JPH0383586A - Novel physiologically active polypeptide - Google Patents

Abstract

PURPOSE:To prepare the subject polypepetide having an antitumor activity by culturing a microorganism transformed with a plasmid containing a DNA region coding a specific polypeptide. CONSTITUTION:A codon-specifying amino acids constituting the peptide of human tumor necrosis factor(TNF) is selected and subjected to a chemical synthesis to give a human TNF gene (A). The component A is combined with a terminator, etc., and cloned in a vector to prepare a human TNF gene expression type plasmid (B) such as pTNF 401NN. The component B is treated with a restriction enzyme to prepare an antitumor activity-polypeptide gene expression type plasmid pTNF 637 (C) containing a DNA of formula I coding the antitumor activity-having polypeptide. The component C is transduced into an Escherichia coli to prepare a transformant (D). The component D is aerobically cultured in a medium containing glucose, etc., at approximately 37 deg.C for 2-36hr and the cultured product is purified by a centrifugal method, an electrophoresis method, etc., to prepare a physiologically active polypeptide having an amino acid sequence of formula II and having a Met bonded to the amino terminal thereof.

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 microorganism transformed with the plasmid, and a recombinant microorganism transformed with the plasmid. The present invention relates to a method for producing a novel physiologically active polypeptide using the microorganism cells. More specifically, a novel polypeptide having anti-II tumor 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 a recombinant microbial cell and a method for producing a novel antitumor active polypeptide using the microbial It cell.

In this specification, amino acids and polypeptides are 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.

AlaL-alanine Ar (IL-arginine ASnL-asparagine Asp L-aspartic acid Cys L-cysteine Gln L-glutamine Glu L-glutamic acid Gly glycine is l-histidine 11el-isoleucine 1-eul-monoleucine Ll/SL-lysine Met L-methionine PheL-phenylalanine Pr0L-proline 38rl--serine Thr L-threonine TrpL-tryptophan TVr L-tyrosine Val L-valine DNA sequences are also abbreviated by the types of bases contained in each deoxyribonucleotide that makes up the DNA sequence. As a matter of fact,
For example, use the following abbreviations.

A adenine (indicates deoxyadenylic acid) C cytosine (indicates deoxycytidylic acid) G guanine (indicates deoxyguanylic acid) T thymine (indicates deoxythymidylic acid) Furthermore, (zN) - and - <COO
(5')- and (3') indicate the 7-mino terminal and carboxy-terminal sides of the amino acid sequence, respectively
) indicate the 5' end and 3' end of the DNA sequence, respectively.

(2) Traitor of invention Carswell et al.
We discovered that the serum collected after administering endotoxin to mice that had been previously stimulated with Ilette-Guer+n (BCG>, etc.) contained a substance that caused hemorrhagic necrosis of the transplanted MethA sarcoma cancer.
This substance is called tumor necrosis factor (tumor necrosis factor).
osisFactor (hereinafter sometimes abbreviated as TNF) [E, A, Carswell et al.
P roc, N atl.

Acad, Sci, USA, 72.3666
(1975) ], this TNF was found in mice, rabbits,
It is found in many animals such as humans, and is specific to 11m cells.
Moreover, since it works across species, it has been expected to be used as an anticancer drug.

Recently, P ennica et al.
We carried out DNA cloning to clarify the primary structure of human TNF1 white matter and to
reported on the expression of the NFW gene [D, Penn
1ca et al., Nature, 312. 724
(1984)], then Oi et al.
ai et al.

Nature, 313. 803 (1985)
], Munemura et al. [Munemura et al., Cancer and Chemotherapy, 12. 1e
o (1985)], Wana et al.
et al., 5science, 228. 149<19
[85)] and MarmenOut et al. [A,
M armenout et al.

Eur, J. Biochem, 152. 51
5 (1985)] have successively reported on the expression of the human TNF3Il gene in E. coli.

Through the use of genetic engineering techniques, pure human TNF protein has become available in large quantities, and the physiological activities of TNF other than its antitumor properties 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 [3,3eulte
r et al., l'Jature.

