WO1993014203A1 - Lymphotoxins, expression vector therefor, and production of lymphotoxins with said vector - Google Patents

Abstract

A vector which can efficiently express lymphotoxins including natural lymphotoxin and lymphotoxin-like peptides having a high homology with lymphotoxin; and a process for producing lymphotoxins by using the vector. The vector has the SD sequence located (13) bases upstream from the initiator codon, a base sequence of TTAACT in the -10 region and a base sequence of TTGACA in the -35 region, and a molecular weight of 2.7 to 3.1 kb except for the gene coding for lymphotoxins. Lymphotoxins are produced mainly be genetic recombination and the use of this vector serves to increase the amount of expression thereof greatly, thus enabling them to be clinically used.

Description

 Specification

 Lymphotoxins, their expression vectors, and methods for producing lymphotoxins using the same

 Technical field

 The present invention relates to a vector for advantageously expressing a lymphotoxin, a method for producing a lymphotoxin using the vector, and a lymphotoxin produced by the vector.

 Background technology

 Conventionally, TNF, lymphotoxin and the like have been known as antitumor peptides, and their clinical use has been studied.

 However, among them, TNF has problems of side effects such as lowering of blood pressure and fever at the time of administration, and is considered to be difficult to apply to actual treatment.

On the other hand, natural lymphotoxins and lymphotoxins such as a lymphotoxin-like peptide having high homology thereto have no side effects unlike TNF, but have problems in their production.

 In other words, at present, lymphotoxins are mainly produced by genetic recombination, but their expression levels are low, resulting in poor production efficiency and securing sufficient amounts of lymphotoxins for actual treatment. It was difficult at present.

 Disclosure of the invention

 The present inventors have made intensive studies to dramatically increase the expression level of lymphotoxins for clinical use. As a result, they found that if a specific vector was used, lymphotoxins, which had been expressed very little in the past, could be efficiently expressed, and completed the present invention.

That is, an object of the present invention is to have an SD region 13 bases upstream of the start codon, a base sequence of 110 region is TT AACT, a base sequence of 135 region is TTGACA, and Expression of lymphotoxins with a size of 2.7-3.1 kb excluding genes encoding The idea is to submit a vector.

 Another object of the present invention is to provide a method for producing lymphotoxins, which comprises transforming a host microorganism with the vector described above and culturing the transformed microorganism.

 Yet another object of the present invention is to introduce a peptide having high homology to a novel natural lymphotoxin advantageously prepared by the method of the present invention.

 Brief explanation of drawings

 FIG. 1 to FIG. 3 are drawings showing steps for constructing the vector of the present invention.

 FIG. 4 = is a drawing showing gene 11 and a peptide having high homology to the natural lymphotoxin encoded by the gene (hereinafter, may be referred to as the peptide of the present invention).

 BEST MODE FOR CARRYING OUT THE INVENTION

 The vector of the present invention uses a promoter in which the nucleotide sequence of the 10 region is TTAACT and the nucleotide sequence of the 135 region is TTGACA, and uses a promoter between the SD region (ribosome binding site) and the initiation codon (ATG). 13 bases, to which a gene coding for a lymphotoxin (hereinafter abbreviated as “LT”) (hereinafter abbreviated as “LT structural gene”) is ligated, and a vector excluding the LT structural gene is ligated. It can be constructed by setting the base length to 2.7 to 3.1 kb.

Specifically, for example, it can be constructed according to any of the granules in FIGS. 1 to 3. According to the method shown in FIG. 1, the vector of the present invention comprises a base sequence encoding the first half of the promoter (gene 1), the second half of the promoter including the SD region and the start codon, and the first half of the LT gene. Nucleotide sequence (gene 2) containing a base E sequence (gene 2) and a stop codon (gene 4), and a base sequence (gene 3) encoding these and most of the LT structural gene cut out from a known LT vector ) And an expression vector (gene 5) It can be built by performing a game.

 According to this method, the distance between the SD region and the start codon can be reduced to 13 bases by using the chemically synthesized gene 2.

 In addition, the expression vector to be ligated requires that the constructed vector of the present invention has a base length of 2.7 to 3.1 kb excluding the LT structural gene. Yes, for example, by using PUC-based PUC, PTZ, Bluescript, etc., the demand can be satisfied.

 Furthermore, even if the LT structural gene used in the vector of the present invention is a natural lymphotoxin gene, a part thereof is modified by utilizing the natural lymphotoxin gene or completely synthesized by organic synthesis. It may be a gene encoding an artificially produced peptide with high homology to natural lymphotoxin.

