JPS638399A - Physiologically active polypeptide - Google Patents

Abstract

NEW MATERIAL:The physiologically active polypeptide having amino acid sequence of formula. USE:An antitumor agent having lymphotoxin(LT) activity. PREPARATION:For example, an LT-producing cell is produced by the cell fusion of a normal human T-lymphocyte and a tumor cell. Whole RNA is extracted from the obtained cell e.g. by guanidine hydrochloride process and mRNA is separated e.g. by gelfiltration process. A complimentary cDNA is synthesized with a reverse transcriptase using the mRNA as template and is integrated into a proper plasmid to obtain a recombinant plasmid, which is inserted into E.coli, etc., to effect transformation of the cell and produce a cDNA library. The library is cloned with a synthetic probe and the cloned DNA is cut from the clone with a restriction enzyme and linked to a manifestation vector. The product is inserted into E.coli and cultured. The compound of formula can be separated from the cultured liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to physiologically active polypeptides, and more particularly to novel polypeptides having lymphotoxin activity.

[Conventional technology]

Lymphotoxin (hereinafter abbreviated as LT) is known as a type of lymphokine that is released either specifically or non-specifically from lymphocytes and lymphoid cell lines and has cytotoxic activity. LT is not only harmful to many cancer cells (such as certain anticancer drugs or interferon molecules)
C! 1 [Since it enhances the apricot effect, it is expected to be applied in medicine as an antitumor agent. [Gran
Ger G, A, et al., 14th International Chemical Law Society Conference,
Kyoto, June 23-28, 1985, Abstracts,
International Congress
ss of Chemotherapy.

Ky:+to, Japan, June 23-28
, Abstractspl 5); ff Tsunaga (M
Atsunaga K, ) et al., Abstracts of the above,
[Page 352] Regarding LT production by human-derived cells, tonsil cells or peripheral blood lymphocytes were treated with phytohemagglutinin (hereinafter referred to as PH).
Method of culturing with A) and obtaining it from the culture supernatant [Peter J, B, et al., Journal of Immunology (J.

Engineering nnmuno1.1) 1 770 (1973):
Walker et al. (Walfer S, M, and Lu
cas Z, J, ), Journal of Immunology (J, ■mmunol, 1091233 (19
72)), a method in which lymphoid cell lines are cultured in the presence of PMA and obtained from the culture supernatant [Yamamoto et al.
amoto R,S, ) et al; Journal of Biological Response Modifiers (J,
Biol, Re5ponse Modifiers)
3 76 (1984)), human T cell hybridomas were treated with phorbol myristate acetate (hereinafter abbreviated as PMA).
and/or a method of culturing in the presence of concanavalin A (hereinafter abbreviated as Con A) [Asada et al.: Cellular Immunol-, 77150<198
3))) etc. are known.

[Problem that the invention seeks to solve]

However, these methods require a small amount of raw noodles and require an expensive nutrient source (for example, fetal bovine serum) in the medium, making it extremely difficult to economically obtain LT with high purity.

Another method for obtaining large amounts of LTi is to use so-called genetic engineering techniques, in which the entire gene corresponding to LT is incorporated into a vector, replicated, transcribed, and translated in bacteria, fungi, yeast, or animal cells, and then transmitted by these cells. It has been considered that the gene corresponding to LT can be produced, and the acquisition of the gene corresponding to LT has been long awaited.

The present inventors previously obtained the human T cell hybridoma clone A-C5-8 strain that produces LT by cell fusion using the emetine-actinomycin D method [Asada et al.; Cellular Immunol.
77 150 (1983) J).

However, A-C5-8 strain'i<Con A %P)a
Even if cultured under LT optimal injection conditions (culture time 30 hours or more) in the presence of
A (hereinafter referred to as mRNA and N)k could not be obtained, and therefore the gene corresponding to LT could not be obtained either.

Therefore, the present inventors independently investigated the conditions for obtaining mRNA corresponding to LT, and found that by culturing A-C5-8 strain with whole PMA and/or Con A within 24 hours (particularly within 4 hours), cells The present inventors have found that it is possible to obtain mRNA f corresponding to the LT polypeptide produced within the LT polypeptide, and by applying this mRNA and gene recombination technology, the present inventors have conducted extensive research and found that ( ) We succeeded in producing the entire LT polypeptide in a microorganism transformed with an expression vector that had been integrated into the human LTrl.
? The present invention has been completed by successfully obtaining the entire human LT polypeptide substantially free of impurities from the repeptide-containing product.

LT using genetic recombination technology? ! - Regarding the production method, report by Gray et al. (P, W, Gray et al.
; Nature 312 721 (1984))
There is.

Gray et al. PMed nonadherent human peripheral blood lymphocytes.
A, Staphylococcus mRNA was extracted from cells after 48 hours of culture with enterotoxin B and thymosin α.
After cDNA was obtained and cDNA was created, it was cut with a restriction enzyme, and after ligation with a synthetic oligonucleotide, LTi was expressed in E. coli. LT estimated from this report has a number of amino acid residues of 171 or 172 and a molecular weight of IB, 60[1].

In the present invention, the mRNA of the present invention has differences such as being obtained from human cell hybridomas and the base sequence of cDNA derived from the mRNA is different.
The number of amino acid residues of T polypeptide V is 152, the molecular weight is different, and the seventh amino acid in the amino acid sequence is different.

[Means for Solving the Problems] That is, the present invention provides a novel polypeptide having LT activity.

More specifically, the cDNA (Table 1) encoding the LT polypeptide obtained in Application No. 60-289249 was cut with restriction enzymes, and a gene expression vector with all four translation initiation sites was cut with the same restriction enzymes. Cut it with
A transformant is obtained by introducing the transformant into a suitable host such as E. coli, and the whole transformant is cultured. The present invention relates to a) LT polypeptide substantially free of all impurities obtained by extraction and purification.

The LT polypeptide has the amino acid sequence of the following formula (1).

Below is the blank formula (1) % formula % The codes in the above-mentioned polypeptides and the base sequences in Table 1 are the following abbreviations.

ALA: alanine, ARC): arginine, ASN: asparagine, ASP: asparaic acid, CYS nigistein, ()LN: glutamine, GLU: glutamic acid, GLY nigericine, HIS: histidine, ILE: isoleucine, LEU
: leucine, LYS: lysine, MET: methionine, PHE: phenylalanine, PRO nigeroline, SER: serine, THR: threonine, TRP:) liptophan, TYR: tyrosine, VAL: valine, A: adenine,
C: cytosine, G nigeanin, T
: Thymine; regarding the amino acid sequence of the polypeptide having human LT activity, refer to the human lymphoblastoid cell line RPM1)
LT of 788 Yumai molecular weight 20.000 and 25.000
A report by Aggarwal et al. [Journal of Biological Chemistry (J, Biol, Chem, 260)]
2 Roro 4 (1985, 1), which differs from the polypeptide of the present invention in the seventh amino acid residue in the above amino acid sequence.

