JPH0817713B2 - Novel bioactive polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Field of Industrial Application The present invention relates to a novel bioactive polypeptide. More specifically, a novel polypeptide having antitumor activity (hereinafter,
It may be abbreviated as a novel antitumor active polypeptide).

In the present specification, amino acids and polypeptides are IUPAC-
Abbreviations are based on the method adopted by the IUB Biochemistry Committee (CBN). For example, the following abbreviations are used.

Ala L-Alanine Arg L-Arginine Asn L-Asparagine Asp L-Aspartic acid Cys L-Cysteine Gln L-Glutamine Glu L-Glutamic acid Gly Glycine His L-Histidine Ile L-Isoleucine Leu L-Leucine Lys L-Lysine Met L- Methionine Phe L-Phenylalanine Pro L-Proline Ser L-Serine Thr L-Threonine Trp L-Tryptophan Tyr L-Tyrosine Val L-Valine Also, the DNA sequence is abbreviated by the type of base contained in each deoxyribonucleotide constituting it And
For example, the following abbreviations are used.

A adenine (representing deoxyadenylic acid) C cytosine (representing deoxycytidylic acid) G guanine (representing deoxyguanylic acid) T thymine (representing deoxythymidylate) Further, (H ₂ N)-and -(COOH) indicates the amino terminal side and the carboxy terminal side of the amino acid sequence, and (5 ')-and (3') indicate the 5'terminal side and the 3'terminal side of the DNA sequence, respectively. is there.

(2) Background of the Invention Carswell et al. Have found that a substance collected from mice that had been previously stimulated with Bacillus Calmette-Guerin (BCG) or the like after administration of endotoxin contained hemorrhagic necrosis of cancer due to transplanted Meth A sarcoma. It was found that the substance was contained and the substance was identified as Tumor Necrosis Factor,
(Hereinafter sometimes abbreviated as TNF) [EACarsw
ell et al., Proc. Natl. Acad. Sci., USA, 72 , 3666 (1975)].
This TNF is found in many animals such as mice, rabbits, and humans, and since it works specifically in tumor cells and across species, it has been expected to be used as an anticancer agent.

Recently, Pennica et al. Performed cDNA cloning of human TNF to elucidate the primary structure of human TNF protein and reported the expression of human TNF gene in Escherichia coli [D. Pennica et al., Nature, 312 , 724]. (1984)].
After that, Shirai et al. [T. Shirai et al., Nature, 313 , 803 (198
5)], Soumura et al. [Soumura et al. Cancer and Chemotherapy, 12 , 160 (198
5)], Wang et al. [AM Wang et al., Science, 228 , 149 (198
5)] and Marmenout et al. [A. Marmenout et al., Eur. J. Bioche
m., 152 , 515 (1985)] successively reported the expression of the human TNF gene in Escherichia coli.

As described above, the use of gene manipulation techniques has made it possible to obtain a large amount of pure human TNF protein, and it is becoming clear that TNF has physiological activities other than the antitumor activity. For example, cachectin, one of the causes of cachexia in terminal cancer and patients with severe infections, is very similar to TNF [B. Beulter et al., Nature, 316 ,
552 (1985)], because cachectin has lipoprotein lipase inhibitory activity, administration of TNF may increase the amount of triglyceride in blood, and as a result, may cause side effects such as hyperlipidemia. Was suggested. In addition, the effect on vascular endothelial cells [JR
Gamble et al., J. Exp. Med., 162 , 2163 (1985)], bone resorption [DR Beltolini et al., Nature, 319 , 516 (1986)], etc. have been reported.

On the other hand, recent advances in gene manipulation technology have made it possible to substitute, add, or delete any amino acid in a protein with another amino acid. In this way, many studies have been conducted to modify naturally occurring proteins to create new proteins that serve specific purposes.