 Further, according to the method of FIG. 2, the LT structural gene of the LT expression vector obtained by the method of FIG. 1 can be exchanged for another LT structural gene. That is, a gene (gene 6) encoding most of the promoter is cut out from the LT expression vector prepared by the method shown in FIG. 1 and cut out from this and another vector or synthesized. A gene (structural gene) that contains a gene (gene 8) encoding most of the LT structural gene, an SD region and an initiation codon (ATG), and is prepared so that this distance is 13 bases. 7 or 10) and an expression vector (gene 3) are ligated to construct the vector of the present invention.

 In addition, the method shown in FIG. 3 incorporates the LT structural gene of the LT expression vector obtained by the method of FIG. 1 into another vector as it is, and the number of copies, etc. The vector of the present invention having a characteristic can be obtained.

The above method is one of the methods for preparing the LT expression vector of the present invention. It is an example and can be prepared by other methods. For example, a promoter that satisfies the condition that the nucleotide sequence of the −10 region is TT AACT and the nucleotide sequence of the 135 region is TT GACA includes T rp pro It is also possible to incorporate the LT structural gene into a vector having the same rp promoter and remove the unnecessary base from the resulting vector to prepare the vector of the present invention.

 The LT expression vector obtained as described above can be used to transform a host microorganism in accordance with a conventional method and culture the transformed microorganism to produce LT.

 Transformants can be cultured in a conventional manner, using L-culture medium, M9 medium, M9-force amino acid medium, high phosphoric acid medium, high phosphoric acid-casamino acid medium, etc. Can be used.

 In particular, in order to obtain a high LT expression, it is preferable to add 3-indoleacrylic acid to the medium. 3 The amount of β-indoleacrylic acid to be added is about 20 to about L O Og per 1 ml of the medium.

 In order to separate and purify the LT obtained by the culture from the culture, the microorganism is destroyed by, for example, sonication or the like, and then a supernatant is obtained by centrifugation or the like. It is carried out by subjecting to a purification method in which purification means such as dialysis, ion chromatography, hydrophobic chromatography, and affinity chromatography are combined.

 Next, the present invention will be described in more detail with reference to Examples, Reference Examples and Test Examples, but the present invention is not limited to these Examples and the like.

 Example 1

 (1) Construction of the head part of the promoter:

-A gene (gene 1) containing a promoter portion whose base sequence in the 10 region is TT AACT, and a base sequence in the 35 region is TTGACA (hereinafter referred to as “trp promoter 1”), and an SD region and Of the LT structural gene (hereinafter abbreviated as "MLT") prepared according to Reference Example (4) described later (Japanese Patent Application No. Hei. The head (gene 2) was organically synthesized using a DNA synthesizer. Gene 1 and gene 2 were synthesized using the following oligonucleotides (1) to (6) and the following oligonucleotides (7) to (14).

 (Oligonucleotide for gene 1 preparation)

 (1) 5 '-AA T T C A T T G T C C GA C A T C A C A A C G

 G T T

 (2) 3 '-G T A A C A G G C T G T A G T G T T G C C AA G

 A C C G T T

 (3) 5 '— C T G G C A A A T A T T C T G A A A T G A G C T

 G T T

 (4) 3 '-T A T AA G A C T T T A C T C G A C AA C T G T

 T A A

 (5) 5 '-G A C A A T T A A T C A T C G A A C T A G T T A

 A C T A G T

 (6) 3 '-T T A G T A G C T T G A T C A A T T G A T C A

(Oligonucleotide for gene 2 preparation)

 (7) 5 '-A C G C A A G T T C A C G T AAAAA G

 (8) 3 '-T G C G T T C AA G T G C A T T T T T C C C A T

 A G C

 (9) 5 '-G G T A T C G A T A A G C T A T G

(10) 3 '-T A T T C G A T A C T T A A T A C

(11) 5 '-A A T T A T G A A T T T C C A G

 (12) 3 '— T T A A A G G T C G G G T C T G

 (13) 5 '-C C C A G A C T G C T C G T C A G C A T C C G A

 A G A T G C A

(14) 3 '— TCGAGCAGTCGTAGGCTTCT Dissolve 0.10 D of each oligonucleotide in sterile water 151, and add 4 polynucleotide kinase (Toyobo), 400 iaM ATP, and 10 times the volume of force-initiating buffer. In addition, the mixture was adjusted to 301 and reacted at 37 for 2 hours. After reaction 65. The enzyme was inactivated by heat treatment with C for 10 minutes. Thereafter, each oligonucleotide 0.050 D was combined into one tube, an equal amount of annealing buffer was added, reacted at 9 CTC for 3 minutes, and cooled slowly. To this was added 200 units of T4 DNA ligase (Takara Shuzo), 1 MDTT 1 ^ 1, 10 mM MATP 10 ^ 1, and annealing buffer 7 ^ 1, to give 1001. Then, 16. C for 16 hours, electrophoresed on 10% PAGE gel, stained with ethidium bromide (EtBr), cut out the target genes 1 and 2, After elution in an elution buffer (0.5 M ammonium acetate-1 mM EDTA), desalting was performed. The obtained genes 1 and 2 respectively had the following nucleotide sequences.