Furthermore, when expressed in E. coli, the methionine residue present in the LT polypeptide is used as a translation initiation codon, so addition of an extra methionine residue can be prevented.

The misfire will be explained in detail below.

■Selection of LT high-producing cells.

LT-producing cells include normal human lymphocytes, CCRF-
CEM, MOLT-AF %JURKAT, etc., human T lymphocyte cell lineage and its cloning strain, RPM
I-1788, etc.) B IJ lymphocyte lineage cell lines can be used, but since the amount of LT production is low and the cells can be passaged, normal human lymphocytes and human T lymphoid proteolytic cell lines can be used. It is preferable to use an LT-producing human T cell hybridoma obtained by cell fusion with human cells.

LTi live human) T cell hybridomas are the parental cells human)
The TIJ member's tip cell line produces LT, and therefore, the amount of mRBTA extracted and separated from the cells is also suitably used.

The analytical method used to measure LT activity was that of Kobayashi (Kobashi).
ayashi Y.) et al. [Journal of
Immunology (J, ImmunoL) 122 7
91 (1979))'! This was done using r. That is, cell culture supernatant or cell extract sample of mouse L/P3 cells (
It was measured as a total indicator of damaging activity against L cell sub-strains). 1 unit/1 of LT was expressed as the concentration that caused total damage to 50% of the target cells.

LTi live human T cell hybridoma can be produced by a known method (Japanese Patent Application Laid-open No. 72520/1983).

In other words, T IJ lymphoid tumor cells are treated with a protein synthesis inhibitor or a combination thereof with an RNA synthesis inhibitor, stimulated with human T lymphocyte whole mitogen or antigen, and both are treated with a fusion promoter (polyethylene (glycol, etc.), and only the resulting fused cells (human T cells (Vibe IJ) F-ma) can be isolated and obtained. Human 'r, m cell hybridoma whole culture solution (for example, basal medium RPM Nee 1640 medium, 10% tallow serum, 5
Cultured at 37°C in an atmosphere of 25% Co-95% air in a culture medium (hereinafter referred to as RPMI medium) supplemented with 10-5M 2-nucleated ethanol and 2mM glutamine to produce LT as described above. h) Screen all T cell hybridomas.

■Cell culture To obtain mRNA from LTi live human T cell hybridomas, at least 10' cells are required.
They are commonly obtained by cell culture. i.e. 105-107 cells/+ in total cell nutrient medium
The o'K prepared product is cultured at 67°C in a Petri dish or tissue culture flask rotating culture solution (spinner flask) in an atmosphere of 25% CO-95% air.

The culture time varies depending on the medium, composition, and initial cell concentration, but 1 to 5 days is appropriate. Centrifuge the culture solution to obtain cells.

The nutrient medium is a basal medium containing one or more selected from saccharides, amino acids, vitamins, hormones, proteins, antibiotics, growth factors, and salts, or a basal medium to which animal serum is added. The appropriate medium is selected and used.

As a basal medium, commercially available RPMI-1640
Medium, MEM medium, Dulbecco's modified MEM medium, etc. can also be used.

Animal serum includes fetal bovine serum, newborn bovine serum, horse serum,
1 to 20 units can be added to the total basal medium such as human serum.

The cells can also be propagated and used in warm-blooded animals other than humans, such as nude mice and hamsters.

LT-producing human T cell hybridoma obtained by amplification of 0mRNA is adjusted to 10" to 10" in a nutrient medium and cultured by adding Tanshu and/or Con A k to LT. It is possible to obtain NoI'(fl cells) containing mRNA in the J1°.
The preferred range of ng (nanograms)/tni% Con A is 5-50 μm/me.

The culture time is suitably within 24 hours, especially within 8 hours.

The reason for this is that the content of mRNA corresponding to lymphotoxin activity in μ, medium-aaat+ cells decreases with the passage of time. Especially if it exceeds 24 hours, LT production
The optimal time for culturing T cell hybridomas and collecting LT from the culture supernatant is 24 to 72 hours, when intracellular LT
The amount of mRNA corresponding to this is extremely small, and it is difficult to recover all of it.

■ Extraction of total RNA from cells Extraction of total RNA from the cells obtained in step (■) was performed using the guanidine hydrochloride method [Deeley F, G, et al., Journal of Biological Chemistry (
J, Biol, Chem, ) 252 8310
(1977)) and other known methods.

-j That is, the cells (109 or more) obtained in ■ are homogenate buffered! 1. (8M guanidine hydrochloride, 51
) containing nM dithiothreitol and 20 mM sodium acetate, adjusted to p)17 with NaOH, and disrupted by homogenization. After extracting all the nucleic acids from the broken tissue by geltanol precipitation and phenol extraction, the total RNA is recovered by lithium chloride precipitation. In order to prevent degradation of RNA by RNase during the extraction operation, it is preferable to sterilize the equipment in an autoclave after treatment with dry heat or diethylpyrocarbonate, and to wear vinyl gloves during the operation.

■Total RNA (F) Separation of mRNA The desired mRNA can be separated from total RNA using known methods such as sucrose density gradient centrifugation, gel filtration, electrophoresis, membrane filtering, and methods using oligo dT columns. It can be carried out by any method or by a combination of these methods.

In order to confirm that the obtained mRNA encodes the desired LTi, it is recommended to translate the protein into the m-state and examine its biological activity. For example, the African frog (Xenopus levis)
1aevis))), reticulocyte lysate, or wheat germ, the whole mRNA is injected or added to a suitable protein synthesis thread, and the protein is translated into protein. This is done by confirming that the test shows i-damaging activity.

The method using African frog oocytes is carried out, for example, as follows.

Approximately 50 to 1)00 n of mRNA per oocyte was injected using the f microinjection method, and 20 of them were injected in total modified Perth salt solution (1 lodi-fied).
Barth 5alt 5solution ) (N
aCtO,13&5KC10,075&%NaHC
O30,22%MgSO4・7H200,2,! ? , C
a(Noz)2・4H200, (38/l % CaC
42.6H200.09g, HEPES 2.38,
! 9. Streptomycin 100.9. Penicillin G (
Dissolve 100,000 units) in 1t; Using this culture supernatant as a sample, LT activity is measured using L- and P3 cytotoxic activity as an indicator.

The mRNA encoding the LT animal of the present invention has the following properties.
be arrested.

■It has an S capacity of 12.6 S to 14.68.

■Has polyadenylic acid Kirizo at the 3' end.

■ LT polypenotide code.

■ Cloning of lymphotoxin cDNA Using the mRNA obtained in step (■) as a template and using oligo (dT) as a primer, (iATP, dGTP).

dCTP% mRNA is produced by reverse transcriptase (e.g. avian myeloid leukemia virus reverse transcriptase) in the presence of dTTP.
Single-stranded cDNA f complementary to A is synthesized and heat-treated to create template m.
Denature RNA. Next, using this single-stranded cDNA as a template, double-stranded DNA 1.f is synthesized using Escherichia coli DNA polymerase I (Klenow fragment).