Some studies have been conducted on the modification of human TNF protein, and in the amino acid sequence of human TNF protein shown in FIG. 1, substitution of either or both Cys ⁶⁹ and Cys ¹⁰¹ with other amino acid residues ( PCT application publication WO86 / 04606,
Japanese Patent Application No. 61-106772), substitution of Gly ¹²² with another amino acid residue (Japanese Patent Application No. 61-106772, Japanese Patent Application No. 61-238048), Ala ¹⁸
Substitution of amino acid residues with other amino acid residues has been reported (JP-A-61-233337). Regarding deletion of amino acid residues on the amino-terminal side, TNF with 6 amino acid deletion has cytotoxic activity (Japanese Patent Application No. 61-50923), and 7 amino acid deletion
TNF has cytotoxic activity (Japanese Patent Application No. 61-90087)
No.), 1-10 amino acid-deleted TNF has cytotoxic activity, and its specific activity reaches its maximum in 6-8 amino acid-deleted TNF (PCT application publication WO86 / 02381), 10-amino acid deletion TNF has cytotoxic activity (Japanese Patent Application No. 61-
114754), and that 11-amino acid-deleted TNF has cytotoxic activity (Japanese Patent Application No. 61-173822).

Therefore, the present inventors have improved specific activity, improved stability,
For the purpose of broadening the reaction spectrum, reducing side effects, etc., we have conducted intensive studies on modification of human TNF protein,
The present invention has been completed.

(3) Object of the Invention An object of the present invention is to provide a novel antitumor active polypeptide.

Other objects of the present invention will become more apparent from the following description.

(4) Structure of the invention According to the studies by the present inventors, the above-mentioned object of the present invention is And a recombinant plasmid comprising a DNA region encoding the novel antitumor polypeptide, which is achieved by providing a novel antitumor polypeptide represented by the following formula or a polypeptide having Met linked to its amino terminus. And a method for producing a desired novel antitumor active polypeptide by using the recombinant microbial cell transformed by the recombinant plasmid thus obtained, and this novel antitumor active polypeptide. It has been found to be achieved by providing a pharmaceutical composition containing.

 The present invention will be described in more detail below.

(A) Cloning of human TNF gene; For the human TNF gene, an appropriate one is selected from several codons designating the amino acids [D. Pennica et al. It can be obtained by synthesizing. When designing the human TNF gene, it is desirable to select the codon most suitable for the host cell to be used, and to provide a cleavage site with an appropriate restriction enzyme at an appropriate position so that cloning and gene modification can be easily performed later. Is desirable. In addition, the DNA region encoding the human TNF protein preferably has a translation initiation codon (ATG) in a form in which the reading frame is aligned upstream, and a translation in a form in which the reading frame is aligned downstream. It is preferred to have a stop codon (TGA, TAG or TAA). It is particularly preferable to link two or more of the translation stop codons in tandem for the purpose of improving expression efficiency. Furthermore, this human TNF gene can be cloned into an appropriate vector by using the cleavage sites of restriction enzymes acting upstream and downstream thereof. An example of the nucleotide sequence of such human TNF gene is shown in FIG.

The human TNF gene designed as described above can be obtained by dividing each of the upper strand and the lower strand into several oligonucleotides as shown in FIG. 2 and chemically synthesizing them. It is desirable to adopt a method of ligating each oligonucleotide. The diester method [HG Khorana, “Some Recen
t Developments in Chemistry of Phosphate Esters of
Biological Interest ", John Wiley and Sons, Inc., New
York (1961)], triester method [RL Letsinger et al.,
J. Am. Chem. Soc., 89 , 4801 (1967)] and the phosphite method [MD Matteucci et al., Tetrahedron Lett., 21 , 719 (198
0)], but the synthesis by the phosphite method using a fully automatic DNA synthesizer is preferable from the viewpoints of synthesis time, yield, easiness of operation and the like. For purification of the synthesized oligonucleotide, gel filtration, ion exchange chromatography, gel electrophoresis, high performance liquid chromatography using a reverse phase column, etc. can be used alone or in combination as appropriate.

The 5'-terminal hydroxyl group of the synthetic oligonucleotide thus obtained is phosphorylated using, for example, T4-polynucleotide kinase and then annealed, for example
Ligate using T4-DNA ligase. As a method for producing a human TNF gene by ligating synthetic oligonucleotides, synthetic oligonucleotides are divided into several blocks and ligated, for example, pBR322 [F. Bolivar et al., Gene, 2 , 95
(1977)] once cloned into a vector such as
The method of ligating the DNA fragments of each of these blocks is preferable. Block DNA that constitutes such human TNF gene
The fragment-containing plasmid is preferably pTNF1BR or pTNF.
2N or pTNF3 is used.