316, 552 (1985)], and since cachectin has riboprotein 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.

Qaable et al., J. EXI), Med, 1
62.2163<1985)], bone resorption effect [D,
R, Beltolini, 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 conducted on the modification of human TNF protein, and in the amino acid sequence of human TNF protein shown in Figure 1, other amino acid residues of either or both of cy s 41 and cy s /I/ (PCT)
Publication application WO36/04606, patent application No. 61106
772), substitution of G l'/''' with other amino acid residues (Japanese Patent Application No. 61-106772, Patent Application No. 61-238
No. 048).

Substitution of A1a18 with other amino acid residues (Patent application 1986-
No. 233337) has been reported. Also, regarding deletion of amino acid residues on the amino terminal side, 6 amino acid deletion T
NF has cytotoxic activity (Japanese Unexamined Patent Application Publication No. 61-5
0923〉, 7 amino acid deletion TNF has cytotoxic activity (Patent application 1987-90087M), 1-
10 amino acid deleted TNF has cytotoxic activity,
The specific activity is maximized in 6 to 8 amino acid deleted TNF (PCT application publication W 086/02381), and 10 amino acid deleted TNF has cytotoxic activity (Japanese Patent Application No. 114754/1982). ), and that 11 amino acid deleted TNF has cytotoxic activity (Japanese Patent Application No. 173822/1983).

Therefore, the present inventors have conducted extensive research into modifying the human TNF protein with the aim of improving specific activity, improving stability, broadening the reaction spectrum, and reducing the 91 action, and have completed the present invention. It's arrived.

0) Object of the invention The object of the present invention is to provide a novel anti-I11 psychotropic 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 produce a novel anti-l1ffl tumor-active polypeptide using a recombinant microorganism transformed with the recombinant plushide and its recombinant microorganism cells.
The objective is to provide a method for J-building. Another object of the present invention is to provide a pharmaceutical composition containing the polypeptide.

Further objects of the invention will become clear from the following description.

(4) Structure of the invention The present invention provides the following anoic acid sequence (+-12N) -Pro-8er-ASD-Lys-
Pro-Vat-A la-His-vat
-Val -A Ia -A sn -Pro-G
In -A la -G lu -G ly -G I
nL eu-GIn-T rp-1eu-A 5ll-
A r(+-A r.

A la -A sn -A la -L eu -L
eu -A Ia -A snG ly-V al-
G lu-L eu-A rg-A sp-A sn-
Examples include a plasmid characterized by containing a double-stranded DNA consisting of a single-stranded DNA represented by CTGATAAGCTT-(3') and a single strand of DNA complementary to the single-stranded DNA.

More specifically, plasmid 1) TNFS37 can be mentioned.

The present invention includes a recombinant microbial cell transformed with a recombinant plasmid containing a DNA region encoding the above-mentioned novel physiologically active polypeptide or a polypeptide having M13t bound to its amino terminus.

Preferably, the microbial cell is Escherichia coli.

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; A new bioactive polypeptide is produced and accumulated, and the resulting culture i! The present invention includes a method for producing a novel physiologically active polypeptide, which is characterized by separating the novel physiologically active polypeptide from a substance.

The present invention includes a pharmaceutical composition containing 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 TNF gene consists of amino acids [D, P
[enn1ca et al., supra]] is selected from among several codons, and it can be obtained by 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 it is desirable to provide a cleavage site with an appropriate restriction WJ compound at an appropriate position to facilitate subsequent cloning and genetic modification.

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 downstream thereof. Translation 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 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 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 placed on a chemical bench or , it is desirable to take a method of linking each oligonucleotide. The synthesis method for each oligonucleotide is the diester method rLl (, G, Khora
na.

“S 01le R13Cent D 13Ve1
01) 0113ntS InChemistry
of Phosphate E 5ters
of B iological intercst
”, John WileVand 3ons,
■nc, New York (1961)
].

