JPH04182497A - Polypeptide - Google Patents

Abstract

NEW MATERIAL:A polypeptide having a sequence wherein 106th Gly is replaced with another amino acid or is deficient in the amino acid sequence from 1st Ser to 155th Leu shown by the formula. USE:An antitumor agent. PREPARATION:For example, a gene encoding an amino acid sequence of human tumor necrosis factor(TNF) is cleft with a restriction enzyme to prepare a DNA fragment encoding an amino acid sequence of 1st Ser to 155th Leu shown by the formula, which is subjected to gene conversion in combination with digestion with a restriction enzyme and connection with T4DNA ligase to give a converted material wherein 106th Gly is replaced with another amino acid or is deficient. The converted material is linked to a manifestation vector, Escherichia coli is transformed with the vector and the converted material is cultured to give a polypeptide of human TNF converted material.

Description

Detailed Description of the Invention "Industrial Application Field" The present invention relates to (a) providing a novel polypeptide as a TNF converter, and relates to the polypeptide itself, a pharmaceutical composition containing the same, a method for producing the same, and It relates to genetically recombinant DNA, plasmids, transformed microbial cells, etc.

“Prior art” TNF (tumor necrosis factor) was developed by Carsw in 1975.
Bacillus Calmet was prepared in advance by Ell et al.
It is a physiologically active substance that was found to exist in the serum of mice infected with B. te Guerin (BCG) and treated with endotoxin (Proc. Natl. A.
cad, Sci, USA 723666 (1975
)), in 1984 by Penn1ca et al.
NF cDNA was cloned and the entire structure (amino acid sequence) of human TNF protein was revealed (Natural
re 312.724 (1984)), TNF has specific antitumor effects such as cytotoxic activity against tumor cells, hemorrhagic necrosis against transplanted tumors, and inhibition of growth. It has been reported that side effects may occur, and research and development are being carried out to find something with better efficacy and side effects. For example, JP-A-61-40221, JP-A-63-119692
, Japanese Unexamined Patent Publication No. 277488 discloses that human TNF transformants are provided by deleting specific amino acids in human TNF protein, substituting with other amino acids, or adding them using genetic engineering techniques. There is.

"Disclosure of the Invention" The present inventors substituted or deleted glycine at position 106 in the amino acid sequence of human TNF protein with another amino acid, and obtained a human TNF converter that has high antitumor activity and low side effects, which has not been described in any literature. He discovered that polypeptides could be obtained, and based on that knowledge he completed his invention.

The present invention relates to a polypeptide having a sequence in which 011 at position 106 in the amino acid sequence represented by Ser 1 to Leu 155 shown in SEQ ID NO: 1 of the sequence listing is replaced with another amino acid or deleted. . Also, a recombinant plasmid containing DNA encoding the polypeptide, a microorganism cell transformed with this recombinant plasmid, a method for producing the polypeptide using this microorganism cell, a pharmaceutical composition, and methionine bonding to the N-terminus of the amino acid sequence. cI! from 316th to 318th in the base sequence represented by the 1st T to 465th G shown in the polypeptide and Sequence Listing SEQ ID NO: 2! It also relates to a DNA having a sequence in which GGC encoding y is replaced or deleted by a codon encoding another amino acid.

Throughout this specification, amino acids, polypeptides, bases,
The following list is used to represent these arrays.

Amino acid: Symbol Afa Cys Asp G
lu meaning Alanine Nstein Ryosubaraginic acid Glutamic acid Symbol Phe Gl
! y His IAe Lys-i taste Phenylalanine Glinone Histidine Iso0ynone Linn symbol Leu Met
Asn Pro Gffin meaning 0 ynone methionine Ryosubaragin proline glutamine symbol Arg Ser Th
r VaiTrp Meaning Arginine Serine Threonine Valine Tryptophan Symbol Tyr Meaning Thionone base Also, the abbreviations used in this specification have the following meanings.

SDS: Sodium dodecyl 5u
lfateDTT Nidithiothreitol (Dithi)
othreitol') PMS F: Phenyl
Methylsulfonyl FluorideCP
G Resin Controlled-Pore Glass
Other amino acids replacing 106th GE)j in the resin in the present invention include Ala, Cys, Asp, G
l! u, circle 1e, His, IA'e, Lys, L
eu, Met, Asn, Pro, Gl! n.

Arg, Ser, Thr, VaCTrp or Ty
Can you name r, Asp, Lys, Gj? u,
Trp, Pro, Arg or AA'a are preferable, and the 106th CI! Deletion of y is also desirable.

Next, the embodiments of the present invention will be described in detail, or
Regarding the genetic manipulation technology according to the present invention, methods and means described in many documents are applied with appropriate adjustment. The documents are listed below for reference.

T., Maniatis et al. (1982):
Molecular Cloning.

Co1d Spring Harbor Labora
toryXR, Wu et al.

(1983): Methods in Enzymo
logy, 100 and +01, R, Wu et a
l, (19871Methods in Enzyrn
ology.

153, 154, and 155 The polypeptides of the present invention can be produced using various methods, means, and machines, and representative production methods are described below.

(1) Acquisition of human TNF by human TNF
As mentioned above, the base sequence and amino acid sequence of the gene are P
enn1ca et al., and by appropriately changing the base sequence, the Hi-T NF gene is designed as shown in SEQ ID NO: 3 in the sequence listing.

At this time, it is desirable to select codons that are suitable for the host cell (E. coli), and to select codons at appropriate positions so that cloning by ligation of DNA fragments and genetic modification for constructing transformants, which will be described later, can be easily carried out. It is desirable to place appropriate restriction enzyme cleavage sites. Of course, it is necessary to install a translation start codon (ATG) upstream of the human TNF gene and a translation stop codon (TAA, TGA, or TAG) downstream to match the reading frame. In addition, it is preferable to install appropriate restriction enzyme cleavage sites downstream of the translation stop codon to improve applicability to vectors and ease of cloning.

The HiI-T NF gene can be produced by chemically synthesizing several oligonucleotides for each of the tangles and lower strands, and sequentially and suitably linking each block.

For example, in SEQ ID NO: 3 and 4 of the sequence listing, human TN
Each strand of the F gene is divided into 10 oligonucleotides of approximately 50 bases each, and a total of 20 oligonucleotides are chemically synthesized. Its synthesis method is the diester method (H,G, Khorana
,”Some Recent Developments
in Chemistry of Phosphat
e Estersof Biological Int.
John Wiley and 5o
ns.

Inc., New York (1961)), triester method [:R,L.

Letsinger et al., J., Am., C.
hem, Soc, 89.4801 (1967))
and phosphite method CM, D, Matteucci
et aL, Tetrahedron Let
t,, 21. 719 (1980)), but the phosphite method using a fully automatic DNA synthesizer is preferably used due to its ease of operation.