After ligating the DNA fragments of each block constituting the human TNF gene cloned as described above, and connecting it to the downstream of an appropriate promoter and SD (Shine-Dalgarno) sequence, an expression type gene can be obtained. . As usable promoters, tryptophan operon promoter (trp promoter), lactose
Operon promoter (lac promoter), tac promoter, P _L promoter, lpp promoter, and the like, especially trp promoter is preferable. The plasmid having the trp promoter is preferably pYS3
1N or pAA41 is used. Furthermore, for the purpose of improving expression efficiency, a terminator that functions efficiently in Escherichia coli can be added to the downstream of the human TNF gene. Examples of such a terminator include lpp terminator, trp terminator and the like. Particularly, trpA terminator is suitable, and pAA41 is preferably used as a plasmid having trpA terminator. An expression type plasmid can be prepared by cloning this expression type human TNF gene into, for example, a pBR322-derived vector. As the human TNF gene expression type plasmid, pTNF401NN or pTNF401A is preferably used.

(B) Cloning of a novel antitumor activity polypeptide gene; the human TNF gene-expressing plasmid thus obtained is cleaved with an appropriate restriction enzyme to remove a specific region in the human TNF gene, and then an appropriate nucleotide sequence. Gene repair using a synthetic oligonucleotide having By using such a method, an expression type plasmid containing a gene encoding a novel antitumor activity polypeptide in the form of substituting, adding or deleting any amino acid in human TNF protein with other amino acid Can be created. As such a novel antitumor activity polypeptide gene expression type plasmid, pTNF489 is preferably used.

(C) Confirmation of expression and evaluation of activity; Examples of the microbial host for expressing the human TNF gene and the novel antitumor activity polypeptide gene include Escherichia coli, Bacillus subtilis, yeast and the like. Among them, Escherichia coli (Escherichia coli coli)] is preferred. The human TNF gene expression type plasmid and the novel antitumor activity polypeptide gene expression type plasmid can be prepared, for example, by using a known method [MV Norgard et al., Gene, 3 , 279 (1978)],
Microbial host, eg Escherichia coli C600r-m-
It can be introduced into the strain (ATCC33525).

The recombinant microbial cells thus obtained are cultured by a method known per se. As the medium, for example, M9 medium containing glucose and casamino acid [edited by T. Maniatis et al.,
"Molecular Cloning", P440, Cold Spring Harbor Labor
atory, New York (1982)], and it is desirable to add, for example, ampicillin, etc., if necessary.
Culturing is carried out at 37 ° C. for 2 to 36 hours under conditions suitable for the target recombinant microorganism, for example, aeration and agitation by shaking. In addition, a drug such as 3-β-indoleacrylic acid may be added for the purpose of efficiently functioning the promoter at the start of the culture or during the culture.

After culturing, the recombinant microbial cells are collected by, for example, centrifugation, suspended in, for example, a phosphate buffer, the recombinant microbial cells are disrupted by, for example, sonication, and a lysate of the recombinant microbial cells is obtained by centrifugation. The 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. The expression of the human TNF gene or the novel antitumor activity polypeptide gene is confirmed by comparing the migration patterns with a lysate of microbial cells containing no expression type plasmid as a control.

The human TNF protein thus obtained and the activity of the novel antitumor active polypeptide were evaluated by transplanting them into mice.
In vivo activity assay (Carswell et al., Supra) to see the effect of necrosis of Meth A sarcoma, in vitro activity assay to see cytotoxicity against mouse L cells [Ruff, J. Immunol., 126 , 23
5 (1981)], etc., but measurement time, quantitativeness,
From the viewpoint of ease of measurement and the like, evaluation by the in vitro activity measuring method is preferable.

Thus, according to the present invention, it becomes 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 pharmaceutical composition for antitumor is obtained. It is now possible to provide.

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 nucleotide sequence shown in Fig. 1 was designed. Upon designing, Pennica et al. [D. Pennica et al. Nature,
312 , 724 (1984)], based on the base sequence of the structural gene portion of human TNF precursor cDNA reported, a cleavage site by an appropriate restriction enzyme was provided at an appropriate position, and a translation initiation codon (ATG) at the 5'side. And two translation stop codons (TGA and TAA) on the 3'side, respectively. In addition, a cleavage site by the restriction enzyme ClaI was provided upstream of the translation initiation codon on the 5'side to allow ligation with the promoter while keeping the SD sequence and the translation initiation codon in an appropriate state. Furthermore, a cleavage site by the restriction enzyme HindIII was provided downstream of the translation stop codon on the 3'side so that it could be easily ligated to the vector / plasmid.