 Gene 1

AATTCATTGTCCGACATCACAACGGTTCTGGCAAATATT GTAACAGGCTGTAGTGTTGCCAAGACCGTTTATAA

 -35 one 10

CTGAAATGAGCTGTTGACAATTAATCATCGAACTGA ^ TTAA GACTTTACTCGACAACTGTTAATTAGTAGCTTGACTkATT

CTkGT

 GJTCA 遗 DENKO 2

 SD region Cla start codon

 ACGCAAGTTCACGTAAAkAGGGTATCGATAAGCTkTGk

TGCGTTCAAGTGCATTTTTCCCATAGCTATTCGA ^ TACT

ATTATGAATTTCCAGCCCAGACTGCTCGTCAGCATCCG TAATACTTAAAGGTCGGGTCTGACGAGCAGTCGTAGGC AAGATGCA

 TTCT

(2) Recovery of MLT gene:

 Pkk223-3-3 MLT DNA disclosed in Reference Example (5) was cut with a restriction enzyme EcoRI-Seal (Toyobo), and a DNA fragment of 495 bp was recovered. The DNA was digested with N sil (NEB) and a DNA fragment (gene 3) of 431 bp was recovered.

 (3) Preparation of an oligonucleotide containing a termination codon and further having a Hind m site:

 Using an ABI DNA synthesizer, two consecutive stop codons from the oligonucleotide shown below, and an oligonucleotide (gene 4) having a HindE site were further organically synthesized. Synthesized.

 (Oligonucleotide for preparation of gene 4)

 (15) 5 '-A C T G T C T T C T T T G G A G C C T T C G C T

 C T G TA G TA GA

 (16) 3'—TGCACAGAAGAGAAACCCTCGGAAAGCGA

 GA C A T C A T C T T C GA

 The above synthetic gene 0.1 OD was dissolved in annealing buffer 100 11 and reacted at 90 ° C for 3 minutes, and then cooled slowly to obtain gene 4 having the following nucleotide sequence. Was.

 ¾denko 4

ACTGTCTTCTTTGGAGCCTTCGCTCTGTAGTAGA

(4) Cutting Blue Script M13SKII (+):

Bluescript M13SKII (+) ns (Toyobo) was digested with restriction enzyme EcoRI-Hindm (Toyobo), and then electrophoresed on a 0.8% low melting point agarose gel. Br staining Then, the desired 2.9 kb plasmid DNA (gene 5) was obtained.

 (5) Construction of a plasmid that possesses the trp promoter overnight and possesses the MLT:

 The gene 5 was reacted with the genes 1, 2, 3 and 4 using T4 DNA ligase to obtain a plasmid (PBTrpMLT) having the MLT gene. Further, the HB101 strain for transformation which had been frozen and stored at 170 ° C. was thawed, and 20 ng of pBTrpMLT was added to the suspension 1001. After reaction on ice for 45 minutes, a heat shock was applied at 42 ° C for 90 seconds, and the mixture was cooled for 2 minutes. After adding SOC Medium 900 ^ 1 and culturing at 37 ° C for 1 hour, the suspension is spread on an LB agar plate containing 0.1 mg Zm1 ambicilin, and then incubated at 37 ° C. After overnight culture in C, transgenic plants having a vector (pB trp MLT) comprising genes 1, 2, 3, 4, and 5 were obtained. DNA was isolated from this transformant, cut with the restriction enzyme EcoRI — Hindi !, and then electrophoresed on a 1% agarose gel. EtBr staining confirmed a fragment of 629 bp. did.

 (6) Expression of pBtrpMLT:

