JPS63267291A - Novel physiologically active polypeptide - Google Patents

Abstract

PURPOSE:To obtain a antitumor and physiologically active polypeptide, consisting of a specific amino acid sequence and having excellent specific activity and stability and useful as a carcinostatic agent, etc. CONSTITUTION:A synthetic oligonucleotide obtained by synthesizing with a codon derived from a human tumor necrotic factor (TNF) protein is linked and cloned in a vector, such as pBR322, to provide a human TNF gene expression type plasmid (e.g. pTNF401A) (A), which is then subjected to treatment with a restriction enzyme, genetic reparative treatment, etc., to afford a recombinant plasmid pTNF478 (B) containing a DNA region capable of coding a physiologically active polypeptide, etc., having an amino acid sequence, such as an amino acid expressed by formula I. The components (A) and (B) are then introduced into Eschericia.coli to afford recombinant microbial cells (C). The resultant cells (C) are subsequently cultivated in a culture medium containing glucose, casamino acid, etc., and the cultivated cells (C) are collected and subjected to treatment, such as crushing, to provide a cell lysate (D), which is then subjected to separation treatment, such as electrophoresis, to recover the aimed physiologically active polypeptide consisting of an amino acid sequence, such as an amino acid expressed by formula II.

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, novel polypeptides having antitumor activity (hereinafter sometimes abbreviated as novel antitumor activity polypeptides), recombinant plasmids containing a DNA region encoding the polypeptides, and constructs transformed with the plasmids. The present invention relates to a modified microbial cell and a method for producing a novel antitumor active polypeptide using the microbial cell.

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-Alanine ArQL-Arginine Asn L-Asparagine Asp L-Aspartate Cys L-Cystine Gln L-Glutamine GIuL-Glutamate Gly Glycine HiSL-Histidine 11eL-Isoleucine eu L-Leucine Lys L-Lysine Met L-Methionine Phe L- Phenylalanine prol-proline 3er L-serine Thrl-threonine Trl) L-t-lyptophan Tyr L-tyrosine Val L-valine The DNA sequence is also abbreviated by the types of bases contained in each deoxyribonucleotide that makes up the DNA sequence. year,
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, (H2N) - and -( COO
H) indicates the amino terminal side and carboxy terminal side of the amino acid sequence, respectively, (5')- and (3'
) indicate the 5' end and 3' end of the DNA sequence, respectively.

('2J Background of the Invention Carswell et al.
They discovered that the serum collected after administering endotoxin to mice that had been previously stimulated with mette-Querin (BCG) contained a substance that causes hemorrhagic necrosis of transplanted MethA sarcoma cancers. Tumor necrosis factor
[E, A. Carswell et al., Proc, Natl.

Acad, Sci, USA, 72.3666
(1975) ], this TNF was found in mice, rabbits,
Since it is found in many animals including humans, and 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)]. After that, Jiben et al. [T, 5hira
i et al.

Nature, Yu13. 803 (1985) ]
, Munemura et al. [Yozaki et al., Cancer and Chemotherapy, 12. 160(
1985) Ko, Wana et al. [A, M., Wang et al., S.
science, Yumi 149 (1985)] and M.
armenout et al. [A, M arms-no
ut et al.

Eur, J. Bio; hel., 152. 51
5 (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 [8, Beulter et al.
et al., Nature.

316, 552 (1985)], since cachectin has riboprotein lipase inhibitory activity,
Administration of TNF increases the amount of triglycerides in the blood,
It was suggested that this may result in side effects such as hyperlipidemia. In addition, there is also a shadow on vascular endothelial cells 1 [J, R.

Qamble et al., J. Exp. Med, 62
．． 2163 (1985)], bone resorption effect [D, R
, Beltolini et al., Nature, 319
．． 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 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 Cys and Cys with other amino acid residues ( POT application publication WO3
6/ No. 04606, patent application Sho 6l-106772),
Substitution of G Iy″ゝ with other amino acid residues (patent application 1986)
No. 1-106772, Japanese Patent Application No. 61-238048).

