JPH02128696A - Novel physiologically active polypeptide - Google Patents

Abstract

NEW MATERIAL:A physiologically active polypeptide losing 1-12 amino acid residues on amino end side and having a sequence containing substitution of 157th Leu to Phe or a polypeptide linking Met to the above-mentioned polypeptide in an amino acid sequence expressed by the formula and shown by sequence from 1st Val to 157th Leu. USE:An antiulcer agent. PREPARATION:For example, in an amino acid sequence expressed by the formula, a recombinant microbial cell transformed by a recombinant plasmid containing a DNA domain coding a physiologically active polypeptide losing 1-12 amino acid residues on amino end side in an amino acid sequence expressed by the formula and having a sequence containing substitution of the 157th Leu to Phe or polypeptide linking Met to amino ends of the above-mentioned polypeptide is cultured and the aimed product is separated from the cultured material to provide the physiologically active polypeptide.

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 IT 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-1tJB Committee on Biochemistry (CBN), for example, the following abbreviations are used.

AlaL-Alanine Arg L-Arginine ASn L-Asparagine AspL-Aspartic acid CVS L-Cystine Gln L-Glutamine Glu l-Glutamate Gly Glycine HIS l-Histidine 11eL-Isoleucine Leu L-Leucine Lys L-Lysine Met 1-Methionine PheL- Phenylalanine Prol-Proline 3erl--Serine Thr 1-Threonine Trl) L-t-LibutofunTyr 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. shall
For example, use the following abbreviations.

A Adenine (indicates deoxyadenylic acid) C Cytosine (indicates deoxycytidylic acid) G Guanine (indicates deoxyguanylic acid) )T Thymine (represents deoxythymidylic acid) Furthermore, (82N)- and -(COO
I-(> indicates the amino-terminal side and carboxy-terminal side of the amino acid sequence, respectively, (5')- and (5')-
3') indicate the 5' end and 3' end of the DNA sequence, respectively.

(a) Background of the Invention Carswell et al.
It was discovered that serum collected after administering endotoxin to mice previously stimulated with metteQuerin (BCG) contained a substance that caused hemorrhagic necrosis of transplanted MethA sarcoma cancers, and this substance was used as tumor necrosis factor. (Tumor N eclipse
Factor (hereinafter sometimes abbreviated as TNF) [E, A., Carswell et al., Pro.
c, Natl.

Acad, sci, USA, 72.3
666 < 1975) Ko. TNF is found in many animals such as mice, rabbits, and humans, and because it acts specifically on tumor cells and across species, it has been expected to be used as an anticancer agent.

Recently, Pennica et al.
We performed DNA cloning to clarify the primary structure of the human h-TNF protein, and reported on the expression of the human h-TNF gene in Escherichia coli [
D. Pennica et al., Nature.
312. 724 (1984)], then Hirai et al. IT, 3hirai et al.

Nature, Yu13. 803 (1985)]
, Munemura et al. [Moku et al., Cancer and Chemotherapy, 12. 160(
1985)], WanQ et al. [A, M. Wang et al., 3science, 228. 149 (1985
)] and MarIIlenout et al. [Δ, M
Armenout et al.

[2 ur, J. Biochain, 1
52. 515 (1985) 1.
Several reports have been made regarding the expression of the TNF gene in E. coli.

As described above, by using genetic engineering techniques, a large amount of pure human TNF protein is now available, and the physiological activities of TNF other than its antitumor activity are becoming clear. For example, Cachectin, which is one of the causes of cachexia seen in early stages of cancer and patients with severe infectious diseases, is very similar to 1NF [B, Beu1℃].
e ``Ra, Nature.

316, 552 (1985)], Cachectin has riboprotein lipase inhibitory activity; Rikihi et al., administration of Ding NF increases blood triglyceride levels, resulting in side effects such as hyperlipidemia. It was suggested that this is a possibility. Also, other than that,
Shadow on vascular endothelial cells'W [J, R.

Gallble et al., J. Exp. Med.
162.2163 (1985)], bone resorption action II
D, R, Beltolini et al., Nature
, 319. 516 (1986) 1st prize has been reported.

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

In this way, the naturally occurring tar protein is modified,
Much research has been carried out to produce new proteins for specific purposes.

Several studies have also been conducted on the modification of human TNF protein, and in the amino acid sequence of the human TNF protein shown in Figure 1, the amino acid residues of either or both of Cys' and Cy3/p/ Substitution of Gly with other amino acid residues (PCT Application Publication No. WO 36/04606, Japanese Patent Application No. 106772/1983), substitution of Gly with other amino acid residues (Japanese Patent Application No. 106772/1981, Japanese Patent Application No. 1983/1983) -238048Q), substitution of A Ia with other amino acid residues (Patent Application No. 1983-233337)
has been reported.

Furthermore, regarding the deletion of amino acid residues on the amino terminal side, 6-amino acid deleted TNF has cytotoxic activity.
F has cytotoxic activity (Patent application 1986-1990)
087), 1 = 10 amino acid deleted TNF has cytotoxic activity, and its specific activity is 6-8 amino acid deleted TN.
Maximum at F (PCT Application Publication WO 36/
02381), rNF with a 10 amino acid deletion has cytotoxic activity (Japanese Patent Application No. 114754/1982)
It has been reported (Japanese Patent Application No. 6,117,3822) that 11 amino acid-deleted TNF has cytotoxic activity.