The synthesized oligonucleotide is purified by conventional purification methods such as high performance liquid chromatography using a reversed phase chromatography column and electrophoresis using polyacrylamide gel. The oligonucleotides are then phosphorylated using, for example, T4 polynucleotide kinase, annealed, and then ligated using T4 DNA ligase. Here, oligonucleotides are divided into several blocks and sequentially ligated to obtain the desired human TNF gene sequence, cut with restriction enzymes or blunted with T4 DNA polymerase, and purified by electrophoresis. Regarding the obtained DNA fragments, for example, pLlc8, pLlc9, pLlc18
, 19 (J. Messing et al.

Gene, 19.259 (1982)), and cloned by transforming competent cells by a conventional method. Plasmid DNA is extracted and purified from the obtained clone according to a known method, and it is checked whether the base sequence of the DNA fragment inserted into the vector has achieved the desired gene sequence. Each part of the human TNF gene that has been achieved is cut out using a restriction enzyme from a plasmid vector containing each part, ligated to the vector again, integrated, and cloned to obtain a plasmid vector containing the desired full-length human TNF gene. . The desired human TNF gene can be obtained by cutting the thus obtained plasmid vector with a restriction enzyme and then separating and purifying it by gel electrophoresis.

On the other hand, for the above-mentioned method, cDNA encoding human TNF is extracted from mRNA derived from human cells expressing TNF.
The methods of producing A and using its cDNA may be combined as appropriate.

(2) Construction of human TNF expression vector The human TNF gene obtained in (1) above is appropriately inserted into an expression vector to construct a human TNF expression vector. The expression vector has a transcription promoter region and a translation signal SD (Shine-Dalgarno) sequence upstream of the translation start codon (ATG), and a transcription terminator region downstream of the translation stop codon (TAA, TGA or TAG). There is a need. In addition, transcription promoters include trp promoter, lac promoter, tac promoter, PL promoter, PH05 promoter, ADC[
A promoter etc. can be used, and transcription terminators include trp terminator-1rrnB terminator-1.
ADCI terminator etc. can be used.

Such expression vectors include, for example, ρKK223-3
(Pharmacia) and pPL-1ambda (same as on the left), which can be easily obtained from commercial products, but it is also possible to use products that have been improved to have higher expressability or handleability.

(3) Human T
Examples of methods for producing DNA encoding an NF converter polypeptide include the following method.

(i) It is produced by appropriately linking oligonucleotides chemically synthesized according to the method described in (1) H) Obtaining the TNF gene. According to this method, modifications such as substitution, addition, or deletion of amino acids, polypeptides, etc. can be made freely.

(ii) After cutting the human TNF gene prepared in (1) above with an appropriate restriction enzyme and removing a specific region within the gene, a synthetic oligonucleotide having a base sequence into which a mutation has been introduced (e.g., annealing the upper and lower sequences) double-stranded DN linked together
A fragment) or other appropriate genes. With this method, as in the case (i) above, modifications can be made freely.

(iii) A synthetic oligonucleotide having a base sequence into which a mutation has been introduced is used as a primer to extend the DNA chain, thereby producing a converted polypeptide gene (
Part-directed mutagenesis (T, A, Kunkel et al.
Al, Methods in Enzymology
, 154.367 (1987)). This method is I
Although it is difficult to apply to the addition and insertion of relatively long DNA strands exceeding O base pairs, other modifications can be made in the same manner as in the case (i) above. It is particularly suitable for replacing any amino acid.

In the present invention, the site-directed mutagenesis method (ii) is preferred, so the following description will focus on this method.

First, in performing the site-directed mutagenesis method, a template DNA is prepared. The human TNF gene obtained in (1) above,
Messing et al. (Methods in Enzym
lol! y.

153, 3 (1987)) for the preparation of single-stranded plasmid DNA <pUc
118, pUc119, etc.) and introduced into an E. coli strain. A clone having the desired plasmid is selected from among the transformants obtained.

This plasmid was used in dut- and ung-mutant E. coli strains (
CJ236 strain, etc.), incorporate uracil into the gene, and infect E. coli strain with a mutant helverphage such as Ml 3KO7 to obtain the desired single-stranded plasmid DN.
Get an A.

On the other hand, an oligonucleotide primer of about 15 to 50 bases having the conversion introduction site according to the present invention and the base sequences before and after the site is chemically synthesized. After annealing this primer and the uracil-introduced double-stranded plasmid DNA obtained in the above step, for example, T4 DNA polymerase and T
Double strand it using 4DNA ligase. In order to inactivate the DNA strand containing uracil as a template and increase the frequency of mutation introduction, the double-stranded plasmid is introduced into an ung+ E. coli strain. Colony hybridization is performed using the aforementioned primer as a probe, and clones having a plasmid containing DNA encoding the desired transformant polypeptide are selected from among the resulting transformants.

A DNA fragment encoding a human TNF converter polypeptide is cut out from the plasmid obtained above by restriction enzyme cleavage treatment, and inserted into an expression vector as in the case of (2) above to obtain the desired human TNF converter expression vector. Can be constructed.

For introducing an expression vector into a host cell such as an E. coli strain, for example, a known method CMo1 using competent cells of an E. coli strain prepared by the calcium chloride method can be used.
ecular Cloning, T., Maniat
iset al. (1982)).

Microbial cells such as Escherichia coli, Bacillus subtilis, and yeast can be used as host cells, and among them, E. coli is JM 8.
3, mutants of E. coli K-12 strains such as JM103.

(4) Obtaining human TNF converter polypeptide In the present invention, the transformed microbial cell described in (3) above is cultured, and the desired human TNF converter polypeptide is produced and accumulated in the culture. To separate. Conventionally known methods for culturing microbial cells, especially Escherichia coli,
For example, a method can be used in which E. coli is inoculated into a culture solution containing the nutrients required by E. coli and cultured in large quantities in a short period of time, usually by shaking or stirring at 32 to 37°C for about 12 to 24 hours. Examples of the medium include L medium, λ (9 medium, etc.)
[Refer to Molecular Cloning] above, or add antibiotics such as ampicillin as necessary, or insert the lac promoter or
Isopropyl-β-D-thiogalactopyranoside can also be added to increase the efficiency of transcription promoters such as the tac promoter.

After culturing the human TNF converter polypeptide of the present invention, the microbial cell aggregate is usually disrupted by ultrasonication while suspended in Tris buffer, and then centrifuged to remove bacterial cell residue. Obtained by removing. Furthermore, the product thus obtained can be layered by combining treatment with a nucleic acid/endotoxin remover, filtration with a filter, anion exchange chromatography, and other conventional protein separation and purification methods.

The human TNF converter polypeptide of the present invention has an antitumor effect similar to the antitumor effect of human TNF, and
It exhibits superior antitumor activity compared to other drugs, and has fewer side effects, so it is effective as an antitumor agent and an active ingredient in medicine. Among the human TNF converter polypeptides of the present invention, those in which Gly at position 106 in the above amino acid sequence is replaced with Pro or Arg, and those in which Gly is deleted are particularly preferred. When formulating the pharmaceutical composition, it can be formulated into a pharmaceutical composition together with a pharmacologically acceptable carrier or diluent.

