JPH0387196A - Novel physiologically active polypeptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention <Industrial Application Field> The present invention relates to a novel physiologically active polypeptide, a silk recombinant plasmid containing a DNA region encoding the polypeptide, and a recombinant microbial cell transformed with the plasmid. and a method for producing a novel physiologically active polypeptide using the microbial cells. More specifically, a novel polypeptide having antitumor activity (hereinafter sometimes abbreviated as novel antitumor activity polypeptide), a D
The present invention relates to a silk recombinant plasmid containing an NA region, a recombinant microbial cell transformed with the plasmid, and a method for producing a novel antitumor active polypeptide using the microbial cell.

In this specification, amino acids and polypeptides are IUPA
(, - shall be abbreviated according to the method adopted by the IUB Committee on Biochemistry (CBN), for example, the following abbreviations are used.

AIaL-Alanine AroL-Arginine ASnL-Asparagine ASp L-Aspartic acid Cys l--Cystine Gln L-Glutamine GluL-Glutamic acid Gll/ Glycine 1-+1sL-Hisdecine 1el--Isoleucine LOUL-Leucine LVSL-Lysine Met L-Methionine PheL -Phenylalanine prol--Proline 3erl--Serine Thrl--Threonine TrpL-t-Rip1-Juan Tyr L-Dyrosine Vat L-valine Also, the sequence of DNA is contained in each deoxyribonucleo polypeptide that constitutes it. It shall be abbreviated by type, for example, the following abbreviations will be used.

A Adenine (represents deoxyadenylic acid) C Cytosine (represents deoxycytidylic acid) G Guanine (represents deoxyguanylic acid) T Amino (represents deoxythymidylic acid) Furthermore, (t12N) - and -( C.O.
011) indicate the amino terminal side and carboxy terminal side of the amino acid sequence, respectively, and (5'〉- and (3')
') indicate the 5' end and 3' end of the DNA sequence, respectively.

BACKGROUND OF THE INVENTION CarSWell et al.
It was discovered that serum collected after administering endotoxin to mice previously stimulated with mette-Quer+n (BCG) contained a substance that causes hemorrhagic necrosis of transplanted MethA sarcoma cancers. Necrosis factor
sisFactor (hereinafter sometimes abbreviated as TNF) [E, A. Carswell et al., procJJ atl.

8cad, Sci, USA, 72.366
6 (1975)], this TNF is found in many animals such as mice, rabbits, and humans, and is specific to tumor cells.
Moreover, since it works across species, it has been expected to be used as an anticancer drug.

Recently, Penn1Ca et al.
We performed CDNA cloning to clarify the primary IN4 structure of the human TNF protein, and reported the expression of the human TNF protein in Escherichia coli [D
, P ennica et al., Nature ,
312. 724 (1984)]. after that,
Jifu et al. [T, 5hirai et al.

Nature, 313. 803 (1985)
], Munetoshi et al. [Tadahan et al., Cancer and Chemotherapy, 12. 16
0 (1985)], Wan (1 et al. [A, M, W
anc+ et al., 3 cicnce, 228. 14
9 (1985)] and Marmenout et al.
, M armenout et al.

Eur, J. Biochem, 152. 51
5 (1985)] subsequently reported on the expression of the human 1-TNF1 gene in Escherichia coli.

As described above, by using genetic engineering techniques, it has become possible to obtain pure human TNF protein in large quantities, and physiological activities other than the antitumor activity of TNF are becoming clear. For example, Kakekujin, which is one of the causes of cachexia seen in patients with terminal cancer or severe infections, is very similar to TNF [13, 3 eulte.
r et al., Nature.

316, 552 (1985)], since kakekudin has riboprotein lipase inhibitory activity,
It was suggested that administration of TN' increases the amount of 1-liglycerides in the blood, which may result in side effects such as hyperlipidemia.
Effects on vascular endothelial cells [J, R.

Gamble et al., J. Exp. Med.
162.2163 (1985)], bone resorption FD
, R. Beltolini et al., Nature.
319. 516 (1986)] etc. have been reported.

On the other hand, recent advances in genetic engineering technology have made it possible to substitute, add, or delete any amino acid in a protein with another amino acid.

In this way, many studies are underway to create new proteins that serve specific purposes by modifying naturally occurring proteins.

Several studies have also been conducted on the modification of human TNF protein, and in the amino acid sequence of human TNF protein shown in Figure 1, substitution of either or both of Cvslf and CyS/'/ with other amino acid residues has been carried out. (PCT Application Publication No. W086/04606, Patent Application No. 61106772
), substitution of G IV"' with other amino acid residues (Japanese Patent Application No. 106772/1982, Japanese Patent Application No. 238048/1982).

Substitution of Ala18 with other amino acid residues (Patent application 1986)
No. 33337) has been reported. In addition, regarding the deletion of amino acid residues on the amino terminal side, 6 amino acid deletion TN
F has cytotoxic activity (Japanese Patent Application Laid-open No. 61-50
923), 7 amino acid deletion - "N1: has cytotoxic activity (Japanese Patent Application No. 61-90087), 1-
10 amino acid deleted TNF has cytotoxic activity,
The specific activity is maximized in 6-8 amino acid deleted TNF (PCT Application Publication No. WO 36/02381), and 10 amino acid deleted TNF has cytotoxic activity (Japanese Patent Application No. 114754/1982). It has been reported that 11 amino acid deletion TNF has cytotoxic activity (Japanese Patent Application No. 173822/1982).

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

<Object of the Invention> An object of the present invention is to provide a novel anti-111i tumor active polypeb 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 method for producing a novel antitumor active polypeptide using a recombinant microbial cell and a recombinant microorganism transformed with the above recombinant plasmid. .

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

<Configuration of the Invention> According to the research of the present inventors, the object of the present invention is to obtain the following amino acid sequence (H2N) -Pro-8er-ASII-LysP
ro -V al -A la-mouth 1s-V al -
V at -A laA sn -Pro-G In
-A Ia -G Iu -G ly -G InL
eu-G 1n-T rp-L eu-A 5n-A
rO-A r.