As a result, the present inventors have been conducting intensive research into the modification of human TNF protein with the aim of increasing specific activity, improving stability, broadening the reaction spectrum, and reducing side effects. As a result, the present invention was completed.

(3) Object of the invention The object of the present invention is to provide a novel polybeb with anti-embryonic 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 viscous 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 of the present inventors, the purpose of the present invention is as follows:
The amino acid sequence shown in the figure from the 1st Val to the 157th Leu includes J3-(, 1 on the amino terminal side, 2], 3, 4X, 5], 6, 7, A novel physiologically active polypeptide having a sequence in which 8, 9, 10, 11, or 11 amino acid residues are deleted and the 157th position of Leu is replaced with Phe, or at its amino terminus. Recombination achieved by providing a recombinant plasmid containing a Met-bound polypeptide and a DNA region encoding the novel antitumor active polypeptide, and further transformed by the thus obtained recombinant plasmid. It has been found that the present invention can be achieved by providing a microbial cell, a method of producing a desired novel antitumor polypeptide using the microbial cell, and a pharmaceutical composition containing this novel antitumor polypeptide.

The present invention will be explained in more detail below.

(A> Cloning of human TNF gene; human TNFm 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 TNFi gene, it is desirable to select the most suitable codons for the host cell used, and to place cleavage sites with appropriate restriction enzymes at appropriate positions to facilitate subsequent cloning and genetic modification. It is desirable to provide

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 TN FiM gene can be cloned into an appropriate vector by using cleavage sites for 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 TN Fm Buddha designed as described above, each of the upper and lower strands is divided into several oligonucleotides as shown in Figure 2.
It is preferable to chemically synthesize them and connect the respective oligonucleotides. The synthesis method for each oligonucleotide was the Tehasier ester method [H, G, Khoran
a.

” S Ole ReCent De Velol
) llents InCheiistry or
Phosphate E 5ters ofB
iological interest”,
John Wileyand 5ons, I
nc, New York (1961)].

Triester method [R, L, L ets+nger et al.
J.

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

Tetrahedron Lett,, 21. 7
19 (1980)], but synthesis by the phosphite method using a Zenshiro 1[1] 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,
B olivar et al., Gene, 'l.
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.

1) TNF2N 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. . Usable dark bu[''[-tar, 1-rib 1 to fan operation []n bu[]motor Brp promoter).

Schiff 1--S A Bellon Promoter (Iac BuD
Examples include tac promoter, tac promoter, P+- promoter, 1pp promoter, and the like, with trp promoter being particularly preferred. As a plasmid containing a TRP protein, preferably pyS
3IN or IIIA A41 is used.

Furthermore, for the purpose of improving expression efficiency, h 1' N
1. It is possible to add a terminator downstream of the gene to E. coli 'C, which functions well. Examples of such terminators include the 1pp terminator, the trp terminator, etc., but the trp A terminator is particularly preferred, and pA41 is preferably used as the plasmid that terminates the tr[l A terminator. This expression type human 1-”'I'-'N gene is
For example, 1) Expression-desired lasmid can be created by []-linking a vector derived from F3R322. As the plasmid desired to express the human TNF gene, pTNF401NN or DTNF4018 is preferably used.

(B) Cloning of a novel anti-liver cancer active polybebubud gene; The human TNFm envelope expression plasmid thus prepared was digested with an appropriate restriction enzyme 'c', and after removing a specific region within the human TN gene, Gene repair is carried out using synthetic oligonucleotides that modify specific base sequences.

By using such a method, it is possible to develop an intervening agent containing a gene encoding a novel anti-tumor active polypeptide in which the last four amino acids in the human TNF protein are replaced with other amino acids, added to, or deleted. It becomes possible to create a busismid in the form of a turret. As such a novel anti-ulcer active polypeptide gene promoter plasmid, pTNF is preferably used.
621. pT N Fe12, p”rNF6
23, [) T N F 624. l)'T N
Fe12, pTNF 626. DTNF62
7. pTNF628, pTNF629.
pTNF630゜pTNF631 or I) TNF63
2 is used.

(C) Expression confirmation and activity evaluation; C is used as a microbial host for expressing the Hil-TN F-gene and the novel antitumor active polypegut gene FG.
Examples include Escherichia coli, Bacillus subtilis, and yeast, but especially Escherichia coli
coli)] is preferred. The human 1-FNF gene expression plasmid and the novel antitumor active polypeptide gene expression plasmid can be obtained by, for example, the known ly method [4, V,
Norgard et al.

Gene, 3. 279 (1978)], the microbial value -F, for example, Niji [Lichia] C600
rm strain (AT CC33525).