The dosage form of the pharmaceutical composition of the present invention includes external preparations, oral preparations, injection preparations, etc., and is administered by an administration method suitable for each dosage form.

Example 1 (Design of human TNF gene) Based on the amino acid sequence of the human TNF structural gene that has already been reported by CPennjca et al., supra, a sequence list is prepared for the nucleotide sequence of the human TNF gene for convenience of gene construction and transformant production. A DNA base sequence of SEQ ID NO: 3 was designed. Here, restriction enzyme cleavage sites were incorporated at appropriate intervals, and cleavage sites for the restriction enzyme EcoRI were placed upstream of the translation start codon (ATG) and downstream of the translation stop codons (TAA and TGA) for easy ligation with the plasmid vector. A cleavage site using the restriction enzyme HindI [[ was provided in each of them.

Example 2 (Chemical synthesis of oligonucleotide) The DNA designed in Example 1 was chemically synthesized by the phosphite method using an automatic DNA synthesizer (Applied Biosystems, model 381A). The synthesis was carried out using U-1 to lO and L- having the base sequence shown in SEQ ID NO: 4 in the sequence listing.
The oligonucleotides were divided into 20 oligonucleotides numbered 1 to 10, and the synthesized oligonucleotides were excised from a CPG resin (sold by Funakoshi Co., Ltd.) and the protecting group was removed according to the manual of Applied Biosystems.

Separation and purification of each oligonucleotide was performed by HPLC (high performance liquid chromatography) using a reverse phase chromatography column or polyacrylamide gel electrophoresis (gel concentration 10-20%) containing 7M urea.

For HPLC method, Nucleozil 5C18 Ram (
Triethylaminoacetic acid (loOmM) buffer (1) H7 containing acetonitrile was purified by reverse phase chromatography using φ4.6X150mm (sold by Chemco).
, O). The above elution was performed using a linear concentration gradient of 5 to 35% (30 minutes) of acetonitrile, and a peak at about 15 minutes was collected.

Regarding the polyacrylamide gel electrophoresis method, each synthetic oligonucleotide sample is separated by electrophoresis, a band portion of the desired size is cut out based on the observation results of the migration pattern by ultraviolet shadowing method, and the polyacrylamide gel fragment is divided into approximately 1 ~Cut into 2mm' pieces, about 2m! ! Elution buffer y (0,5M NH4
0Ac and 1mM EDTA) was added and shaken overnight at 37°C. The elution buffer containing each oligonucleotide was collected and phenol extracted (50% phenol 15
(using 0% chloroform solution), isobutanol extraction, and ethanol precipitation to obtain purified samples of each oligonucleotide.

Some of the synthesized and purified oligonucleotides were analyzed using the Maxam-Gilbert method (A, M, Nlaxam et al.
al.

Methods in EnzymologY, 654
99 (1980)) I' knee, it was confirmed that it had the desired base sequence.

In the operations related to genetic recombination described below (Examples 3.4 and 5), the reaction conditions for restriction enzymes and other related enzymes were mainly in accordance with the method described in Molecular Cloning (supra). The enzymes mentioned above were mainly obtained from Takara Shuzo, and Takara Shuzo's manual was also used as reference.

Example 3 (Human T by ligation of synthetic oligonucleotides)
Construction of NF gene) (1) First, an attempt was made to construct a human TNF gene according to Figure 1. - The synthetic oligonucleotides obtained in Example 2 were divided into three groups (U and L-1 to 4, U and L-5 to 7, and U and L-8 to 10) and cloned. That is, the 5' end of each oligonucleotide (1 to 2 μg) of U-2, 3, 4, 6, 7, 9, and 10 and L-1, 2, 3, 5, 6, 8, and 9 was Unit T
4 polynucleotide kinase (Takara Shuzo), each is phosphorylated separately. The phosphorylation reaction was carried out in 10μl of an aqueous solution (50mM Tris-HCI! pH 7,6,1
0mM MgClt, 0.1mM Spermidin
e, 0.1mM EDTA, 10mK (DTT and 1mM ATP) at 37°C for 1 hour, and after the reaction was completed, T4 polynucleotide kinase was inactivated by treatment at 70°C for 10 minutes. Add 1 to 2 μg of U-
Aqueous solution 1 having the same composition as above, containing each of the oligonucleotides 1, 5 and 8 and L-4, 7 and IO separately.
Prepare 0 μl and add each oligonucleotide (U-1 to 10 and L-1 to 10) with the same numbers of U and L.
The aqueous solution was mixed (20 μl), boiled at 100° C. for 5 minutes, and then slowly cooled for annealing. Next, the obtained 1
0 annealed bodies (double-stranded DNA fragments) were added to each group in an aqueous solution (66mM Tri
s-HCffipH7,6,6,6mM MgCl2
．． 10mM DTT, 1mM ATP and 1100u
/me BSA) total liquid volume 120-160μl
), after annealing by heating to 40°C and slow cooling, 70
0 units of T4 DNA ligase (Takara Shuzo) was added, and the ligation reaction was carried out at 16°C for 15 hours. After the reaction is complete,
Each reaction solution was separated by polyacrylamide gel electrophoresis (gel concentration 6%), and the desired size (176b
p, 150 bp, and 153 bp) were cut out, and the target 3
DNA fragments from the book were recovered. Furthermore, each collected sample was subjected to phenol extraction (using a 50% phenol-150% chloroform solution) and isobutanol extraction, and the target DNA was purified by ethanol precipitation. According to the above method, three purified double-stranded DNA fragments were separately phosphorylated at their 5' ends using T4 polynucleotide kinase, mixed in an aqueous solution for ligation reaction, and then Annealing is performed by heating to T4
DNA ligase was added for ligation. This ligated DNA was recovered by ethanol precipitation and mixed with 1mM DTT and l
50μl of high salt buffer (50mM Tris-HC, j7 pH7,
5,100mM NaC7, 10mM MgcA□), 15 units of restriction enzyme EcoRI (Takara Shuzo) and 15 units of restriction enzyme Hind N (Takara Shuzo) were added, and a cleavage reaction was carried out at 37°C for 2 hours. After the reaction, the desired DNA fragment (
about 480 bp) was separated and purified.

On the other hand, 5 μg of plasmid vector pUC9 (distributed from Kyushu University Genetic Information Laboratory) was added according to the method described above.
Digested with restriction enzymes EcoRI and Hind III, and subjected to agarose gel electrophoresis (gel concentration 1%) to obtain approximately 2.7
The Kbp DNA fragment was isolated and purified.

