacrylamide gel and according to the method of Example 3,
A DNA fragment of approximately 3.0 Kbp containing most of pTNF 471 (Nco IHi
nd III) was recovered from the agarose gel.

In addition, the approximately 140 bp DNA fragment obtained above (Nco IHind
III) 50 μm of 10 mM Tris-HCl (pH 7.4), 10 mM MgS
The product was dissolved in O ₄ , 1 mM dithiothreitol aqueous solution, 10 units of restriction enzyme Acc I (Takara Shuzo) was added, and the cleavage reaction was carried out at 37 ° C. for 1 hour. After completion of the reaction, polyacrylamide gel electrophoresis (gel concentration 8%) is performed. According to the method of Example 2, a DN of about 110 bp containing a part of the human TNF gene
The A fragment (Nco I Acc I) was recovered from polyacrylamide gel.

Further, an oligonucleotide having the base sequence shown in FIG. 10 was synthesized and purified according to the method of Example 2. 0.5 μg of each of the obtained two synthetic oligonucleotides was subjected to terminal phosphorylation and annealing according to the method of Example 3.

After annealing, the obtained double-stranded oligonucleotide was added to the previously obtained DNA fragment of about 3.0 Kbp (Nco IHind II
I) and a DNA fragment of about 110 bp (Nc
o IAcc I) and ethanol precipitation followed by Example 3
The ligation reaction with T4-DNA ligase was performed according to the method described in 1. After the reaction was completed, it was introduced into the Escherichia coli C 600r-m-strain according to the method of Example 3, and a clone having the plasmid pTNF617 (about 3.2 Kbp) was selected from the transformants. This plasmid has the following amino acid sequence _{(H 2 N) -Arg-Lys} -Arg-Lys-Pro-Val-Ala-His-V
al-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-L
eu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-L
eu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-A
sn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-T
yr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-G
ly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-L
eu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-S
er-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-A
la-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-P
ro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-G
lu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-L
eu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-I
le-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-G
lu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-Ile-Ile-A
la-Trp- (COOH) is a novel antitumor polypeptide gene-expressing plasmid that encodes a novel antitumor polypeptide represented by la-Trp- (COOH) or a polypeptide in which Met is bound to its amino terminus. I showed you how to create it.

Example 6 (Confirmation of expression) Human TNF gene expression type plasmid pTNF 401A obtained in Example 4 and expression type plasmid pTN obtained in Example 5
Escherichia coli C 600r having F471 or a novel antitumor activity polypeptide gene expression type plasmid pTNF617
-M- strain was used in M9 medium [0.6% Na ₂ containing 30 to 50 μg / ml ampicillin, 0.2% glucose and 4 mg / ml casamino acid].
HPO ₄ −0.3% K ₂ HPO ₄ −0.05% NaCl−0.1% NH ₄ Cl aqueous solution (p
H7.4) is autoclaved, and then autoclaved MgSO ₄ aqueous solution and CaCl ₂ aqueous solution are added separately to a final concentration of 2 mM and 0.1 mM, respectively. ] 250 ml was inoculated and shake culture was carried out at 37 ° C until the OD ₆₀₀ reached 0.7. Then, 3-β-indoleacrylic acid having a final concentration of 50 μg / ml was added to the culture medium, and shaking culture was further continued at 37 ° C. for 12 hours.

After collecting the Escherichia coli cells by centrifugation, the cells were washed with PBS buffer (20 mM phosphate buffer containing 150 mM NaCl, pH 7.4). Wash the cells with 10 ml of PB
Suspend in S buffer, ultrasonic generator (Kubota, 200M
After destroying the bacterial cells by using the (type), the bacterial cell residue was removed by centrifugation.

Tris-HCl was added to a part of the obtained E. coli lysate.
Buffer (pH 6.8), SDS, 2-mercaptoethanol,
Glycerol, final concentration 60 mM, 2%, 4%, 10%, respectively
In addition, SDS-polyacrylamide gel electrophoresis [Suzuki, K., 31 , 43 (1977)] was performed. The separation gel was 15%, and the migration buffer was SDS, Tris-glycine system [UK Laemmli, Nature, 227 , 680 (1970)]. After completion of the electrophoresis, the protein in the gel was stained with Coomassie-R-250 (Bio-Rad) to confirm the expression of human TNF gene and novel antitumor activity polypeptide gene. A copy of part of the results is shown in FIG.

In addition, the stained gel is a chromatographic scanner (Shimadzu, CS
-930 type), the ratio of the produced antitumor active polypeptide in the Escherichia coli cytoplasmic protein was calculated. As a result, human TNF gene expression type plasmid pTNF 40
About 16.2% of total cytoplasmic proteins in E. coli with 1A
, About 23.2% of total cytoplasmic proteins were produced in Escherichia coli having the expression type plasmid pTNF 471, and about 18.7% of the antitumor activity polypeptide was also produced in Escherichia coli having the novel expression type plasmid pTNF 617. However, each was recognized.