The recombinant microbial cells thus obtained are cultured in a manner known per se. Examples of the medium include M9 medium [T, Mani] containing glucose and casamino acids.
“Molecular Ctoning”, edited by atis et al.
”, P 440. Co1d Sprong
Harbor 1 laboratory, New
'10rk (1982) Reference 1, and it is desirable to add, for example, 7-mubicillin, etc., depending on the necessity. Cultivation is carried out at 37° C. for 2 to 36 hours under conditions suitable for recombinant microorganisms, such as aeration by shaking and stirring. In addition, at the start of culture or during culture, 3-β-
Agents such as indole acrylic acid can also be added.

After culturing, the recombinant microbial line +a is collected by, for example, centrifugation, suspended in, for example, a phosphate buffer, the recombinant microbial cells are disrupted by, for example, ultra-A wave treatment, and a lysate of the recombinant microbial cells is obtained by centrifugation. The protein in the obtained Lysee 1~ was mixed with sodium lauryl acid (
The proteins in the gel are separated by electrophoresis using a polyacrylamide gel containing (hereinafter sometimes abbreviated as SDS), and the proteins in the gel are stained using an appropriate method.

Lyse 1- of microbial cells without expression plasmid
The expression of the human TNF gene or the novel anti-tumor active polypeptide gene is confirmed by comparing the electrophoresis pattern using the sample as a control.

The anticancer activity of the human TNF gene and the novel antitumor active polypeptide thus obtained was evaluated by in v
In vitro activity assay (Carswell et al., supra) and cytotoxicity against mouse cells.
Activity measurement method [Ruff, J, I m5unol
,, 126. 235 (1981)], etc.

Human TNF protein and novel antitumor active polypeptides can be isolated and purified from E. coli lysate according to known and commonly known protein separation and purification methods.
Affinity column chromatography using antibodies against proteins etc. is advantageous. Among them, human T
Particularly suitable is affinity column chromatography using a mouse monoclonal antibody against NF protein or the like. By using the purified human TNF protein and novel antitumor active polypeptide thus obtained, it becomes possible to study in vivo anticancer activity (described above) and side effects.

Evaluation of the side effects of human TNF protein and novel antitumor active polypeptides is carried out using in vitro methods such as h-actin activity measurement.
This can be carried out by an in vitro method, or an in vivo method in which the agent is administered to an experimental animal such as a mouse to determine its lethality and degree of reduction in blood pressure.

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 TN Fl gene having the base sequence shown in FIG. 1 was designed.

Pennlca et al., Nature, 312.
724 (1984)], based on the nucleotide sequence of the structural gene portion of the human TNF gene body cDNA, a cleavage site with an appropriate restriction enzyme was set at an appropriate position ([position 7.5') and a translation start codon ( ATG) and two translation stop codons (TGA and TAA) on the 3' side.
were given to each. Furthermore, a cleavage site using the restriction enzyme CfaI was provided upstream of the 5' translation initiation codon, allowing for ligation to the promoter while maintaining an appropriate state between the SD sequence and the translation initiation codon. Furthermore, a cleavage site using the restriction IIJ element Hindl was provided downstream of the 3' translation stop codon to facilitate ligation with a vector plasmid.

Example 2 (Chemical Synthesis of Oligonucleotides) The human TNF3m gene designed in Example 1 is synthesized in 17 oligonucleotides as shown in FIG. Oligonucleotides were synthesized using a 1.11 DNA synthesizer (Applied Biosystems).

Purification of the synthetic oligonucleotide was carried out according to the manual of Applied Biosystems. In other words, an ammonia aqueous solution containing the synthetic oligonucleotide was heated at 55°C overnight. The protecting group of the DNA base is removed by keeping the DNA base, and a high molecular weight synthetic oligonucleotide fraction is separated by gel filtration using Sephadex G-50 Fine Gel (Pharmacia).
After performing electrophoresis on a polyacrylamide gel containing M urea (gel concentration 20%), the migration pattern is observed by an ultraviolet shadowing method. After cutting out a band portion of the desired size and crushing the polyacrylamide gel fragments into small pieces, 2 to 5 #Ii! Hara 7F for elution of -[
500mM NH40AC-1ffiMEDTA-0
, 1% SDS (1)H7,5) was added thereto, 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 through a gel filtration column (Sephadex G-50).
A purified product of the synthetic oligonucleotide was obtained. Note that, if necessary, polyacrylamide gel electrophoresis was repeated to improve the purity of the synthetic oligonucleotide.

Example 3 (Cloning of chemically synthesized human I-TNF gene) The 17 synthetic oligonucleotides prepared in Example 2 (
TNF-1 to TNF-17),
Divide the F m transmission into three blocks and clone it.

0.1 = 1.0 μJ of synthetic Aligomecleodide TNF
-2~TN[-6's 5' end side is 5~15 units I~
1-4-polynucleotide kino-1ze ([.

cot:s type, Takara Shuzo), and phosphorylate each separately. Phosphorylation reaction is 10-20 Ilfi 5
0IBM King rlS-to1 (J (DH9,5
), 10mM M! j
Cj2.

The test was carried out at 37° C. for 30 minutes in an aqueous solution containing 5 mM dithi A threitol and 10 mM ATP. After completion of the reaction, an aqueous solution of the synthesized A-ly I nucleotide was mixed, and T4-vorinucle A-butokinase was inactivated and removed by phenol extraction and -chill extraction.

