JPS63188396A - Novel and biologically active polypeptide - Google Patents

Abstract

PURPOSE:To obtain a biologically active polypeptide consisting of a specific amino acid consequence, stable, having high specific activity, low side effects and broad reaction spectrum range and useful in anticancer agent, etc. CONSTITUTION:The desired and appointed codon of constitutive amino acid of human tumor necrosis (TNF) protein is selected and chemically synthesized to provide a human TNF gene (A). Then a terminator is provided to the gene (A) to prepare an expression type A gene, which is cloned into a vector derived from pBR322 to afford an expression type plasmid (B) (e.g. pTNF401A) of the A gene. Then the B plasmid is subjected to treatment by restriction enzyme to give a recombinant plasmid (e.g. pTNF471) (C) containing DNA domain coding a biologically (antitumor) active polypeptide having amino acid conse quence such as amino acid, etc., expressed by the formula or the polypeptide with which Met (L-methionine) is combined at the amino end of the polypeptide. Then Escherichia coli, etc., is transformed with the C plasmid and the resultant recombinant cell is cultivated in a culture medium and a biologically active polypeptide produced and accumulated in the culture medium is separated.

Description

Detailed Description of the Invention (1) Industrial Application Field The present invention relates to a novel physiologically active polypeptide, a recombinant plasmid containing a DNA region encoding the polypeptide, a recombinant microbial cell transformed with the plasmid, and a recombinant microbial cell transformed with the plasmid. The present invention relates to a method for producing a novel physiologically active polypeptide using the microbial cells. More specifically, a novel polypeptide having anti-embryonic activity (hereinafter sometimes abbreviated as novel anti-tumor active polypeptide), a recombinant plasmid containing a DNA region encoding the polypeptide, and a recombinant plasmid transformed with the plasmid. The present invention relates to recombinant microbial cells and methods for producing novel antitumor active polypeptides using the microbial cells.

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

AIAL -Alginine AROL -Arginine ASN L -Asparagin ASP L -Asparagin CYS L -Sistin GLN L -glutamine GLU L -glutamate LY Glytamic acid 1 IS L -Histidine 11EL -Lewish LEUL Ne Lysl -Lizin MET L -methionine PHEL -Phenyl Alanine PROL -Proline 3er l-serine Thr L-threonine Trial-tryptophan TVr L-tyrosine Val l-valine Furthermore, the sequence of DNA shall be abbreviated by the type of base contained in each deoxyribonucleotide constituting it,
For example, use the following abbreviations.

A Adenine (represents deoxyadenylic acid) C Cytosine (represents deoxycytidylic acid) G Guanine (represents deoxyguanylic acid) T Thymine (represents deoxythymidylic acid) Furthermore, (82N) - and -( Coo
l-1) indicates 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.

(2) Background of the invention Q Arswell et al.
It was discovered that serum collected after administering endotoxin to mice previously stimulated with almette-Guerin (BCG) contained a substance that causes hemorrhagic necrosis of transplanted MethA sarcoma cancers. Necrosis factor
ROSISFactor (hereinafter sometimes abbreviated as TNF) [E, A, Carswell et al.
proc, Natl.

Acad, sci, USA, 72.36
66 (1975)]. TNF is found in many animals such as mice, rabbits, and humans, and since it acts specifically on tumor cells and across species, it has been expected to be used as an anticancer agent.

Recently, Penn1ca et al.
We carried out NA cloning to clarify the primary structure of human TNF protein and
reported on the expression of the F gene [D, Penn1
ca et al., Nature, 312. 724(
1984)], then Jiben et al.
rai et al.

Nature, 313. 803 (1985)
], Munemura et al. l Chemistry 1 [Law, 12.
160 (1985) ], Wan! 1 et al. [A, M,
Wano et al., 5science, Yu28. 149 (1
985) Ko and Marmenout et al. [A, M
Armenout et al.

[Eur, J. Biochem, 152. 5
15 (1985)] have successively reported on the expression of the human TNF 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 the physiological activities of TNF other than its antitumor activity are becoming clearer. For example, cachectin, which is one of the causes of cachexia seen in patients with terminal cancer or severe infections, is very similar to TNF [B, Belter et al., Nature.

316, 552 (1985)], and since cachectin has lipoprotein lipase inhibitory activity, T.
It has been suggested that administration of NF increases the amount of triglyceride in the blood, which may result in side effects such as hyperlipidemia. In addition, there are also effects on vascular endothelial cells [J, R.