Substitution of A I a /J' with other amino acid residues has been reported (Japanese Patent Application No. 233337 (1981)). Regarding the deletion of amino acid residues on the amino terminal side, it has been shown that 6-amino acid-deleted TNF has cytotoxic activity (JP-A-61-50923), and that 7-amino acid deleted TNF has cytotoxic activity. (Patent Application No. 61-90087)
, 1 to 10 amino acid deleted TNF has cytotoxic activity, and the specific activity is maximum in 6 to 8 amino acid deleted TNF (PCT application publication W 086/023
81), that 10 amino acid deleted TNF has cytotoxic activity (Japanese Patent Application No. 114754/1983), and 1.
It has been reported that TNF with a single amino acid deletion 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 the following amino acid sequence (82N)-Pro-5er-ASI)-L'
/5-Pro-Vat-Ala-His-Val-Va
l-Ala-A sn-Pro-G In-A
la -G lu -G ly -G In -L e
u −G In −T rp −L eu −A sn
-A r(] -A rg-A Ia-Asn-A
Ia -Leu -Leu-A 1a-Asn -Q
ly-V al -G Iu -L eLl-A r
Q -A Sp-A Sn -G In -L eu
-Vat -Val -Pro -Ser -G I
u −G +y−Leu−, Tyr−Leu −11e
-ryr-Ser -G In -Val -Leu-
Phe -Lys-G ly-G In -G Iy
-CVS -Pro -Ser -T hr -H
is −V at −1eu −1eu −T hr
-@ is -Thr -■le -Ser -A r
g -11e -A la-Val-5er-Tyr-
G ln-T hr -L ys -V al -A
sn -1eu -L eu -A la -I l
e-Lys-3er-Pro-Cys-Gln=Arg
-Glu-Thr-Pro-Glu-Gly-Ala-
Glu-A Ia-Lys-Pro-Trp-
Tyr-G Iu-Pro ro-I le-Tyr-Le
u-G +y-G +y-Val-Phe-G In-
L eu-G Iu-L yS-G ly-A SO-
A r(1-Leu-8er-A 1a-Glu-1
le-Asn-Arg-Pro-A so-TVr-
Leu-A sp-Phe-A 1a-G Iu-S
er -G ly -G In -V al -T
yr -P he -GIV -1le -11e-A
This is achieved by providing a novel anti-tumor active polypeptide represented by la-Leu -(COOH) or a polypeptide having Met bound to its amino terminus, and also by providing a DNA encoding the above-mentioned novel anti-tumor active polypeptide.
This is achieved by providing a recombinant plasmid containing the region, and furthermore, a recombinant microorganism cell is transformed with the thus obtained recombinant plasmid, and the microorganism cell is used to produce the desired novel antitumor active polypeptide. It has been found that this has been achieved by providing a method and a pharmaceutical composition containing this novel anti-tumor active polypeptide.

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 [0, p
ennica et al., supra] by selecting an appropriate codon from among several codons and chemically synthesizing it. When designing the human TNF gene,
It is desirable to select the most suitable codon for the host cell to be used, and it is desirable to provide a cleavage site with an appropriate restriction enzyme at an appropriate position to facilitate subsequent cloning and genetic modification.

Furthermore, it is preferable that the DNA region encoding the human TNF protein has a translation start codon (ATG> in a format that matches the reading frame in the upstream direction, and a translation initiation codon (ATG) in a format that matches the reading frame 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 TNF gene can be cloned into an appropriate vector by using restriction and enzyme cleavage sites 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. Synthesis method for each oligonucleotide Toshite Hashiester method [H, G, Khorana
.

“One Recent Development
ents 1nChen+1stry of p
hosphate E 5ters of B 1
oloaical interest”,
John W 1leyand 3ons,
(NC, New York (1961)]
.

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

Am, Chelm, Soc, 89.4801 (196
7)] and the phosphite method [M, D, Matte
ucci et al.