Example 7 (Evaluation of activity) The in vitro anticancer activity measurement of the novel antitumor activity polypeptide was carried out according to the method of Ruff. That is, 100 μl of the material E. coli lysate containing the novel antitumor active polypeptide obtained in Example 6 was serially diluted with a medium and 4
× 10 ⁵ cells / ml mouse concentration L-929 fibroblasts (ATCC C
CL-929) suspension circles 100 μm were mixed in a 96-well tissue culture microplate (coaster). At this time,
Actinomycin D (cosmegen, final concentration 1 μg / ml)
(Manyu Pharmaceutical) is added. As a medium, 5% (vol /
vol) Eagle's minimum essential medium containing fetal bovine serum (Nissui Pharmaceutical) was used. After culturing the above microplate in air containing 5% carbon dioxide at 37 ° C for 18 to 20 hours, crystal violet solution [5%
(Vol / vol) 0.5% (wt / vol) crystal violet dissolved in an aqueous methanol solution] was used to stain the living cells. After washing away excess crystal violet and drying, remove the remaining crystal violet 10 times.
Extract with 0μ 0.5% SDS aqueous solution, and measure the absorbance at 595nm with an ELISA analyzer (Toyo Sokki, ETY-96 type). This absorbance is proportional to the number of surviving cells.
Therefore, the dilution ratio of E. coli lysate corresponding to 50% of the absorbance of the control without the addition of the diluted solution of E. coli lysate containing the antitumor activity polypeptide, etc. was determined by a graph (for example, FIG. 12), and the dilution ratio was calculated as a unit. It is defined as From FIG. 12, 100 μl of E. coli lysate containing human TNF protein encoded by expression type plasmid pTNF 401A
Is an expression-type plasmid with an activity of about 3.3 × 10 ⁶ units.
E. coli lysate containing the antitumor active polypeptide encoded by pTNF 471 100 μ has an activity of about 3.7 × 10 ⁷ units, and the E. coli lysate containing the novel antitumor active polypeptide encoded by the expression plasmid pTNF617.
It was revealed that 100μ each had an activity of about 4.0 × 10 ⁷ units.

The total amount of protein contained in each of the various E. coli lysates obtained in Example 6 was determined by the protein assay kit (Bio-
Rad), and calculated from a calibration curve using bovine serum albumin. When the specific activity of the antitumor active polypeptide or the like was calculated from the expression level, activity value and protein quantification result obtained above, the values shown in Table 1 were obtained. From Table 1, the novel antitumor activity polypeptide encoded by pTNF617 has about 12 times the specific activity of human TNF protein, and pTNF617
It can be seen that it has about 1.4 times the specific activity of the antitumor polypeptide encoded by F471.

[Brief description of drawings]

Figure 1 shows the base sequence of the designed human TNF gene, and Figure 2 shows the base sequence of chemically synthesized synthetic oligonucleotides.
These are shown respectively. 3, 4, and 5 show plasmid pTNF1BR, p containing a part of human TNF gene.
The methods for preparing TNF2N and pTNF3 are shown respectively. Figure 6 shows the human TNF gene expression plasmid pTNF 401N.
FIG. 7 shows the method for constructing N, the method for constructing the expression vector pAA41, and FIG. 8 shows the plasmid for expressing the human TNF gene expression type pTN.
It shows how to make the F 401A, respectively. 9th
The figure shows the antitumor activity polypeptide gene expression type plasmid pT.
It shows how to create NF 471. Fig. 10 shows a novel antitumor activity polypeptide gene expression type plasmid pTNF617
It shows how to create. FIG. 11 shows the results of confirming the expression of human TNF gene and novel antitumor activity polypeptide gene. FIG. 12 shows the results of in vitro anticancer activity measurement of human TNF protein and novel antitumor active polypeptide.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12N 15/09 // (C12P 21/02 C12R 1:19) (72) Inventor Kazuo Kitai Tokyo 4-3-2 Asahigaoka, Hino City, Teijin Ltd., Institute of Biotechnology (72) Inventor Masami Fukuoka 4-3-2, Asahigaoka, Hino City, Teijin Ltd., Institute of Biotechnology (72) Inventor, Yataro Ichikawa Tokyo 4-3-2 Asahigaoka, Hino City Teijin Limited Biotechnology Research Institute

Claims (10)