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

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

Tetrahedron 1-ett,, 21
．． 719 (1980), but synthesis by the phosphite method using a fully automatic DNA synthesizer is preferred from the viewpoints 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 water Mll at the 5' end of the synthetic oligonucleotide thus obtained is phosphorylated using, for example, 14-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,
B of 1var et al., Qene, 2.
95 (1977)], and then the DNA fragments of each block are ligated together. A plasmid containing a DNA fragment of a block constituting the human TNF gene is preferably pTNF1BR.

+1NF2N 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 lpp promoter, but especially the tr
The p promoter is preferred. I) YS31N or pA A 41 is preferably used as the plasmid having the trp promoter.

Furthermore, for the purpose of improving expression efficiency, it is possible to add a terminator that functions efficiently in E. coli downstream of the human TN gene.
1pp terminator, trp A terminator, etc., among which trl) A terminator is particularly preferred, and pA A 41 is preferably used as a plasmid having a trp terminator. This expressed human TNF3i gene, for example, I]BR322
By cloning into Yamago's vector, an expression plasmid can be created. As a human TNF gene expression plasmid, preferably pTNF401NN or pTN
F 401A is used.

(B) Cloning of the frMA antitumor active polypeptide gene; The thus obtained human TNF3i3i genotype plasmid was cut with an appropriate restriction enzyme to remove a specific region within the human TN F3i3i gene. Gene repair is performed using synthetic oligonucleotides having the base sequence.By using this method, it is possible to replace or add any amino acid in the human TNF protein with another amino acid,
Alternatively, it becomes possible to construct an expression plasmid containing a deleted gene encoding a novel anti-malignant active polypeptide. Such a novel antitumor active polypeptide gene expression plasmid is preferably pTNF416.
DTNF416A, 1) TNF 611 or 1
) TNF637 is used.

(C) Expression confirmation and activity evaluation; Examples of microbial hosts for expressing the human TNFffl trochanteric and novel antitumor active polypeptide genes include Escherichia coli, Bacillus subtilis, and yeast, but especially Escherichia coli [Escherichia coli]. Escherichia col
i) ] 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, Nor
gard et al.

Gene, 3. 279 (1978)] using a microbial host such as Escherichia coli C600r.
-m 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.
”, P 440. Co1d 3pring mouth a
rbor 1 laboratory, New
York (1982)], and it is desirable to add, for example, ampicillin, if necessary. Cultivation is performed at 37°C for 2 to 3 hours under conditions suitable for the target recombinant microorganism, such as aeration with shaking and stirring.
Do it for 6 hours. 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 antitumor m-active polypeptide thus obtained was evaluated in vivo by examining the effect of necrosis on MethA sarcoma transplanted into mice.
oActivity measurement method (CarSl#el+ et al.

(mentioned above), and examined the toxicity of m-cells against cells in mice.
VitrO activity assay [Ruff, J.

l m1uno1. , Yu26. 235 (1981)
] etc., but from the viewpoint of measurement time, quantitative nature, simplicity of measurement, etc., evaluation by in Vitr'0 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 (Creation of human TNFm gene expression type plasmid) Human TNF gene expression type plasmid (plus (do) I) TNF401A was created by the method described in JP-A-63-267290. This plasmid has the ability to express the human TNF gene more efficiently, and the method for its construction is shown in FIG.

Example 2 <Creation of novel anti-tumor active polypeptide gene expression plasmid> Human TNF gene expression plasmid I) described in JP-A-63-267290 TN F 401N N 20
μ9 was cleaved with the restriction enzyme C1aI and indm according to the method described in JP-A No. 63-267290, and subjected to polyacrylamide gel electrophoresis (gel 11° 5%) and agarose gel electrophoresis (gel concentration 0.5%). 8%), two DNA fragments (approximately 490 bD and approximately 4.7 K bp, both C1aI4-10indI [[
) was recovered from the gel.