Tetrahedron Lett,, 21. 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., Gene, 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 pTNFIBR.

pTNF2N 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 (Iac promoter), tac promoter, PL promoter,
Examples include the lpp promoter, but especially the tr
The p promoter is preferred. As a plasmid having a trp promoter, preferably l) Y S 31N
, or pA A 41 is used.

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,
Examples include 1pp terminator, 9trpA terminator, etc., but trpA terminator is particularly suitable, and pA A 41 is preferably used as the plasmid having trpA terminator. By cloning this expressed human TNF gene into, for example, I) a BR322-derived vector, an expression plasmid can be created. As a human TNF gene expression plasmid, preferably pTNF401NN or pTNF
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 Such a novel antitumor active polypeptide gene expression plasmid is preferably flTNF 41B,
pTNF416A or pTNF478 is used.

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

Gene, 3. 279 (1978)] using a microbial host such as Escherichia coli C6C60.
0r- strain (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, Mania
tis et al., eds., “Molecularc l0n”
ino”, P 440.Col 5prin
.

)-1arbor 1laboratory,
New York (1982)], and it is desirable to add, for example, ampicillin, if necessary. Cultivation is carried out under conditions suitable for the target recombinant microorganism, such as aeration by shaking and stirring, for 37 days.
℃ for 2 to 36 hours. In addition, at the start of culture or during culture, 3-
Agents such as β-indole acrylic acid can also be added.

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.

Previously, in which the cytotoxicity against mouse L cells is examined.
Vitro activity assay [Ruff, J.

l mmunol, 26. 235 (1981)
However, from the viewpoints of measurement time, quantitative properties, 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 different from the conventionally known human TNF protein, and by using this novel antitumor active polypeptide, an excellent antitumor pharmaceutical composition can be obtained. It is 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.

Penn1ca et al., Natur'e, ≦11
2. 724 (1984)] reported human TN.
Based on the nucleotide sequence of the structural gene part of the F precursor CD N A, cleavage sites with appropriate restriction enzymes are set at appropriate positions, and a translation initiation codon (ATG) is placed on the 5' side and two on the 3' side. Translation stop codons (TGA and TAA) were respectively provided. 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 HindlI was provided downstream of the 3' translation stop codon to facilitate ligation with a vector plasmid.

Example 2 (Chemical synthesis of oligonucleotide) The human TNF gene designed in Example 1 was
Synthesize it in seven oligonucleotides. Oligonucleotide synthesis was performed using a fully automatic DNA synthesizer (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 portion of the desired size and finely crushing the polyacrylamide gel fragments, a 2- to 5-id elution membrane 17-[5001
RM NH40Ac-1mMEDTA-0,1%S
DS (pH 7,5)] was added and photographed overnight at 37°C. The aqueous phase containing the target DNA was recovered by centrifugation. Finally, a purified product of the synthetic oligonucleotide was obtained by applying the solution containing the synthetic oligonucleotide to a gel filtration column (Sephadex G-50).

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 (
TNF-1 to TNF-17), human TN Fi
The ff gene was cloned in three blocks.

0.1-1.0 μ9 synthetic oligonucleotide TNF-
2 to 5' to 15 units of T on the 5' end of TNF-6.
4-polynucleotide kinase (E.

E. coli type 3, Takara Shuzo) to phosphorylate each separately. Phosphorylation reaction is 50i with 10-20μ polishing.
MTris-HCf (1)t-19,5), 10
1M M-! It C12° in 5mM dithiothreitol, 101M ATP aqueous solution at 37°C, 3
This was done for 0 minutes. After the reaction is completed, all the synthetic oligonucleotide aqueous solutions are mixed together, and T4-polynucleotide kinase is inactivated and removed by phenol extraction and ether extraction.