[Claims] 1. The following amino acid sequence (H ₂ N) -Arg-Lys-Arg-Lys-Pro-Val-Ala-His-V
al-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-L
eu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-L
eu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-A
sn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-T
yr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-G
ly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-L
eu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-S
er-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-A
la-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-P
ro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-G
lu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-L
eu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-I
le-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-G
lu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-Ile-Ala-T
A novel bioactive polypeptide represented by rp- (COOH). 2. The polypeptide according to claim 1, wherein Met is bound to the amino terminus. 3. The following amino acid sequence (H ₂ N) -Arg-Lys-Arg-Lys-Pro-Val-Ala-His-V
al-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-L
eu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-L
eu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-A
sn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-T
yr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-G
ly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-L
eu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-S
er-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-A
la-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-P
ro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-G
ln-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-L
eu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-I
le-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-G
lu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-Ile-Ile-A
D encoding a novel bioactive polypeptide represented by la-Trp- (COOH) or a popipeptide having Met bound to its amino terminus
Recombinant plasmid containing NA region. 4. The DNA region has the following base sequence (5 ')-CGTAAGCGCAAGCCTGTAGCCCATGTTGTAGCAAACCCTC.
AAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTGCTG
GCCAATGGCGTGGAGCTGAGAGATAACCAGCTGGTGGTACCATCAGAGGG
CCTGTACCTCATCTACTCCCAGGTCCTCTTCAAGGGCCAAGGCTGCCCGT
CGACCCATGTGCTCCTCACCCACACCATCAGCCGCATCGCCGTCTCCTAC
CAGACCAAGGTCAACCTCCTCTCTGCGATCAAGAGCCCCTGCCAGAGGGA
GACCCCAGAGGGGGCTGAGGCCAAGCCATGGTATGAGCCCATCTATCTGG
GAGGGGTCTTCCAGCTGGAGAAGGGTGACCGACTCAGCGCTGAAATCAAT
CGGCCCGACTATCTCGACTTTGCCGAGTCTGGGCAGGTCTACTTTGGGAT
4. The plasmid according to claim 3, which contains a double-stranded DNA consisting of a single-stranded DNA represented by TATTGCCTGG- (3 ') and a complementary single-stranded DNA. 5. The DNA region has the following base sequence (5 ')-CATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTT.
GACAATTAATCATCGAACTAGTTAACTAGTACGCAAGTTCACGTAAAAAG
GGTATCGATAATGCGTAAGCGCAAGCCTGTAGCCCATGTTGTAGCAAACC
CTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTG
CTGGCCAATGGCGTGGAGCTGAGAGATAACCAGCTGGTGGTACCATCAGA
GGGCCTGTACCTCATCTACTCCCAGGTCCTCTTCAAGGGCCAAGGCTGCC
CGTCGACCCATGTGCTCCTCACCCACACCATCAGCCGCATCGCCGTCTCC
TACCAGACCAGGTCAACCTCCTCTCTGCGATCAAGAGCCCCTGCCAGAGG
GAGACCCCAGAGGGGGCTGAGGCCAAGCCATGGTATGAGCCCATCTATCT
GGGAGGGGTCTTCCAGCTGGAGAAGGGTGACCGACTCAGCGCTGAAATCA
ATCGGCCCGACTATCTCGACTTTGCCGAGTCTGGGCAGGTCTACTTTGGG
The plasmid according to claim 3, which comprises a double-stranded DNA consisting of a single-stranded DNA represented by ATTATTGCCTGGTGATAAGCTT- (3 ') and a single-stranded DNA complementary thereto. 6. The plasmid according to claim 3, which is the plasmid pTNF617. 7. The following amino acid sequence (H ₂ N) -Arg-Lys-Arg-Lys-Pro-Val-Ala-His-V
al-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-L
eu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-L
eu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-A
sn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-T
yr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-G
ly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-L
eu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-S
er-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-A
la-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-P
ro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-G
lu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-L
eu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-I
le-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-G
lu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-Ile-Ile-A
D encoding a novel physiologically active polypeptide represented by la-Trp- (COOH) or a polypeptide having Met bound to its amino terminus
A recombinant microbial cell transformed with a recombinant plasmid containing an NA region. 8. The microbial cell is Esch.
erichia coli), The microbial cell according to claim 7. 9. The following amino acid sequence (H ₂ N) -Arg-Lys-Arg-Lys-Pro-Val-Ala-His-V
al-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-L
eu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-L
eu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-A
sn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-T
yr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-G
ly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-L
eu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-S
er-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-A
la-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-P
ro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-G
lu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-L
eu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-I
le-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-G
lu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-Ile-Ile-A
D encoding a novel physiologically active polypeptide represented by la-Trp- (COOH) or a polypeptide having Met bound to its amino terminus
Characterized by culturing a recombinant microbial cell transformed with a recombinant plasmid containing an NA region, producing and accumulating a novel bioactive polypeptide in the culture, and isolating the novel bioactive polypeptide from the obtained culture And a method for producing a novel bioactive polypeptide. 10. An antitumor effective amount of the following amino acid sequence (H ₂ N) -Arg-Lys-Arg-Lys-Pro-Val-Ala-His-V.
al-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-L
eu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-L
eu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-A
sn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-T
yr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-G
ly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-L
eu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-S
er-Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-A
la-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-P
ro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-G
lu-Pro-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-L
eu-Glu-Lys-Gly-Asp-Arg-Leu-Ser-Ala-Glu-I
le-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-G
lu-Ser-Gly-Gln-Val-Tyr-Phe-Gly-Ile-Ile-A
A pharmaceutical composition comprising a novel physiologically active polypeptide represented by la-Trp- (COOH) or a polypeptide having Met bound to its amino terminus.