For PB trp MLT, the expression of the protein was confirmed under the conditions of 30 ° C and 37 ° C. A single colony of p B trp MLT, 2. 5 μ . g / m 1 preparative rib preparative file containing emissions M 9 CA medium (M 9 minimum culture areas, 1% _{glucose, I mM M g S 0 4} , 0.1 m MC a Cl ₂ , 0.5% casamino acid, 0.1 mg / ml thiamine monohydrochloride, 0.1 mg Zm 1 ambicilin, 100 mM Hepes) Overnight. The next day, _{add 1Z50 to a fresh M9CA} medium, culture until OD _eoD = 0.5, add 20 g of 3-indoleacrylic acid, and further culture at each temperature for 16 hours. . After completion of the culture, 1 ml of the bacterial cell fluid was transferred to a 1.5 ml centrifuge tube, and the cells were obtained by centrifugation. The cells were released into a 2001 buffer for SDSP AG E. The mixture was dissolved in the buffer solution, heat-treated at 90 ° C for 5 minutes, and electrophoresed by 15% SDSPAGE. After completion of the electrophoresis, the cells were stained with Kumashi Blue and then decolorized. After decolorization, the expression level of MLT per total protein was measured with an LKB laser scanner (Pharmacia LKB). At culture conditions of 30 ° C, 37% of total protein and 37 ^e Under the culture conditions of C, expression of 25% of the MLT protein per total protein was confirmed. This corresponds to a protein mass of 40 to 80 mg per little under culture conditions at 30 ° C.

 (7) Protein production and purification:

pB trp MLT was cultured overnight in 200 ml of M9CA medium containing 2.5 ^ g / ml tritophan, and this culture was inoculated into 71 M9CA medium and OD The cells were cultured at 30 ° C until BO _D = 0.5. Thereafter, 3 ^ -indoleacrylic acid was added to a final concentration of SO ^ gZml, and the cells were further cultured for 16 hours, followed by centrifugation to collect the cells.

 The obtained cells (33.7 g / wet) were suspended in 50 ml of 50 mM Tris-HCl (pH 8.0) containing 30 mM NaCl and 0.1 mM p-APMSF, and This cell suspension was passed through a high-pressure homogenizer (RANNIE) three times at 600 psi to destroy the cells. Further, the supernatant was obtained by removing the cell residue by centrifugation.

Polyethyleneimine was added to 500 ml of the obtained supernatant to a final concentration of 0.1%, and the mixture was stirred at 4 ° C for 10 minutes, centrifuged, and the nucleic acid was removed. Done. 480 ml of the supernatant was recovered, and ammonium sulfate was added to 50% saturation, followed by stirring at 4 ^{eC for} 6 hours, followed by centrifugation to obtain a crude protein as a precipitate.

This crude protein was dissolved in 45 ml of 5 mM phosphate buffer (pH 7.4), and dialyzed against the same buffer. After further centrifugation, the insoluble protein was removed, and 52 ml of the supernatant was recovered. Further, a heat treatment was performed at 50 ° C for 10 minutes to remove proteins to be precipitated. Prepare 50 ml of the recovered solution in advance The column was applied to a column of DE AE Sepharose Fast Flow (Pharmacia LKB) equilibrated with 5 mM phosphate buffer (pH 7.4), and 5 mM phosphate buffer (PH 7.4). Was eluted.

 The eluate was purified by an ultrafilter (YM10 membrane) manufactured by Amicon, to obtain a concentrated solution of 130 ml. The concentrated solution was dialyzed against 5 mM phosphate buffer (pH 7.0) to obtain a 132 ml protein solution. The obtained protein solution was adsorbed onto a column of CM Sepharose Fast Flour (Pharmacia LKB II) equilibrated with 5 mM phosphate buffer (PH 7.0), After washing with a phosphate buffer (pH 7.0), elution was carried out with a stepwise concentration gradient with 5 mM phosphate buffer (pH 7.0) containing NaCl.

Fractions containing the peptide were collected from the eluate, and after passing through a phosphate buffer, the activity was determined by the L929 test, and the specific activity and the recovery in each purification step were determined. In addition, the protein quantification was measured using a protein 'Arcey' system manufactured by Biorad. Table 1 shows the results.

Table 1 Protein port degree iff ¾ H-i «:

(ml) (mg / ml) (mg) (u / ml) (u) (%) (mg) ( fold) cell disruption fluid ^{500 3862.5 1.26X 10 B 6.3 X 10} '0 100 1.63 XIO 7 1 Bok o

J Ό J, NONO 4 «0J 5.75 7 ¾ 0 9 1 9 q 1 丄 n ^{B B} J 丄 Q Q o A 1 n ⁷ 1 · o ¾ 7 / renimine treatment

52 781.5 1.15X 10 ° 6 x 10 ¹⁰ 95 7.66 IO ⁷ 4.7 m

50 12.34 617.3 1.18X 10 ° 5.9 X 10 ' ⁰ 94 9.56 IO ⁷ 5.9

DEAE area 130 1.57 203.7 4 Χ 10 ^Β 5.2 X10 ¹⁰ 83 2.55 X 10 ^B 15.6

195.5 3.82Χ 10^Β 5.04X10^ID 80 2.58 X 10^B 15.8 DEAE 132

195.5 3.82Χ 10 ^Β 5.04X10 ^ID 80 2.58 X 10 ^B 15.8

X

 〇

CM division white 70 1.96 136.85 4.43X10 ⁸ 49 2.27 10 ⁸ 14

(8) Purity test of purified protein and confirmation of its structure:

 2 g of the purified protein was electrophoresed on 15% SDS PAGE, stained with Coomassie blue, decolorized, and then subjected to purity test using an LKB laser-scanner. As a result, the protein purified by the present invention was recovered at a purity of 95% or more.