Galble et al., J. EXI), Med, 1
62.2163 (1985)], bone resorption effect [D,
RoBeltolini et al., Nature, 3
19. 516 (1986)] etc. have been reported.

On the other hand, recent advances in genetic engineering technology have enabled the substitution or addition of arbitrary amino acids in proteins with other amino acids,
or allowed to be deleted.

In this way, many studies have been carried out to create new proteins that serve specific purposes by modifying naturally occurring proteins.

Several studies have also been carried out on the modification of the human TNF protein, and in the amino acid sequence of the human TNF protein shown in Figure 1, other amino acid residues have been added to either or both of the amino acid residues Cys If and cys. Substitution to groups (
POT application publication WO36/04606, patent application Sho 6l
-106772), substitution of GIV''' with other amino acid residues (Japanese Patent Application No. 106772/1983,
38048) and substitution of Ala with another amino acid residue (Japanese Patent Application No. 61-233337).

Also, regarding the deletion of amino acid residues on the amino terminal side,
6 amino acid deleted TNF has cytotoxic activity (JP-A No. 61-50923), 7 amino acid deleted TNF
has cytotoxic activity (patent application 1986-900)
87), 1 to 10 amino acid deleted TNF has cytotoxic activity, and the specific activity is 6 to 8 amino acid deleted TNF.
(PCT application publication WO 36/
02381), that 10 amino acid deleted TNF has cytotoxic activity (Japanese Patent Application No. 114754/1982),
It has also been reported that 11 amino acid deleted 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.

(3) Purpose of the Invention The purpose of the present invention is to provide a novel polypeptide with antitumor activity.

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 cells.

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

(4) Structure of the Invention According to the research conducted by the present inventors, the object of the present invention is to obtain the following amino acid sequence (H2N>-Ar(1-LyS-Ar(lLy
s -Pro -Val -A Ia -His -
Val - Vat - A la - A sn - P r
o -G In -A Ia -G Iu -G Iy
-G In -L elJ-G In-T rp-L
eU-A Sn-A rll-A r! 1l-AIa
-A sn -A Ia-1eu-1eu -A l
a -A sn -GIV-Val-Gltl-Leu
-ArO-ASII-ASn-〇In-L eu-Va
t-Val-Pro-8er-Glu-Gly-L
eu-Tyr-L eu-I le-Tyr-5er-
G In -V al -L eu -P he -L
VS -G ly -G In -G Iy-Cys
-Pro-5er-Thr-His-Val-Leu
-Leu-Thr-H1s-Thr-I 1e-8e
r-Aro-11e-Ala-Val-8er-T
yr-Gin-Thr-Lys-Val-Asn-L
eu-Leu-8er-A la -11e -L y
s -S er -Pro -Cys -G In
-A rQ-G Iu-Thr-Pro-G Iu-
G +y-A 1a-G lu -A 1a-L ys
-Pro -Trp -Tyr -G Iu
-Pro-I Ie-Tyr-Leu-G ly-G
ly-Val -P he -G In -L eu
-G Iu -L VS -G Iy -A 5L-A
ro-Leu-8er-A 1a-Glu-I Ie-
Asn-A ra-P ro-A sp-Tyr-L
eu-A sp-P he-A la-G Iu-S
er-G ly-G In-Val-Tyr-Phe
-Gly-11e-11e-Ala-Leu-(COO
H) To provide a recombinant plasmid containing a novel physiologically active polypeptide represented by or a polypeptide having Met bound to its amino terminus, and a DNA region encoding the novel antitumor active polypeptide. A recombinant microbial cell transformed by the recombinant plasmid thus obtained, a method for producing the desired novel antitumor polypeptide using the microbial cell, and a method for producing the novel antitumor polypeptide. It has been found that this can be achieved by providing a pharmaceutical composition containing.

The present invention will be explained in more detail below.

(A) Cloning of the human TNF gene; the human TNF gene consists of amino acids [D,
It can be obtained by selecting an appropriate codon from among several codons specifying [Pennica et al., supra] 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 enzymes at appropriate positions to facilitate subsequent cloning and genetic modification. 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).

TAG or TAA).

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 TNFM 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 TNF gene designed as described above, each of the upper and lower strands is divided into several oligonucleotides as shown in Figure 2, and these are chemically synthesized. It is desirable to use a method of linking each oligonucleotide. The synthesis method for each oligonucleotide is the diester method [HoG, Khorana
.