To this synthetic oligonucleotide mixture, add 0.1~
1.0μ9 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, 661MT of 30 μm
ris-H(J(1)H7,6), 6.6 m
M MCI C12゜1On+M dithiothreitol, 1a dissolved in MATP aqueous solution, 300 units of T
Add 4-DNA ligase (Takara Shuzo) and incubate at 11℃ for 15 minutes.
A time ligation reaction was performed. 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, 3 μm of E. coli plasmid 1) BR322 (approximately 4.4 K bp) was added to 30 μm of 10 mM Tri
s-HC1(ll'87.5), 60 mM N
a C1,7mMQCfz aqueous solution was added, 10 units of restriction enzyme CfaI (New England Biolabs) was added, and a cleavage reaction was performed at 37°C for 1 hour.

After cleavage with restriction enzyme C18, phenol extraction was performed.

Ether extraction is performed and DNA is recovered by ethanol precipitation. This DNA was polished by 30 μm with 501M Tris-
1-1(J (DH7,4), 1001M N
a C1,101M M<1804 was dissolved in an aqueous solution, 10 units of restriction enzyme 5alI (Takara Shuzo) was added, and a cleavage reaction was carried out at 37°C for 1 hour. After the reaction is completed, agarose gel electrophoresis (gel concentration 0.8%) is performed, and the cutting pattern is observed by ethidium bromide staining. Plasmid 1) Cut out the band corresponding to the approximately 3.7K bp DNA portion containing most of BR322, and divide the agarose gel fragment into three times the amount (vat 7
wt) in an 8M NaC10* aqueous solution. c.
The glass filter method of Hen et al. [C, W.

Chen et al., Anal, 3iochea+, 101
．． 339 (1980)], an approximately 3.7 Kbp DNA fragment <C1a I H8al I> was recovered from an agarose gel.

Approximately 220b containing part of the previously obtained human TNF gene
The DNA fragment of p is subjected to a terminal phosphorylation reaction according to the method described above, and then mixed with an aqueous DNA solution of about 3.7 Kbp containing most of the plasmid +) BR322. After ethanol precipitation, both DNAs were separated according to the method described above.
A ligation reaction of the fragments was performed.

Escherichia coli (:, 600r-m- strain) was transformed using the usual CaCR2 method (M, V, Nor(la
A modified method of the method of rd et al.) was used. That is, 5Id
Rice culture medium (1% tryptone, 0.5% In extract, 0.
5% Na CL 1) H7, 2) Escherichia-D
The turbidity (OD, <-) of the culture solution containing Kokutai at 600rv was inoculated with an 18-hour culture medium of C6C600r- strain.
Grow until the value reaches 0.3. The bacterial cells were soaked in cold magnesium buffer [0,1M Na C1,51
M M(J C12゜51M TriS-H(J
(1) H7,6,0°C)] and washed twice in 2d cold calcium buffer [1001Mca C12,2
501M KCf, 51MMQ C10,51M
Resuspend in Tris-H (J(1)H7,6°0°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 mixed with 2=1 (vol.
l, : vol,) mix. Stir this mixture for 60 minutes.

After keeping at 0°C, 1 d of LBG medium (1% tryptone,
Add 0.5% yeast extract, 1% NaCj, 0.08% glucose, 1)H7,2), and culture for 1 hour at 37°C. Transfer the culture solution to a selective medium [L medium plate containing 30 μ'j/d of ampicillin (Sigma)].
Inoculate at a rate of 00 μU/plate. 37 plates
The transformed strain is grown by culturing overnight at °C. DNA was prepared from the obtained ampicillin-resistant colonies using a known method, and acquisition of the target plasmid pTNF1BR (approximately 4.OK bp) was confirmed by agarose gel electrophoresis. FIG. 3 shows the method for constructing plasmid pTNF1BR.

By the same method as above, synthetic oligonucleotide TN
Plasmid pTNF2N using F-8 to TNF-13
(approximately 3.IKbp) was added to the synthetic oligonucleotide TNF
Plasmid pTNF3 (
Approximately 2.4K bl)) were created, respectively. Figures 4 and 5 show plasmids pTNF2N and I) TNF3.
We will show how to create each.