The previously purified approximately 480 bp DNA fragment (containing the human TNF gene) and this pUc9 fragment were mixed in a 20 μl ligation reaction solution according to the method described above, and 350 units of T4 DNA ligase was added. The ligation reaction was carried out at 16°C for 3 hours. Calcium chloride method (Molecula
r Cloning]
ChloIecuIarC1 competent cells of the K-12JM83 strain (distributed by the Kyushu University Genetic Information Laboratory) were transformed using the above ligation reaction solution according to a conventional method.
oning]Q From the obtained ampicillin-resistant clone, prepare a plasmid using a known method, treat it with restriction enzymes (EcoRI and HindIII) according to the method described above, and analyze its migration pattern by agarose gel electrophoresis. By doing so, we investigated the insertion of the human TNF gene into the ptJc9 vector. As a result, it was confirmed that a gene of approximately 250 bp had been inserted, and the nucleotide sequence of the inserted gene for that clone was determined using the dideoxy method CF, Sanger, 5c.
214.1205 (1981)), approximately 130 b downstream from the EcoRI site.
It was confirmed that this gene fragment had the target human I-T NF gene nucleotide sequence of about 90 bp upstream from the p and old ndI site. This clone and plasmid were named pUA41/JM83 and pUA41, respectively.

(2) Subsequently, an attempt was made to construct a human TNF gene according to FIG.

Two groups (
U and L-3 to 6 and U and L-6 to 9), and each oligonucleotide was phosphorylated and annealed in the same manner as in step 11 above, and then ligated using T4 DNA ligase. The method described above. The two DNA fragments (201 bp and 200 bp) obtained were separated and purified by polyacrylamide gel electrophoresis (gel concentration 6%) according to 100 μl of 67 mM Tris-HC.
A' (pH 8,8), 6.7mMMgCj? 2.16
．． 6mM (NH4)2SO4, 6,7μM EDTA
, 1mM DTT, 200μg/ml BSA and 330μM each deoxyribonucleotide triphosphate (dA
TP, dGTPSdCTP, and TTP) were dissolved in an aqueous solution, 2 to 5 units of T4 DNA polymerase (Takara Shuzo Co., Ltd.) were added, and both ends of the DNA fragment were blunted by reacting at 37°C for 30 minutes. After the reaction, 68
T4 DNA polymerase was inactivated by treatment at °C for 10 minutes, and two DNA fragments of interest were recovered by ethanol precipitation.

Meanwhile, 5 μg of pUC9 vector was added to 50 μl of medium sultbuff containing 1 mM DTT and 100 μg/ml BSA (10 nt (Tris-HCI,
pH 7, 5, 50ml (Na(J' and 10mM Mg
Cl! 2) and 15 units of restriction enzyme Hin.
After adding cI (Takara Shuzo) and reacting at 37°C for 2 hours,
It was recovered by ethanol precipitation. The resulting cleaved and ring-opened pUc9 vector was inserted with the two D
Each NA fragment was separately treated with T4 according to the method described above.
Integrate using DNA ligase into E, coli K-
12JM83 strain was transformed. For each clone obtained, the nucleotide sequence of the inserted DNA was examined in the same manner as in step (1) above, and it was confirmed that it was the desired nucleotide sequence. These clones were each pUA42
/JM83 and pUA43/JM83, and the plasmids were named ρυA42 and pUA43.

The restriction enzyme Ec
oRI (High Salt Buffer) and 5acI (Takara Shuzo Jiro Salt Buffer), restriction enzyme Hae M (
pUA42 obtained in step (2) above was digested with restriction enzyme 5acI (low salt buffer) and Hpa
I (Takara Shuzo: KC/Buffer) and pUA43
was digested with restriction enzymes Hpa I and Hae I [(KCf buffer), respectively, according to the method described above. Lowe
Salt Buff-r-(10mM Tris-HCI
pH 7,5 and 1゜mM MgCj'2) and high salt buffer or KCI buffer-r- (20mM T
ris-HCf pH 8,5, 100mM KCI and 10mM MgCl! Combination 2) was handled by dividing the cleavage reaction into two steps and performing an ethanol precipitation operation between the reactions. EcoR from pLIA41
l-3acl DNA fragment (127 bp) and HaeI [
-)1indlII DNA fragment (80bp), pLIA
Sac I-Hpa I DNA fragment from 42 (
147 bp) and Hpa I − from pLIA43
Each HaeII DNA fragment (126 bp) was separated and purified by polyacrylamide gel electrophoresis (gel concentration 6%) according to the method described above.

On the other hand, 5 μg of pUc19 plasmid vector (distributed from Kyushu University Genetic Information Laboratory) was digested with restriction enzymes EcoRI and Hindl [about 2,7
A kbp DNA fragment was separated and purified by agarose gel electrophoresis (gel concentration 1%). The four Ds refined earlier
The NA fragments were sequentially added as shown in the diagram (Figure 2) and ligated using T4 DNA ligase according to the above method, and finally the purified pUc19 vector (approximately 2.7
Kbp fragment) was transformed into JM83 strain.

Regarding the plasmid DNA contained in this transformant,
When the nucleotide sequence of the inserted DNA was examined according to the above method, it was found that a plasmid vector (approximately 3.2 K
It was confirmed that the clone had the following bp). This clone was named PUA44/JM83 and the plasmid was named pUA44.

Example 4 (Construction of human TNF expression vector) (1)
Expression vector pKK with E. coli tac promoter
In order to make 223-3 (obtained from Pharmacia) easier to handle, the following improvements were attempted.

i) Reduce the molecular weight of the expression vector.

ii) The restriction enzyme BamHI cleavage site is unique.

1i) The direction of the tac promoter is reversed to that of the ampicillin resistance gene.

The method is illustrated in FIG.

5 μg of plasmid vector pBR322 (distributed from Kyushu University Genetic Information Laboratory) was cut with restriction enzymes EcoRI and old ndI according to the method of Example 3, and both ends thereof were blunted using T4 DNA polymerase. did.

According to the method of Example 3, a DNA fragment of approximately 4.4 Kbp was separated and purified by agarose gel electrophoresis (gel concentration 1%), and a 1100n BglII linker (restriction enzyme BglII cleavage site was inserted into the open ring site). A 1Obp double-stranded DNA fragment containing (obtained from Takara Shuzo) was inserted and ligated using T4 DNA ligase. Among the transformants obtained according to the method of Example 3, the plasmids were examined to see if they could be cleaved with restriction enzymes. Plasmid pB with new site
A clone with R9333 (approximately 4.4 Kbp) was obtained.

5 μg of the plasmid pBR9333 obtained above was dissolved in high salt buffer according to the method of Example 3, and cut with restriction enzymes BglII (Takara Shuzo) and PvuII (Takara Shuzo) to obtain approximately 2.3 Kbp containing the origin of replication. DNA of
The fragments were separated and purified by agarose gel electrophoresis (gel concentration 1%). On the other hand, expression vector pKK223-3C
5 μg of the protein (approximately 4.6 kbp) was dissolved in high salt buffer in the same manner as above and digested with restriction enzymes BamHI (Takara Shuzo) and 5caI (Takara Shuzo). Restriction enzyme BamHI
In the cleavage reaction, the amount of enzyme added is about 1/2 of the usual amount,
Partial cutting was performed with a reaction time of 5 to 30 minutes.