“8 ome Recent D evelopm
ents in - Chemistry of Phosphat
e E 5ters of3 iologica
I'm interested'', John W.
ileyand 5ons,' Inc.
New York (1961)].

Triester method [R, L, L etsinger et al., J.

Am, Chem, Soc, 89.4801 (
1967)] and the phosphite method [M, D, Ma
tteucci et al.

Tetrahedron Lett,, 2L 7
19 (1980)], but synthesis by the phosphite method using a fully automatic DNA synthesizer is preferred from the viewpoint of synthesis time, yield, simplicity of operation, etc. 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 annealed and ligated using, for example, T4-DNA ligase. A method for constructing the human TNF gene by ligating synthetic oligonucleotides involves dividing the synthetic oligonucleotides into several blocks and ligating them, for example, pBR322[F,
Bolivar et al., Qene, 2.95 (
A preferred method is to clone the DNA fragments into a vector such as [1977] and then ligate the DNA fragments of each block. A plasmid containing a DNA fragment of a block constituting the human TNF gene is preferably IITNFIBR.

tlTNF2N or pTNF3 is used.

After ligating the DNA fragments of each block constituting the human TNF gene cloned as described above, an expressed gene can be obtained by ligating downstream of an appropriate promoter and SD (Shine-Dalgarno) sequence. . A usable promoter is the tryptophan operon promoter (trp promoter).

Lactose operon promoter (lac promoter), tac promoter, PL promoter,
Examples include the Ipp promoter, but especially the tr
The p promoter is preferred. I) YS3IN or ERA A 41 is preferably used as the plasmid having the trp promoter.

Furthermore, for the purpose of improving expression efficiency, a terminator that functions efficiently in E. coli can be added downstream of the human TNF gene. As such a terminator,
til+1 terminator, 2 trp terminator, etc., but trp A terminator is particularly suitable, and pA A 41 is preferably used as the plasmid having trp A terminator. By cloning this expressed human TNF gene into a DBR322-derived vector, for example, an expression plasmid can be created. As a human TNF gene expression plasmid, preferably pTNF401NN or p,"r
NF 401A is used.

(B) Cloning of a novel antitumor active polypeptide gene; The human TNF gene expression plasmid thus obtained is cut with an appropriate restriction enzyme to remove a specific region within the human TNF gene, and then an appropriate base sequence is extracted. We perform gene repair using synthetic oligonucleotides with 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, pTNF471 is preferably used.

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

Gene, 3. 279 (1978) using
Microbial hosts, such as Escherichia coli C6C600
It can be introduced into the r-1 Otsu 1 strain (ATCC33525).

The recombinant microbial cells thus obtained are cultured in a manner known per se. As a medium, for example, M9 medium [T, ManiaN
“Molecular Cloning”, edited by S et al.
P440. Co1d 5prin(1Harbo
r Laboratory, New York
(1982)], and it is desirable to add, for example, ampicillin, if necessary. 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. 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.

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

The activity of the human TNF protein and the novel anti-tumor active polypeptide thus obtained was evaluated by in vivo observation of the effect of necrosis on MethA sarcoma transplanted into mice.
o activity assay (Carswell et al.

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

(111mLIn01., Yu 26. 235 (1981
)], but from the viewpoint of measurement time, constant stability, simplicity of measurement, etc., evaluation by in VitrO activity measurement method is preferable.

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

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 by Pennica et al. [D.

P ennica et al., Nature, 312
．． 724 (1984)], a cleavage site with an appropriate restriction enzyme was placed at an appropriate position, and a translation initiation codon (ATG> and two translation stop codons (TGA and TAA) were added to the 3' side.In addition, a cleavage site with the restriction enzyme CjaI was provided upstream of the 5' translation start codon, and a cut site between the SD sequence and the translation start codon was added. In addition, a cleavage site with the restriction enzyme Hindl [[] was provided downstream of the 3' translation stop codon, allowing for easy ligation with vectors and plasmids. I made it.

Example 2 (Chemical synthesis of oligonucleotide) The human TNF gene designed in Example 1 was
Synthesize it in seven oligonucleotides. Oligonucleotides were synthesized using fully automatic DNA synthesis tI (Applied Biosystems).