Add o, (-) to this synthetic oligonucleotide mixture.
i, oμJ of synthetic oligonucleotides TN Fl and TNF-7 are added, and the mixture is slowly cooled to the invasive temperature heated at 90° C. for 5 minutes to perform 7-ringing.

Next, after drying this under reduced pressure, 30μρ of 66mM
Tris-H(J (pi-17,6), 6.6
1MM Mg (J2゜10-M Jiji A Threatol,
Dissolve in 1 mM Al-P aqueous solution, add 300 units of Tl-DNA ligase (Takara Shuzo),
The ligation reaction was carried out at 1°C for 15 hours. After the reaction is completed, polyacrylamide gel electrophoresis (gel concentration 5%) is performed, and the migration pattern is observed by edidium bromide staining. A band portion of the desired size (approximately 220 bp) is cut out, and DNA is recovered from the polyacrylamide gel according to the method of Example 2.

On the other hand, plasmid l11BR322 (approximately 4.4 Kbl) for 3 μ9 of Oitai was added to 10 mM T of 30/lfi.
ris・-t-1C1 (pH 7.5>, 60
mm Na C, 7mMM! J (J2) was dissolved in aqueous solution, 10 units of restriction enzyme (New England Pi4 Labs for JaI) was added, and the mixture was incubated at 37°C for 1 hour.
A time cleavage reaction was performed.

Restriction enzyme (after cleavage with JaI) 1-Nol extraction.

Perform ether extraction and recover DNA by Tethanol precipitation. Transfer this DNA to 30 μ9 of 501 M Mrri
s-)1cJ (1)H7,4),
100 mM Na(J, 1
0...MM! Dissolve in 1JsOn aqueous solution, 10 units 1
- restriction enzyme 5afe (Takara Shuzo Co., Ltd.) was added, and a dissection reaction was performed at 37°C for 1 hour. After the reaction is completed, agarose gel electrolysis (gel concentration 0.8%) is performed, and the cutting pattern is observed by the ]-budium zo1mite staining method. Plasmid I) Approximately 3,7K containing most of BR322
The band corresponding to the bp DNA portion was cut out, and the agarose gel fragment was washed with 3x a(vo!'Wt) of 8M
Na (υ- dissolved in Jo4 aqueous solution). The glass filter method of Cben et al. [C, W.

Chen et al., Anal, [3ioehe3 10
1.339 (1980)], approximately 3.7K bp
70,000 and 1-
Recovered from Sgel.

Previously obtained human 1N [approximately 220 genes containing part of the gene]
After phosphorylating the terminals of the bp DNA fragment according to the method described above, it is mixed with an aqueous solution of approximately 3.7 Kbp DNA containing most of plasmid DBR322. After 1 ethanol precipitation, both [)N
The ligation reaction of the A1gi piece was (j).

Transformation of the 1 Schielichia]suQ 600r-m- strain was carried out by a modified method of the usual Ca(J2 method (method of Norgard et al., M.V.). Hitob 1 hen, 0.5% Yeast 1 kiss, 0.5%
NaC, pH 7,2) and Niji I Lychia coli C600
An 18Fgf medium M base of the r1- strain is inoculated and grown until the turbidity at 600 nm (OD 1top ) of the culture solution containing the bacterial cells reaches 0.3. The bacterial cells were soaked in cold magnesium buff 7'-[0, IM NaCl, 5
111M Mo C12゜5mM Tris-H
(J (pH 7, 6, 0°C)] and 2 d of cold calcium buffer [1001Mca C1
2, 250sM K(J, 5 1MM(l
Cjz, 5 1M Tris-Fl
Resuspend in C.O.(p)l 7.6°C) and leave at 0°C for 25 minutes.

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

After keeping at 0°C, 1 d of LBG medium <1% tributone,
Add 0.5% yeast extract and 1% Na (J, 0.08% glucose, l) 87.2), and culture with shaking at 37°C for 1 hour. Transfer the culture solution to a selective medium [medium plate containing ampicillin (Sigma) 30゜μ9/d] for 10 minutes.
Inoculate at a rate of 0 μJ2/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 the acquisition of the target plasmid pTNFIBR (approximately 4.0 K bp>) was confirmed by agarose gel electrophoresis. We will show you how to create it.

By the same method as above, synthetic oligonucleotide TN
Plasmid pTNF2N using F-8 to TNF-13
(approximately 3.IKbll) was added to the synthetic oligonucleotide TN
Plasmid pTNF3 using F-14 to TNF-17
(approximately 2.4K bp) were created. Figures 4 and 5 show the methods for constructing plasmids pTNF2N and pTNF3, respectively.

The thus obtained plasmids pTNFIBR, 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, l
ylaxam et al., Methods Enzymol,
65. 499 (1980) 1.