A Ia-Asn -A 1a-L eu -L eu
-A 1a-AsnG 1y-V al -G Iu
-L eu -A ro -A 5n-A 5nSer
・-L eu-Val-Vat-Pro-8er-Gl
u -G ly-Leu-Tyr-L eu -I
1e-Tyr-3erG 1n-Val-Leu-Ph
e-L'/S-G +y-G InG my -CVs
-Pro -Ser -T hr-mouth 1s-Va
Leu -L eu -Thr -l-1is -Th
r -I le -5erA rQ-I Ie-A
la-Val-3er-Tyr-G InT hr-
L vS-V al-85n-l-eu -1-cu-
3crA la-11e-lys-3er-Pro-C
VS-G InA ro -G lu -T hr -
Pro -G lu -G ly-81aG Iu-
A la-Lys-Pro-Trp-Tyr-
G luP ro-I Ie-Tyr-Leu-G
ly-G ly-VaP he-G In-L eu
-G Iu -L Vs -G ly -A 5pAr
o-Leu-3er-A 1a-G lu -I 1
e-AsnA rq-Pro-Asp-'Tyr-Le
u-Asp-PheA Ia-G IU-5er-G
Iy-G In-Val-TyrPhe-Gly
-I Ie-I le-81a-phe (Co○11
The present invention includes a novel anti-tumor active polypeptide represented by the following formula or a polypeptide having Met bound to its amino terminus. In addition, D
Includes recombinant plasmids containing the NA region.

The following base sequence (5')-C as a recombinant plasmid
CGAGTGAC AAGCCTGTAGCCCATGTTGTA G C
A A A CCC-r CA A G CT G A
G GG G CA G CT' CCA G T
G G CT G to ACCG CCG G G CCA
A ”r G CCCT G CTGGCC8ATG
to CGTGGAGCTGAGAAACAACTCACT
GGTGGT8CC AT CA G A G G
G CCT G T'ΔCCTCATCTACTCCC
AGGTCCTCTTCAAGGGCCAAGGCTG
CCCGTCGACCCATGTGCTCCTCACC
CACACCATCAGCCGCATCGCCGT
CT CCT A CC GACCAAAGGTCAAC
CTCCTCTCTGCGATCAAGAGCCCCT
GCCAGAGGGGAGACCCCAGAGGGGGGC
TGAGG CCA A G CCA T G G T
TOTGAGCCCA T CT AT CT G G
GA G G GG T CTTCCAGCTGGA
GAAGGGTGACCGACTCAGCGCTG8A
ATCAATCGGCCCGACTATCTCGACT
To T T G CG A G T CT G G G
CA G G T CTACTTTGGGATTAT
Single-stranded DNA represented by TGCCTTC-(3') and its complementary single-stranded DNA
Examples include plasmids containing double-stranded DNA that differ from A.

Or to the next base sequence (5')-C TCATAACGG TTCTGGCAAATATTCTGAAATG GC
G to TGTTGACAATTAATCATCGAACT
TTAACTAGTACGCAAGTTCACGTAA
AAAGGGTATCGATATGCCGAGTGAC
To A G CCT G T A G CCCA T G
T T G -rGCAAACCCTCAAGCT
GAGGG G CA G CT CCA G T G
G CT G A A CCGCCGGGCCA8T
GCCCTGCTGCCAATGGCGTGGAGC
TGAGAAACAACTCACTGGTGGTACC
ATCAGAGGGGCCTGTACCTCATCTAC
TCCCAGGTCCTCTTCAAGGGCCAAG
GCTGCCCGTCGACCCATGTGCTCCT
CACCCACACCATCAGCCGCATCGCC
GTCTCCTACCAGACCCAAGGTCAACC
TCCTCTCTGCGATCAAGAGCCCTG
To CCAG to GGGAGACCCC to GAGGGGGCT
GAGGCCAAGCCATGGTATGAGCCCA
TCTATCTGGGGAGGGGTCTTCCAGCT
GGAGAAGGGGTGACCGACTCAGCGCT
GAAATCA8TCGGCCCGACTATCTCG
ACTTTGCCGAGTCTGGGCAGGTCTA
CTTTGGGATTATTGCCTTCTGΔTAA
Examples include plasmids containing double-stranded DNA consisting of a single-stranded DNA represented by GCTT-(3') and a single-stranded DNA complementary thereto.

A more specific example is plasmid pTNF641.

The present invention includes a recombinant microbial cell transformed with a recombinant plasmid containing a DNA region encoding the novel physiologically active polypeptide described above or a polypeptide having Met bound to its amino terminus. Here the microbial cells are Escherichia co
li) is preferred.

The present invention involves culturing recombinant microbial cells transformed with a recombinant plasmid containing a DNA region encoding the above-mentioned novel physiologically active polypeptide or a polypeptide having Met bound to its amino terminus. The present invention includes a method for producing and accumulating a bioactive polypeptide, producing and accumulating a novel bioactive polypeptide from the obtained culture, and producing a novel bioactive polypeptide from the obtained culture.

The present invention includes a pharmaceutical composition containing an antitumor-effective amount of the above-mentioned novel physiologically active polypeptide or a polypeptide having Met bound to its amino terminus.

The present invention will be explained in more detail below.

(A) Cloning of human TNF gene; human TNF31
One child is an amino acid [D,
It can be obtained by selecting an appropriate codon from among several codons specifying 1 and chemically synthesizing it. When designing the human TNF gene, it is desirable to select codons that are most suitable for the host cell used, and to provide cleavage sites with appropriate restriction molecules at appropriate positions to facilitate subsequent cloning and genetic modification. This is desirable.

Furthermore, the DNA region encoding the human TNF protein preferably has a translation initiation codon (ATG) with matching reading frames upstream thereof, and a translation initiation codon (ATG) with matching reading frames in the downstream direction. Stop codon (TGA).

T, AG or TAA) is preferred.

The above translation stop codon is used to improve expression efficiency.
It is particularly preferred to connect two or more in tandem.

Furthermore, this human TNFI gene can be cloned into an appropriate vector by using the cleavage sites of restriction enzymes that act upstream and downstream thereof. An example of the base sequence of such a human TNF gene is shown in FIG.

To obtain the human TNF311 gene designed as above,
For each of the upper chain and lower chain, for example, the second
It is preferable to separate the oligonucleotides into several oligonucleotides as shown in the figure, chemically synthesize them, and then link each oligonucleotide together. The synthesis method for each oligonucleotide is the diester method [1-1, G, Kho
rana.

“Some Recent [) evelopm
ents in Chemistry of p
hosphate E 5ters of3 io
Logical Interest”, John
Wileyand 3ons, Inc.
New York (1961)].