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 an ammonia aqueous solution containing synthetic oligonucleotides at 55°C overnight, the protecting groups of the DNA bases are removed, and the DNA bases are removed from the ammonia solution containing Sephadex G-50 Fine Gel (
A high-molecular-weight synthetic oligonucleotide fraction is collected by gel filtration using a high-molecular-weight synthetic oligonucleotide (Pharmacia). Next, 7M
After polyacrylamide gel electrophoresis containing urea (gel concentration 20%), the migration pattern is observed by ultraviolet shadowing. After cutting out a band of the desired size and finely crushing the polyacrylamide gel fragments, 2 to 5Id elution buffer: p-[500
mM Nt-1a OAc-1n+MEDTA-0
, 1% SDS (EIH7,5)] was added, and the mixture was shaken at 37°C 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-50).
) 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) The 17 synthetic oligonucleotides prepared in Example 2 (
The human TNF gene was divided into three blocks and cloned using TNF-1 to TNF-17).

0.1-1.0μ synthetic oligonucleotide TNF-
2 to 5' to 15 units of I on the 5' end of TNF-6.
4-polynucleotide kinase (E.

E. coli B type, Takara Shuzo) to phosphorylate each separately. Phosphorylation reaction is carried out at 5°m of 10-20 μm.
MTris-H(J (pt-+ 9.5>, 10
mM MOCR2゜5 mM dithiothreitol,
The test was carried out in a 10mM ATP aqueous solution at 37°C for 30 minutes. After the reaction is completed, all aqueous synthetic oligonucleotide solutions are mixed together, and I4-polynucleotide kinase is inactivated and removed by phenol extraction and ether extraction.

To this synthetic oligonucleotide mixture, add 0.1~
1.0 μg of synthetic oligonucleotides TNF-1 and T
NF-7 is added, heated at 90° C. for 5 minutes, and then slowly cooled to room temperature for annealing.

Next, after drying this under reduced pressure, 66 mM
Tris-)I Cj(1)H7,6), 6.
6mM MgCjz.

The mixture was dissolved in an aqueous solution of 10 mM dithiothreitol and 1 mM ATP, and 300 units of I4-DNA Liga (Takara Shuzo) was added thereto, followed by a ligation reaction at 11°C for 15 hours. After the reaction is completed, polyacrylamide gel electrophoresis (gel concentration 5%) is performed, and the migration pattern is observed by ethidium bromide staining. A band portion of the desired size (approximately 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, plasmid 1) BR322 (approximately 4.4K bp) for E. coli of 3 μm was added to 30.cz, 1L of lo mM
Tris-HCR(+)H7,5), 60 m
MNa CL 7111 was dissolved in an aqueous solution of MM0CI2, 1 unit of the restriction enzyme CfaI (New England Bioraps) was added, and a cleavage reaction was carried out at 37°C for 1 hour.

After cleavage with restriction enzyme CfaI, phenol extraction.

Ether extraction is performed and DNA is recovered by ethanol precipitation. Transfer this DNA to 30μ ships of 50111MTri
s-H(J(DI-17,4), 100 mM
It was dissolved in an aqueous solution of NaCj and 10mM MC+804, 1 unit of restriction enzyme 3alI <Takara Shuzo Co., Ltd.) was added, and a cleavage reaction was carried out at 37°C for 1 hour. After the reaction is complete, agaro gel electrophoresis (gel concentration 0.8%) is performed, and the cutting pattern is observed by ethidium bromide staining. Approximately 3.0% containing the majority of plasmid IIBR322.
Cut out the band corresponding to the 7K bp DNA part,
Three times the agarose gel fragment (vol, 'wt)
was dissolved in an 8M NaCf0a aqueous solution. Chen
[C, W.

Chen et al., Anal, 3iochem, 101
．． 339 (1980)], approximately 3.7K bp
A DNA fragment (Cja IH8alI) was recovered from the agarose gel.

Approximately 220b containing part of the previously obtained human TNF gene
After the terminal phosphorylation reaction of the DNA fragment of pB is performed according to the method described above, it is mixed with an aqueous solution of about 3.7 Kbp DNA containing most of the plasmid pB R322. 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 C,600r-111- strain was performed using the usual CaCR2 method (M, V, Noroar
A modified method of the method of d et al.) was used. That is, L medium of 5Id-, j4ney (1% tryptone, 0.5% yeast extract, 0.5% Na
(J, DH1,2) Nijieri l; Near] C600
An 18-hour culture medium of the r-to strain is inoculated and grown until the turbidity at 600 nm (OD6Iσ) of the culture solution containing the bacterial cells reaches 0.3. The bacterial cells were soaked in cold magnesium buffer F-[0, IM Na CR, 5mM M(l
CRz.