The thus obtained plasmids pTNF1BR, pRNF2N and pTNF3 containing part of the human TNF gene,
It was confirmed that the base sequence of the synthetic oligonucleotide used was as designed using the Maxam-Gilbert method [A, M, Ma
xam et al., Methods Enzymol, 65.
499 (1980)].

Example 4 (Human TNF gene expression type - Creation of plasmid) Plasmid pTNF1BR10μ9 obtained in Example 3
was added to the restriction enzymes CfaI and 5al in the same manner as in Example 3.
After cutting with I and polyacrylamide gel electrophoresis (gel concentration 5%), a DNA fragment of about 220 bl containing part of the human TNF gene <C1a> was obtained according to the method of Example 2.
l-8alI) was recovered from polyacrylamide gel.

Next, 10 plasmids pTNF2 obtained in Example 3
μ9 was added to 100 μu of 10 mM Tris-HCj
(DH7,5), 60i M Na CR,7m
MMfJ (J2) was dissolved in an aqueous solution, 40 units of the restriction enzyme PVIJI (Takara Shuzo) was added, and the cleavage reaction was carried out at 37°C for 1 hour. After polyacrylamide gel electrophoresis (gel concentration: 5%), approximately 170 bp of DNA containing part of the human TNFm gene was extracted according to the method of Example 2.
Fragment A (SalI''PVIJII) was recovered from the polyacrylamide gel.

In addition, plasmid pTNF3 obtained in Example 3 10
u9 also 100ul of 10mM Tris-HC
R (pH 7,5), 60 mM Na CL 7
Dissolved in an aqueous solution of mMMgCj2 and added 40 units of the restriction enzyme pvJ and 40 units of the restriction enzyme HindI [
[(Takara Shuzo) was added, and the cleavage reaction was carried out at 31°C for 1 hour. After polyacrylamide gel electrophoresis (gel concentration 5%), human TNF was purified according to the method of Example 2.
A DNA fragment of approximately 110 bEl containing part of the gene (pv
LIII MHind m) Bo IJ 7ri IJ /
L, 7 was recovered from midgel.

On the other hand, plasmid p with E. coli trp promoter
Ys31N (approximately a, yKbp) 5μ9 was cut with the restriction enzymes CfaI and l-1indl[I 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 4.7 Kbp (CaI→
Hindn[) was recovered from the agarose gel.

Approximately 2 cells containing part of the human TNF gene obtained in this way
Three DNs of 20bp, about 170bp and about 110bp
A! ? The li piece and a DNA fragment of approximately 4.7 Kbp containing most of plasmid 1) 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, Escherichia coli C600r-11 was added according to the method of Example 3.
- Introduce the target human TNF gene expression plasmid pTNF401NN (approximately 5.2K
bp) was selected. FIG. 6 shows the method for constructing the plasmid 1) TNF401NN.

In addition, 5μ of the above plasmid pYS 31N was partially digested with the restriction enzyme PvuII according to the above method, and then further digested with the restriction enzyme HindI and subjected to agarose gel electrophoresis (gel concentration: 0.8%). , approximately 2.7K b containing the trp promoter, according to the method of Example 3.
DNA fragment of p [PvuII[2] HHindI[
[ ] Recovered from 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 [pvuIF(2)” Hind III
] The mixture was mixed, and a ligation reaction using T4-DNA ligase was performed according to the method of Example 3. After the reaction was completed, the target plasmid 1) AA41 (approximately 2.7K
bp) was selected. Such a plasmid is produced by removing the copy number control region from the plasmid pYs31N and inserting the E. coli (trl) A terminator downstream of the cloning site located downstream of the trp promoter.
This is a multi-copy, high-efficiency expression vector that has been given the following: Figure 7 shows how to create it.

This plasmid pAA41 2μ9 was cut with the restriction enzymes CfaI and Hindu in the same manner as above, and after agarose gel electrophoresis (gel concentration 0.8%), most of the plasmid pAA41 was extracted according to the method of Example 3. An approximately 2.7K bp DNA fragment containing (CfaI-HindII
I) was recovered from the agarose gel.