 Example 2

 Construction expression of Ink LT

 (1) Acquisition of the trp promoter from the ptrpMLT vector:

 The pt r p M L T DNA obtained in Example 1 was replaced with a restriction enzyme BamHI.

 (Toyobo Co., Ltd.) After digestion with ClaI (Pharmacia LKB), the mixture was electrophoresed on a 1.5% low-melting point agarose gel, and a promoter region (gene 6) consisting of 129 bp was recovered. This gene 6 had the following nucleotide sequence.

 Gene 6

 GATCCCCCGGGCTGCAGGAATTCATTGTCCGACATCACAA GGGGCCCGACGTCCTTAAGTAACAGGCTGTAGTGTT

CGGTTCTGGCAAATAATCTGAAATGAGCTGTTGACAATTA GCCAAGACCGTTTATTAGACTTTACTCGACAACTGTTAAT

ATCATCGAACTAGTTAACTAGTACGCAAGTTCACGTAAAA TAGTAGCTTGATCAATTGATCATGCGTTCAAGTGCATTTT

AGGGTAT TCCCATAGC

(2) Construction of L T head

 From the following six oligonucleotides, a gene (逭 7) that encodes the head of LT was prepared while keeping the SD region and initiation codon separated by 13 bases.

(Oligonucleotide for preparation of Gene 7) (1) 5 '-CGAT AA GCTATGCTTCCAGGAAGT

(2) 3 '-T A T T C G A T A C G AA G G T C C T G A A C C

 A G A A

 (3) 5 '-A G G T C T T A C A C C A T C A G C T G C C C C A

 G

 (4) 3 '— C G T G G T A G T C G A C G G G T C T G A C G A

 G

 (5) 5 '-A C T G C T C G T C A G C A T C C G A A G A T G

 C A

 (6) 3 '-CAGGCTCGTAGGGCTTTCTT

Among the above-mentioned nucleotides, 0.1 OD of each of the oligonucleotides (2) to (5) is dissolved in sterilized water 151, and T4 polynucleotide kinase 40 is dissolved. Add 37 mM 0 mM ATP and 10 volumes of Kinase Buffer to bring the volume to 30 zl. The reaction was performed at C for 2 hours. After the reaction, 65. The enzyme was inactivated by heat treatment with C for 10 minutes. Then, combine 0.05 OD of each of the oligonucleotides (1) to (6) into a single tube, add an equal volume of annealing buffer, and react at 90 ° C for 3 minutes. It cooled down. To this, T4 DNA ligase (2000 units) (Takara Shuzo), 1M DTT11, 10mM ATP10 ^ 1, and annealing ring buffer 71 were added, and the mixture was added. Then 16. The mixture was reacted for 16 hours with C, electrophoresed on a 10% PAGE gel, stained with EtBr, the target gene 7 was excised, eluted in an elution buffer, and desalted. The nucleotide sequence of the obtained gene 7 is as follows.

 Gene 7

CGATAAGCTATGCTTCCAGGAGTAGGTCTTACACCAT TATTCGATACGAAGGTCCTCATCCAGAATGTGGTA

CAGCTGCCCAGACTGCTCGTCAGCATCCGAAGATGCA GTCGACGGGTCTGACGAGCAGTCGTAGGCTTCT (3) Acquisition of LT gene:

 After cleavage of ptrpMLTDNA using a restriction enzyme Nsil (NEB)-Hindm (Toyobo), it was electrophoresed on a 1% low melting point agarose gel, and a gene consisting of 465 bp (gene 8) was recovered.

 (4) Cutting the blue script M 13 S Κ + (+):

 Blue script M13SII (+) 2 {JLS (Toyobo) was cut with restriction enzyme BamHI-Hind EI (Toyobo), and then electrophoresed on a 0.8% low melting point agarose gel. After EtBr staining, the desired plasmid DNA (gene 9) consisting of 2.9 kb was obtained.