5 n1M Tris-HCf (IIH7,6,0
°C)] twice in 2 m cold calcium buffer [100 mMCa Cf2.250 111 M
KGN, 5mM MOCI2, 5mM Tris
-t-1cj (1)H7,6°0°C)] and left 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, add 1% tryptone, 5% yeast extract, 1% NaCf, 1% LBG medium, 0.
08% glucose, pH 7,2) was added and heated to 37°C.
Incubate with shaking for 1 hour. The culture solution is inoculated onto a selective medium [medium plate containing 30 μg/ml ampicillin (Sigma)] at a rate of 100 μρ/plate. The plates are incubated overnight at 37°C to grow the transformants. From the obtained ampicillin-resistant colonies, DNA was prepared using a known method, and the target plasmid pTNF1BR (approximately 4.OK bp
) was confirmed. In Figure 3, plasmid pTNF1
A method for creating a BR will be shown.

By the same method as above, synthetic oligonucleotide TN
Plasmid pTNF2N using F-8 to TNF-13
(approximately 3.1 KI) + 1), synthetic oligonucleotide T
Plasmid pTNF using NF-14 to TNF-17
3 (approximately 2.4K bp) were created. Figures 4 and 5 show plasmids pTNF2N and pTNF3.
0 creation methods are shown below.

It was confirmed that the base sequences of the synthetic oligonucleotide parts of the thus obtained plasmids pTNFIBR, pRNF2N and +1TNF3 containing part of the human TNF gene were as designed using the Maxam-Gilbert method [A, M, M
axam et al., MethodsEnzymol, 65
．． 499 (1980)] Niyotte confirmed.

-36-Example 4 (Creation of human TNF gene discovery type plasmid) 10 μl of the plasmid DTNFIBR obtained in Example 3 was treated in the same manner as in Example 3 with restriction enzymes CfaI and 5al.
After cutting with I and polyacrylamide gel electrophoresis (gel concentration 5%), human TNF3
A DNA fragment (about 220 bl) containing a part of the i gene (Ca<5alI) was recovered from the polyacrylamide gel.

Next, 10 plasmids pTNF2 obtained in Example 3
10 +11M Tris-H with μy of 1ooμm
CI(DH7,5), 60mM Na(J,
7 mMMU (J2 aqueous solution was dissolved, 40 units of restriction enzyme PVt (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, cleavage with restriction enzyme 3al , after polyacrylamide gel electrophoresis (gel concentration 5%), approximately 170b11+ containing part of the human TNF gene was obtained according to the method of Example 2.
7) DNA fragment (SalI+PvulI) was recovered from polyacrylamide gel.

In addition, plasmid pTNF3 obtained in Example 3 10
μSF was also 100 μfi of 10 mM Tris-1.
-ICI (pH 7,5), 60 mM Na
It was dissolved in cL 7 mM MgC12 aqueous solution, 40 units of restriction enzyme PvuII and 40 units of restriction enzyme HindI (Takara Shuzo) were added, and a cleavage reaction was carried out at 37°C for 1 hour. After polyacrylamide gel electrophoresis (gel concentration: 5%), approximately 110 bl of DN containing a part of the human TNF gene was purified according to the method of Example 2.
Fragment A (PvuII HHind ■) was recovered from the polyacrylamide gel.

On the other hand, plasmid 1 with E. coli trp promoter
) YS31N (approximately 4.7 Kbp) was cut with restriction enzymes + JaI and ) (indlll in the same manner as above,
After agarose gel electrophoresis (gel concentration 0.8%), a DNA fragment of approximately 4.7K bl containing most of the plasmid pYs31N ((JaI←
HindIII) was recovered from the agarose gel.

Approximately 2 cells containing part of the human TNFi gene thus obtained
3 D of 20bp, about 170bp and about 110b11
The NA fragment and a DNA fragment of approximately 4.7 Kb+1 containing most of plasmid I) YS31N were mixed, 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-1 strain according to the method of Example 3, and the desired human TNF gene expression plasmid pTNF401NN (approximately 5.2K bl) was selected from among the transformed strains.
Clones with >1 were selected. FIG. 6 shows the method for constructing the plasmid I) TNF401NN.