In addition, the previously obtained human TNF gene expression plasmid pTNF4o1NN5μ9 was digested with the restriction enzymes CjaI and )lindI in the same manner as above, and after polyacrylamide gel electrophoresis (gel concentration 5%), the Approximately 490 bl containing the entire human TNF gene according to the method
) was recovered from the polyacrylamide gel.

The thus obtained DNA fragment of about 2.7 bp containing most of the plasmid pA A 41 and the DNA fragment of about 490111 bp containing the entire human TNF gene were mixed, and after ethanol precipitation, Example 3 T4 according to the method of
- 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
p) was selected. This plasmid is
It has the ability to express human TNFm trochanter more efficiently, and the method for its preparation is shown in FIG.

Example 5 (Creation of a novel anti-tumor active polypeptide gene expression plasmid) The human TNF gene expression plasmid pT NF 4oIN N 20μ9 obtained in Example 4 was digested with restriction enzymes CjaI and CjaI according to the method of Example 4. Cut with Hindll and perform polyacrylamide gel electrophoresis at a gel concentration of 5%.
) and agarose gel electrophoresis (gel concentration 0.8%), two DNA fragments (approximately 490 bp and approximately 4,7K bp) were generated according to the methods of Examples 2 and 3, respectively.
, both C1a I MHind [[) were recovered from the gel.

Approximately 490b including the entire human TNFl trochanter obtained here
Polish the DNA fragment of p with 50μ of 1M Tris.
-I-lCf (p+ 7.5), 60111M
Na (J, 7mM 5%)
Then, according to the method of Example 2, a DNA fragment of about 460 bl (Ava
I 4-48 ind■) was recovered from the polyacrylamide gel.

Further, a double-stranded oligonucleotide having the base sequence shown in FIG. 9 was synthesized and purified into an upper strand and a lower strand according to the method of Example 2. 0.5μ9 of each of the two synthetic oligonucleotides obtained were subjected to terminal phosphorylation according to the method of Example 3, and then annealed.

The double-stranded oligonucleotide after annealing was prepared in 1.
: 41 tc approx. 4.7K bp (7) D N
A fragment (CjaI-1-1indn[) and human TNF
A DNA fragment of approximately 460 bp containing most of the gene (Av
After mixing with aI+Hind I[[) 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 C600 r-to strain according to the method of Example 3, and the desired plasmid 1) TNF41 was selected from among the transformed strains.
6 (approximately 5.2K bD) was selected.

This plasmid is a novel antitumor active polypeptide gene expression plasmid that encodes a novel antitumor active polypeptide in which the amino terminal seven amino acids of TNF are deleted, and the method for its construction is shown in FIG.

Furthermore, 1) TNF401NN to 1) TNF401
By the same method as in the case of creating A, an expression plasmid pTNF 416A (approximately 3.2 Kbl )
)It was created. Figure 8 shows the method for making it.

Expression type plasmid 1) TNF416A obtained above,
According to the method of Example 3, the restriction enzyme Hind [[ and 5a
Cut with lI and perform polyacrylamide gel electrophoresis (gel concentration 5%) and agarose gel electrophoresis at a gel concentration of 0.
8%), then according to the methods of Examples 2 and 3, respectively.
Two DNA fragments (approximately 270 bl) are generated and approximately 2.
9K bp, both 3alJ+)1indn[) were recovered from the gel.

3 of the novel antitumor active polypeptide genes obtained here
Approximately 270 bl of DNA fragment containing the '' side half was added to 50μ of 10 mM Tris-HCj (pt-+7.
4), 10IM MCI 804, 1mM
Dissolved in dithiothreitol aqueous solution, added 10 units of restriction enzyme 5au3AI (Takara Shuzo),
The cleavage reaction was carried out for 1 hour at °C. After completion of the reaction, polyacrylamide gel electrophoresis (gel concentration 5%) was performed according to the method of Example 2 to obtain an approximately 190 bp DNA fragment (
Sau3AI+IHind III)
Collected from J Ruamide gel.