Triester method [R,L, Letsinger et al., J
Am, Chem, Soc,, 89.480
1 (1967)] and the phosphite method [M, D,
Matteucci et al.

Tetrahedron Lett,, 21. 7
19 (1980)], but from the viewpoint of synthesis time, yield, simplicity of operation, etc., synthesis by the phosphite method using a fully automatic DNA synthesis machine is preferable. To purify the synthesized oligonucleotide, gel filtration, ion-exchange chromatography, gel electrophoresis, high-performance liquid chromatography using a reversed phase column, etc. can be used individually or in combination as appropriate.

The hydroxyl group at the 5' end of the synthetic oligonucleotide thus obtained is phosphorylated using, for example, T4-polynucleotide kinase, and then ligated using an annealing process, such as T4-DNA ligase. A method for constructing the human TNF gene by linking synthetic oligonucleotides is to divide the synthetic oligonucleotides into several blocks and link them, for example, pBR322[F, B
olivar et al., Qene, 2. 95<
1977)], a method is preferred in which the DNA fragments of each block are ligated after cloning into a vector. A plasmid containing a DNA fragment of a block constituting the human 1 to TNF gene is preferably pTNFIBR.

pTNF2N or pTNF3 is used.

After ligating the DNA fragments of each block constituting the human-TNI gene,
By ligating downstream of an appropriate D'E-tar or SD (cytoine-Dalgarno) sequence, it can be made into an expressable gene. As a usable promoter, 1
-Lyptophan operon promoter (trp promoter).

Lactose operon promoter (lac promoter), tac promoter, PL promoter,
Examples include the lpp promoter, but especially the tr
The p promoter is preferred. As a plasmid with a trp promoter, preferably IIY S 31N
, or I) A A 111 is used.

Furthermore, for the purpose of improving expression efficiency, it is possible to add a terminator that functions efficiently in E. coli downstream of the Hi-T N gene. Examples of such terminators include the 1pp terminator, the trp-hataheminator, etc. Among these, the TRP A terminator is particularly preferred, and 11A A41 is preferably used as a plasmid containing the TRP A terminator.
By cloning into the derived vector, an expression plasmid can be created. As a human TNF gene expression plasmid, preferably pTNF401NN or 11T
NF 401A is used.

(B) Cloning of a novel antitumor active polypeptide gene; The human TNFW Nobuko expression plasmid thus obtained was cut with an appropriate restriction enzyme to remove a specific region within the human TNF3i gene, and then an appropriate base Gene repair is performed using synthetic oligonucleopolypeptides having the sequence. By using such a method, an expression plasmid containing a gene encoding a novel anti-tumor active polypeptide in which any amino acid in the human TNF protein is replaced with another amino acid, added, or deleted is obtained. It becomes possible to create As such a novel antitumor active polypeptide gene expression plasmid, preferably p”rNF
416. 1) TN U4168 or 1) TN “495
is used.

(C) Expression confirmation and activity evaluation; Microorganism hosts to avoid expression of the TNF gene and novel antitumor active polypeptide gene include Escherichia coli,
Examples include Bacillus subtilis and yeast, but especially Escherichia coli [Escherichia coli]
) is preferred. The human TNFI Nobuko expression plasmid and the novel antitumor active polypeptide gene expression plasmid can be obtained by, for example, known methods [M, V, Norg
ard et al.

Gene, 3. 279 (1978)] using a microbial host such as Escherichia coli C600r.
-ma (ATCC33525).

The recombinant microbial cells thus obtained are cultured in a manner known per se. As a medium, for example, M9 medium containing glucose and casamino acids [T, M an
Edited by Iatis et al.
lonir+g”, P 440. Co1d 5
prinql-1arbor LabOratOrV
, New York (1982)], and it is desirable to add, for example, ampicillin, if necessary. 18 Cultivation is carried out at 37° C. for 2 to 36 hours under conditions suitable for the desired recombinant microorganism, such as aeration by shaking and stirring. Furthermore, a drug such as 3-β-indole acrylic acid can be added at the start of culture or during culture in order to make the promoter function efficiently.

After culturing, the recombinant microbial cells are collected by, for example, centrifugation, suspended in, for example, a phosphate buffer, the recombinant microbial cells are disrupted by, for example, sonication, and a lysate of the recombinant microbial cells is obtained by centrifugation. The white matter in the obtained lysate was separated by electrophoresis using a polyacrylamide gel containing sodium lauryl sulfate (hereinafter sometimes abbreviated as SO3), and the fluorescent matter in the gel was separated using an appropriate method. Dyeing using.

Human T
Confirm the expression of NFI gene or novel antitumor active polypeptide gene.

The activity of the human TNF gene and the novel antitumor active polypeptide thus obtained was evaluated by in viv to determine the effect of preventing necrosis of MethA sarcoma transplanted into mice.
o activity assay (Carswell et al.

), to examine cytotoxicity to mouse L cells.
Vitro activity assay [Ruff, J.

Immunol, 126. 235 (1981)
], etc., but in terms of measurement time, quantitative properties, simplicity of measurement, etc., evaluation by in VitrO activity measurement method is preferable.

<Effects of the Invention> Thus, according to the present invention, it is possible to obtain a novel physiologically active polypeptide that is different from the conventionally known human 1 to TN fluorescent white matter, and by using this novel antitumor active polypeptide, antitumor activity can be achieved. It has become possible to provide superior pharmaceutical compositions for tumors.

<Examples> 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 base sequence shown in FIG. 1 was designed.

Penn+ca et al., Nature, 312.
724 (1984)], a cleavage site with an appropriate restriction enzyme was set at an appropriate position, and a translation initiation codon ( ATG), and 3
Two translation stop codons (TGA and TAA') were added to the ' side. In addition, a cleavage site using the restriction enzyme CjaI was provided upstream of the 5' translation initiation codon, making it possible to link the SD sequence and the translation initiation codon to the promoter while maintaining an appropriate state. Furthermore, a cleavage site using the restriction enzyme indm was provided downstream of the 3' translation stop codon to facilitate ligation with vectors and plasmids.

Example 2 (Chemical synthesis of oligonucleotide) The human TNF gene designed in Example 1 was
Seven oligonucleotides are synthesized in minutes G'J 1. Oligonucleotide synthesis is performed by Applied Biosystems, a fully automated DNA synthesis company.