Example 4 (Creation of human TNFi trochanter expression plasmid) Plasmid pTNF1BR10μ9 obtained in Example 3
were treated with restriction enzymes (Jal and 3al) in the same manner as in Example 3.
After cutting with a polyacrylamide gel electrophoresis (gel concentration 5%), human TNFm
Approximately 220 bp DNA fragment containing part of the trochanter <C9a
I H8alI ) was recovered from the polyacrylamide gel.

Next, 10 plasmids pTNF2 obtained in Example 3
μg to 100 μN of 101M Trls-11(J(
pH 7.5>, 60mM NaC, 7mM
The mixture was dissolved in an aqueous MgCl2 solution, 40 units of restriction enzyme PVL1m (Takara Shuzo) was added, and a cleavage reaction was carried out at 37°C for 1 hour. Then, according to the method of Example 3, υ
After cutting with IW@sal for one time and polyacrylamide gel electrophoresis (gel concentration 5%), a D of about 170 bp containing part of the human TNFm trochanter was extracted according to the method of Example 2.
The NA fragment (SalI+PvuI[) was recovered from the polyacrylamide gel.

In addition, plasmid pTNF3 obtained in Example 3 10
μ9 also iooμN 10 mM Tris-11C
I(DH7,5), 60mM NaCL7
Dissolve in an aqueous solution of mM OC72, add 40 units of restriction enzyme pvull and 40 units of restriction enzyme Hi
ndl [(Takara Shuzo) was added, and the cleavage reaction was carried out at 37°C for 1 hour. After polyacrylamide gel electrophoresis (gel concentration 5%), a DNA fragment of about 1iobp containing a part of the human TNF gene (
Pvu[”Hind III] Lab'J 7 RiI
Collected from J/L/7 midgel.

On the other hand, plasmid p with E. coli trp promoter
Ys31N (approximately 4.7 Kbp > 5μ9 was digested with 1.II restriction enzyme C1aI and HindllIt' in the same manner as above, and after agarose gel electrophoresis (gel concentration 0.8%), according to the method of Example 3, Plasmid l) Y S 3
A DNA fragment of about 4.7K bp containing most of 1N (C
jaI+Hindl[[) was recovered from the agarose gel.

Thus obtained human TNF)i! Three DNA fragments of approximately 220 bp, approximately 170 bp, and approximately 110 bp containing part of the m trochanter were mixed with a DNA fragment of approximately 4.7) (bp) containing most of plasmid I) YS31N, and after ethanol precipitation, A ligation reaction using Tl-DNA ligase was carried out according to the method of Example 3. After completion of the reaction, Example 3
Escherichia CC600r-+ according to the method of
-, and selected from among the transformed strains the desired human TNF gene expression plasmid pTNF401NN (approximately 5.2K
bp) was selected. FIG. 6 shows the method for creating the plasmid 1) TN F 401NN.

In addition, after partially decomposing the plasmid pYS 31N 5μ9 with the restriction enzyme pvu according to the method described above,
Furthermore, after cutting with restriction enzyme 1-(indnl) and agarose gel electrolysis vJ (gel concentration 0.8%), approximately 2.7 Kb containing the trp promoter was obtained according to the method of Example 3.
DNA fragment of p [PVuII (214-4Hind m
] was recovered from the agarose gel.

Next, an oligonucleotide having the fi group sequence shown in FIG. 7 was synthesized and purified according to the method of Example 2. 0.5μ9 each of the two synthetic oligonucleotides obtained
The terminal was phosphorylated according to the method of Example 3, and after annealing, the previously obtained approximately 2.7K
bp DNA 116 piece [PvuI[(2)”Hind
According to the method of Example 3, a ligation reaction using T4-DNA ligase was carried out. After the reaction was completed, it was introduced into Escherichia coli C600r-ta strain according to the method of Example 3, and the target plasmid pΔΔ41 (approximately 2.7K bp) was extracted from the transformed strain.
Clones with > were selected. Such a plasmid is derived from the plasmid pYS 31N] Bee number & 1lt
It is a multi-copy, high-efficiency expression vector in which the 1D region has been removed and the E. coli TRP A terminator has been added downstream of the cloning site, which is located just downstream of the TRP pro. Figure 7 shows how to create it. Indicated.

This plasmid 11AA41 2μ7 was cleaved with till restriction elements CfaI and HindI [[r] in the same manner as above.
After 7110-Sgelumi pneumophoresis (gel concentration 0.8%),
According to the method of Example 3, plasmid 11A A 41
An approximately 2.7K bp DNA fragment ((Ja
I-1-1indIl was recovered from the agarose gel.

In addition, 5μ of the previously obtained human TNF gene expression plasmid pT NF 401N N was cleaved with ill restriction enzymes CfaI and Hindll in the same manner as above, and subjected to polyacrylamide gel electrophoresis (gel concentration 5%). ,
According to the method of Example 2, a DNA Alli piece of approximately 490 bp ((Ja T ”Hin
d m ) was recovered from the polyacrylamide gel.

The thus obtained DNA fragment of about 2.7 Kbl containing most of the plasmid pΔA41 and the DNA fragment of about 490 bp containing the entire area of the Hi-T NF 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 expressing p) were selected. This plasmid is
Human] has the ability to express the NF gene more efficiently, and the method for its production is shown in Figure 8.