After cutting, tac promoter and rrnBT+T2
Approximately 1. including terminator etc. The IKbp DNA fragment was separated and purified by agarose gel electrophoresis in the same manner as above. Approximately 2.3 kbp DNA containing the previously purified origin of replication
Approximately 1. E. coli K-12JM103 strain (Kyushu University Genetic Information Laboratory) was prepared by inserting and ligating the I Kbp DNA fragment using T4 DNA ligase as described above and using the calcium chloride method as in Example 3. were introduced into competent cells. Among the obtained transformants, a clone having an expression vector (approximately 3.4 Kbp) containing the desired tac promoter etc. was selected, and this expression vector was named pKKlol.

(2) The next step will be explained according to FIG.

5μ of the expression vector pKK101 obtained in step (1) above
g was treated with restriction enzymes EcoRI and old n according to the above method.
d) and cut into approximately 3 pieces containing the origin of replication, transcriptional regulatory region, etc.
．． A 4 kbp DNA fragment was subjected to agarose gel electrophoresis (
Separation and purification were performed using a gel concentration of 1%). In addition, the plasmid pUA containing the human TNF gene obtained in Example 3
44 (approximately 3.2 kbp) was similarly digested with the restriction enzymes EcoRI and HindI, and a DNA fragment of approximately 480 bp containing the entire human TNF gene was separated and purified by agarose gel electrophoresis. This DNA fragment containing the entire human TNF gene was first inserted into the expression vector pKK1.
The DNA fragment of about 3.4 kbp purified from E. coli K-12JM1 was inserted and ligated using T4 DNA ligase according to the above method, and the DNA fragment of about 3.4 kbp purified from 01 was inserted into E. coli K-12JM1 according to the above method.
It was introduced into 03 strains. A clone having the desired human TNF expression vector (approximately 3.9 Kbp) was selected from among the resulting transformations, and this expression vector was named pKF4102.

Example 5 (human) Construction of TNF transformant expression vector) (1
) This will be explained according to FIG.

The plasmid pUA44 obtained in Example 3 was cut with restriction enzymes EcoRI and HindllI according to the method described above, and the D
The NA fragment was separated and purified by agarose gel electrophoresis. On the other hand, Messing et al.
nzymology.

153, 3 (1987)), a plasmid vector ptJc for the preparation of single-stranded plasmid DNA.
Restriction enzyme Eco was added using lI9 (obtained from Takara Shuzo).
About 3. cut with RI and old ndIII and containing the IG region.
A 2 Kbp DNA fragment was separated and purified by agarose gel electrophoresis. Due to the presence of this IG region (intergenic region of M13 phage DNA), plasmid pUc119 is a helper phage M13.
After KO7 infection, the DNA is preferentially turned into single-stranded DNA, wrapped in phage particles, and released outside the bacterial cell. The approximately 480 bp DNA fragment containing the entire human TNF gene purified above and I
The approximately 3.2 Kbp pUc119 fragment containing the G region was ligated using T4 DNA ligase as described above, and then transformed into E. coli K-12JM8 as described in Example 3 above.
It was introduced into 3 stocks.

From among the obtained transformants, the desired plasmid (approximately 3
．． 7 Kbp) was selected, this clone was named pUcl 19-hTNF/JM83, and the plasmid was named I) UC119-hTNF.

In order to incorporate and retain uracil into the DNA of the plasmid pUcl 19-hTNF obtained above, Escherichia coli strain CJ236 (dut-, ung-) was prepared by the calcium chloride method according to the method of Example 3. were introduced into competent cells. The CJ236 strain has a deletion mutation (dut-
), a competitive reaction occurs, making it possible to create DNA that partially incorporates uracil instead of thymine, and furthermore, ung
- It is deficient in uracil N-glucosylase due to mutation, and retains uracil in its DNA. This CJ236 strain was obtained from Bio-Rad.

The clone obtained by the above introduction (pUcl 19-h
TNF/CJ236) with helper phage 113KO7
(obtained from Takara Shuzo), 2XYT broth (1,6%
Tryptone, 1% yeast extract and 0.5% NaCf5p
Single-stranded plasmid DNA (approximately 3.7 Kbas) into which the desired uracil was introduced was cultured in
es) was encapsulated in phage particles and released outside the bacterial cells. The released phage particles are collected from the culture supernatant,
- Target single-chain plasmid DNA was prepared according to the method for preparing heavy-chain phage DNA.

(2) Next, explanation will be given according to FIG.

In order to introduce mutations into the human TNF gene using positive-strand oligonucleotides, brimers 4123, 4124, 4125, 4150. He designed 4222, 4223, 4267 and 4268. Chemical synthesis and purification of this oligonucleotide were performed according to the method of Example 2 above.

Site-specific mutation introduction into the human TNF gene is a biotechnology
Rad's system (Muta-Gene" in vit
r.

mutagenesis kit). That is, about 0.5 μg of brimer prepared above
A portion of the 'terminus phosphorylated with T4 polynucleotide kinase according to the above method and the previously prepared uracil-introduced heavy chain plasmid DNA (pLIc 119-hT
NF) and 10 μl of annealing buffer y-(20 mM Tris-14C1'
pH 7, 4, 2mM lilgcl 2 and 50m
Annealing (heating to about 70°C and then slow cooling) was performed in M NaCf ). After annealing, add 1Ox synthesis buffer (5mM each deoxyribonucleotide triphosphate (dATPSdGTP, ticTP and T
TP), 10mM ATP, 100mM Tris
- Add 1/10 volume of HCl2 pH 7,4, 50mM MgCf2 and 20mM DTT), l unit of T4 DNA polymerase and 2-4 units of T4
Double-stranded reaction using DNA ligase (37°C19
0 minutes). TE buffer (10mM Tri
The reaction was stopped by adding about 8 volumes of s-H (J' pH 7.5 and 1 mM EDTA) and freezing.

According to the method of Example 3, E. coli K-12TGI strain (ung"
The above reaction solution was treated with the double-stranded DNA into competent cells (Amersham). By introducing hetero double-stranded DNA into the ``Ung'' strain, the DNA strand containing uracil, which is a template, is inactivated and becomes no longer a target for replication.

Therefore, the mutation introduction frequency becomes highly efficient, exceeding 50%. Among the obtained transformants, a clone having a plasmid (approximately 3.7 Kbp) containing the desired transformant DNA was selected using the colony hybridization method using the primer used for mutation introduction as a probe. did. For the selected clone, the nucleotide sequence around the mutation introduction site of the plasmid was analyzed by dideoxy method CF.
, Sanger, supra], and it was confirmed that the transformant DNA had mutated as designed.

These plasmids were designated as pUc119-F412, respectively.
3, pUcl 19-F4124, pUcl 19-F
4125, pUc 119-F4150, p(JC11
9-F4222, pLlclI9-F4223, pU
Cl 19-P4267 and pUcl 19-F426
It was named 8.