 (5) Construction of a plasmid that possesses the trp pro 乇 overnight and LT

 T4 DNA ligase was generated for genes 6, 7, 8, and 9 to obtain a plasmid having the LT gene (pBTrpLT). Further, the HB101 strain for transformation, which had been frozen and stored at -70, was thawed, and 20 ng of pBTrpLT was added to the suspension lOOJl. After 45 minutes of ice-cooling, a heat shock was applied at 42 ° C for 90 seconds, followed by ice-cooling for 2 minutes. To this, add S0C medium 9001 and incubate at 37 for 1 hour.Pour the suspension onto LB agar plate containing O.lmgZml of ampicillin, and place it at 37 ° C. After culturing overnight at C, a transformant having pB trp LT was obtained. DNA was isolated from this transformant, digested with the restriction enzyme BamHI-Hindm, electrophoresed on a 1% agarose gel, and subjected to EtBr staining to a 665 bp fragment. Was confirmed.

 (6) The current of p B t r p L T:

The expression of the protein under the condition of pB trp LT at 37 ° C was confirmed. A single colony of pB trp LT was transferred to M9CA medium containing 2.5 tg / ml tritophan (M9 minimal medium, 1% glucose, 1 mm MMgSC, 0.1 mM MCaCl). ₂ , 0.5% casamino acid, 0.1 m & / m 1 thiamine monohydrochloride, 0.1 mg gZm l ambisilin, 100 mM MH epes) and cultured overnight at 37 ° C. For example intends 1 Roh 5 0 amount the next day the new M 9 CA medium, OD _BD. After culturing to 0.5, 3> ff-indoleacrylic acid (20 gZml) was added, and the cells were further cultured at 37 ° C for 16 hours. After completion of the culture, 1 ml of the bacterial cell fluid was transferred to a 1.5 ml centrifuge tube, and the cells were obtained by centrifugation. The cells were lysed in 200 1 SDSPAGE buffer, heat treated at 90 ° C for 5 minutes, and electrophoresed on 15% SDSPAGE. After the electrophoresis, the cells were stained with Kumashi Blue and then decolorized. After decolorization, LKB laser scanner was used to measure the expression level of LT per total protein, and it was confirmed that 10% of LT protein was expressed per total protein under the culture condition of 37 ° C. It was done. This corresponds to a protein content of 10 to 20 mg per liter under the culture condition of 37 ° C.

 (7) Protein production and purification:

pB trp LT was cultured overnight in 100 ml of M9CA medium containing 2.5 g / ml tryptophan, and the culture was inoculated into 3.51 M9CA medium. The _cells were cultured at 37 ° C until OD _BDD = 0.5. Thereafter, 3β-indoleacrylic acid was added to a final concentration of 20 pg / m 1, and the cells were further cultured for 16 hours, followed by centrifugation to collect the cells.

 The obtained bacterial cells (15, 95 g / wet) were added to 30 mM NaCl and

 O. lm Mp — The suspension was suspended in 300 ml of 50 mM Tris-hydrochloric acid (pH 8.0) containing APMSF, and this suspension was placed in a high-pressure homogenizer (RANNIE) for 600 p.m. The cells were disrupted three times by si.i. Further, the inside of the high-pressure homogenizer was washed with 200 ml of the same buffer, and the recovered liquid and the disrupted liquid were centrifuged to obtain a crude extract.

Polyethyleneimine is added to 500 ml of this crude extract to a final concentration of 0.1%, stirred at 10 minutes for 10 minutes, centrifuged, and nucleic acid is removed. Was. Collect 480 ml of the supernatant, add ammonium sulfate to 50% saturation, and stir at 4 ^{eC for} 5 hours. Subsequently, centrifugation was performed to obtain a crude protein as a precipitation source. This protein was dissolved in 45 ml of 5 mM phosphate buffer (PH 7.4) and dialyzed against the same buffer. After further centrifugation, insoluble proteins were removed, and 52 ml of the supernatant was recovered. Further, a heat treatment was performed at 50 ° C. for 10 minutes to remove proteins to be precipitated. 50 ml of the recovered solution was applied to a column of DE AE Sepharose Fast Flow (Pharmacia LKB), which had been flattened with 5. mM phosphoric acid buffer solution (PH 7.4). Elution was performed with M phosphate buffer (PH 7.4).

The eluate was concentrated using an ultrafilter (YM10 membrane) manufactured by Amicon, to obtain 130 ml of a concentrated solution. This concentrated solution was dialyzed against 5 mM phosphoric acid buffer solution (PH 7.0) to obtain 132 ml of a protein solution. The protein solution was adsorbed onto a column of CM Sepharose Fast Flow (Pharmacia LKB 钍) equilibrated with 5 mM phosphate buffer (pH 7.0), and the solution was treated with 5 mM phosphate buffer (PH After washing with 7.0), elution was carried out with 5 mM phosphate buffer (pH 7.0) containing NaCl according to a stepwise concentration gradient. The fraction containing the peptide was collected, dialyzed against a phosphate buffer, and the activity was determined by an L929 test, and the specific activity and the recovery in each purification step were determined. In addition, the protein quantification was measured using a Protein Attay system manufactured by Piolado. Table 2 shows the results.