Example 5 (Creation of novel anti-tumor active polypeptide gene expression plasmid) The human h TN F gene expression plasmid pTNF 401A 20μ obtained in Example 4 was digested with t and lI restriction enzymes according to the method of Example 4. (JaI and l-1indll
IT” cutting, polyacrylamide gel electrophoresis vJ (gel concentration 5%) and agarose gel electrophoresis running gel concentration 0
．． 8%), following the methods of Examples 2 and 3, respectively, 162 DNA breaks (about 490b[) and about 2
．． 7K bp, both coins (Ja IHHind III)
was recovered from the gel.

Approximately 490b including the entire human TNF gene obtained here
p DNA fragment in 50 μl of 10 mM Tris.
-HCj (p)-17,5), 60 mM N
a. Dissolve C in 7mM MgCj2 aqueous solution, add 1 unit of 11 endonuclease AVa (Takara Shuzo), and add 3
The cleavage reaction was carried out at 1°C for 1 hour. After the reaction was completed, polyacrylamide gel electrophoresis (gel concentrate 5%) was performed.
According to the method of Example 2, a DNA fragment of about 460 bp (AvaI + Hind
) was recovered from polyacrylamide gel.

In addition, a double-stranded oligonucleotide having the base sequence shown in FIG. 9 was synthesized and purified separately into an upper strand and a lower strand according to the method of Example 2, and the resulting four strands were Example 3 for each 0.5 μ9 of synthetic oligonucleotides of
The terminals were phosphorylated and annealed according to the method described in .

The double-stranded oligonucleotide after annealing is first
Approximately 2.7K bp (7) DNA fragment (cI
An approximately 460 bEl DNA fragment (AvaII”) containing all-→Hind[) and most of the human TNFW gene
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 Scherichia coli (:, 600r-■- strain) according to the method of Example 3,
Target plasmid from the transformed strain I) TNF 41
A clone with 6A (approximately 3,2K bp) was selected. This plasmid is a form of anti-i! in which the amino-terminal 7 amino acids of T-NF have been deleted. This is a novel antitumor active polypeptide gene expression plasmid that encodes a tumor active polypeptide, and the method for its construction is shown in Figure 9.

On the other hand, the human TNF gene expression plasmid 1) TNF 416A20μ9 obtained above was cut with the restriction enzyme) 1indl[I according to the method of Example 4, and then 50m
M Tris -HCf (+)H7,4),
A cleavage reaction using the restriction enzyme NC0I (Takara Shuzo) was carried out in a 100 mM NaC and 101 M Mg804 aqueous solution for 3 minutes.
Perform at 7°C for 1 hour. After completion of the reaction, agarose gel electrophoresis (gel concentration 0.7%) and polyacrylamide gel electrophoresis (gel concentration 5%) were performed according to the method of Example 2, and approximately 140 bp containing part of the human TNF gene was detected. DNA fragment (NCoI4-+Hind ir) was extracted from polyacrylamide gel and according to the method of Example 3,
Approximately 3.0 Kbp of D containing most of DTNF 416A
Each NA fragment (NcoIHHind [1) was recovered from the agarose gel.

Furthermore, the approximately 140 bp DNA fragment obtained above (Nc
oI4-Il-1ind m > 101M of 50μ sentences
Tris-HCf (DH7,4), 10
+1M M(] SO4, IRIM didiothreitol 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, polyacrylamide gel electrophoresis (
A DNA fragment of approximately 110 bp (Nc.

IMACCI) was recovered from the polyacrylamide gel.

Furthermore, an oligonucleotide having the base sequence shown in FIG. 10 was synthesized according to the method of Example 2.

Purified. For each of the two synthetic oligonucleotides obtained, 0.5μ9, the terminals were phosphorylated and annealed according to the method of Example 3.

After annealing, the resulting double-stranded oligonucleotide was combined with the approximately 3.0K bp DNA fragment (Nc
oI + Hind I[
After mixing with 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, the target plasmid pTNF621 (approximately 3.2K
bD) was selected.

This plasmid has the following amino acid sequence (82N) -Pro-8er-ASI) -Lys-
Pro-V al-A la -His-V al
-V at -A la -Asn'-Pro-Gl
n-Ala-Glu-Gly-Gln-Leu-G
In -T rD -L eu -A sn -A
rg-A ro -Ala-Asn-Ala-L eu
-Leu-Ala-AsnG ly -V al -G
lu -L eu -A rq -A sp -A
sn −G In −L eu −Vat −Vat
-Pro -Ser -G lu -G IV-L13
1J-Tyr-Leu-11e-Tyr-Ser-
G ln-V al-L eu-P he-L ys-
Gly-GIn-GIy-Cys-Pro
-Ser -Thr -His -Val -L e
u-L eu-Thr-His-Thr-1le-Se
r-Arg-11e-Ala-Val-8er-Tyr
-Gln-Thr-Lys-Val-Asn-Leu
-L eu-5er-A Ia -I Ie -Lys
-Ser -Pro -Cys -G In -A
rq-G lu-Thr-P ro-G lu-G I
y-A 1a-G ILI-A Ia-1yS-P r
O-T rl)-T Vr-G 1u-Pro -I
1e-Tyr-L eu-Gly-Gly-Val
-P he-G In-L eu-G lu-L ys
-G Iy-A s.