The test was carried out by the phosphite method using Model 380A). Purification of the synthetic oligonucleotide was performed according to the manual of Applied Biosystems.

That is, by keeping the ammonia aqueous solution containing the synthetic oligonucleotide at 55°C overnight, the protecting groups of the DNA bases are removed, and the high molecular weight synthetic oligonucleotide is removed by gel filtration using Sephadex G-50 Fine Gel (Pharmacia). Separate the nucleopolypeptide fraction. Next, polyacrylamide gel electrophoresis containing 7M urea (
After the gel concentration is 1 to 20%, the migration pattern is observed using the ultraviolet seed winging method. After cutting out a band of the desired size and finely crushing the polyacrylamide gel fragment, add 2 to 5 days of elution buffer.
[500mM N+140AC-1mMEDTA-0
, 1% SDS (pl-17,5> ) was added, and the mixture was heated at 37°C.
It was shaken overnight. The aqueous phase containing the target DNA was recovered by centrifugation. Finally, the solution containing the synthetic oligonucleotide was filtered onto a gel filtration column (Sephadex G-5).
0) to obtain a purified synthetic oligonucleotide. Note that, 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 created in Example 2 (
The human TNF gene was divided into three blocks and cloned using TNF-1 to TNF-17).

0.1-1.0 μ9 synthetic oligonucleotide TN “-
2 to 5' to 15 units of T4 on the 5' end of TNF-6.
- Polynucleotide kinase (E.

E. coli type 3, Takara Shuzo), and phosphorylate each separately. Phosphorylation reaction takes place at 50 m for 10-20 μm.
MTriS-mouth C9 (pl-19,5), 10
mM M(JCB.

The test was carried out in an aqueous solution of 5 mM dithiothreitol and 10 mM ATP at 37° C. for 30 minutes. After the reaction is complete, mix all the synthetic oligonucleotide aqueous solutions,
T4-polynucleotide kinase is inactivated and removed by phenol extraction and needle extraction.

Add 0.1 to 1.0μ9 of the synthetic oligonucleotide TNF1 to this synthetic oligonucleopolypeptide mixture.
, T, and NF-7 are added, heated at 90° C. for 5 minutes, and then slowly cooled to room temperature to perform annealing.

Next, after drying this under reduced pressure, 66 mM
Tris-l-1(J (pl-17,6), 6
．． GIIIM (IC92) Dissolved in 10mM dithiothreitol, 1mM A-rP aqueous solution, 300 units of T4-DNA ligase (Takara Shuzo)
was added, and the ligation reaction was carried out at 11°C for 15 hours. After the reaction is completed, the gel concentration for polyacrylamide gel electrophoresis is 5.
%) and observe the migration pattern using ethidium bromide staining. Target size: approx. 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 μq of E. coli plasmid 1) BR322 (approximately 4
．． 4K bll) with 30μ of 10mM Tri
s-HC9(pl-17,5), 60 mM N
a CL 7 mM MgC92 aqueous solution and 10
A cleavage reaction was carried out at 37° C. for 1 hour by adding the unit restriction enzyme CjaI (New England Biolabs).

After cleavage with restriction enzyme CjaI, phenol extraction.

Ether extraction is performed and DNA is recovered by ethanol precipitation. This DNA is 30μ of 5On+MTris
-1-1fJ (pth 7.4), 100
mM Na (1,10 mM (lsO4) was dissolved in an aqueous solution, 10 units of restriction enzyme 5alI (Takara Shuzo) was added, and the cleavage reaction was carried out at 37°C for 1 hour. After the reaction was completed, agarose gel electrophoresis (gel s1 degree 0
．． 8%), and the cutting pattern is observed by ethidium bromide staining. Plasmid 1) BR32
A band corresponding to a portion of approximately 3.7K bll of DNA containing most of 2 was cut out, and the agarose gel fragment was
It was dissolved in 8M NaCjO4 aqueous solution of Baimura (vol 7wt). The glass filter method of Chen et al. [C,
W.

Chen et al., Anal, Biochem, 1
01. 339 (1980)], approximately 3.7K
A bp DNA fragment (CjaI←5alI) was recovered from the agarose gel.

Approximately 220 cells containing a portion of the previously obtained human TNF3m Nobuko
After phosphorylating the terminals of the bp DNA fragment according to the method described above, it is mixed with about 3.7 Kbill of an aqueous DNA solution containing most of the plasmid pBR322. After ethanol precipitation, both DNAs were separated according to the method described above.
A ligation reaction of the fragments was performed.

Transformation of Escherichia coli C600r-m- strain
Normal Ca (J2 method (M, V, N orgar
d et al. carried out the method using the improved method of 1). That is, a 5-component L medium <1% tribone, 0.5% yeast extract, 0.
Escherichia to 5% Na CR, 1ll-17,2)
]C600r-m- strain was inoculated with an 18-hour culture medium, and the turbidity at 600 nm (OD6.,
) is grown until it reaches 0.3. The bacterial cells were soaked in cold magnesium buffer [0, IM Na C9,5
mM M(l CRz .

5111M TriS-11cf (1) l-17,
6,0°C)] and washed twice in 2- cold calcium chloride solution.
Buffer [100mMCa C12, 250mM
KCR, 5mM MCl CJz, 5mM
Resuspend in 7.6°C) and leave at 0°C for 25 minutes.

Next, the bacterial cells were concentrated to 1/10 of this volume in a calcium buffer, and the ligated DNA aqueous solution was 2:1 (v
ol, : vol,) mix. Stir this mixture for 60 minutes.

After keeping at 0°C, 1-LBG medium (1% tryptone,
Add 0.5% yeast extract, 1% NaCJ, 0.08% glucose, p.7.2), and culture with shaking at 37°C for 1 hour. Transfer the culture solution to a selective medium [medium plate containing 30 μg/- of ampicillin (Sigma)] at 100%
Inoculate at a rate of μm/plate. 1 plate at 37℃
The transformed strain is grown by late culture. From the obtained ampicillin-resistant colonies, DNA was extracted using a known method.
was prepared, and the acquisition of the target plasmid pTNFIBR (approximately 4.OK bp) was confirmed by agarose gel electrophoresis. FIG. 3 shows the method for constructing plasmid pTNFIBR.