Furthermore, an oligonucleotide having the base sequence shown in FIG. 10 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 completion of the reaction, the obtained double-stranded oligonucleotide was combined with the previously obtained approximately 2.9K bp DNA fragment (SalI”
Hind m) and the 3' side portion of the novel antitumor active polypeptide gene (S
After mixing with au3AI''Hind III) 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-m- strain according to the method of Example 3, and the desired plasmid I) TNF478 (approximately 3.2 Kbl) was selected from among the transformed strains.
A clone with the following was selected. This plasmid has the following amino acid sequence (82N)-Pro-3er-ASI)-Lys
-Pro -V al-A Ia-His-V a
l-V al-A la -A sn -Pro -
G In -A Ia -G lu -G Iy -G
In -L eu -G In -Trl) -L
eu -A Sn -A rlJ-A rg-A l
a -A sn-A Ia -L eu -L eu
-A Ia -A 5n-G +y -V al -G
Iu-L eu-A I'o -A St) -A
Sn -G In -LetJ -Val -vat
-Pro -Ser -G Iu -G ly -L
eu -Tyr -Leu -11e -Tyr -S
er -G In-Val-Leu-Phe-Lys-
G ly -G In-G Iy-Cys-Pro-
S er-T hr -His-V al -L eu
-L eu -Thr -His -Thr -1l
e -Ser -A ra-I Ie -A Ia-V
al-Ser -Tyr-G 1n-Thr -Lys
-Val-Asn -Leu -Leu-Ala -1
le -Lys -Ser -Pro -CVS
-G In -A ra -G lu -T hr
-Pro -G Iu -G Iy -A Ia -
G Iu -A la -L VS -P rO-T
rp-T yr -G Iu -P rQ -11e-
Tyr-Leu-Gly-Gly-Val-Phe-G
ln-L eu-G lu-L ys-G ly-A
sp-A rg-Leu-8er-A Ia-G l
u-11e-Asn-Ara-Pro-A sp
-T yr -L eu -A sp -P he -
A la -G lu -S er -G my -G
In -V al -T yr -P he -iy
-11e-11e-Ala, -Leu -(COOH
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 FIG.

Example 6 (Confirmation of expression) Novel antitumor active polypeptide gene expression plasmid obtained in Example 5 1) TNF416゜1) TNF
416A or 1) Escherichia with TNF478
DSC600r-m- strain was treated with 30-50 μg/fd ampicillin, 0.2% glucose and 4 m9/mf
M9 medium containing 1 of casamino acids [0.6% NazHP0
4 0.3% to 2 HPO4-0,05% NaC1-
After sterilizing the 0.1% N84Cj aqueous solution (1) H7,4) in an autoclave, M
(1804 aqueous solution and CaCl2 aqueous solution
1i1a degrees 211M and 0.1111M. The cells were inoculated at 1,250 days and cultured at 37°C until OD6 reached 0.7. Next, 3-β-indoleacrylic acid at a final concentration of 50 μg/rd was added to the culture solution, and the shaking culture was continued at 37° C. for 12 hours.

After collecting E. coli cells by centrifugation, the cells were washed using PBS buffer (20111M phosphate buffer containing 1501M NaCl, DH7,4).After washing, the cells were washed with 10-PBS buffer. Suspended in ultrasonic generator (Kubota, 200M type)
After the bacterial cells were disrupted using a microorganism, the bacterial cell residue was removed by centrifugation.

A portion of the obtained E. coli lysate was treated with Tris-
1-1cf buffer (1)H6,8), SDS,
2-mercabutoethanol and glycerol were added to final concentrations of 601 M, 2%, 4%, and 10%, respectively, and 5DS-polyacrylamide gel electrophoresis [Suzuki, Genetics, 31.43 (1977)] was performed. .

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

Nature, Yu27. 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 novel antitumor active polypeptide gene.