The desired human TNF converter gene obtained by mutagenesis was transformed into an expression vector pKK having a tac promoter according to the method for constructing a human TNF expression vector in Example 4.
101 to construct a TNF transformant expression vector. The human TNF converter gene (approximately 480 bp) is contained in the approximately 3.7 Kbp plasmid pUc119 obtained above.
-F4123, pUc119-F4124, pUc1
19-P4125, PUC119-F4150, pUc
l19-F4222, pUc119-F4223, p
Uc119-F4267 and pUcl 19-F426
8, the restriction enzyme Eco
It was separated and purified after cleavage with RI and old ndl. Each desired human TNF transformant expression vector (pKF
4123, pKF4124, pKF4125, pKF4
150, pKF4222, pKF4223, pKF4
267 and pKF4268) as a host, similar to the human TNF expression vector (pKF4102).

It was obtained using E. coli K-12JM103 strain.

The above eight types of transformant expression vectors can be
A novel bioactive polypeptide with the transformations shown below is produced in E. coli.

Vector pKF4123: Encodes the 106th czy or Asp polypeptide F4123 shown in SEQ ID NO: 1 of the sequence listing.

Vector pKF4124: Encodes polypeptide F4124 in which the 106th Gly shown in SEQ ID NO: 1 in the sequence listing is converted to Lys.

Vector pKF4125: Encodes polypeptide F4125 in which the 106th Gly shown in SEQ ID NO: 1 of the sequence listing is converted to Glu.

Vector pKF4150: Encodes the 106th Gly or Trp polypeptide F4150 shown in SEQ ID NO: 1 of the sequence listing.

Vector pKF4222・1 shown in Sequence Listing SEQ ID NO: 1
It encodes polypeptide F4222 in which Gly at position 06 is converted to Pro.

Vector pKF4223: Encodes polypeptide F4223 in which the 106th Gly shown in SEQ ID NO: 1 in the sequence listing is converted to Arg.

Vector pKF4267: Encodes polypeptide F4267 in which the 106th Gly shown in SEQ ID NO: 1 in the sequence listing is converted to Aj7a.

Vector pKF4268: Encodes polypeptide F4268 in which the 106th aiy shown in SEQ ID NO: 1 of the sequence listing is deleted.

Example 6 (Expression and purification of human TNF and human TNF converter using E. coli) The human TNF expression vector obtained in Example 4 (pKF4
102) and human TNF transformant expression vectors (pKF4123, pKF4124, pKP4 obtained in Example 5)
125, pKF4150, pKF4222, pKF42
23, pKF4267 and pKF4268), E. coli K-12JM103 strain, 25-5
0 μg/m (! M9 medium containing ampicillin and O, OQ 1% vitamin Bl (0,6% Na2HPO4,
0.3% KH2PO, 0.05% Nacl, 0.
1% NH4Cz, 2mM (gsO+, 0.2% glucose and 0.1mh4 CaCA2) 20ml
and cultured at 37°C for 18 hours. Add 20 ml of this culture solution to 1 liter of the above medium,
Shaking culture was performed at 7°C for 12 to 3 hours. Then, isopropyl-β-D-thiogalactopyranoside (IPTG
) was added to a final concentration of 1mM and further incubated at 37°C.
The shaking culture was continued for 118 hours.

After collecting E. coli cells by centrifugation, TP buffer
The bacterial cells were washed using r-(10 mM Tris-H (J7 pH 8,0 and 100 μM PMSF). After washing, the bacterial cells were suspended in TP buffer at a volume of 10 mf per gram of bacterial cells. Ultrasonic generator (heat/
The microbial cells were subjected to ultrasonic disruption using a model W-225 (Model Systems: Model W-225). The resulting suspension was centrifuged to remove bacterial cell residue and both upper and lower grooves were collected. The purification process after this ultrasonic crushing treatment is mainly carried out at low temperatures (0°C to 4°C).
°C).

After filtering this supernatant with a 0.45 μm filter, anion exchange chromatography (column size: φ2.
5 x 1.5 cTn and flow rate: 0.5m! ! / minute)
An active fraction was obtained by assaying for the presence or absence of activity according to the method described in Example 7 using 5DS-polyacrylamide gel electrophoresis and L929 cells described below. For elution, a TP buffer containing NaCl was used, and the NaCl concentration was varied from 0.05M to O, 1M, 0.2M, 0.5M, I
0.1 for human TNF and human TNF converters F4123, F4124, and F4125.
M NafJ', human TNF converter F4150 is I
MNaCf, human TNF converter F4222, F4
267 and F 4268 are 0.2 M NaCf,
Human TNF converter F 4223 in 0.05 M NaC
Each eluted with A'. Furthermore, in order to remove endotoxin etc. derived from E. coli cells, treatment with a nucleic acid/endotoxin removal agent C-9 (manufactured by Kurita Kogyo, sold by Dainippon Pharmaceutical) was performed according to the attached manual. Thus, one-step purified samples of human TNF and human TNF converters were obtained.

Using this sample, the antitumor activity of Examples 7 and 8 described later was evaluated.

After filtering the above sample with a 0.20 μm filter, F
Things Q■ (HR10/1) under control by PLC system
0 and HR515) prepacked column (Pharmacia L
TPQ buffer containing NaCl was analyzed by anion exchange chromatography using KB Biotechnology Co., Ltd.
-(20mM Tris-)1c1pH8,0 and 10
MM PMSF), and the fractions were eluted with 5DS-
An active fraction was obtained by assaying for the presence or absence of activity according to the method described in Example 7 below using polyacrylamide gel electrophoresis and L929 cells. The elution method was performed according to the following program under the control of the FPLC system. The final step in each sample is 5 to increase purification purity.
DS-polyacrylamide gel electrophoresis was repeated several times until a single band was obtained.

1st step: Using MonoQ (I (R515) column (
Flow rate: l-7 min) 0-2 min; OMNa(J) 2-15 min; O-0,15M NaCl! Linear concentration Linear concentration - Placement 5-20 minutes 15 - 0.5 M NaCl
Linear concentration gradient 20-30 min; 0.5 M Na(J'
The fractionation results (NaCI! concentration and retention time) of the active fraction according to the above method showed that human TNF converter F4123 and 0.0.
2 M, 18.2 min, and the same F4124 was the non-adsorbed fraction.

2nd step: Use MonoQ■ (HR10/10) column (flow rate: 4ml 11min) 0-5min, O-0,2M NaCj7 linear concentration gradient 5
~16 minutes; 0.2 M Na(J) 16-20 minutes;
0.2-0.5 M NaC/linear concentration gradient 20-24
minutes; 0.5 M NaCj7 The fractionation results (NaCA' concentration and retention time) of the active fraction according to the above method are human TNF, 0.2 M, 5.3 minutes, human TNF converter F 4
222 is 0.2M, 4.4 minutes, F 4223 is 0.1
M, 1.8 minutes, same F 4267 or 0.2M.

4.5 minutes and F 4268 or 0.2 M, 4.5 minutes.