By the same method as above, synthetic oligonucleotide TN
10 pieces of Arasumi NF2N using F-8 to TNF-13
(approximately 3.IK bp) was added to the synthetic oligonucleotide T
Plasmid pTNF using NF-14 to TNI-17
3 (approximately 2.4K bp) were created. Figures 4 and 5 show plasmids pTNF2N and pTNF3.
We will show how to create each.

The thus obtained human ``N1: plasmids containing -8 ((() pTNF1BR, pRNF2N and 1]T
The fact that the base sequence of the synthetic oligonucleotide-using portion of NF3 is as designed was confirmed by the Magizam-Gilbert method [A
, M. Maxam et al., MethodsE IIZ
VIII+,,65. 499 (7980)].

Example 4 (Creation of human TN F gene expression plasmid) 10 μg of plasmid pTNFIBR obtained in Example 3
were added to the restriction enzymes CRa■ and 3al in the same manner as in Example 3.
After cutting with I and performing polyacrylamide gel electrophoresis (gel concentration 5%), C1 human TNF was purified according to the method of Example 2.
Approximately 220 bp DNA fragment containing part of the 31t gene <C
RaI→5alI) was recovered from the polyacrylamide gel.

Next, 10 plasmids pTNF2 obtained in Example 3
μ9 to 10 (I u 10 mM Tr + s-mouth CJ (D
7.5), 60m M
Na CR, dissolved in 7mM MOCj 2M solution,
40 units of the restriction enzyme PVLII (Takara Shuzo) was added, and the cleavage reaction was carried out at 37°C for 1 hour. After cleavage with restriction enzyme 5alI according to the method of Example 3 and polyacrylamide gel electrophoresis (gel m degree 5%), approximately i.i. 70bp DNA fragment (SalIHPvull)
was recovered from polyacrylamide gel.

In addition, plasmid pTNF3 obtained in Example 3 10
, czg was also 100 μJ2 of 10 mM Tris-
l-1 (J (p<7.5), 60 mM NaC
9.7mM MOCJ2M solution, 40 units of restriction enzyme pvu [and 40. :lnit restriction enzyme H
ind [1 (Takara Shuzo) was added, and the cleavage reaction was carried out at 37°C for 1 hour. After polyacrylamide gel electrophoresis (gel concentration 5%), according to the method of Example 2,
A DNA fragment of approximately 110 bD (PvuII(he)1ndI[l) containing a portion of the human TNF gene was recovered from a polyacrylamide gel.

On the other hand, plasmid l carrying the E. coli trp promoter
]Ys31N (approximately 4.7 Kbl)>5 μg was cut with the restriction enzymes cnal and indm in the same manner as above, and after agarose gel electrophoresis (gel concentration 0.8%), the following procedure was performed according to the method of Example 3. An approximately 4.7K bp DNA fragment (CfaI4-
11-1ind■) was recovered from the agarose gel.

The thus obtained approximately 2
3 D of 20bp, about 170bp and about 110b11
The NA fragment and a DNA fragment of approximately 4.7 Kbp containing most of plasmid 1) YS3IN were mixed, and after ethanol precipitation, a ligation reaction using T4-DNA ligase was performed according to the method of Example 3. After completion of the reaction, the desired human TNF Nobuko expression plasmid pTNF401NN<approximately 5.2 Kb+1 was introduced into Escherichia coli C6C600r- strain according to the method of Example 3, and selected from among the transformed strains.
) were selected. FIG. 6 shows the method for constructing the plasmid 11TNF401NN.

In addition, the above plasmid pYs31N5μ3 was partially digested with the restriction enzyme pvu[ and then further cut with the restriction enzyme)-1indlll according to the above method, and after agarose gel electrophoresis (gel concentration 0.8%), Example According to the method of 3, an approximately 2.7 Kbp DNA fragment [PvuII(2)''1ndI[[] containing the trp promoter was recovered from an agarose gel.

Next, an oligonucleotide having the base sequence shown in FIG. 7 was synthesized and purified according to the method of Example 2. The ends of each of the two synthetic oligonucleotides (0.5 μ9) obtained were phosphorylated according to the method of Example 3, and after annealing, the previously obtained approximately 2.7 K b
DNA fragment of p [PvuII (2) ←)l-1i
After mixing with nd ll [] and precipitating with ethanol (D), a ligation reaction using T4-DNA ligase was performed according to the method of Example 3. After the reaction was completed, according to the method of Example 3. The target plasmid pΔA41 (approximately 2.7
K bp) was selected. Such a plasmid is a multi-copy, high-efficiency expression vector in which the copy number control region has been removed from the plasmid pYS31N and an E. coli TRP A terminator has been added downstream of the cloning site located downstream of the trp promoter. Figure 7 shows how to create it.

After cutting 2 μg of this plasmid pAA41 with the restriction enzymes CjaI and Hindl [and performing agarose gel electrophoresis at a gel concentration of 0.8%] in the same manner as described above, A41 was added to the plasmid according to the method of Example 3. Approximately 2 including the majority
．． 7K bp DNA fragment (cpa engineering, -) l-li
nd (2) was recovered from the agarose gel.

In addition, 5 μq of the previously obtained human TNFJ Nobuko expression plasmid pTNF 401NN was cleaved with the restriction enzymes CRa and Ind m in the same manner as above, and after polyacrylamide gel electrophoresis (gel concentration 5%), Example 2 (approximately 490 bl containing the entire human TNF gene)
A DNA fragment ((Ja ``Hind 11'') was recovered from the polyacrylamide gel.

The approximately 2.7K bp DNA fragment containing most of the plasmid pΔA41 thus obtained and the approximately 490 bp DNA fragment containing the entire human TNF gene were mixed, and after ethanol precipitation, the mixture was prepared according to the method of Example 3. , T4-DNA
A ligation reaction using ligase was performed.

After completion of the reaction, according to the method of Example 3, Escherichia
The target plasmid IITN F 401A (approximately 3.2K
bl')) were selected. This plasmid has the ability to express the human TNF gene more efficiently, and the method for its construction is shown in FIG.