Example 2 (Chemical synthesis of oligonucleotide) The human TNF gene designed in Example 1 is divided into 17 oligonucleotides and synthesized as shown in FIG.
Oligonucleotides were synthesized by the phosphite method using a fully automatic DNA synthesizer (Applied Biosystems, model 380A). Purification of the synthetic oligonucleotide was performed according to the manual of Applied Biosystems. That is, by keeping an aqueous ammonia solution containing a synthetic oligonucleotide at 55 ° C. overnight, the protecting group of the DNA base was removed, and Sephadex G-50
The high molecular weight synthetic oligonucleotide fraction is collected by gel filtration using Fine Gel (Pharmacia). Then, after polyacrylamide gel electrophoresis containing 7 M urea (gel concentration 20%), the migration pattern is observed by the ultraviolet shadowing method. Cut out a band of the desired size, crush the polyacrylamide gel fragment into small pieces, and then add 2-5 ml of elution buffer [5
00mM NH ₄ OAc-1mM EDTA-0.1% SDS (pH7.5)] and add 37
Shake overnight at ° C. The aqueous phase containing the target DNA was recovered by centrifugation. Finally, the solution containing the synthetic oligonucleotide was applied to a gel filtration column (Sephadex G-5
The purified product of the synthetic oligonucleotide was obtained by subjecting to (0). 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 (TNF-1 to TNF-17) prepared in Example 2 were used to clone into 3 blocks of the human TNF gene. did.

0.1-1.0 μg of synthetic oligonucleotide TNF-2 to TNF
Using 5 to 15 units of T4-polynucleotide kinase (E. coli B type, Takara Shuzo) on the 5'end side of -6,
Each is phosphorylated separately. Phosphorylation reaction is 10-20μ
It was carried out in an aqueous solution of 50 mM Tris-HCl (pH 9.5), 10 mM MgCl ₂ , 5 mM dithiothreitol and 10 mM ATP at 37 ° C. for 30 minutes. After completion of the reaction, all the synthetic oligonucleotide aqueous solutions are mixed, and the T4-polynucleotide kinase is inactivated and removed by phenol extraction and ether extraction. Add 0.1-1.0 μm to the synthetic oligonucleotide mixture.
Synthetic oligonucleotides TNF-1 and TNF-7 (g) are added, and the mixture is heated at 90 ° C. for 5 minutes and then gradually cooled to room temperature, followed by annealing. Next, after drying this under reduced pressure, 30μ
1 of 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl ₂ , 10 pmM dithiothreitol and 1 mM ATP aqueous solution, dissolved in 300 units of T4-DNA ligase (Takara Shuzo) and ligated at 11 ° C for 15 hours. Was done. After completion of the reaction, polyacrylamide gel electrophoresis (gel concentration 5%) is performed, and the migration pattern is observed by the ethidium bromide staining method.
A band having a desired size (about 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 μg of E. coli plasmid pBR322 (about 4.4 Kb
p) 30 μl of 10 mM Tris-HCl (pH 7.5), 60 mM NaCl, 7 mM
Dissolve in MgCl ₂ aqueous solution and add 10 units of restriction enzyme ClaI.
(New England Biolabs) added, 37
Cleavage reaction was carried out at ℃ for 1 hour. After digestion with the restriction enzyme ClaI, phenol extraction and ether extraction are performed, and the DNA is recovered by ethanol precipitation. 30 μl of this DNA
It was dissolved in mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM MgSO ₄ aqueous solution, 10 units of restriction enzyme SalI (Takara Shuzo) was added, and a cleavage reaction was carried out at 37 ° C. for 1 hour. After the reaction,
Perform agarose gel electrophoresis (gel concentration 0.8%),
The cutting pattern is observed by the ethidium bromide staining method. Approximately 3.7 Kbp DN containing part of plasmid pBR322
The band corresponding to the portion A was cut out, and the agarose gel fragment was dissolved in a 3-fold amount (vol / wt) of 8M NaClO ₄ aqueous solution. Chen et al.'S glass filter method [CW Chen et al., Anal.
Biochem. 101 , 339 (1980)], and a DNA fragment (ClaISalI) of about 3.7 Kbp was recovered from the agarose gel.