3rd step: Using MonoQ (HR515) column (flow rate: 1-7 min) 0-2.5 min; 0-0.2 M NaCl linear concentration gradient 2.5-8 min; 0.2 M Na( J'8~lO min; 0.2-0.5 M NaC17 linear concentration gradient lO
~12 minutes; 0.5 M NaCl The active fraction fractionation results (NaCl concentration and retention time) according to the above method are as follows: human TNF is 0.2 M, 3.7 minutes; human TNF converter F4
123 is 0.2M, 6.6 minutes, the same F4124 is the non-adsorbed fraction, the same F4125 is 0.2M, 7.8 minutes, the same F4150
is 0.2M, 5.0 minutes, same F 4222 is 0.2M, 3
．． 5 minutes, the same F4223 is the non-adsorbed fraction, the same F4267
is 0.2M, 3.4 minutes and F 4268 is 0.2M,
It was 3.3 minutes.

4th step: Using MonoQ @ (HR515) column (flow rate: 1 ml 11 minutes) 0-6 minutes, 0-0.15M NaC/linear concentration gradient 6
~11 min; 0.15-0.2 M NaCl linear gradient 1-13 min; 0.2-0.5 M NaCl linear gradient 3-15 min; 0.5 M NaCf Activity according to the above method The fractionation results (NaCf concentration and retention time) are as follows: human TNF is 0.16M, 6.5 minutes, human T
NF converter F 4222 or 0.16M, 5.7 minutes, F 4267 or 0.15M, 5.5 minutes and F 42
It was 68 or 0.16M, 5.9 minutes.

Purified samples of human TNF and human TNF converters were thus obtained. This sample was used to evaluate the antitumor activity in Example 7 described below.

5DS-polyacrylamide gel electrophoresis was performed on the purification process and the purified sample to confirm the expression and purification of human TNF and converter polypeptide. Each sample was washed with Laemml i' containing 10 mM DTT.
s sample buffer y - (62,5mM Tris-
HCI! pH 6,8, 2% SO3, 0,01% BPB and 10% glycerol), as well as Laemmli (
Electrophoresis was performed using a 15% separation gel according to the method of Nature, 227°680 (1970)).

After the electrophoresis was completed, the proteins in the separation gel were confirmed by staining with Coomassie brilliant blue. Figure 7 shows some of the results. The expression levels of human TNF and converter polypeptide by each expression vector were approximately the same, and the expression efficiency was calculated by applying the obtained stained gel to a chromato scanner (Shimadzu, model C5-920). It was about 20% of the total bacterial protein. Moreover, the final purified sample showed a single band in the resulting stained gel. The molecular weights of human TNF and converter polypeptide were calculated from the electrophoresis positions and are shown in Table 1.

As one of the physical properties of proteins, the isoelectric points of human TNF and converter polypeptide were measured by isoelectric focusing gel electrophoresis using Ampholine (manufactured by LKB), and the obtained results are shown in Table 1. It was shown to.

Table 1 Isoelectric points and molecular weights of human TNF and converter polypeptides Human TNF 5.6 17.
0F4123 (10'Gj7y →Asp) 5.3
17.0F4124(”'Gfy-Lys)
6.4 17.0F4125(10'Gβy-
”GA u) 5.3 17.0F4150(
”'Gj7y-Trp) 5.5 17.0F
4222 (10'Gfy-Pro) 5.6
17.0F4223 (10@Gi-Arg) 5.6
,6.3"17.0F4267("'Gfy-A
la) 5.6 17.0 Example 7 (in
Evaluation of in vitro antitumor activity) The cytotoxic activity against mouse-derived connective tissue cell L 929 (ATCCCCLI) was evaluated using the Aggarwal assay for the one-step purified sample and purified sample of human TNF and human TNF converter polypeptide obtained in Example 6. et al (J.

Biol, Chem, 260.2345 (19
85)). That is, a 96-well tissue culture microplate (manufactured by Corning)
L929 at 3X10' cells/0.1 ml/well
Cells were planted and cultured overnight at 37°C in the presence of 5% carbon dioxide. As a medium, Dul containing 10% fetal bovine serum was used.
Eagle's minimum essential medium (DME medium, manufactured by Sigma) modified by Becco was used.

The next day, the medium was replaced with the above medium supplemented with actinomycin D at a final concentration of 1 μg/d, and samples serially diluted in this medium were treated in each well (total medium volume 0.1 mj
'). After further culturing for 20 hours, 0.5% crystal violet solution (0.5% crystal violet/
The living cells attached to the plate were stained with 20% methanol (room temperature, 15 minutes).

Phosphate buffer-PBS (10mM Na-K
Phosphate, 0.8% NaCI! and 0
．． After thoroughly washing with 0.02% KCj7), the crystal violet remaining on the plate was extracted using 0.17711 0.0IN hydrochloric acid solution containing 30% ethanol, and its absorbance (492 nm) was measured using an ErA reader (Bio-Rad). (Model 2550). This absorbance is proportional to the number of viable cells. Therefore, the final dilution rate of the sample in the well showing the absorbance equivalent to 50% of the absorbance of the sample-untreated well is determined, and the reciprocal of this sample dilution rate is calculated as the number of units per 1 m, f of the sample (units/ml).
).

From the L929 cytotoxic activity (units/me) determined according to the above method, the specific activity of each sample (units/■・
In order to calculate protein), protein quantification of each sample was performed. Quantification was performed using the Bradford method (Anal, B
iochem.

72, 248 (1976)), protein concentration (■/ml) was determined using bovine serum albumin as a standard sample. From these results, human TNF and human TNF
The specific activities calculated for the converter samples are shown in Table 2.

Table 2 Human TNF and converter polypeptide L929
Cytotoxic activity one-step purified sample Purified sample human TNF 2.7X1078.0XIO
7F4123 2.0X1073.6X107
F4124 2.2XI074.6X107F
4125 1.8XIO' 3.2X1
0'F4150 4.0X10" 4.
0XI07F4222 2.7X10@2.7
XIO7F4223 7.0XIO'
3.9X107F4267 1.2X1073
．． 5X107F4268 1.0x1072.
5x107 From Table 2 above, it can be seen that the human TNF converter polypeptide has the same cytotoxic activity on L929 cells as human TNF.

Example 8 (Evaluation of in vivo antitumor activity) Regarding the one-step purified sample of human TNF and human TNF converter obtained in Example 6, mouse transplantable fibrosarcoma MethA
Antitumor therapeutic activity against tumors was determined. The test consisted of 10.2 ml of Meth
A tumor cells (distributed from Sasaki Institute) were subcutaneously transplanted into the back of BALB/c mice (male, 5 weeks old, Charles River), and 8 days later, it was confirmed that the tumor diameter had reached 6 to 10 mm. , Samples serially diluted with physiological saline (0,2rnj
7/mouse) was administered through the tail vein. The lethal dose was taken as the highest dose, and each dose sample was prepared by dilution in several stages.

Approximately 2 weeks after administration, observation of tumor growth, etc. was continued.