Approximately 490 genes including the entire human TNF3W gene obtained here
bl) DNA fragment in 50μ of 10mM Tri
s-mouth C1 (pi-17,5), 60 mM
It was dissolved in an aqueous solution of NaCl,7giMMgCjz, 10 units of restriction enzyme AvaI (Takara Shuzo) was added, and a cleavage reaction was carried out at 37°C for 1 hour. After the reaction, polyacrylamide gel electrolysis a (gel concentration 5%)
Human TNF3! ! Approximately 46 genes containing most of the genes
01) l) DNA fragment (Ava engineering + Hind II [
> was recovered from polyacrylamide gel.

Further, a double-stranded oligonucleotide having the salt base sequence shown in FIG. 4 was synthesized and purified into an upper strand and a lower strand according to the method described in JP-A-63-267291.

The two synthetic oligonucleotides obtained were each 0.5
About μ9, Jikai Il! According to the method described in 163267290, the terminals were phosphorylated and then annealed.

After annealing, the double-stranded oligonucleotide was converted into a DNA fragment of about 4.7 Kbp (CffiaI+-
A DNA fragment (AvaI Ml-
1 ind m), and after ethanol precipitation, T4
- Perform a ligation reaction using ONΔ ligase to create an anti-I25#
After S, the target plasmid 1) TNF4 was introduced into Escherichia coli C600r-1- strain and selected from among the transformed strains.
16 (approximately 5.2 Kbp) 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 7-acino acids of TNF have been deleted. Figure 4 shows the method for its construction. Ta.

Furthermore, pTNF401NN to 1) TNF401Δ
An expression plasmid pT NF 416A (approximately 3.2 Kbl), which has the ability to efficiently express a novel antitumor active polypeptide gene in which the amino-terminal 7 amino acids of TNF are deleted, was created using the same method used to create )
)It was created. Figure 3 shows the method of making it.

Expression type plasmid 1) TNF416A obtained above,
Disgusted by the method described in JP-A No. 63-267290!
Two DNA fragments (approximately 560 bp and approx. 2.6K
bp, both 3alI+EcoRI) were recovered from the gel.

5 of the novel antitumor active polypeptide genes obtained here
A DNA fragment of approximately 5eobp including the ' side half was added to 50μ of 101!1M Tris-Cf (C1-Cf 7.
4) It was dissolved in an aqueous solution of 10 mM MC1804 and 1 mM dithiothreitol, 10 units of restriction enzyme KpnI (Takara Shuzo) was added, and a cleavage reaction was performed at 37°C for 1 hour. . After the reaction was completed, polyacrylic acid gel electrophoresis (gel 81°, 5%) was performed, and
According to the method described in Japanese Patent Publication No. 3-267290, approximately
A 00 bp DNA fragment (EcoRI HKpnI) was recovered from the polyacrylamide gel.

Furthermore, an oligonucleotide having the base sequence shown in FIG. 5 was synthesized and purified according to the method described in JP-A-63-267290. The terminals of 0.5 μg of each of the two synthetic oligonucleotides obtained were phosphorylated, and after annealing, a ligation reaction using T4-DNA ligase was performed.