Approximately 220 bp DN containing part of the previously obtained human TNF gene
The A fragment was subjected to terminal phosphorylation reaction according to the method described above, and then approximately 3. containing most of the plasmid pBR322.
Mix with a 7 Kbp DNA solution in water. After ethanol precipitation, both DNA fragments were ligated according to the method described above.

Transformation of the Escherichia coli C600r-m- strain was carried out by an improved method of the ordinary CaCl ₂ method (the method of MV Norgard et al.). That is, 5 ml of L medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH7.2) was added to Escherichia coli C600.
The rm-strain is inoculated with a culture medium for 18 hours, and the culture containing the cells is grown until the turbidity at 600 nm (OD ₆₀₀ ) reaches 0.3. Bacteria in cold magnesium buffer [0.1M
NaCl, 5 mM MgCl ₂ , 5 mM Tris-HCl (pH 7.6, 0 ° C)] 2
Wash twice, 2 ml of cold calcium buffer [100 mM C
_{aCl 2, 250mM KCl, 5mM MgCl} 2, 5mM Tris-HCl (pH7.6,0
℃)] and leave at 0 ℃ for 25 minutes. Next, the cells are concentrated to 1/10 of this volume in a calcium buffer and mixed with the ligated DNA aqueous solution in a ratio of 2: 1 (vol.:vol.). After maintaining this mixture for 60 minutes at 0 ° C, 1 ml of LBG medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 0.08% glucose, pH7.2) was added and shaken at 37 ° C for 1 hour. Incubate. Add the culture medium to the selection medium [ampicillin (Sigma) 30μ
L medium plate containing g / ml] at a rate of 100 μl / plate. Plates are cultured overnight at 37 ° C. to grow transformants. From the obtained ampicillin-resistant colonies, DNA was prepared by a known method and subjected to agarose gel electrophoresis to obtain the desired plasmid pTNF1BR (about 4.0 Kb
p) was confirmed. FIG. 3 shows the method for constructing the plasmid pTNF1BR.

Synthetic oligonucleotide TN was prepared in the same manner as above.
Plasmid pTNF2N (about 3.1 Kbp) using F-8 to TNF-13
Using the synthetic oligonucleotides TNF-14 to TNF-17, plasmid pTNF3 (about 2.4 Kbp) was prepared. 4 and 5 show the method for constructing the plasmids pTNF2N and pTNF3, respectively. The plasmids pTNF1BR, pRNF2N and pTNF3 containing a part of the human TNF gene thus obtained
The fact that the nucleotide sequence of the synthetic oligonucleotide-use part of the product is as designed is that the Maxam-Gilbert method [AMMaxa
M. et al., Methods Enzymol., 65 , 499 (1980)].

Example 4 (Preparation of human TNF gene expression type plasmid) 10 μg of the plasmid pTNF1BR obtained in Example 3 was cleaved with restriction enzymes ClaI and SalI in the same manner as in Example 3, and polyacrylamide gel electrophoresis (gel concentration 5 %)After,
According to the method of Example 2, a DNA fragment (ClaISalI) of about 220 bp containing a part of human TNF gene was recovered from polyacrylamide gel.

Next, 10 μg of the plasmid pTNF2 obtained in Example 3 was
100 μl of 10 mM Tris-HCl (pH 7.5), 60 mM NaCl, 7 mM MgCl
_It was dissolved in ₂ aqueous solution, 40 units of restriction enzyme PvuII (Takara Shuzo) was added, and the cleavage reaction was carried out at 37 ° C for 1 hour. Then, according to the method of Example 3, cleavage with the restriction enzyme SalI,
After polyacrylaminode gel electrophoresis (gel concentration 5%), according to the method of Example 2, a DNA fragment of about 170 bp (SalIPvuII) containing a part of human TNF gene was recovered from polyacrylamide gel.

In addition, 10 μg of the plasmid pTNF3 obtained in Example 3 was also
100 μl of 10 mM Tris-HCl (pH 7.5), 60 mM NaCl, 7 mM MgCl
₂ Dissolve in an aqueous solution to obtain 40 units of the restriction enzymes PvuII and 40
Add the unit's restriction enzyme HindIII (Takara Shuzo) to 37 ℃
The cleavage reaction was carried out for 1 hour. Then, after polyacrylamide gel electrophoresis (gel concentration 5%), according to the method of Example 2, a DNA of about 110 bp containing a part of the human TNF gene.
The fragment (PvuIIHindIII) was recovered from polyacrylamide gel.