In addition, the gel after staining should be scanned using a chromatography scanner (Takaguruma,
C8-930 type), the proportion of the produced novel antitumor active polypeptide in E. coli cytoplasmic protein was calculated. As a result, the expression plasmid pTNF41
The production amount of the novel antitumor active polypeptide in E. coli with 6 is only about 50% of that in the case of expression type plasmid I) TNF 416A, and the production amount of the novel antitumor active polypeptide in E. coli with 6
1 and the expression plasmid IITNF 416A were 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, the E. coli lysate containing the novel antitumor active polypeptide obtained in Example 6 was sequentially diluted with a medium, and a 4×1
9294I fibroblasts (ATCCCCCL-929) suspension at a concentration of 0” cells/IIrR.
The mixture was mixed in a 6-well tissue culture microplate (coaster). At this time, actinomycin D (Cosmegen, Banyu Pharmaceutical) was added at a final concentration of 1 μg/−. Eagle's Minimum Essential Medium (Hinaga Pharmaceutical Co., Ltd.) containing 5% (vat/vol) fetal bovine serum was used as the medium. 5% of the above microplate
After culturing at 37°C for 18 hours in air containing carbon dioxide,
Crystal violet solution [5% (vol/■01
) Living cells were stained using 0.5% (wt/vol) crystal violet dissolved in an aqueous methanol solution. After washing away the excess crystal violet and drying it, extract the remaining crystal violet with 100 μg of 0.5% SDS aqueous solution.
The absorbance at 95rv is measured using an ELISA analyzer (Toyo Sokki, Model ETY-96). This absorbance is
proportional to the number of surviving cells. Therefore, the dilution factor of the E. coli lysate containing the novel antitumor active polypeptide, which corresponds to 50% of the absorbance of the control to which no diluted solution is added, is calculated using a graph (for example, Fig. 11), and the dilution factor is determined by the unit. It is defined as From FIG. 11, the expression plasmid 0TNF47 obtained in Example 6
It was revealed that 100 μm of E. coli lysate containing the human TNF protein encoded by 8 had an activity of about 540 units.

The total amount of protein contained in the E. coli lysate containing the novel antitumor active polypeptide encoded by the expression plasmid pTNF478 obtained in Example 6 was determined using a protein assay kit (Bio-Rad). Calculated from a calibration curve using bovine serum albumin. The specific activity of the E. coli lysate containing the novel antitumor active polypeptide was calculated from the activity values and protein quantification results obtained above, and it was found to have a specific activity of approximately 680 (units/rrtg-protein). Recognize.

[Brief explanation of the drawing]

Figure 1 shows the designed human TNF3! Figure 2 shows the base sequence of the gene, and Figure 2 shows the base sequence of the chemically synthesized synthetic oligonucleotide. Figures 3, 4 and 5 show plasmid p containing part of the human TNF gene.
The methods for producing TNF1BR, pTNF2N, and pTNF3 are shown. Figure 6 shows the method for creating the human TNF gene expression plasmid pTNF401NN, Figure 7 shows the method for creating the expression vector 1)A A41, and Figure 8 shows the human TNF gene expression plasmid pTNF401A and its novel antitumor activity. The method for producing polypeptide gene expression plasmid 1) TNF 416A is shown. Figure 9 shows novel antitumor active polypeptide gene expression plasmid 1) TNF416
Figure 10 shows the method for constructing the novel anti-tumor active polypeptide gene expression plasmid pTNF476. FIG. 11 shows the results of measuring the activity of the novel antitumor active polypeptide. Patent applicant Teijin Ltd.〈-ΦWQ! Q k O #
Q cI ト e c) 5 birds -4-rooster Pvu ]I 謬DAl CY)Lf') B Pyu'Jl(lnhang=8 口Figure 91ctα construction land tallri==hB page 1L

Claims (10)

[Claims] (1) The following amino acid sequence [There is an amino acid sequence] A novel physiologically active polypeptide represented by (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 pTNF478. (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.