In addition, the above plasmid pYs31N5μg was partially digested with the restriction enzyme PvuII according to the above method, further cut with the restriction enzyme )(indll[, and after agarose gel electrophoresis (gel concentration 0.8%), Approximately 2.7K bp containing the trp promoter according to the method of Example 3
DNA fragment [P vuI[f21” Hind m
] 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. The ends of 0.5 μg of each of the two synthetic oligonucleotides 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) HHind [[
], and after ethanol precipitation, a ligation reaction using T4-DNA ligase was performed according to the method of Example 3. After the reaction, Escherichia
The clone was introduced into E. coli CEOor-m- strain, and a clone having the target plasmid 1) AA41 (approximately 2.7 K bp) was selected from among the transformed strains. Such plasmids are
The copy number control region was removed from the plasmid flYs31N, and the cloning region located downstream of the trp promoter was
It is a multi-copy, high-efficiency expression vector with an E. coli trDA terminator downstream of the 7th site.
The figure shows how to create it.

2 μg of this plasmid DAA41 was cut with the restriction enzymes C#aI and Hindl in the same manner as above, and after agarose gel electrophoresis (gel concentration 0.8%), plasmid l was digested according to the method of Example 3. )A DNA fragment of about 2.7K bp containing most of A41 (CRaIHl-(
indl[[) was recovered from the agarose gel.

In addition, the previously obtained human TNF gene expression plasmid I) 5 μg of TNF 401NN was cut with the restriction enzymes CfaI and Hindll in the same manner as above, and after polyacrylamide gel electrophoresis (gel concentration 5%), Example 2 Approximately 490 bp containing the entire human TNF gene
The DNA fragment <CRa I"Hind I [[) was recovered from the polyacrylamide gel.

The approximately 2.7 K bp DNA fragment containing most of the plasmid 11A A 41 obtained in this way and the approximately 490 bp DNA fragment containing the entire human TNF gene were mixed,
After ethanol precipitation, T4-
A ligation reaction using DNA ligase was performed.

After completion of the reaction, according to the method of Example 3, Escherichia
The target plasmid pTN F 401A (approximately 3.2 Kb
Clones with p) were selected. This plasmid is
It has the ability to express the human TNF gene more efficiently, and the method for its production is shown in Figure 8.

Example 5 (Creation of a novel antitumor active polypeptide gene expression plasmid) 20 μg of the human TNF gene expression plasmid pTNF 401A obtained in Example 4 was cut with restriction enzymes CjaI and Hindll according to the method of Example 4. Then, after polyacrylamide gel electrophoresis (gel concentration 5%) and agarose gel electrophoresis (gel concentration 0.8%), two DNAs were generated according to the methods of Examples 2 and 3, respectively.
Fragment (approximately 490 bp and approximately 2.7 K bp, both CR
a IMHindll[) was recovered from the gel.

Approximately 490b including the entire human TNF gene obtained here
The DNA fragment of p.
HCj (1)H7,4), 10mM Mg5o
n, was dissolved in a 1 mM dithiothreitol aqueous solution, 10 units of restriction enzyme HapU (Takara Shuzo) was added, and a cleavage reaction was performed at 37°C for 1 hour. After the reaction was completed, polyacrylamide gel electrophoresis (gel concentration 5%) was performed, and an approximately 390 bp DNA fragment containing most of the human TNFm trochanter (Hap■←H
indDI) was recovered from the polyacrylamide gel.

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

Purified. The terminals of 0.5μ9 of each of the four synthetic oligonucleotides obtained were phosphorylated according to the method of Example 3, and after annealing, T4-DN
A ligation reaction using A ligase was performed.

After the reaction, the obtained double-stranded oligonucleotide was combined with the previously obtained approximately 2.7K bp DNA fragment (C1a I
``A DNA fragment of about 390b11 (Hapl[4-1l
After mixing with -1indI[l] and 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-n+- strain according to the method of Example 3,
Target plasmid +1 TNF471 from among the transformed strains
(approximately 3.2K bp) was selected. This plasmid has the following amino acid sequence (H2N>-
Ar(] -Lys-Ar(1-Lys-Pro-
Val-A Ia-His-Vat-Val-A Ia
-A sn-Pro-G In-A la-G lu
-G Iy-G In-L eu-G In-Trl
)-L 13u-A Sn-A r(]-A rg-A
la-A sn-A la-L eu-L eu-A
la-A 5n-G ly-Val-G Iu-L
eu-A ra-A sp-A 5n-Gln-Leu
-Val-Val-Pro-8er-Glu-G +y
-Leu-Tyr-Leu-1le-Tyr-5er-
G In-Val-Leu-Phe-Lys-G IV
-G 1n-G ly -CVS -Pro -S
er -T hr -His -V al -1eu-
1eu -Thr -His -Thr -1le -
S er -Aro-I 1e-Ala-Val-3e
r-Tyr-Gln-Thr-Lys-Val-A s
n-L eu-L eu-5er-Ala-I Ie-
LIS-8er-PrO-CyS-Gln-A ro
-G lu -T hr -Pro -G lu -
G ly -A la -G lu -A Ia -L
ys -Pro -Trp -Tyr -G
lu -Pro-I Ie-Tyr-Leu-G IV
-G ly-Val-Phe-G ln-Leu-G
lu-LVS-G +y-A 5F)-Aro-Leu
-8er-Ala-Glu -11e-Asn -A
ro -P ro-A sp -Tyr-L eu-
A sp -P he -A Ia-G Iu-S e
r-G ly-G In-Val-Tyr-Phe-G
ly-Ile-11e-Ala-Leu-(COOI-
1) This is a novel antitumor active polypeptide gene expression plasmid encoding a novel antitumor polypeptide represented by or a polypeptide having Met bound to its amino terminus, and the method for its construction is shown in Figure 9. .