Alkali treatment and phenol extraction on this double-stranded DNA? and separate it from denatured mRNA and protein. This double-stranded DNA is subjected to full action of reverse transcriptase to further synthesize complete double-stranded DNA. Since the DNA obtained here has a hairpin structure, S enclease [Aspergillus
Aspergillus oryzae
The hairpin structure is cleaved using rice malt and rice S1 nuclease to obtain DNAt with a complete double-stranded structure. The DNA obtained here was obtained using the poly(dG)-poly(dC) or poly(dAJ-poly(dT) homopolymer elongation method [Noda(N
oda M, ) et al., Nature 29
5 202 (1982); Maniatis (Mani-at
is T, et al., “Molecular Cloning (A Library Manual) J”Molecular
rcxoning (a 1 laboratory man
ual ) 217 (1982) Cold Spring
Nova Laboratory (Co1d Spring
Harbor Laboratory) New York]
For example, plasmid pBR322
into the restriction enzyme Pst I cleavage site. The obtained recombinant plasmid, for example, Perbal
"A Practical Guide to Molecular Cloning" by B.)
Guide to Mo1ecu1ar Clonin
g″268 (1984), John Wiley &amp;
A host such as the Sun 101 strain is transformed, and all tetracycline-resistant strains are selected to create a cDNA library.

Colony no-bridization tests using all synthetic probes for this cDNA library [Montgomery D, L, et al., Cel'1 14 673 (1978);
Goeddel, D.V., et al., Nucleic Acids Research.
Res, ) 84057 (1980)) to screen the clone of interest. That is, Gray et al. Nature 312 721 (19
404 to 42 of lymphotoxin reported in 84)
A complementary base sequence corresponding to the 18 bases of the first aroma and the 18 bases from 500 to 517 was chemically synthesized, and the 5' end of the probe was synthesized using polynucleotide kinase (Escherichia coli white rice T4 polynucleotide kinase infected with T4 phage). Transferring the phosphoric acid of γ-”2P-ATP to the hydroxyl group, 3
2P: Prepare all labeled 2a probes. The above C
A clone 'kA that strongly binds to both probes is selected from the DNA library.

Plasmi v DNA is isolated from the clones obtained here, and fixed to a nitrocellulose filter as single-stranded DNA by heating or alkaline denaturation. Lymphotoxin mRNA k Miyamu mRNA fraction was added to this and hybridized, and the bound mRNA f was eluted and recovered. This was injected into African frog oocytes, and the recovered mRNA encoded lymphotoxin. (hereinafter referred to as "Ibridization/Translation testing" and "When").

Cloned D into which a DNA fragment containing a base sequence complementary to lymphotoxin mRNA was incorporated by the above method.
NA i can be obtained.

The cloned DNA fragment of this transformed strain was excised with an appropriate restriction enzyme, labeled with 32p, and used as a total zorope, and rescreened with the aforementioned cDNA library IJ to obtain a larger size cDNA fragment. may be selected.

For the thus obtained cloned DNA fragments, a complete restriction enzyme map was prepared and cloned using M13 phage, as described by Sanger F. et al. [Proceedings of the National Academy of Sciences].
Op Science of Day USA (P
roc, Natl Acad, Sci, USA
745463 (1977)), and by extracting the already known amino acid sequence of lymphotoxin, the base encoding a polypeptide containing the amino acid sequence of lymphotoxin was obtained. Cloned DNA with sequence
i can get it.

■Preparation of dust vector containing the base sequence encoding the amino acid sequence of LT polypezotide ■cDNA encoding the amino acid sequence of LT polypezotide
Cleavage of A with a restriction enzyme For the production of DNA encoding the amino acid sequence of the present invention, all cDNA shown in Table 1.
Extract and collect DNA fragments. This cDNA fragment includes the entire cDNA shown in Table 1 from position 225 to position 840.

Ligation of synthetic oligonucleotide and L'I' cDNA fragment A synthetic oligonucleotide having the following structure AATTCTAT() CA AT is then digested with EcoR and a 623 bp EcoR fragment is recovered according to a conventional method.

○Translation of LT cDNA fragment into dust vector Hijin expression vector pKK 223-3 (manufactured by Pharmacia) 'k
After digestion with EcoR, the 5' end is dephosphorylated with alkaline phosphatase derived from bovine intestinal mucosa. Ligation of the EcoRI fragment created in step 0 and the above circular open vector is performed. After the reaction, the closed vector is recovered according to a conventional method and transformed into E. coli.

(2) Screening with synthetic oligonucleotide probes Colony hybridization is performed between the 32p-labeled synthetic oligonucleotide probe shown in (2) and the transformed strain of E. coli prepared in θ, and all strains that hybridize with the synthetic oligonucleotide probe are selected. Next, a vector is prepared from this colony, cut with a restriction enzyme, and subjected to agarose scale electrophoresis623.
Obtain the entire vector containing the bp cDNA fragment.

(2) Removal of the 6' Song end non-coding region of the LT cDNA After converting the vector obtained in step (2) into a linear DNA using Hinam, the 6' end non-coding region of the LT cDNA was removed using BAL 31 nuclease. Next, DNA polymerase 1
After making the end smooth with (Flenow Fragment), E
It is digested with coRI and fractionated by polyacrylamide gel electrophoresis to recover a fragment of about 4'60 bp.

@LT Rider 1 John with CDNA and linker ■
Hindl Linker to the 460 bp fragment obtained in
(Manufactured by Zenshuzo Co., Ltd.) After seven irradiations, it was digested with Hindl.

Lidar 1-John pKK 223-3 k EcoRI M Hini Hind with θLT cDNA and expression vector
A fragment of 4555 bp is obtained. Next, it was ligated with the approximately 465 bp EcoRl-Hindu fragment obtained in
A vector containing the pLT cDNA fragment is obtained.

■Creation of multi-copy expression vector pAT 153 (manufactured by Amajam) f Baz H
Digested with Pvu 1 and fractionated by agarose gel electrophoresis to obtain all 2655 bp fragments. On the other hand, the vector obtained in step (3) is also digested with Bam HI and Pvu I, and the entire fragment of approximately 980 bp is obtained in the same manner. B of the above two oak species
Am H knee Pvu I fragment was completely replicated and L
A vector containing a TcDNA fragment is obtained.

@) Expression of LT cDNA in E. coli The E. coli transformant obtained in step ① was incubated with O in a medium containing isopropyl-β-D-thiogalactoside (hereinafter abbreviated as PTC).
Culture until D 550 nm is approximately 1.