Example 5 (Creation of novel antitumor active polypeptide gene expression plasmid) 20 μg of the human TNF gene expression plasmid tlT NF 401A obtained in Example 4 was mixed with 100 μg of 10 mM Tris-1 (J ( pl
-17,4), 10 mM M(
1304 and 1111M dithiothreitol aqueous solution, 40 units of restriction enzyme Kpnl (Takara Shuzo) was added, and a cleavage reaction was carried out at 37°C for 1 hour. Further, cleavage with the restriction enzyme CRaI was performed according to the method of Example 3, and the gel concentration for polyacrylamide gel electrophoresis was 5%.
) and agarose gel electrophoresis (gel concentration 0.8%), two DNA fragments (approximately 160 bp and approximately 3.0 K bp) were generated according to the methods of Examples 2 and 3, respectively.
, CjaIHKiln■) were recovered from the gel.

Approximately 16 human TNFm including the first half of the trochanter obtained here
0bp DNA fragment into 50 μg of 10 m1ylTr
is-mouth CB (pl-17,4), 10111
M
pII (Takara Shuzo) was added and the cleavage reaction was carried out at 37°C for 1 hour. After the reaction, polyacrylamide gel electrophoresis (gel concentration: 5%) was performed, and a DNA fragment of about 100 bp containing the first half of the human TNF gene (CβaJ+ → ap■) was transferred to polyacrylamide gel according to the method of Example 2. The gel was collected.

Furthermore, an oligonucleotide having the base sequence shown in FIG. 9 was synthesized according to the method of Example 2.

Purified. 0.5 μg of each of the two synthetic oligonucleotides obtained were subjected to terminal phosphorylation and anyl ring according to the method of Example 3.

After the reaction, the obtained double-stranded oligonucleotide was combined with the previously obtained approximately 3,000 bp DNA fragment <CJa I
'-'K11nI) and an approximately 100bp DNA fragment containing the first half of the human TNF gene (CjaI"Hat)
(2) and after ethanol precipitation, a ligation reaction using T4-DNA ligase was performed according to the method of Example 3. After the reaction was completed, it was introduced into Escherichia coli C600r-m- strain according to the method of Example 3, and the target plasmid pTNF489 (<approximately 3.2 Kbp) was selected from among the transformed strains.
We selected clones with the following values.

This plasmid (yo, the following amino acid sequence (fi2N)
-Val-8rg-3er -3erS er-A
rG-T hr-P ro -3er -A Sp -
L VSP ro-Val -A la -His
-Val -Val-A IaA sn -pr
o-Q In-8la -G lu -G IV-G
In1-eu-Gln-Trp-1-cu-Asn-
Ar(1-ArtlAla-8sn-A la-L
eu-l-eu -A 1a-A snG ly-V
al-G 1u-l-cu-to rg-to sn-5nS
er-Leu-Vat-Val-Pro-8er-G
uGly-Heu-Tyr-Lcu-1le
-Tyr -3erG In -Val-Leu-Ph
e-Lys-G ly-G InG1'/Cys
Pro 5er--Thr 1lls 7a eu -L eu-Thr -1-11s-Thr -
I le -3erA ra-11e-A la -V
al-3er-Tyr-G InThr-Lys-
Vat-Asn-Leu-Leu-5er
A la -11e -1-ys -Sar -p r
o -Cys -G InA r(J-G lu-T
hr-Pro-G lu-G IV-A laG
1u-A 1a-LVS-Pro-Trl)-ryr
-QluPro-1le-'ryr -Leu-GIV
-GIV-ValP he-G In-L eu-G
lu-Lys-Gly-A 5p-Ara-Le
u-8er-A 1a-G lu -11e-AsnA
rg-Pro-A sp-Tyr-L eu-A
sp-P heA 1a-G lu-S er-G
Iy-G In -V al-TyrPhe-G l
y −(le −11e −A 1a−Leu(COO
This is a novel antitumor active polypeptide gene expression plasmid that encodes a novel antitumor polypeptide represented by (1) or a polypeptide with Met bound to its amino terminus, and the method for its construction is shown in Figure 9. .

Next, the plasmid obtained here 1) TNF4895μ
9 was added to 50 μL of 10 mM Tris-1-1 (
J (D 7.5), 60 mM Na CR,
Dissolved in 7111M MCI CR2 aqueous solution, 20
One unit of restriction enzyme C1aI and 20 units of restriction enzyme AvaI (Takara Shuzo) were added, and the cleavage reaction was carried out at 37°C for 1 hour. After agarose gel electrophoresis (gel concentration 0.7%), approximately 3.2K cells containing the majority of plasmid 111 TNF488 were collected according to the method of Example 3.
A bp DNA fragment (CRaIHAvaI) was recovered from the agarose gel.

On the other hand, an oligonucleotide having the base sequence shown in FIG. 10 was synthesized and purified according to the method of Example 2. 0.5μ each of the four synthetic oligonucleotides obtained
The tusks are mixed in the combination shown in FIG. 10, annealed, and then mixed with the previously obtained DNA fragment of approximately 3.2 Kbp (CRa IHAvaI). After ethanol precipitation, a ligation reaction using T4-DNA ligase was performed according to the method of Example 3. After completion of the reaction, Example 3
The target plasmid 11T was introduced into Escherichia coli C600r-m- strain according to the method of
Clones with NF497A or I) TNF 497B (both approximately 3.2 K bp) were selected. These plasmids had the following amino acid sequence (+-(2N)-Pro-3ep-ASD-11sP
ro-V al -A Ia-Is -V al
-V al -A IaA Sn -Pro-G I
n-8la -G lu-'G IV -G InL
eu-Gln-Trp-L eu-Asn-ArO-A
r(1-Ala-Asn-Ala-l-eu-1-eu
-Ala-Asn -Q ly -V al -G I
u -L eu -A rg -A 5n-A 5nS
er-Leu-Vat-Val-Pro-8cr-Gl
uG lv -1-eu -Tyr -Leu-1le
-Tvr -5erG In -V, at -Leu
-Phe-LyS-G 1y-G In -Gly-
Cys-Pro-3et-Thr-1-1is-val
--Leu-L eu-Thr-His-Thr -I
1e-8erAro-11e-Ala-Val-8
er-Tyr-GinThr-Lys-Val-As
n -Leu-L eu-8erA 1a-I Ie-
Lys-5ar-P, ro-CyS-G 1n-A
ro -G lu-T hr -Pro -G lu
-G 1y-A la -〇 IU-A 1a-L l
/S-P rO-T rL-T Vr -G 11Pr
o -I 1e-Tyr -L eu-Gly-Gly
-VaP he -G In-L eu-G 1u-L
l/S-G IV-A St) Arg-1eu-3e
r-Ala-Glu-11e-8sn-A ra-P
ro -A 3p-T yr-L eu-A sp
-P heA Ia-G lu-S er -G ly
-G In -V al-T yr-Phe-Ctly
-11e -11e-Ala-Leu (COO mouth) A novel antitumor active polypeptide gene-mediated plasmid encoding a novel antitumor polypeptide expressed by -11e -11e-Ala-Leu (COO mouth) or a polypeptide with Met attached to its amino terminus FIG. 10 shows the method for making it.