Ara-Leu-8er-Ala-Glu-11
e-Asn-A r(+-P rO-ASll-TV
r-L eU-A Sp= Phe-A la -G
lu −S er −G ly −G In −V a
l-T yr -Phe-Gly -11e -I I
This is a novel anti-tumor active polypeptide gene expression plasmid encoding a novel anti-tumor polypeptide represented by e-Ala-Phe-(COOH> or a polypeptide in which Met is bound to its amino terminus. shows how to create it.

Furthermore, by using the synthetic NA shown in FIG. 11 instead of the synthetic N having the nucleotide sequence shown in FIG.
Create a plasmid containing a DNA region encoding a deleted TNF having a sequence in which 1.2, 3, 4.5, or 6 amino acid residues on the amino terminal side are deleted from the amino acid sequence represented by ()Ta. On the other hand, by the method described in FIG. 12, from the first Val shown in FIG.
In the amino acid sequence up to the 57th Leu, the 8,9,10,11,11 amino acid residues on the amino terminal side are deleted.
A plasmid containing the desired DNA region was constructed.

In FIG. 10, instead of the 1 lasmid DTNF 416A, the D
By using a plasmid containing the NA sequence as a starting material, 157 from the first Val shown in FIG.
In the amino acid sequence expressed up to Leu,
1.2.3.4, 5゜6, 8.9.10 on the amino terminal side
．． A novel physiologically active polypeptide having a sequence in which amino acid residues 11 and 11 are deleted and Leu at position 157 is replaced with Phe, or Me at the amino terminus.
Plasmid pTNF 622. containing a DNA region encoding polybebidide to which t is attached. DTNF62
3. pTNF 624゜pTNF 625. 1) T
NF626. 1) TNF 627°DTNF 62
8. I) TNF 629. I) TNF 630
゜+) TNF631 or pTNF632 could be obtained.

Example 6 (Confirmation of expression) Expression vector pAA41゜hi1~TNF gene expression plasmid DTNF401N obtained in Example 4
N or DTNF 401A, or obtained in Example 5,
Escherichia coli C600r1- strain carrying each novel antitumor active polypeptide gene expression plasmid was
~50 μg/ml of M9 medium containing 2 ampicillin, 0.2% gluten and 4 Q/le of casamino acids [
0.6% Na 2 HPO4 - 0.3% to 2 tons (PO4
-0,05%Na(J-0,1%NHi(J aqueous solution(t)
) After sterilizing H7, 4) in an autoclave, separately
-1-Crepe sterilized M! 3804 aqueous solution and Ga
C12 aqueous solutions are added to a maximum of 11111°2 wM and 0.1 mM, respectively. ] 250j1
1! 0[)me, 3 times until ρ reaches 0.7.
Shaking culture was performed at 7°C. Then a final concentration of 50μ
g/d of 3-β indole acrylic acid was added to the culture solution, and culture with shaking was continued at 37° C. for 12 hours.

After collecting E. coli cells by centrifugation, PBS buffer p
- (201M phosphate buffer containing 150mM Na (J, I) H7°4) was used to wash the bacterial cells. After washing, wash the bacterial cells with 10 d of PBS buffer? -, the bacterial cells were disrupted using an ultrasonic generator (Kubota, 200M type), and the bacterial cell residue was removed by centrifugation.

A portion of the obtained E. coli lysate was treated with TriS-
11 (J-buff-y-(1)H6,8), SDS
, 2 mercaptoethanol each final +11r! 160mM, 2%24%, 1
0% Ni'J Ruyo plus SDS-polyacrylamide gel electromethod ll] [Suzuki, Genetics. 31.43 (19
77)].

The separation gel was 12.5%, and the running buffer was SC2.
, Tris-glycine system [U. K. LaemmlN
ature, 227.680 (1970)
] was used. After the electrophoresis was completed, the proteins in the gel were stained with Coonsie Blue R-250 (Bio Rat) to confirm the expression of the human DIN gene and the novel anti-IN tumor active polypeptide gene.

Example 7 (Inv No. 0 Evaluation of anticancer activity) In VitrO anticancer activity measurement of 1i antitumor active polypeptide,
This was carried out according to the method of Ruff.

That is, when 100μ of E. coli Lyse 1-containing the novel anti-tumor active polypeptide obtained in Example 6 was diluted in a medium, 4 x IO5 cells/Id of grade A mice, 929 fibroblasts, Ill cells. (ATCC CCL92
9) 100 μl of the suspension was mixed in a 9G hole tissue culture microplate I (]-star). At this time, actinomycin D at a final concentration of 1 μg/terminal was added to Smegen. Banyu Pharmaceutical) is added.