After collection, the bacterial cells are crushed by ultrasonication, and treated with Tris-hydrochloric acid (#I).
After extraction with a solution (pH 8, 0) and sterile filtration, total LT activity is measured and the transformant expressing the highest amount of LT polypeptide is screened.

■Culture and purification of LT polypeptide-producing E. coli (LT-producing E. coli) by IPT()'i
Culture in a medium containing i until LT polypezotide can be sufficiently produced. Next, the culture is digested with 'k IJ zozyme and disrupted by freeze-thawing, ultrasonic disruption, French press, Dynomill, etc., and then the extract is collected by centrifugation or filtering. This extract is purified by a combination of ammonium sulfate salting out, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, gel filtration, and 5DS-polyacrylamide gas phoresis to obtain the LT polypeptide as a substantially pure product. be able to.

(2) Regarding the LT polypeptide obtained in [phase], the amino acid composition and N-terminal amino acid sequence were measured, and as shown in the Examples, the results matched the values estimated from the formula ([).

The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples.

〔Example〕

Example 1 (1) Preparation of lymphotoxin-producing human T cell ipridoma 106 human peripheral blood lymphocytes (hereinafter referred to as PBL)/concanavalin A (hereinafter referred to as Co
abbreviated as n A) 20μ: After treatment with i/m for 2 days, 0.
Con Af bound to the cells was removed as much as possible with 2M α-methyl-D-mannoside.

On the other hand, human CCRF-CEM (hereinafter abbreviated as CEM) abscess cells in the proliferation phase in RPMT medium were collected by centrifugation, and RPMI 164.0-10
Emetine hydrochloride (Hani Kagaku Co., Ltd.) and actenomycin D (PL Biochemicals Co., Ltd.) were each suspended at 2X10' cells/WLl in mM HEPES medium at 5X.
After adding 10-5 M and 0.25 p9/rug and treating at 37°C for 12 hours, emetine hydrochloride and actinomycin Di in the culture solution were removed by centrifugation.

After mixing PBL prepared as above and CEM'a at a ratio of 10:1, centrifugation was performed to obtain a cell pellet with 0.5
Door g LD 46% tyrene glycol (PEG
-1540, Wako Pure Chemical Industries, Ltd.], 5μ9/trtl of poly-L-arginine and Southern MgM medium containing 15% dimethyl sulfoxide were added, and the mixture was stirred slowly for 67°0145 seconds to fuse. 25 mM HEPE in Ll
Ten volumes of MEM medium buffered with S (PH 7,2) were slowly added and centrifuged.

Add RPMI medium to the cell pellet to bring the number of cells to 106/-, and add 100 μt of the cell pellet and feeder cells (fee
der ceFl) was mixed with 100 μt of RPMI medium containing mitomycin C-treated OEM (4 x 10 cells/ytl), added to a 96-well culture plate, and incubated at 67°C for about 3 to 3 hours in an atmosphere of 25% CO and 95% air. After 4 weeks of culture, all of the proliferated fused cells were cloned as mitomycin C-treated CEM i feeder cells by the limiting dilution method, and after proliferating each clone, lymphotoxin activity was measured.

In addition, unless otherwise specified, the culture conditions are C025
%-Carried out at 67°C in an atmosphere of 95 air.

Lymphotoxin-producing cloned human T cell hybridoma strain A-05-8 used in the present invention Gold, 2.5 x 10
Can A 20 μj at a cell concentration of 5 cells/punishment! The lymphotoxin activity obtained by stimulation with 20 ng/punishment and PMA 20 ng/punishment and culturing for 24 hours was 250 units/punishment. On the other hand, the lymphotoxin activity of the unstimulated A-05-8 strain was 4.0 units/me.

Example 2 (1) Lymphotoxin-producing human T cell hybridoma (
*-05-8) culture A-05-3 strain at a cell concentration of 5 x 1 Q6 ~ 107 cells/particle, PMA and Con A k each at a final concentration of 10 n
g/rttls was added at 20 μg/rIte and cultured at 2.4.8° for 24.48 hours.

(2) Preparation of total RNA Total RNA of strain A-C5-8 was extracted mainly by the salt-guanidine method. That is, Example 2-(1)
A-C5-8 cells after each culture time of 1) 00 Or
The cells were collected by centrifugation for 5 minutes, suspended in PBS (5 mM phosphate buffer, containing 0.15 M NaCt, p)i 7.4), and further centrifuged at 1000 rpm for 5 minutes to wash the cells.

This cell [Example 2-4 x 108.6 x 108.3.2 x 10B for each culture time of (1),
1.8×109 and 2×109) were suspended in t-homogenate buffer and homogenized. 0 for this homogenate
．． After adding and mixing 025 equivalent of 1M acetic acid and 1.5 times the amount of cold ethanol (-20°C), the mixture was left at -20°C for more than 6 hours. This was heated to -10°C and 1500Orpm (R
Centrifuge for 30 minutes using a PR18-20-ter, manufactured by Hitachi, Ltd., and add a washing buffer (in which 2 [1 nMEDT] is further added to the homogenate buffer, hereinafter abbreviated as sword) to the resulting precipitate. After dissolving it uniformly by pipetting, add 1M gold acetate to adjust the pH to 5, and add 1 liter of cold ethanol.
．． A 5x needle was added and the mixture was left at -20°C for more than 6 hours. This was centrifuged at -10°C and 1500 rpm for 30 minutes, and the precipitate was dissolved in wB, pH adjusted with 1M acetic acid, and cold ethanol precipitation was repeated 76 times.