Next, 20 μU of the human TNF1 trochanteric expression plasmid pTNF 49713 obtained above was digested with the restriction enzyme HindI according to the method of Example 4, and then
mM Tris-Flcf (D port 7
．． 4), 100 m MNa (J,
Restriction enzyme NC in 10 mM MC+ 304 aqueous solution
The cleavage reaction using 0I (Takara Shuzo) is carried out at 37°C for 1 hour. After the reaction is completed, the gel concentration for agarose gel electrophoresis is 0.
7%> and polyacrylamide gel electrophoresis (gel concentration 5%) according to the method of Example 2.
Approximately 140 bp DNA fragment containing part of the gene (Nc
oI <-+ II ind I [[) was obtained from polyacrylamide gel and pT
Approximately 3.0 KbD DNA containing most of NF 497B
The fragments (NcoT←+1-find I[[) were each recovered from an agarose gel.

Furthermore, the approximately 14 obp DNA fragment obtained above (Nc
oI+l-1ind I[[) to 101+1 of 50μ sentence
M Trisl-1cfCp mouth 7.4), 10 m
MMCI SO4 was dissolved in a 1mM didiothreitol aqueous solution, 10 units of restriction enzyme AccI (Takara Shuzo) was added, and a cleavage reaction was carried out at 37°C for 1 hour. After the reaction was completed, a polyacrylamide gel was run and an approximately 110 bp DNA fragment (NC) containing a portion of the human TNF gene was obtained according to the method of Example 2.

IWACCI) was recovered from the polyacrylamide gel.

Further, an oligonucleotide having the base sequence shown in FIG. 11 was prepared according to the method of Example 2.

Purified. 0.5 μq of each of the two synthetic oligonucleotides obtained were subjected to terminal phosphorylation and annealing according to the method of Example 3.

After annealing, the resulting double-stranded oligonucleotide was attached to the previously obtained approximately 3,000 bp DNA fragment (Nc
oI+H ind m ) and a DNA fragment of about iobp (NcoI+AccI) containing a portion of the human TNF gene, and after ethanol precipitation, a ligation reaction using T4-DNA ligase was performed 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 target plasmid pTN "641 (approximately 3.2 K bp) was selected from among the transformed strains. This plasmid has the following amino acid sequence (112N)-Pro-Ser-ASI)-
LysPro-Val-Ala-eyes-
V al - V al - A laΔsn-Pr
o-Gln-Ala-Glu-Gly-Gln-
Leu-Gln-Trp-Leu-Asn-ArO
-ArgA la - A sn - A Ia -
L eu - L eu-Δla-AsnG IV-V
al- G lu- L eu-A rL- AS
D-A SnGln-Leu-Val-Val-
Pro-Ser-GluG ly-t eu-Tyr-
Leu-1te-Tyr-SerGIn
- Val - Leu - Phe - Lys
- G ly - G InG IV - Cys -
Pro-3er-Thr-His-
Vaseu - L eu-Thr - 1-l i
s-Thr-I le-3erA ro-1 1e
-Ala-Val-Ser-Tyr
-G InThr - Lys - Val-Asn
-Leu -Leu -SerAla - 1 1
e-Lys-Ser-Pro-Cys-GlnA
ro-G Iu-Thr-Pro-G
lu- G ly- A laG lu - A I
a - Lys - Pro - Trp -
Tyr-Glu-Pro-Ile-T
yr-Leu-Gly-Gly-V
aPhe-Gln-L eu-GILI-LVS-GI
V-ASFIArQ-Leu-3er-A la-
Glu-1 1(!-A3nA rq-P r
o-A sp-Tyr-l-eu-A sp
- Phe eight l - Glu - Ser- G l
y-Gln-Val-Tyr-Ph
e-Gly-11e-Ile-Ala-Phe(COO
This is a novel antitumor active polypeptide gene expression plasmid that encodes a novel antitumor polypeptide represented by (1) or a polypeptide with lylet attached to its amino terminus, and the method for its construction is shown in Figure 11. .

Example 6 (Confirmation of expression) Expression vector obtained in Example 4 1) AA41.

pTNF 401NN or pTNF401A or the novel anti-11i1i tumor-active polypeptide gene expression plasmid proposed in Example 5 above.
M9 medium [0,6%
Naz+-I PO4-0,3%K2hl PO
4-0,05% NacRO11%N4C9 aqueous solution (p
After crepe sterilizing 1-17,4) into O1, M(]SO4 aqueous solution and CaCRz aqueous solution, which were separately sterilized by O1 to crepe, were added to a final concentration of 2 mM and 0.1 mM, respectively.
Add to 1 mM. ]250-, O
Shaking culture was performed at 37°C until Dl reached 0.7. Next, 3-β-indole acrylic acid at a final concentration of 50 μg/− was added to the culture solution, and further incubated at 37°C.
The shaking culture was continued for 12 hours.

After collecting Escherichia coli cells by centrifugation, the cells were washed with PBS buffer (20 mM phosphate buffer containing 150 mM NaC9, DI-17,4). After washing, the bacterial cells were suspended in 10-PBS buffer and placed in an ultrasonic generator (Kubota, 200M model).
After the bacterial cells were disrupted using a microorganism, the bacterial cell residue was removed by centrifugation.

Tri
s-l-ICj buffer (pl-16,8), SD
S, 2-mercaptoethanol, and glycerol were added to final concentrations of 60 mM, 2%, 4%, and 10%, respectively, and 5DS-polyacrylamide gel electrophoresis ``Meiki, Genetics, 31.43 (1977)'' was performed. .

The separation gel was 12.5%, and the running buffer was SD3.
, Tris-glycine system [U, K, l-aemmN
ature, 227. 680 (1970)]
was used. After electrophoresis, the proteins in the gel were stained with Coomassie Blue R-250 (Bio-Rad) to confirm the expression of the novel antitumor active polypeptide gene. A portion of the results are shown in Figure 12. Ta.