Regarding tumor growth, the tumor volume (major axis x minor axis of tumor mass 2)
/2), calculate the tumor volume ratio to the tumor volume on the day of sample administration (day 0), and calculate the number of days from the day of sample administration (D2 or D5) at which this value becomes 2 or 5. Then, the ratio to the control group (physiological saline administration) was calculated, and the D2% control value and the D5% control value were determined. The larger the value, the lower the tumor growth ability and the higher the antitumor activity.

The results obtained according to the above are listed in Table 3.

As a result, human TNF converter F 4222, human TNF converter F 42
Compared to human TNF, 23 and F4268 showed a higher tumor volume rate at the dose when the mortality rate was 0%, indicating that they have a high antitumor therapeutic effect on Meth A tumors transplanted in mice.

lbl 155 Sequence number: 2 Sequence length: 465 Sequence type: Number of nucleic acid strands Knee heavy chain topology: Linear sequence type: Other nucleic acid synthesis NA Haen GA stone ■ Hit country A snore α weak button A GGTCTACTTrT section πA CC mountain number -3 Length of distribution = 474 Type of chain ・Number of nucleic acid strands Double-stranded topology Type of linear sequence: Other nucleic acid synthesis NA sequence [Note 1] Number: number from the start codon ('ATG) Note 2] Ma or Mu, cutting position for oligonucleotide production Note 3] Restriction enzyme, restriction enzyme cleavage site provided for human TNF gene construction [Note 4] Lowercase letters: ramp truncated base sequence prepared for linking the human 1-TNF gene to a plasmid vector Sequence number = 4 Sequence length: 27 (U-1), 50 (U-2), 49
(U-3), 50 (U-4), 50 (U-5), 52 (
U-6), 48 (U-7), 49 (U-8), 51 (
U-9), 53 (U-1's, 29 (L-1), 52 (L
-2), 50 (L-3), 50 (L-4), 49 (L
-, 5), 49 (L-6), 51 (L-7), 49 (L
-8), 51 (L-9), 49 (L-10) Sequence type: Number of nucleic acid strands Knee-hon silk topology: Linear sequence type: Other nucleic acid synthetic NA Sequence number = 5 Sequence length S = 21 (Blymer 4123) 21 (Blymer 4124) 21 (Blymer 4125) 21 (Blymer 4150) 21 (Blymer 4222) 21 (Blymer 4223) 21 (Blymer 4267) 21 (Blymer 4268) Sequence type: Number of nucleic acid strands Knee-hon silk topology: Type of linear sequence: Other nucleic acid synthetic NA Note: *Mutation-introduced base] (Effect of the invention) According to the present invention, 1 in the amino acid sequence of human TNF.
By converting or deleting the 06th amino acid to another amino acid, a novel human TNF converter with higher antitumor activity and lower side effects than human TNF is provided.

[Brief explanation of the drawing]

Figures 1 and 2 show the method for constructing the human TNF gene by ligation of synthetic oligonucleotides, Figures 3 and 4 show the method for constructing the human TNF expression vector, and Figures 5 and 6 show the method for constructing the human TNF gene by ligation of synthetic oligonucleotides. FIG. 7 is a diagram for explaining the method for producing a gene for a human TNF converter by the mutation method, and FIG.
It is a figure which shows the electrophoresis result of the purified sample of F converter. Figure 1 11-1υ-2υ-3υ-4υuυ-6u-7υ-EI
Ll-9Ll-10shi-I L-21-31-4
L-5L-6L-Ryo L-8L-9L-10
↓ p[UA41 to be incorporated into JC9 [Note] O-5° terminal phosphorylation (-) ・-〃(+) Obtained clone pLj Incorporate into c9 pL
)Incorporate into C9 Obtain or O-on pUA41
p4JA42 pUA43
pUA41 Fig. 4 pKK+oI pUA44 EcoRI HindlllpKF4102 Fig. 5 Fig. 7 (A) 1. Protein molecular weight marker 2, ultrasonication total sample 3, ultrasonication supernatant 4, human TNF/- stage purification assay 4 5. Human TNF/purification trial - fl - Continuation of amendment (voluntary) June 14, 1991 Wataru Fukasawa, Commissioner of the Japan Patent Office1, Indication of case 1990 Patent Application No. 3111292, Name of invention Polypeptide name ( 035) In Column 5 of the Detailed Description of the Invention in the Ishihara Sangyo Co., Ltd. Specification, r5.3J on page 44, line 14 of the Statement of Contents of Amendment is changed to r6. 6J
, r4.4J on the 15th line to r5.5J, rl on the 16th line, 8J to r2. 3J and two r4.5J on the 17th line are each corrected to r5.6J. that's all

Claims (1)

[Claims] 1. Ser 1 to 15 shown in sequence number 1 in the sequence listing
A polypeptide characterized in that, in the amino acid sequence up to the 5th Leu, Gly at the 106th position is substituted with another amino acid or deleted. 2. Gly at position 106 is deleted or other amino acids substituted at position 106 are aspartic acid, lysine,
2. The polypeptide of claim 1, which is glutamic acid, tryptophan, proline, arginine or alanine. 3. 15 from the 1st Ser shown in sequence number 1 in the sequence table
1. A recombinant plasmid comprising a DNA encoding a polypeptide having an amino acid sequence up to the 5th Leu in which Gly at the 106th position is replaced or deleted with another amino acid. 4. The recombinant plasmid is pKF4123, pKF4
124, pKF4125, pKF4150, pKF42
22, pKF4223, pKF4267 or pKF42
68. The recombinant plasmid of claim 3, which is 5. 15 from the 1st Ser shown in sequence number 1 in the sequence table
A recombinant microbial cell transformed with a recombinant plasmid containing a DNA encoding a polypeptide having an amino acid sequence up to the 5th Leu in which Gly at the 106th position is replaced or deleted with another amino acid. 6. The recombinant microbial cell according to claim 5, wherein the recombinant microbial cell is Escherichia coli. 7. Ser 1 to 15 shown in sequence number 1 in the sequence table
A recombinant microbial cell transformed with a recombinant plasmid containing a DNA encoding a polypeptide in which Gly at position 106 has been replaced or deleted with another amino acid in the amino acid sequence up to the 5th Leu. A method for producing a polypeptide, which comprises culturing in a medium to produce and isolate a polypeptide having the amino acid sequence. 8. 15 from the 1st Ser shown in sequence number 1 in the sequence table
1. A pharmaceutical composition comprising, as an active ingredient, a polypeptide having an amino acid sequence up to the 5th Leu in which Gly at the 106th position is substituted or deleted with another amino acid. 9. 15 from the 1st Ser shown in sequence number 1 in the sequence table
A polypeptide characterized in that in the amino acid sequence up to the 5th Leu, Gly at the 106th position is substituted or deleted with another amino acid, and has a Met at its N-terminus. 10, 465 from the 1st T shown in sequence number 2 in the sequence listing
316 to 3 in the base sequence represented by G.
A DNA characterized by having a sequence in which GGC encoding Gly at position 18 is replaced or deleted by a codon encoding another amino acid.