On the other hand, the plasmid pYS containing the E. coli trp promoter
5 μg of 31N (approximately 4.7 Kbp) was added to the restriction enzyme ClaI as above.
And HindIII, and after agarose gel electrophoresis (gel concentration 0.8%), according to the method of Example 3, a DNA fragment of about 4.7 Kbp (ClaIHin containing most of the plasmid pYS31N) was prepared.
dIII) was recovered from the agarose gel.

About 220 containing part of the human TNF gene thus obtained
Three DNA fragments of bp, about 170 bp and about 110 bp and plasmid pY
After mixing with a DNA fragment of about 4.7 Kbp containing most of S31N and performing ethanol precipitation, the ligation reaction with T4-DNA ligase was performed according to the method of Example 3. After completion of the reaction, it was introduced into the Escherichia coli C600r-m- strain according to the method of Example 3, and a clone having the desired human TNF gene expression type plasmid pTNF401NN (about 5.2 Kbp) was selected from the transformants. . FIG. 6 shows the method for constructing the plasmid pTNF401NN.

In addition, 5 μg of the above plasmid pYS31N was partially digested with the restriction enzyme PvuII according to the method described above, and further digested with the restriction enzyme HindIII, followed by agarose gel electrophoresis (gel concentration 0.8%), and then according to the method of Example 3. , A 2.7 Kbp DNA fragment containing the trp promoter [PvuII (2) HindII
I] was recovered from the agarose gel.

Next, an oligonucleotide having the base sequence shown in FIG. 7 was synthesized and purified according to the method of Example 2. 0.5 μg of each of the two synthetic oligonucleotides obtained was subjected to terminal phosphorylation according to the method of Example 3 and after annealing, about 2.7 Kbp of the previously obtained DNA was obtained.
After mixing with the fragment [PvuII (2) HindIII] and ethanol precipitation, a ligation reaction with T4-DNA ligase was performed according to the method of Example 3. After completion of the reaction, it was introduced into the Escherichia coli C600r-m- strain according to the method of Example 3, and the desired plasmid pAA41 (about 2.7 Kbp) was selected from the transformants.
A clone with was selected. Such a plasmid is obtained by removing the copy number control region from plasmid pYS31N and tr
This is a multicopy, high-efficiency expression vector in which E. coli trpA terminator is added to the downstream of the cloning site existing in the downstream of p promoter, and its formation method is shown in FIG. 7.

2 μg of this plasmid pAA41 was cleaved with the restriction enzymes ClaI and HindIII in the same manner as described above, and after agarose gel electrophoresis (gel concentration 0.8%), about 2.7 parts of the plasmid pAA41 containing most of the plasmid pAA41 was prepared according to the method of Example 3. Kbp DNA fragment (ClaI
HindIII) was recovered from the agarose gel.

In addition, the previously obtained human TNF gene expression type plasmid p
5 μg of TNF401NN was treated with the restriction enzymes ClaI and HindI in the same manner as above.
After cutting with II and polyacrylamide gel electrophoresis (gel concentration 5%), according to the method of Example 2, a DNA fragment (ClaIHindIII) of about 490 bp containing the entire human TNF gene was recovered from the polyacrylamide gel.

The thus obtained DNA fragment of about 2.7 Kbp containing most of the plasmid pAA41 and about 490 bp containing the entire human TNF gene.
Example 3 was mixed with the DNA fragment of Example 3 and precipitated with ethanol.
The ligation reaction with T4-DNA ligase was performed according to the method described in 1. After the reaction was completed, it was introduced into the Escherichia coli 600r-m- strain according to the method of Example 3, and a clone having the desired plasmid pTNF401A (about 3.2 Kbp) was selected from the transformants. This plasmid has the ability to more efficiently express the human TNF gene, and its construction method is shown in FIG.

Example 5 (Preparation of novel antitumor activity polypeptide gene expression type plasmid) Human TNF gene expression type plasmid p obtained in Example 4
20 μg of TNF401A was added to 100 μl of 10 mM Tris-HCl (pH7.4), 1
Dissolve in 0 mM MgSO ₄ , 1 mM dithiothreitol aqueous solution,
Add 0 units of the restriction enzyme KpnI (Takara Shuzo) at 37 ℃
The cleavage reaction was carried out for 1 hour. Cleavage with the restriction enzyme ClaI was performed according to the method of Example 3, and after polyacrylamide gel electrophoresis (gel concentration 5%) and agarose gel electrophoresis (gel concentration 0.8%), Example 2 was performed.
2 DNA fragments (about 160
bp and about 3.0 Kbp, both ClaIKpnI) were recovered from the gel.