In addition, the gel after staining should be processed using Chroma 1~ Scanner (Shimadzu,
C8-930 type>, the proportion of the produced human TNF protein or novel antitumor active polypeptide in E. coli cytoplasmic protein was calculated. As a result, human TNF protein accounted for approximately 17% of the total cytoplasmic protein in E. coli harboring the human TNF gene expression plasmid pTNF401A, and approximately 10% in E. coli harboring the novel antitumor active polypeptide gene expression plasmid 1) TNF641. % of new antitumor active polypeptides were observed. In addition, the amount of human TNF protein produced in E. coli containing the human TNF gene expression plasmid pTNF 401NN is
It is only about 40% of the case of A, and the expression vector +1A A
The usefulness of 41 was shown.

Example 7 (Evaluation of activity) The activity of the novel antitumor active polypeptide was measured using the Ruf
It was carried out according to the method of f. That is, the E. coli lysate containing the novel antitumor active polypeptide obtained in Example 6 was diluted with a culture medium at 100 μm, and 4×105
mouse L929 fibroblast cells (ATCCC
100μ of the C-929) suspension was mixed in a 96-well II culture microplate (Costar). At this time, actinomycin D (final concentration 1 μg/-) was added.
] Sumegean Banyu Pharmaceutical) is added. As the medium, Eagle's Minimum Ethical Medium (Hinaga Pharmaceutical Co., Ltd.) containing 5% (vol/vol) fetal bovine serum was used. After culturing the above microplate at 37°C for 18 hours in air containing 5% carbon dioxide gas, a crystal violet solution [5% (vol/vol) methanol aqueous solution and 0.5% (vt/vol) crystal・
[Dissolved violet] was used to stain living cells. After washing off and drying the excess crystal violet, the remaining crystal violet was extracted with 100 μm of 0.5% SDS aqueous solution, and its absorbance at 595 nm was measured using an ELISA analyzer (Toyo Sokki, ET).
Y-96 type). This absorbance is proportional to the number of surviving cells. So, the new anti-II! r! The dilution factor of E. coli lysate that corresponds to 50% of the absorbance of the control to which no diluted solution of E. coli lysate containing tumor-active polypeb polypeptide is added is determined using a graph (for example, Fig. 13), and that dilution factor is defined as a unit. do. 13th
From the figure, Escherichia coli lyse-1 containing the human TNF protein encoded by the expression plasmid l'1 TNF 401A.
100μ summer has an activity of about 1.3X 10' units and contains E. coli lyse 1 containing a novel antitumor active polypeptide encoded by the expression plasmid DTNF641.
It was revealed that ~100 μm had an activity of about 1.2×10′ conils, respectively.

Expression type plasmid pTNF401A obtained in Example 6
Human TNF protein or expression plasmid encoded by ) and calculated from a calibration curve using bovine serum albumin. The specific activities of the human h TNF protein and the novel antitumor active polypeptide were calculated from the expression levels, activity values, and protein standard results obtained above, and the values shown in Table 1 were obtained. Table 1 shows that the novel antitumor active polypeptide has a specific activity approximately 1.7 times that of human TNF protein.

Table 1 Comparison of human TNF protein and the novel antitumor active polypeptide of the present invention

[Brief explanation of drawings]

Figure 1 shows the designed base sequence of the human TNFI gene, and
The figures show the base sequences of chemically synthesized synthetic oligonucleotides. Figures 3, 4 and 5
The figure shows plasmid pTN containing a portion of the human TNF gene.
How to create FIBR, pTNF2N and pTNF3,
They are shown below. Figure 6 shows the method for creating the human TNF gene expression plasmid pTNF401NN.
Figure 7 shows how to create the expression vector rlA A41.
Figure 8 shows human TNFm Nobuko expression plasmid +1.
Each method for creating the TNF 401 is shown. Figure 9 shows the method for constructing the novel anti-tumor active polypeptide gene expression plasmid pTNF489, and Figure 10.
The figures show the methods for producing novel antitumor active polypeptide gene expression plasmids pTNF497B and IITNF497A, respectively. Figure 11 shows novel antitumor active polypeptide gene expression plasmid t) TNF
641 is shown. FIG. 12 shows the expression results of the novel antitumor active polypeptide gene. FIG. 13 shows the results of measuring the activity of the novel antitumor active polypeptide.

Claims (1)

[Claims] (1) The following amino acid sequence [There is a gene sequence. ] [There is a gene sequence. ] A novel physiologically active polypeptide represented by (2) The polypeptide according to claim 1, characterized in that Met is bound to the amino terminus. (3) The following amino acid sequence [There is a gene sequence. ] [There is a gene sequence. ] A recombinant plasmid containing a DNA region encoding a novel physiologically active polypeptide represented by the following formula or a polypeptide having Met bound to its amino terminus. (4) The DNA region has the following base sequence [there is a gene sequence]. ] [There is a gene sequence. ] Single-stranded DNA represented by and its complementary single-stranded DNA
The plasmid according to claim 3, characterized in that it contains a double-stranded DNA consisting of A. (5) The DNA region has the following base sequence [there is a gene sequence]. ] [There is a gene sequence. ] Single-stranded DNA represented by and its complementary single-stranded DNA
The plasmid according to claim 3, characterized in that it contains a double-stranded DNA consisting of A. (6) The plasmid according to claim 3, wherein the plasmid is plasmid pTNF641. (7) The following amino acid sequence [There is a gene sequence. ] [There is a gene sequence. A recombinant microbial cell transformed with a recombinant plasmid containing a DNA region encoding a novel physiologically active polypeptide represented by the following formula or a polypeptide having Met bound to its amino terminus. (8) The microbial cell according to claim 7, wherein the microbial cell is Escherichia coli. (9) The following amino acid sequence [There is a gene sequence. ] [There is a gene sequence. ] A recombinant microbial cell transformed with a recombinant plasmid containing a DNA region encoding a novel physiologically active polypeptide represented by 1. A method for producing a novel bioactive polypeptide, which comprises producing and accumulating the bioactive polypeptide and separating the novel bioactive polypeptide from the resulting culture. (10) There is the following amino acid sequence [gene sequence] in an effective amount for antitumor. ] [There is a gene sequence. ] A pharmaceutical composition containing a novel physiologically active polypeptide represented by the following formula or a polypeptide having Met bound to its amino terminus.