Table Ϊ

(8) Purity test for purified protein:

 Purified protein 2; Cig was electrophoresed in 15% SDS PAGE, stained with Coomassie blue, decolorized, and tested for purity with an LKB laser scanner. As a result, the protein purified by the present invention was recovered with a purity of 95% or more.

 Example 3

 Construction and expression of recombinant LT (TLT)

 (1) Acquisition of the trp promoter from the trpMLT vector:

 In the same manner as in (1) of Example 2, a promoter region (gene 6) of 129 bp was obtained from PtrpMLTDNA.

 (2) Construction of L T head:

 From the following four oligonucleotides, a gene (gene 10) was prepared which kept the SD region and the initiation codon at a distance of 13 bases and simultaneously encoded the head of LT.

 (Oligonucleotide for preparation of gene 10)

 (1) 5 '-CGA T AA G C T AT G C C T G C C C GA

 (2) 3 '-T A T T C GA T A C G GA C G GG T C T G A C G

 G

 (3) 5 '-A C T G C C C G T CA G CA C C C C AA GAT G

 C A

(4) 3'-GCAGTCGTGG GG TTCT Of the above oligonucleotides, 0.1 OD of each of (2) and (3) is dissolved in 15 l of sterile water, and T4 polynucleotide kinase, 40 0 mM MATP, 10 times the volume of kinesin buffer was added to make 30 ^ 1, and reacted at 37 ° C for 2 hours. After completion of the reaction, heat treatment was performed at 65 ° C for 10 minutes to inactivate the enzyme. Thereafter, 0.0550 D of each of the oligonucleotides (1) to (4) is combined into one tube, and annealing buffer is applied. An equal volume of the solution was added, and the mixture was reacted at 90 ° C for 3 minutes and cooled slowly. To this was added 200 units of T4 DNA ligase (Takara Shuzo Co., Ltd.), 1 MDTT 1 ^ 1, 10 mM MATP 10 jl, and 7 il of Annealing buffer, and the mixture was adjusted to 100 / il. Then, react at 16 ° C for 16 hours, run on a 10% PAGE gel, stain with EtBr, cut out the target gene 10 and elute it in the elution buffer. Desalted. The obtained gene 10 had the following nucleotide sequence.

 Gene 1 0

 CGATAAGCTATGCCTGCCCAGACTGCCCGTCAGCACCCC

TATTCGATACGGACGGGTCTGACGGGCAGTCGTGGGG

AAGATGCA TTCT

(3) Acquisition of LT gene:

 PtrpMLTDNA was digested with a restriction enzyme Nsil (NEB) — HindII (Toyobo), and then electrophoresed on a 1% low-melting point agarose gel to recover a gene consisting of 4655 bp (gene 8).

 (4) Cutting Blues Script M13SKII (+):

 Bluescript M13S Π (+) 2 / g (Toyobo) was digested with restriction enzyme BamHI-Hindll and then electrophoresed on a 0.8% low-melting-point agarose gel. After the r-staining, the desired plasmid DNA (gene 9) consisting of 2.9 kb was obtained.

 (5) Construction of a brasmid containing the trp promoter and TLT:

T4 DNA ligase was allowed to act on genes 6, 8, 9, and 10 to obtain a plasmid (pB trp TLT) having the TLT gene. Further, the HB101 strain for transformation, which was frozen and stored at 170 ° C, was thawed, and 200 ng of pBTrp TLT was added to 100 μl of this suspension. did. After 4 5 minutes water cooled, 4 give 9 0 seconds heating preparative tio click at 2 ^e C, cooled for 2 minutes on ice. Add 900 ^ 1 SOC medium and leave at 37 ° C for 1 hour After the culture, the suspension was spread on an LB agar plate containing 0.1 ml / ml of ampicillin, and cultured at 37 ° C overnight to obtain a transformant having pBTrp TLT. . DNA was isolated from this transformant, cut with the restriction enzyme BamHI—HindlE, and then electrophoresed on 1% agarose gel. The fragment of 637 bp was confirmed by EtBr staining. did.