Approximately 160 bp including the first half of human TNF gene obtained here
50 μl of 10 mM Tris-HCl (pH7.4), 10 mM MgS
It was dissolved in O ₄ , 1 mM dithiothreitol aqueous solution, 10 units of restriction enzyme HapII (Takara Shuzo) was added, and a cleavage reaction was carried out at 37 ° C. for 1 hour. After completion of the reaction, polyacrylamide gel electrophoresis (gel concentration 5%) was carried out, and according to the method of Example 2, about 100 bp containing the first half of human TNF gene.
DNA fragment (ClaIHapII) was recovered from the polyacrylamide gel.

An oligonucleotide having the nucleotide sequence shown in FIG. 9 was synthesized and purified according to the method of Example 2. 0.5 μg each of the obtained two synthetic oligonucleotides
Regarding the above, according to the method of Example 3, terminal phosphorylation was performed and annealing was performed.

After the reaction was completed, the obtained double-stranded oligonucleotide was
The approximately 3.0 Kbp DNA fragment (ClaIKpnI) obtained above and the approximately 100 bp DNA fragment (ClaIKpnI) containing the first half of the human TNF gene.
HapII) and mixed with ethanol, followed by ligation reaction with T4-DNA ligase according to the method of Example 3. After the reaction was completed, it was introduced into the Escherichia coli C600r-m- strain according to the method of Example 3, and a clone having the desired plasmid pTNF489 (about 3.2 Kbp) was selected from the transformants. This plasmid contains the following amino acid sequence It is a novel antitumor active polypeptide gene expression type plasmid that encodes the novel antitumor polypeptide represented by or a polypeptide in which Met is bound to the amino terminus thereof, and its construction method is shown in FIG. 9.

Example 6 (Confirmation of expression) Expression vector pAA41 and human TNF obtained in Example 4 above
The gene expression type plasmid pTNF401NN or pTNF401A, or the Escherichia coli C600r-m strain having the novel antitumor activity polypeptide gene expression type plasmid pTNF489 obtained in Example 5 was treated with 30 to 50 μg / ml of ampicillin, 0.2.
% Glucose and 4 mg / ml casamino acid in M9 medium [0.6% Na ₂ HPO ₄ -0.3% K ₂ HPO ₄ -0.05% NaCl-0.1% NH ₄ Cl
After autoclaving the aqueous solution (pH 7.4), separately add autoclave-sterilized MgSO ₄ aqueous solution and CaCl ₂ aqueous solution to final concentrations of 2 mM and 0.1 mM, respectively. ] Inoculate 250 ml and 37 ℃ until OD ₆₀₀ reaches 0.7
Shaking culture was performed. Then, 3-β-indoleacrylic acid having a final concentration of 50 μg / ml was added to the culture medium, and shaking culture was further continued at 37 ° C. for 12 hours.

After collecting the Escherichia coli cells by centrifugation, the cells were washed with PBS buffer (20 mM phosphate buffer containing 150 mM NaCl, pH 7.4). Wash the cells with 10 ml of PB
Suspend in S buffer, ultrasonic generator (Kubota, 200M
After destroying the bacterial cells by using the (type), the bacterial cell residue was removed by centrifugation.

Tris-HCl was added to a part of the obtained E. coli lysate.
Buffer (pH6.8), SDS, 2-mercaptoethanol,
Glycerol, final concentration 60 mM, 2%, 4%, 10%, respectively
In addition, SDS-polyacrylamide gel electrophoresis [Suzuki, K., 31 , 43 (1977)] was performed. The separation gel was 15%, and the migration buffer was SDS, Tris-glycine system [UK Laemmli, Nature, 227 , 680 (1970)]. After completion of the electrophoresis, the protein in the gel was stained with Coomassie Blue R-250 (Bio-Rad) to confirm the expression of human TNF gene and novel antitumor activity polypeptide gene. A copy of part of the results is shown in FIG.