Add a small amount of 0.1% SDS'i to the ethanol precipitate, suspend it, and add an equal volume of extraction buffer (0.5% SDS'i) to the ethanol precipitate.
DS, 0.1 M NaCt, 50 mM sodium acetate, 5 mM EDTA (containing 2Na, 2 days 5.2) and 2 equivalents of water-saturated phenol [containing 0.1% 8-quinolite, 100 mg Tris-HCl buffer ( p) 18
．． 0) Saturated]-chloroform-isoamyl alcohol solution (volume ratio 50:50:1) was added, shaken for 10 minutes, and then centrifuged at 6000 rpm for 10 minutes. The lower layer separated into three layers was removed, and an equal volume of chloroform-isoamyl alcohol solution (volume ratio 50:1) was added to the upper layer and the middle oil. After shaking for 10 minutes, centrifugation was performed at 3000 rpm for 10 minutes to remove all of the lower layer. Removed. This extraction operation using chlorophor L-isoamyl alcohol solution was repeated three times. Add 1 equivalent of 6M sodium acetate (pH 5,2) to the upper layer containing DNA% RNA, sugar, etc., add 2 parts of cold ethanol and leave at 20°C for more than 6 hours, then heat to 150°C.
Centrifugation was performed for 60 minutes at 00 rpm (RPR18-20-ter). Add a small amount of political distilled water to the resulting precipitate. After dissolving, add an equal amount of cold 4M lithium chloride to % 0
Leave at °C overnight, centrifuge at 15,000 rpm for 30 minutes, wash the precipitate with a small amount of 2M lithium chloride, dissolve in sterile distilled water, and add 1/108 of 6M sodium acetate (pH 5,2). After that, 2 equivalents of cold ethanol was added, and the mixture was left at -20°C for 3 hours or more. 15000
Centrifuge for 30 min at rpm to precipitate
M) IJ female acid buffer (0,5M NaCL,
Contains 0.1% SDS, 1mM EDTA3Na,
pH 7.5). Next, oligo (dT) 12-15 cellulose (P
L Biochemicals Co., Ltd.) was packed into a 1 m diameter column to supply total RNA. After washing with the same buffer until the absorbance at 260 nm is 0.05 or less, further o, o I
M Tris-HCl buffer (0.1M NaC1.0.1%
SDS-1rnMEDTAAr5Na-containing, -7,5)
Elution was started and washing was carried out until the absorbance at 260 nm became 0.05 or less. Finally, add 0.01 M Tris-HCl buffer (0.1% SDS, 1 mM EDTA3N)
containing a, pH 7,5) with polyadenylic acid linkage @RNA (m
RNA) All RNA was eluted from the column, fractionated using a 7-lux collector, and fractions with detectable absorbance at 260 nm were collected. 58°160.180. from A-C5-8 cells cultured for 2, 4, 8, 24 and 48 hours, respectively
12[], 258μ, ? One mRNA ', was recovered.

(3) Confirmation that mRNA corresponding to LT is translated in oocytes A two-year-old African frog (female, weighing over 50 g, purchased from the Japan Center for Biological Materials) was injected with serum gonadotropin ( Veterinary scoop Pimex injection (Sankyo)) 2 []
It was injected intramuscularly into the thigh at a rate of OU/mouse. The next day, after being immersed in ice water and anesthetized, the abdomen? Incision was made to collect all oocytes, and M
Isolated in BS.

Example 2-(
Dissolve the mRNA k obtained in 2) in K fungal distilled water (1 m9/
ml) and injected 20 oocytes (ftMk) under a practical microscope using a microcapillary and a microinjector (Narishige Scientific Instruments Research Institute table) each using a microcapillary and a microinjector (Table 1). Cultured in C. At the same time, 53 nl of sterile distilled water was injected.
Twenty oocytes were also cultured at 23°C in 0.2-MBS.
did (control).

As a result of measuring the LT activity of the culture supernatant after 24 or 48 hours of culture, it was found that 48 hours of culture is the optimal culture time for oocytes. Furthermore, Con of A-C5-8 cells
The culture time under stimulation of A and PMA is 2,4.8.2
The translated LT activities of each mRNA in cells cultured for 4.43 hours were 7.8, 4.6, and 4.1, respectively.
, 2.7, and 0 units/d, indicating that 2-hour culture is the optimal culture time for A-05-8 cells in order to obtain %mRNA. LT in control
Since no activity was detected, it was confirmed that LT mRNA was translated in oocytes.

(4) Mass culture of A-C5-8 cells and acquisition of mRNA Example 2- (Total culture of A-05-8 cells under the optimal culture conditions under Con A and PMA stimulation determined in 31)
9 cells? collected. mRNA was isolated and purified from these cells according to the method of Example 2-(2), and 512 μy of mRNA was obtained.

(5) Fractionation of mRNA and L by sucrose density gradient centrifugation
T Total mRNA 512μ, UK [1,01M] Lys-HCl buffer (0,1M EDTA2Na, 0.2% SDS included, pH 7,5) K Dissolved in the same buffer with 5-30% sucrose. Layer the density gradient solution on top of the RP
The whole sample was centrifuged at 28,000 rpm for 16 hours at 15° C. using an R55T-2080-ter (manufactured by Hitachi).

The contents were then fractionated into 25 tubes (216 μt each). Add 1/10 volume of 3M acetic acid (3M acetic acid) and 2 equivalents of cold ethanol to each fraction, and leave at 20 °C overnight.
A was precipitated and collected.

The recovered mRNA was dissolved in sterile distilled water (C0.5 m9/wound) and 100 nt' was injected into 20 oocytes according to the method of Example 2-31, and mixed with MBS of 0.2 to 6. 48
After culturing for an hour, the LT activity of the culture supernatant was measured.

As a result, fraction 413 showed the maximum total LT activity and standard markers of sedimentation constant (5S, 18S, 28B)
The standard curve using +IJ revealed that the sedimentation constant of mRNA corresponding to LT was 12.6 S to 14.68.

The purified mRNA' obtained here was used in the experiment below.

Example 6 Synthesis of fil eDNA Using 5 μgk of purified mRNA, reverse transcriptase system (
3ZP) New England Nuclear Ltd.)
cDNA was synthesized with all partial changes. 20 μt of reverse transcriptase reaction buffer 10 μt of deoxynucleoside triphosphate mixture 20 units of ribonuclease A inhibitor, 12-1810 μt of oligo(dT)
600mM β-mercaptoethanol 5μ, 9.3
2p-labeled dCTP [specific activity 800 C1/mmot (
100 μci)), mRhJA 5 t4. 50 units of avian myeloid leukemia virus-derived reverse transcriptase system and 100
After reacting for 42°01) hours in a volume of μt, the reaction was stopped by cooling on ice, and after centrifugation, the mRNA and cDNA hybrids were separated by heat treatment for 6 minutes in a boiling water bath, and then in an ice water bath. The mixture was rapidly cooled for 5 minutes.

The denatured protein i12000x was pelleted by centrifugation for 9.2 minutes and cooled on ice again. Add 99 μt of this reaction completed solution to 16.2 μt of sterile distilled water under water cooling, 46.8 μt of DNA polymerase reaction buffer, 10.4 μt of deoxynucleoside triphosphate mixture, 32p labeled dc'rp (800 Ci/nmot (100 μ Maru Hatoyama) C1)] and 15.6 μt of DNA polymerase 1 (8000 units/tug), plus 1
After stirring and centrifuging, incubate at 200 µm at 15°C.
Time reacted. Add 0.2 ME to 197 μt of this reaction-completed solution.
After stopping the reaction by adding DTA2Na (39.4 μL), add 1N NaOH (49.25 μL) and
After 1 hour alkaline heating treatment at °C, it was decomposed into mRNA', cooled with water, and centrifuged.
d 9.25 μt) and neutralized.

This was extracted with 172 equivalents of water-saturated phenol and 172 equivalents of chloroform-isoamyl alcohol (50:1), and after centrifugation, the upper/Mi fraction was separated, and the lower layer was
M Tris-HCl buffer (1a o mMNact.