Example 6 (Confirmation of expression) Expression of the expression vector flAA41° human TNF gene expression type plasmid pTNF401NN or IIT NF 401A 1 obtained in Example 4 or the novel antitumor active polypeptide gene obtained in Example 5 Escherichia coli C60 carrying type plasmid 11 TNF471
Or-111- strain was treated with 30-50 μg/IIIfl ampicillin, 0.2% glucose and 4#iF/ae
M9 medium containing casamino acids [0,6% l'Ja 2
HPO4-0,3% to 2HPO40,05% NaC#-
After sterilizing the 0.1% N84Cf aqueous solution (1) l (7,4) in an autoclave, separately sterilized MO8O4 aqueous solution and CaCj2 aqueous solution are added to the final concentration of 2111M and 0.111IM, respectively. ] The cells were inoculated at 250 d, and cultured with shaking at 37° C. until ODbσ reached 0.7. Next, 3-β-indoleacrylic acid at a final concentration of 50 μg/d was added to the culture solution, and the shaking culture was continued at 37° C. for 12 hours.

After collecting Escherichia coli cells by centrifugation, the cells were washed with PBS buffer (20111M phosphate buffer containing 150mM NaCj, l)87.4). 10#ll of bacterial cells after washing! suspended in PBS buffer and placed in an ultrasonic generator (Kubota, 20
After the bacterial cells were disrupted using 0M type), the bacterial cell residue was removed by centrifugation.

A portion of the obtained E. coli lysate was treated with Tris-
HCf buffer (pH6,8), SDS, 2
- Add mercaptoethanol and glycerol to final concentrations of 60 mM, 2%, 4%, and 10%, respectively, and perform 5O8-polyacrylamide gel electrophoresis [Suzuki, 3
Ilden, Tei, 43 (1977)].

The separation gel was 12.5%, and the running buffer was SD3.
, Tris-glycine system [U, K, Laemmli
.

Nature, 227. 680 (1970)
] was used. After the electrophoresis was completed, the proteins in the gel were stained with Coomassie Blue R-250 (Bio-Rad) to confirm the expression of the human TNF gene and the novel antitumor active polypeptide gene. Copy part of the result and
It is shown in Figure 0.

In addition, the gel after staining was subjected to a chromatography scanner (Shimadzu, C
8-930 type), the proportion of the produced human TNF protein or the novel antitumor active polypeptide in E. coli cytoplasmic protein was calculated. As a result, human TN
F gene expression plasmid 1) In E. coli harboring TNF401A, human TN accounts for approximately 11% of the total cytoplasmic protein.
Similarly, approximately 17% production of the novel antitumor polypeptide was observed in E. coli containing the F protein and the novel antitumor polypeptide gene expression plasmid pLTNF2. Also, human TNF3! The amount of human TNF protein produced in E. coli carrying the lit gene expression plasmid pTNF4o1NN is
Only about 40% of the case with F401A, expression vector I
) The usefulness of AA41 was demonstrated.