After the reaction was completed, the obtained double-stranded oligonucleotide was combined with the previously obtained approximately 2.6K bp DNA fragment (SalI”
EcoRI) and an approximately 500 bp DNA fragment (Ec
oRI-KpnI) and after ethanol precipitation,
According to the method described in JP-A No. 63-267290,
A ligation reaction was performed using T4-DNA ligase. After the reaction was completed, the target plasmid I) TN was introduced into the Escherichia coli strain (600R) and selected from among the transformed strains.
A clone with F611 (approximately 3.2K bp) was selected. This plasmid has the following amino acid sequence (82N) -Pro-3er-ASD-Lys -P
ro-V al -A la-口is -V al
-V al -A Ia -A Sn-P rO-G
In-A la-G lu-G +y-G 1n-L
eu-G ln-T rc+-L eu-A sn-A
rv-A rg-A la-A sn-A la-L
eu -L eu -A la-Asn-G 1y-
V al-G lu-L eu-A ri)-A S+
)-A 5n-G In-L eu-V al -V
al-Pro-Ser-G 1u-Asp-Le
u-Tyr-Leu-11e-Tyr-3er-G l
n-Val-Leu-Phe-LyS-G IV-G
1n-G ly -CVS-Pro-Ser
-T hr-mouth 1S-Val-seu -L eu-
T hr -His -T hr -11e -S e
r -A ro-I Ie-A Ia-Val-5er
-Tyr-G In-Thr-Lys-Val-ASn
-Leu-Leu-5er-A la-11e -Ly
s -Ser -Pro -Cys -G In -A
rg-G Iu -T hr -Pro -G I
u -G Iy -A Ia -G lu-A la
-L VS-P rO-T rp-T Vr-G lu
-Pro-11e-Tyr-Leu-Gly-Gly
-Val-P he-G In-1elj-G lu-
L VS -G +y-A 5p-Aro-Leu-8
er-Ala-Glu-11e-Asn-A rg-P
ro-A S+)-Tyr-L eu-A sp-
P he-A la-G lu-5er-G Iy-G
1n-Val-Tyr-Phe-Gly-11e
-I Ie -A Ia -Leu- (COOl-1>) This is an expression plasmid encoding a novel anti-tumor polypeptide represented by or a polypeptide having Met attached to the terminal end thereof, and is an expression plasmid shown in FIG. shows how to create it.

On the other hand, the human TNF3fl trochanteric expression plasmid pTNF 611 2oμ9 obtained above was used in JP-A No. 63-2
Restriction enzyme 1-1i according to the method described in Publication No. 67290
After cutting with nd■, 50 mM Tris-HC
j (p+-+7.4), 1100I
The cleavage reaction using the restriction enzyme NcoI (Takara Shuzo) was carried out in an aqueous solution of Na C1, 101 M Mg 804.
℃ for 1 hour. After the reaction, agarose gel electrophoresis (gel concentration 0.7%) and polyacrylamide gel electrophoresis (gel concentration 5%) were performed.
According to the method described in Publication No. 290, a DNA fragment of approximately 140 bp (NcoI+Hi
nd m) from polyacrylamide gel and according to the method described in JP-A-63-267290.
Approximately 3,000 bp of DNA containing most of TNF611
The fragments (NCOI4-10 indl [[) were each recovered from an agarose gel.

Furthermore, the DNA fragment (about 140 bl) obtained above (N
coI”Hind m) at 50 μm IOIM T
riS-mouth CI<D eyes 7.4>, 101M Mg
SO4, dissolved in 1mM dithiothreitol aqueous solution,
10 units of restriction enzyme AccI (Takara Shuzo) was added, and the cleavage reaction was carried out at 37°C for 1 hour. After the reaction is complete, run polyacrylamide gel electrophoresis on the moving gel l1lJ! 8%)
According to the method described in JP-A No. 63-267290, approximately 110 bp containing part of the human TNF gene was extracted.
A DNA fragment (NcoI+AccI) was recovered from polyacrylamide gel.

Furthermore, an oligonucleotide having the base sequence shown in FIG. 6 was synthesized and purified according to the method described in JP-A-63-267290. The terminals of 0.5μ9 of each of the two obtained synthetic oligonucleotides were phosphorylated and annealed.