The medium used was Eagle's Minimum Essential Medium containing 5% (vol/vol) fetal bovine serum.
Nippon Pharmaceutical Co., Ltd.) was used. After culturing the above microplate at 37°C for 18 to 20 hours in air containing 5% carbon dioxide, a crystal violet solution [5% (vol/v
ol) 0.5% (wt/vol) in methanol aqueous solution
) was used to stain living cells. After washing away the excess crystal violet and drying it, the remaining crystal violet was extracted with a 0.5% SDS aqueous solution of 100μ polishing.
The absorbance at 595nl was measured using an ELISA analyzer (Toyo Temperature Measurement).

ETY-96 model). This absorbance is proportional to the number of surviving 1Ilrf&. 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, and that dilution factor is defined as a unit. do. Expression type plasmid I) Escherichia coli lysate containing the novel antitumor active polypeptide encoded by TNF621 100 μl is 1
It had an activity of about 0.08 units, which was higher than E. coli lysate containing human TNF protein.

In addition, people with other novel anti-tumor active polypeptide gene expression plasmids l&! Significant anticancer activity was also observed in the bacterial lysates.

[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 nine plasmids containing the human TNF gene.
How to create FIBR, pdNF2N and pTNF3,
They are shown below. Figure 6 shows how to create the human TNF gene expression plasmid pT NF 401N N, Figure 7 shows how to create the expression vector pA A 41, and Figure 8 shows how to create the human TNF trochanteric expression plasmid +1 TNF 401A. The methods are shown below. FIG. 9 shows the method for producing the antitumor active polypeptide gene expression plasmid I) TNF 416A. FIG. 10 shows a method for constructing the novel antitumor active polypeptide gene expression plasmid 11TNF621. Figure 11 shows D encoding the deletion TNE.
Figure 12 shows the base sequence of the synthetic NA used to create the plasmid containing the NA region, and Figure 12 shows the method for creating the plasmid containing the DNA region encoding the deleted TNF. . @Figure 4 TNF-8 TNF-10 TNF-12 Patent applicant Teijin Ltd. Vul Figure 7 A (11-1---------→ AGCTTAGCCCGCCTAATGAGCGGGC
TTTTTTTT-(3”)(3-)-ATCGGGC
GGATTACTCGCCCGAAAAAAAAA-(5
-) (21--→ Fig. 1ndDI Pvu II (1) Verse of C□ω (Den-(TAC77%GA'TT, ATT4CCT
TC dingθ^TA-ty)C3)-T6AAACCC"T
A/'I cho eight ACG x Ha Δ6 ha CTAT7Ce8-σ
') 02ノ □ 110 L/n1/after/ lE １晃i '4u@nF C2ノ06ノ

Claims (6)

[Claims] (1) In the amino acid sequence represented by the 1st Val to the 157th Leu shown in Figure 1, the 1, 2, 3, 4, 5, 6, and 7 amino acid sequences on the amino terminal side ,
A novel physiologically active polypeptide having a sequence in which 8, 9, 10, 11 or 12 amino acid residues are deleted and the 157th Leu is replaced with Phe, or at its amino terminus. A polypeptide to which Met is bound. (2) In the amino acid sequence represented by the 1st Val to the 157th Leu shown in Figure 1, the 1, 2, 3, 4, 5, 6, and 7 amino acid sequences on the amino terminal side ,
A novel physiologically active polypeptide having a sequence in which 8, 9, 10, 11 or 12 amino acid residues are deleted and the 157th Leu is replaced with Phe, or at its amino terminus. A recombinant plasmid containing a DNA region encoding a polypeptide to which Met is bound. (3) In the amino acid sequence represented by the 1st Val to the 157th Leu shown in Figure 1, the 1, 2, 3, 4, 5, 6, and 7 amino acid sequences on the amino terminal side ,
A novel physiologically active polypeptide having a sequence in which 8, 9, 10, 11 or 12 amino acid residues are deleted and the 157th Leu is replaced with Phe, or at its amino terminus. A recombinant microbial cell transformed with a recombinant plasmid containing a DNA region encoding a polypeptide to which Met is bound. (4) The microbial cell according to claim 3, wherein the microbial cell is Escherichia coli. (5) In the amino acid sequence represented by the 1st Val to the 157th Leu shown in Figure 1, the 1, 2, 3, 4, 5, 6, and 7 amino acid sequences on the amino terminal side ,
A novel physiologically active polypeptide having a sequence in which 8, 9, 10, 11 or 12 amino acid residues are deleted and the 157th Leu is replaced with Phe, or at its amino terminus. A culture obtained by culturing recombinant microbial cells transformed with a recombinant plasmid containing a DNA region encoding a polypeptide to which Met is bound, producing and accumulating a novel physiologically active polypeptide in the culture. 1. A method for producing a novel bioactive polypeptide, which comprises separating the novel bioactive polypeptide from. (6) In the amino acid sequence represented by the 1st Val to the 157th Leu shown in Figure 1, the 1, 2, 3, 4, 5, 6, and 7 amino acid sequences on the amino terminal side ,
A novel physiologically active polypeptide having a sequence in which 8, 9, 10, 11 or 12 amino acid residues are deleted and the 157th Leu is replaced with Phe, or at its amino terminus. A pharmaceutical composition containing a polypeptide to which Met is bound.