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, 100 µ samples of the E. coli lysate containing the novel antitumor active polypeptide obtained in Example 6 were diluted in a medium, and 4 × 1
Mouse-929 fibroblasts (AT
CCCCL-929) suspension was mixed in a 96-well tissue culture microplate (Costar). At this time, actinomycin D (Cosmegen, Banyu Pharmaceutical Co., Ltd.) with a final concentration of 1 μg/Id is added. As the medium, Eagle's Minimum Essential Medium (Hakusui Pharmaceutical Co., Ltd.) containing 5% (vol/vol) fetal bovine serum was used. After culturing the above microplate at 37°C for 18 to 20 hours in air containing 5% carbon dioxide,
Crystal violet solution [5% (vol-A 7
- /vol) methanol aqueous solution, 0.5% (wt/v
Live cells were stained using a solution of crystal violet. After washing away the excess crystal violet and drying it, the remaining crystal violet was extracted with 100 μm of 0.5% SDS aqueous solution, and its absorbance at 595n11 was determined as EL l5A7.
Measure with ty+f- (Toyo side device, ETY-96 type). This absorbance is proportional to the number of surviving cells. Therefore, 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 human TNF protein or a novel antitumor active polypeptide is added is calculated using a graph (for example, Fig. 11). Define the dilution factor as a unit. From Figure 11, 100 μA of E. coli lysate containing the human TNF protein encoded by the expression plasmid pTNF401A is 1.5×1.
0” units of activity, and expression type plasmid 1
)Escherichia coli lysate containing a novel antitumor active polypeptide encoded by TNF471 100 μl is approximately 1.2X
It was revealed that each had an activity of about 10 G units.

Expression type plasmid pTNF401A obtained in Example 6
Human TNF protein or expression plasmid encoded by It was quantified and calculated from a calibration curve using bovine serum albumin. When the specific activities of the human TN'F protein and the novel antitumor active polypeptide were calculated from the expression levels, activity values, and protein determination results obtained above, the values shown in Table 1 were obtained. Table 1 shows that the novel antitumor active polypeptide has a specific activity approximately 8 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 the drawing]

Figure 1 shows the nucleotide sequence of the designed human TNF gene, and
The figures show the base sequences of chemically synthesized synthetic oligonucleotides. Figures 3, 4 and 5
The figure shows plasmid pT containing part of the human TNF gene.
The methods for producing NF1BR, pTNF2N, and pTNF3 are shown. Figure 6 shows the method for creating the human TNF gene expression plasmid pTNF401NN, and Figure 7 shows the method for creating the expression vector pA A41.
Figure 8 shows human TNF gene expression plasmid 1)
Each method for creating TNF401A is shown. FIG. 9 shows a method for constructing the novel antitumor active polypeptide gene expression plasmid pTNF471. FIG. 10 shows the results of expression confirmation of the human TNF gene and the novel antitumor active polypeptide gene. FIG. 11 shows the results of measuring the activity of human TNF protein and a novel antitumor active polypeptide.

Claims (10)

[Claims] (1) The following amino acid sequence [There is an amino acid sequence] A novel bioactive polypeptide represented by peptide. (2) The polypeptide according to item 1, characterized in that Met is bound to the amino terminus. (3) A recombinant plasmid containing a DNA region encoding a novel physiologically active polypeptide represented by the following amino acid sequence (amino acid sequence available) or a polypeptide having Met bound to its amino terminus. (4) Single-stranded DNA whose DNA region is represented by the following base sequence [there is a gene sequence] and its complementary single-stranded DNA
A third characterized in that it contains a double-stranded DNA consisting of A.
Plasmids described in section. (5) Single-stranded DNA whose DNA region is represented by the following base sequence [there is a gene sequence] and its complementary single-stranded DNA
A third characterized in that it contains a double-stranded DNA consisting of A.
Plasmids described in section. (6) The plasmid according to item 3, wherein the plasmid is plasmid pTNF471. (7) A recombinant transformed with a recombinant plasmid containing a DNA region encoding a novel physiologically active polypeptide represented by the following amino acid sequence [amino acid sequence is available] or a polypeptide with Met bound to its amino terminus. modified microbial cells. (8) The microbial cell according to item 7, wherein the microbial cell is Escherichia coli. (9) Recombinant plasmid transformed with a recombinant plasmid containing a DNA region encoding a novel physiologically active polypeptide represented by the following amino acid sequence [amino acid sequence is available] or a polypeptide with Met bound to its amino terminus. 1. A method for producing a novel bioactive polypeptide, which comprises culturing microbial cells, producing and accumulating a novel bioactive polypeptide in the culture, and isolating the novel bioactive polypeptide from the resulting culture. (10) A pharmaceutical composition containing an antitumor-effective amount of a novel physiologically active polypeptide represented by the following amino acid sequence [amino acid sequence is available] or a polypeptide having Met bound to its amino terminus.