After anyl ring, the resulting double-stranded oligonucleotide is combined with the previously obtained approximately 3. OK bp DNA fragment (Nc
oI + Hind 11) and an approximately 110 bp DNA fragment (NcoIHAccI) containing part of the human TNF gene, and after ethanol precipitation, JP-A-63-26729
A ligation reaction using T4-DNN soligase was performed according to the method described in Publication No. 0. After the reaction is complete, Escherichia
The target plasmid pTNF637 (approximately 3.2) was introduced into the transformed strain.
bp) was selected. This plasmid contains the following anoic acid sequence (82N>-Pro-8er-Asa-Lys-P r
o-V al-A Ia-口is -V al-V
al -A laA sn-pro-G ln-A
Ia-G Iu-G ly-G 1n-L eu -G
In -T rt)-L eu -A sn-A r
g-A PaA la -A sn -A Ia -L
eu -L eu -A Ia -A 5n-G l
y-V al-G lu-L 13u-A rlJ-A
5l)-A 5n-G In-L eu-V at-
Val-Pro-Ser-Glu-ASI)-
LelJ-Tyr-Leu-11e-Tyr-3er-
Gln-Val-Leu-Phe-Lys-Gly-G
ln-Gly-Cys-Pro-8et-Thr-)-
1i=s-Val-Leu-Leu-Thr-1-1i
s-Thr-1le-8er-Arg-11e-Ala
-Val-8er-Tyr-Gln-T hr -L
Vs −V al −A sn −L eu −L e
u-Ser-Ala-11e-Lys-5er
-Pro-Cys-G In-A r(+-G lu
-T hr-Pro-G Iu-G IV-A 1a
-G Iu-A la-LVS-P rO-T rl)
-TVr-G 1u-Pro-11e-Tyr-Leu
-Glv-Gly-Vat -P he-G In-L
eu-G Iu-L Vs-G Iy-A 5p-A
rg-L eu-S er-A la-G lu-I
Ie-A 5n-A r(1-P rO-A 5l
l-T'/r-L eLI-A 5l)-P he-A
la-G lu-S er-G ly-G In-V
al-Tyr-P he-G ly-11e-11e-
This is a novel antitumor active polypeptide gene expression plasmid that encodes a novel antitumor polypeptide represented by Ala-Phe-(COO) or a polypeptide with Met attached to its amino terminus. We showed how to create it.

Example 3 (Confirmation of Expression) The vfr-specific antitumor active polypeptide gene expression plasmid IITNF 41G obtained in Example 2.

DTNF 416A, pTNF 6N or p
Escherichia coli Ceoorl with TNF637
- Strains were treated with 30-50 μg/- of ampicillin, 0.2%
M9 medium containing glucose and 4q/-〇casamino acids [0,0% Naz PO4-0,3% to 20PO40,
05% NaCj-0,1% NH*Cj aqueous solution (pi-1
After sterilizing 1 and 4) in an autoclave, a MO8O4 aqueous solution and a CaCjz aqueous solution, which were separately sterilized in an autoclave, are added to a maximum n concentration of 2 mM and 0.11M, respectively. ] 250Infl and OD i
Shaking culture was performed at 37°C until pa reached 0.7. Next, 3-β-indoleacrylic acid with a final S degree of 50 μ9/ai) was added to the culture solution, and the culture was continued at 37° C. for 12 hours.

Larger size due to centrifugation! ! ! ! After collecting the bacterial cells, PBS buffer (201
The bacterial cells were washed using M phosphate buffer, proa, 4). After washing, the bacterial cells were suspended in 10 ae PBS buffer and placed in an ultrasonic generator (Kubota, 200M).
After destroying the bacterial cells using a mold, the bacterial cell residue was removed by centrifugation.

A portion of the obtained E. coli lysate was treated with Tris
-mouth C1 buffer p- (DH6,8), SO8,2-
Mercabutethanol and glycerol were added to final concentrations of 601M, 2%, 4%, and 10%, respectively.
5DS-polyacrylamide gel electromethod e [Suzuki, Genetics, 31.43 (197i>1) was performed.

The separation gel was 12.5%, and the running buffer was SO3.
, Tris-glycine system [Ll, K, Lae 1 ml
l+.

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 novel antitumor active polypeptide gene.

The stained gel was transferred to a chromato scanner (Shimadzu, O
8-930 type>, the proportion of the produced novel antitumor active polypeptide in Escherichia coli 1[1 cytoplasmic protein was calculated. As a result, the expression plasmid I) TNF
The production amount of the wiT antitumor active polypeptide in E. coli with 416
It is only about 50% of the case of 6A, and the expression vector pA A
The usefulness of 41 and expression plasmid f) TNF 416A was demonstrated.

Example 4 (Evaluation of activity) The activity of the 1iM antitumor active polypeptide was measured using the Ru
It was carried out according to the method of ff. That is, 100 μl of the E. coli lysate containing the novel antitumor active polypeptide obtained in Example 3 was diluted in a medium, and 4×10
-929 fibroblasts on mice at a concentration of 5/-<ATC
100μ of CCCL-929) suspension was mixed in a 96-well tissue culture microplate (Costar).