In addition to 1 mM EDTA (containing 2Na, pH 8,0), re-extraction was performed and the upper layer was also collected. The collected Amejo TV was extracted with chloroform-isoamyl alcohol, and the lower layer (organic) was removed after centrifugation. After performing this extraction operation four times, extraction was performed six times with an equal amount of water-saturated ethyl ether, and after the organic layer was completely removed, the mixture was heated in a 6 cfC water bath to remove the mixed ethyl ether.

This aqueous layer was concentrated with sec-butanol to a final concentration of 0.01.
) MgC42 from A and 2 equivalents of cold ethanol (-20°C
) was added and left overnight at -80°C.

After centrifuging for 10 minutes at 1200 [IX, 9, the precipitate was dried under reduced pressure, dissolved in 50 μt of sterile distilled water, and mixed with 20 μt of reverse transcriptase reaction buffer, 5 μt of 600 mM β-mercaptoethanol, and deoxynucleoside/triphosphate mixture IDAt. and 32p-labeled dcTP (800
Ci/mmol ('I 0 0 μ Ci
) ) k After stirring well and centrifuging, 5 μt of the above-mentioned reverse transcriptase was added, and the mixture was reacted at 42° C. for 1 hour. The reaction was stopped under water cooling to obtain cDNA f having a hairpin structure.

99μt of the above reaction solution, 92.1μt of sterile distilled water, and 23.4μt of the above-mentioned DNA polymerase I reaction buffer.

S1 Nuclease reaction buffer 55μt1 Aspergillus oryzae
5.5 μt of Sl nuclease (50 units/d) derived from ) was added and reacted for 67°0160 minutes to cleave the hairpin structure and obtain double-stranded cDNA f. To this solution was added 0.1 M female hydrochloric acid buffer (0.1 M EDTA, containing Na,
pH 7,5) Add t46 μ, bed volume 20 ru
ε Sephacryl S-200 column (1,0X25C1
n) and 10% Tris-HCl buffer (0.1M N).
It was eluted with aC4, containing 1mM EDTA2na, pH 7.5). The fraction was fractionated into 600 μt portions, and the fraction eluted near the porosity was concentrated with secondary butanol, and cpNA was recovered by ethanol precipitation.

(2) Preparation of oligo(dC) tailed cDNA Add reaction buffer 1 of the following composition to the double-stranded cDNA obtained above.
Add 60μt2 and react at 37°C for 5 minutes to remove double-stranded cD.
An oligo(dC) tail was added to NA.

The reaction buffer was 2mM DTT, 5mM CaCl2.
0.25 mt;i/rILe BSA, 5 μM
ac'rp, 3H-labeled dCTP (25Ci/m
mole (15 μCi) :) and 30 units of terminal deoxynucleotisol trans7-erase in 0.2M potassium cacodylate-25mM Tris-HCl buffer (pH 6.9).

The reaction was stopped under water cooling and mixed with water-saturated phenol-chloroform-isoamyl alcohol (50:50:1.
l', extracted again with chloroform-isoamyl alcohol (50:1), and extracted with 40 μy of E. coli ribosomal ANA and 1) 50 amounts of 5 M NaCL.
f and 2 equivalents of cold ethanol were added, and the mixture was left at -80°C overnight. Add oligo(d retail) by centrifugation.
NA was collected and dissolved in sterile distilled water at 0.5 ng/
The concentration was μt.

(3) Preparation of recombinant plasmid Oligo (dC) tailed cDNA 1.375 ng
k oligo (dc)) 1o-2o tailed pBR32
2DNA 10 ng (Amarjam Tsutocare Neil solution (100mM NaCl; 0.1mM EDT)
A2Nak containing 10 mM) Lis-HCl buffer 1 (
pH 7,8)) 25μ in 65°01) After incubating for 5 minutes, set the incubator to 45°C,
After the temperature reached °C, the mixture was further incubated for 2 hours, cooled on ice, and subjected to annealing to prepare a recombinant plasmid solution.

- Absorbance (6500n) in 10ml of broth at 37°C
0.05, add this 5m/i to 500 ml of L-broth containing 3.1% glucose, and add 2"C
The cells were cultured at 1, which gave an absorbance (650 nm) of 0.6. After cooling on ice for 30 minutes, 3000 rpm (RpR9-2
(manufactured by Hitachi) and centrifuged at 4°C for 5 minutes to collect bacteria. This bacterial body was cooled with 5 Q mlA CaCl225.
0 punishment and placed on ice for 15 minutes. 4°C, 5 minutes, 2
50 Orpm (RPR9-2o1) Collect bacteria and add 20% glycerol to 50 mM C.
It was dispersed in aCl225WLε, dispensed into 1 ml portions, frozen with dry ice powder, and stored at -80°C. Thaw this preserved bacterial cell dispersion under water cooling, and add 0.15 atl and 20 mM of the above-mentioned recombinant plasmid solution to 0-5 ml of it.
cacz2-6゜mM MnC22-20mM Rb
Mix 0.15a/,' of C1 solution, let it stand at 0℃ for 20 minutes, then let it stand at room temperature for 10 minutes, and then heat it to 37℃ in advance.
0.1% glucony-containing L paste heated to 2.4
ytek was added and mixed, and cultured with shaking at 37°C for 1 hour. A portion of this culture solution was spread on an L-broth agar plate containing 15μi/ltt of tetracycline in addition to the above-mentioned components, and cultured at 67°C for about 12 hours to select tetracycline-resistant bacteria and construct a cDNA library. Created.

(5) Hybridization test For the above cDNA library, a colony hybridization test using all 32P-labeled synthetic cDNA probes was performed in order to screen for transformants carrying a plasmid containing the cDNA encoding LTi.

A synthetic c-DNA probe was developed by Gray et al.
The 18th base (5'-oTc'rAcTcccAooTooTc-
3') as well as a complementary base sequence corresponding to the 18 bases from 500 to 517 (5'-CACATGGAAG
Chemically synthesize G()C)TACTG-3'), Part 1
6.6 pmO2'k 5 units T477 IshiM staining m-derived T4 polynucleotide kinase and rP ATP
(5μCi/pmole (20μCi) )'
(50 mM) Lis-HCl buffer (10 mM
g, C10, containing 5mM DTT, 0.1mM spermidine, 0.1mM EDTA2Na, pH 7,
6) The reaction was carried out at 37°C for 30 minutes.

The reaction was stopped by adding EDTA solution and cooling on ice to 0°C. A recombinant plasmid that hybridizes to both of these two 32p standard cDNA zolobes under weak conditions? The transformants having the following properties were selected.

Six colonies were selected from approximately 10,000 colonies.

Next, all recombinant celestial plasmids were isolated from the six selected strains, and restriction enzyme Hindll! Then, the cDNA was cleaved, subjected to Akaroske luminophoresis seven times, transferred to a nitrocellulose filter, and re-labeled with 32p.
A friend that hybridized under harsh conditions with the Eight Probes. As a result, one clone was selected.