The stained gel was subjected to Chromatoscanner (Shimadzu, CS-930 type) to calculate the ratio of the produced human TNF protein or the novel antitumor active polypeptide in Escherichia coli cytoplasmic protein. As a result, in Escherichia coli having the human TNF gene-expressing plasmid pTNF401A, about 28% of the total cytoplasmic proteins were human TNF proteins, and in Escherichia coli having the novel antitumor activity polypeptide gene-expressing plasmid pTNF489, about 28% of the new anti-tumor proteins were also present. Production of tumor-active polypeptides was observed respectively. Also human
The production amount of human TNF protein in Escherichia coli having the TNF gene-expressing plasmid pTNF401NN was only about 40% of the case of the above pTNF401A, demonstrating the usefulness of the expression vector pAA41.

Example 7 (Evaluation of activity) The activity of the novel antitumor polypeptide was measured by the above-mentioned Ruff.
The method was performed according to the method described above. That is, 100 μl of a sample prepared by serially diluting the Escherichia coli lysate containing the human TNF protein or the novel antitumor active polypeptide obtained in Example 6 with a medium.
And mouse L-929 fibroblasts (A at a concentration of 4 × 10 ⁵ cells / ml)
100 μl of TCCCCL-929) suspension was mixed in a 96-well tissue culture microplate (coaster). At this time, actinomycin D (cosmegen, Banyu Pharmaceutical Co., Ltd.) having a final concentration of 1 μg / ml was added. 5% as a medium
Eagle's minimum essential medium (Nissui Pharmaceutical) containing (vol / vol) fetal bovine serum was used. The above microplate was placed in air containing 5% carbon dioxide at 37 ° C for 18
After culturing for ~ 20 hours, crystal violet solution [5% (vol / vol) 0.5% (wt / vo
Live cells were stained with [1) a solution of crystal violet]. After washing away excess crystal violet and drying, the remaining crystal violet was extracted with 100 μl of 0.5% SDS aqueous solution, and its 595 nm
Absorbance at ELISA analyzer (Toyo Sokki, ETY-96
Type). This absorbance is proportional to the number of surviving cells. Therefore, the dilution ratio of E. coli lysate corresponding to the value of 50% of the absorbance of the control without the addition of the diluted solution of E. coli lysate containing the human TNF protein or the novel antitumor active polypeptide is determined by a graph (for example, FIG. 11)
The dilution ratio is defined as a unit. From FIG. 11, 100 μl of Escherichia coli lysate containing human TNF protein encoded by expression type plasmid pTNF401A has an activity of about 4.9 × 10 ⁵ units, and Escherichia coli containing a novel antitumor activity polypeptide encoded by expression type plasmid pTNF489. It was revealed that 100 μl of the lysate each had an activity of about 2.0 × 10 ⁶ units.

The total protein amount contained in the E. coli lysate containing the human TNF protein encoded by the expression type plasmid pTNF401A obtained in Example 6 or the novel antitumor activity polypeptide encoded by the expression type plasmid pTNF489 was determined by protein assay. It was quantified using a kit (Bio-Rad) and calculated from a calibration curve using bovine serum albumin. From the expression level, activity value and protein quantification results obtained above, human TN
When the specific activities of the F protein and the novel antitumor activity polypeptide were calculated, the values shown in Table 1 were obtained.

[Brief description of drawings]

Figure 1 shows the base sequence of the designed human TNF gene, and Figure 2 shows the base sequence of chemically synthesized synthetic oligonucleotides.
These are shown respectively. 3, 4, and 5 show plasmid pTNF1BR, p containing a part of human TNF gene.
The methods for preparing TNF2N and pTNF3 are shown respectively. Figure 6 shows the human TNF gene expression plasmid pTNF401NN
Fig. 7 shows the construction method of the expression vector pAA41, and Fig. 8 shows the human TNF gene expression plasmid pTN.
The methods of creating the F401A are shown respectively. Ninth
The figure shows the method for constructing a novel antitumor activity polypeptide gene expression type plasmid pTNF489. FIG. 10 shows a photograph of gel electrophoresis which confirmed the results of confirming the expression of human TNF gene and novel antitumor activity polypeptide gene. FIG. 11 shows the activity measurement results of human TNF protein and novel antitumor polypeptide.

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Claims (2)

[Claims] 1. The following amino acid sequence A novel bioactive polypeptide represented by: 2. The novel bioactive polypeptide according to claim 1, wherein Met is bound to the amino terminus.