At this time, 1 μg/- of actinomycin D (Cosmegen, Banyu Pharmaceutical) was added to the culture medium (Eagle's Minimum containing 5% (vol/vol) fetal bovine serum).・Essential medium (Hinaga Pharmaceutical Co., Ltd.) was used. After culturing the above microplate at 37°C for 18 hours in air containing 5% carbon dioxide, crystal violet solution [5% (vol/v01) methanol aqueous solution] Live II cells were stained using 0.5% (wt/vol) crystal violet dissolved in the solution. After washing away excess listal violet and drying, the remaining crystal violet was
Extracted with 0 μm 0.5% SDS aqueous solution, and the 595t
The absorbance at v is measured using an ELISA analyzer (Toyo lateral organ, ETY-96 type). This absorbance is proportional to the number of surviving cells. The dilution factor of the E. coli lysate corresponding to 50% of the absorbance of the control to which no diluted solution was added is determined from a graph (for example, Fig. 7), and that dilution factor is defined as a unit.From Fig. 7, in Example 3, The resulting expression plasmid pTNF637
It was revealed that 100μ of Escherichia coli lysate containing the human TNF protein encoded by E. coli had an activity of about 2×103 units.

The total amount of protein contained in the E. coli lysate containing the novel antitumor active polypeptide encoded by the expression plasmid pTNF637 obtained in Example 3 was quantified using a protein assay kit (Bio-Rad). Calculated from a calibration curve using bovine serum albumin. Activity value and protein quantification obtained above
The specific activity of the E. coli lysate containing the tumor active polypeptide was calculated to be approximately 8.3X 102 (units/
#ly-protein).

[Brief explanation of drawings]

Figure 1 shows the nucleotide sequence of the designed human TNF gene, and
The figures show the base sequences of chemically synthesized synthetic oligonucleotides. Figure 3 shows the human TNF gene expression plasmid pTN1” 401A and the new anti+
t1m active polypeptide gene expression plasmid I)T
Each method for producing NF416A is shown. Figure 4 shows the method for creating the novel antitumor active polypeptide gene expression plasmid pTN'416, and Figures 5 and 6 show the creation method for the novel antitumor active polypeptide gene expression plasmid I) TNF611 and IITNF637. , respectively. Figure 7 shows the results of measuring the activity of the novel antitumor active polypeptide.

Claims (1)

[Scope of Claims] (1) A novel physiologically active polypeptide represented by the following amino acid sequence [there is a gene sequence]. (2) The polypeptide according to claim 1, characterized in that Met is bound to the amino terminus. (3) Recombinant plasmid containing a DNA region encoding a novel physiologically active polypeptide represented by the following amino acid sequence [gene sequence available] [gene sequence available] or a polypeptide with 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
The plasmid according to claim 3, characterized in that it contains a double-stranded DNA consisting of A. (5) The plasmid according to claim 3, wherein the DNA region is characterized by a single-stranded DNA represented by the following base sequence (there is a gene sequence) and one complementary to the single-stranded DNA. (6) The plasmid according to claim 3, wherein the plasmid is plasmid pTNF637. (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 [gene sequence is available] or a polypeptide with Met bound to its amino terminus. modified microbial cells. (8) The microbial cell according to claim 7, wherein the microbial cell is Escherichia coli. (9) A recombinant plasmid transformed with a recombinant plasmid containing a DNA region encoding a novel physiologically active polypeptide represented by the following amino acid sequence [gene 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 modified microbial cells, producing and accumulating a novel bioactive polypeptide in the culture, and isolating the novel bioactive polypeptide from the resulting culture. (10) Contains an antitumor-effective amount of a novel physiologically active polypeptide represented by the following amino acid sequence [gene sequence available] [gene sequence available] or a polypeptide with Met bound to its amino terminus. Pharmaceutical composition.