Next, this selected strain was subjected to a hybridization translation test.
Molecular cloning (
“Mo1ecu/lar Cloning” 329
(1980), Cold Spring Harbor Laboratory). Chilasmi DNA i was extracted from this transformant, heat-denatured and fixed on a nitrocellulose filter, and mRNA containing r and TmRNA k obtained in Example 2-(41)
Add fraction A and react at 50°C for 3 hours.)1 Hybridization? went. After the bound mRNA was eluted and collected, it was removed.

As a result, for pLTl 3, 10 units/me of L
Although T activity was detected, LT activity was not observed in the control using pBR322k.

This cDNA was cut with f restriction enzyme H1ndu and its size was examined by agarose gel electrophoresis.
Mouth cDNA had a 5 Kbp cDNA portion
Regarding the transformant containing f (bacteria cell number: LT16, cloned DNA number: pLTis), cloned D
NAi was isolated and the entire base sequence was determined by the method described below.

Example 4 Strain (LT13)k obtained in Example 3-+5)
Bacterial cells were obtained by culturing in L-broth containing 0.1 tiglucose and 15μ/m and tetracycline. Plasmid DNA was recovered from this bacterial body, and M13m, which will be described in the next section, was
Restriction enzymes EcoR1 and Sma that can be introduced into F 8 and 9
I.

Bam Hl, Pst I, Sal I, H
I created all the ind l restriction # raw maps. See Figure 1.

(2) Determination of base sequence of cloned DNA Cloning D
Determination of the base sequence of NA is done by Messing
) et al. [Gene (C)ene) 19269 (1
After subcloning in cDNA fragment iM13mp13 or mp9 according to Sanger (San 982))
get) et al.'s method
National Academy of Sciences
De USA (Proc, Nat'1. Acad.

Sci, USA) 74 5463 (1977)
Journal of Molecular Biology (J.

Mo1. Biol) 162 729 (1982
)) dideoxy chain termination method, primer annealing and complementary strand synthesis using the Flenow fragment of DNA polymerase I (32P-guided dCTP (800Ci/mmol (20μC1)
) was determined from lumi electrophoresis and autoradiography.

FIG. 2 shows the restriction enzyme cleavage sites used for determining the base sequence and the direction and range in which the base sequence was determined, all indicated by arrows. The rectangular area indicates the area encoding the LT translation region.

Its base sequence is shown in Table 1. There are 16 G-linked tails upstream of the 1st C and 18 C-linked tails downstream of the 1310th T, and is a homopolymer synthesized when added to the vector. 63rd to 677th
The number is the predicted nucleotide sequence encoding all the polypezotides necessary to constitute the LT precursor.

Of these, it is thought that the LT 71i-code is present from the 165th position, and Gray (P, W, ) et al. [Nature 312 721 (198
4)) The 26th amino acid is Th.
The difference is that it is Asn (AJC) while it is r (ACC). Following the codon for the C-bed end amino acid (leucine), there is a stop codon (TAG).In addition to the peptide coding site, the first to fourth GCTCs are CG()
In G, the 862nd to 865th C'ACA are different from ACAC, and the 1606th to 1310th tests T()AAA are different in CCCCT.

Example 5 - (1) Restriction enzyme cleavage of cDNA fragment of pLT 13 Among the restriction enzymes considered from the structure of cDNA, Nsil (222nd) and EcoRl (8,
38) and pL'r 13' according to the usual method.
Cut into 1.5% agarose del electric upheaval? conduct 61
A 6 bp cDNA fragment was extracted from an agarose gel,
The cDNA fragment was purified by a conventional method. This cDNA fragment lacks 60 bases corresponding to the 20 amino acid residues on the N-terminal side of LT.

The base oligonucleotide of the following structure was prepared using T4 DNA ligase (66 mM) using T4 DNA ligase.
IJ female hydrochloric acid buffer (p) 17.6), 6.6 mM MgC
l2.10mM DTT, 16 in ImM ATP
React overnight at °C, synthesize the oligonucleotide 'iL
It was ligated to a TcDNA fragment. This first reaction product was purified by a conventional method and then digested with EcoRl to recover a 623 bp EcoR1 fragment.

Expression vector pKK 223-3 (Pharmacia)
'1EcoR (bovine intestinal mucosa) using white rice alkaline phosphatase (manufactured by Pharmacia) and 50 pressure Tris-HCl buffer (pH 9,0), 1 mM MgCl2.
67 in 0.1 mM ZnCA2.1 Sushi Spermidine
React for 130 minutes at °C to dephosphorylate the 5' end.
It was purified by a conventional method. The above-treated vector and Example 5- (623 bp EcoR obtained from 2+
After ligating the pieces, they were transformed into ←-Yo JM 105 strain according to a conventional method, and 8,700 transformants were obtained due to ampicillin resistance.

(4) Synthetic cDNA DNA probe screening Example 5-(3) The transformant obtained in Example 3-(51
A 3Kp-labeled synthetic cDNA probe was screened by colony hybridization test according to the method described in , and 150 colonies were selected. Twenty-four of these colonies were randomly selected, a plasmid was prepared according to a conventional method, and after cutting with an appropriate restriction enzyme, agarose gel electrophoresis was performed to search for the target fragment and all its directions.

As a result, all seven target colonies were selected and this plasmid was named i pDK 10. pDK IQ
The entire construction process is shown in Figure 3.

Plasmid pDK 10 obtained in Example 5-(4)? l-
After digestion with Hlndl, 20 mM Tris-HCl buffer (PH
8, []) 1) 2mMCaCl2.12mM Mg(
ll't2.1mM EDTA, 600mM N
React for 60 minutes at 30°C in aCt, and
A The wood end was completely removed. Next, the ends were blunt-ended with DNA polymerase (Frenow fragment), digested with EcoR1, and fractionated by 5% polyacrylamide gel electrophoresis.
A fragment around 80 bp (preferably 460 bp) was recovered.

Example 5- (DNA Ifr piece obtained in 51 and Bind
Hindl linker (manufactured by Zenshuzo Co., Ltd.) After complete linkage, Hindl
It was digested and produced privately using a conventional method.

pKK 223-3 k FCORI and Hind
EcoRI-Hind obtained in Example 5-(6)
Combines with I DNA fragment and LT cDNA fragment? FIG. 4 shows the entire construction process of pDK1).

Example 5 - Plasmid f Bam Hl obtained in (7)
After digestion with Pvu, the fragment containing LT cDNA f was fractionated and purified by agarose gel electrophoresis. On the other hand, plasmid pAT 153 (purchased from Amajam) was similarly digested with Ban H1 and Pvu I, and the fragment containing the DNA replication origin was fractionated and purified.

After the Ban Hl-Pvu I fragments of the above two glazes were dried, they were transferred to JM 105a using the usual method, and 4,300 traits were obtained due to the anti-silicin resistance. Transformants were selected. Twenty-four transformants were selected at random from among them, and plasmid DNA was prepared using a conventional method. The desired LT cDNA was isolated using an appropriate restriction enzyme.
The number and orientation of fragments were searched. As a result, the steps for constructing all 24 colonies of pDK 12 are shown in FIG.

? Ampicillin 50 μ9/ynl, excluding proline 19
One preculture was performed in M9 medium containing $1000 essential amino acids. The next day, add LB medium containing 25 μ/dk of ampicillin so that the culture solution is diluted 10 times (composition:
NaC1)o9: pH 7-7.5].

After reaching A55 = 0.6, IPTC) was added to a final concentration of 1 mM, and cultured until fi-ssa = 1. Cultures were disrupted by sonication and total LT activity of cell extracts was measured.

Culture 1) Approximately 3.5 x 105 units of activity per Ll were obtained.

The JM'llJ Koku strain, which had been completely transformed with pDK 12, was cultured overnight in M9 medium containing ampicillin 50μ9/me1 and 19 essential amino acids excluding proline.
The cells were cultured in the following medium containing 20 times the total amount of this culture solution.

Na2HPO47.0 KH2P○46.0 (NHa)2sO45.0 Na-citrate 1.0Mg
SO40,1 Glucose 25. OCas
amino acid 4,0Yeas
t Extract 4. OThiam
ine 0.1 ampicillin
When the A350 of the 0.02 pH 7.5 medium reached approximately 10, IPT()'i was added to a final concentration of 5 ml and cultured with 1 to reach the A350 of 60. The entire culture solution was collected by ultrafiltration and centrifugation, and approximately 75 bacterial cells were collected.
0, ji' (wet) f were collected.

Bacterial cells t 50mM) Liss-HCl buffer (pH 8.0)
, 60mM NaCt and 0.01mM (p-amidinophenyl)methanesulfonyl fluoride hydrochloride ((p
-Amidinophenyl) methanes
ulfonyl F'1uorideHydroch'
1oride; manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 2;000 m7, and after crushing with Dynomill, a crude extract was obtained by centrifugation. The total LT activity of 3.700 d of crude extract was 60 x 1012 units, and the specific activity was 5 x 10' units/■ protein.

Example 8 (1) Ammonium sulfate fractionation To 3.700 mg of the crude extract of Example 7, ammonium sulfate was added to 40% saturation at 5°C, and after 60 minutes,
Centrifuge and precipitate the precipitate with 5 mM IJ acid buffer (pi-17,8) C, hereinafter abbreviated as PB, 1500+x
The sample was dissolved in J and subjected to concentration-dilution and repeated desalination using an ultraconcentrator to obtain an ammonium sulfate salting-out sample. Ammonium sulfate salting out sample 18
The total LT activity of 5ml was 5.2 x 1012 units and the specific activity was 3.7 x I Q6 units/mQ protein.

DEAE-Sepharose CL-6B (Pharmacia Co., Ltd. column (4.5 cm x 5 [1 cm)] pre-equilibrated with ammonium sulfate salting sample 184 dk was pre-equilibrated with s mM pEl (pH 7,8), and then 5 mM PB
3. After washing the column with Ot, the salt concentration was adjusted from 0 to 0.6.
It was eluted by increasing the concentration continuously to M. Eluent is 100mJ
The LT activity of each fraction was measured, and the fractions having all the activities were collected. Recovered liquid approx. 3. Ot k fraction molecule 1t1
1 using an ultrafiltration membrane (Millipore'A) with a diameter of 0.
70WL! ! condensed into. This concentrated solution was used as a DEAE fraction solution.

The total LT activity of DEAE fraction is 2.9 x 10i2
unit, specific activity was 2.4 x 10' units/mq protein.

Phenyl Sepharose CL-6B (Pharmacia) pre-equilibrated with saline buffered with PB
column (5,0cfnX i 5cfn)
Adsorb fraction 1691n, then 10mMPB (pH 7,8) 6001 containing ethylene glycol filtrate%.
After washing with
Amino acid composition of LT polypeptide ILE
2.86 VAL 8.9 9LEU
22.0 21PHE
io, 1 10cys <
o,i.

MET 3.1 3ALA
1). 8 12GLY
9, 1 9THR7, 37 TRP 1.7 2SER19
,420'rYR6,87 PRO12,21) HIS10.19 LYS 5.0 5Question
The 1.92 analysis value was in good agreement with the amino acid composition inferred from the base sequence of the DNA encoding the LT polypeptide.

2 Amino acid sequence The N-terminal amino acid sequence was determined using the Edman method (P
, Edman; Architecture, Biochemistry and Biophysics (Arch.

Biochem, Biophys, ), 22
475 (1949)).

LT polypeptide (approximately 100 μg) was subjected to a coupling reaction with phenylthiocyanate, and then the N-terminal amino acid was cleaved as a 2-anilino-5-thiazolinone derivative, further converted to phenylthiohydantoin-amino acid, and this was subjected to a 018 reverse phase column. The amino acids at the end of the N bed were determined by HPLC (manufactured by Waters). Furthermore, by repeating this operation sequentially, N
The amino acid sequence of the terminal portion was determined.

As a result, the amino acid sequence of the N-bed end portion of the LT polypeptide is MET-HIS-LEU-ALA-HIS-3ER-A
SN-LEU-LYS-PRO-ALA-ALA-HI
It was S-LgU-ILE-OLY-ASP-.

〔Effect of the invention〕

As explained above, the obtained physiologically active polypeptide of the present invention is a novel physiologically active polypeptide that has lymphotoxin activity and is substantially free of impurities. Further, addition of excess methionine to the bioactive polypeptide of the present invention can be prevented.

[Brief explanation of drawings]

Figure 1 is a schematic diagram showing a restriction enzyme map according to an example of the present invention, and Figure 2 shows the restriction enzyme cleavage sites used in determining a base sequence according to an example of the present invention, and the direction and range in which the base sequence was determined. It is a rough route map shown. B---Bam Hl, E・-gcoR[P
...-Pst I FIG. 6 shows the construction steps of the tac promoter-equipped expression plasmid pDK 40 of Example 5. The arrow indicates the function of the promoter (the direction shown in FIG. 4 is the construction step of the tac promoter-equipped expression plasmid pDK1 of Example 5). Arrows indicate the direction of promoter action. Patent applicant Denki Kagaku Kogyo Co., Ltd. Figure 1 Figure 2 Figure 3 ILT13 ↓Ns l I+EcoRI ↓Sigation ↓EcoRI Figure 4, lBAL31 jDHApolymerase I (Klenow
Frugmenmun↓EcoRI Recovering DNA fragment around 460bp Figure 5 pDKll pAT153 Procedural Amendment Procedure Amendment Department August 21st

Claims (1)

[Claims] (1) The following formula [There is a gene sequence] A physiologically active polypeptide having the amino acid sequence represented by