 (6) Expression of pBtrpTLT:

For PB trp TLT, protein expression was confirmed at 37 ° C. A single colony of p B trp TLT, 2. 5 gZm l preparative Ributofan containing M 9 CA medium (M 9 minimal media, 1% glucose, lm MM g SO "0. 1 m MC a C l 2, 0.5% casamino acid, 0.1 mg / ml thiamine-hydrochloric acid, O. lm gZm l ampicillin, Ι π πιΜ Hepes) and cultured overnight at 37 ° C. _Add 1> _{50 to M9CA} medium, culture to OD _BaD = _0.5 , _add 3> 5-indoleacrylic acid 20_gZml, and further at 37 ° C for 16 hours After completion of the culture, 1 ml of the bacterial cell fluid was transferred to a 1.5 ml centrifuge tube to obtain cells by centrifugation, and the cells were dissolved in 200 il of SDSPAGE buffer. The mixture was heat-treated at 90 ° C for 5 minutes and electrophoresed on 15% SDS PAGE.After completion of electrophoresis, the cells were stained with Kumashi Blue and then decolorized. When the expression level of TLT was measured It was confirmed that 10% of the TLT protein (the peptide of the present invention) was expressed per 10% of the total protein under the culture condition of 37 ° C. This was 10 to 20 mg per liter under the culture condition of 37. Of protein.

 (7) Production and purification of protein:

PB trp TLT was cultured overnight in 200 ml of M9CA medium containing 2.5 gZm1 tritophan, and this culture was inoculated into 71 M9CA medium, and OD _BDD = 0, The cells were cultured at 37 ^eC until 5. Then add 3 yff —indoleacrylic acid to a final concentration of 2 Q p.S / ml After culturing for another 16 hours, the cells were collected by centrifugation.

 The obtained bacterial cell 16.92 gZwet (3.5 1 min) was transformed with 50 mM Tris-hydrochloric acid (pH 8.0) 120 containing 30 mM NaCl and O.lmMP-APMSF. The suspension was then passed through a high-pressure homogenizer (RANNIE) three times at 600 psi to destroy the cells. Further, the inside of the high-pressure homogenizer was washed with 120 ml of the same buffer, and the recovered liquid and the disrupted liquid were centrifuged to obtain a crude extract.

The crude extract 2 5 O ml to as the final concentration was added to port Riechiren'i Mi emissions to be 1% 0. After stirring for 1 0 minutes at 4 ^e C, centrifuged, subjected to removal nucleic acid Was. Collect 235 ml of the supernatant, add ammonium sulfate to 50% saturation, stir at 4 ° C for 4 hours, centrifuge, precipitate as crude protein I got This crude protein was dissolved in 45 ml of 5 mM phosphate buffer (PH 7.4) and dialyzed against the same buffer. After further centrifugation, the insoluble protein was removed, and 64.5 ml of the supernatant was recovered. The supernatant was subjected to a heat treatment at 50 ° C. for 10 minutes to remove precipitated proteins. 6 3.5 ml of the recovered solution was applied to a column of DEAE Sepharose Fast Flow (Pharmacia LKB) equilibrated in advance with 5 mM phosphate buffer (PH 7.4), and 5 mM phosphate was added. Elution was carried out with an acid buffer (pH 7.4).

The eluate (flow-through fraction) 2501111 was adjusted to 117.0. The resulting protein solution was adsorbed on a column of CM Sepharose Fast Flow (Pharmacia LKB), flattened with 5 mM phosphate buffer (PH 7.0), and 5 mM phosphate was added. After washing with a buffer solution (PH 7.0), elution was carried out with a stepwise concentration gradient using 5 mM phosphate buffer (pH 7.0) containing NaCl. Fractions containing the peptide were collected, dialyzed against a phosphate buffer, and the activity was determined by an L929 test. The specific activity and the recovery in each purification step were determined. The protein quantification was measured using a protein assay system manufactured by Piolado. Was. The results are shown in Table 3.

Table 3

t

CO

(8) Purity test for purified protein:

 After 2 µg of the purified protein was electrophoresed with 15% SDS PAGE, the protein was stained with Coomassie-I and decolorized, and then the purity was assayed with an LKB laser scanner. As a result, the peptide of the present invention was recovered with a purity of 95% or more.

 Example 4

 (1) Isolation of trp promoter overnight and MLT structural genes

 pB trp MLT plasmid DNA was digested with restriction oxygen EcoRI—Hindm, electrophoresed on a 1% low melting point agarose 'gel, and stained with EtBr. The gene 11) was obtained. FIG. 4 shows the nucleotide sequence and the peptide of the present invention which it encodes.

(2) p uc 9 vector vector

 After cutting Puc92iis (Pharmacia LKB) with the limiting oxygen Eco EI — Hindm, run on a 0.8% low melting point agarose 'gel, stain with EtBr, and purify the target 2.7 Kb. Was obtained (gene 12).

 (3) Construction of a brassmid that possesses trp bromo overnight and MLT

A T4 DNA ligase was allowed to act on genes 11 and 12, and a brassmid (p9trpHLT) having the MLT gene was obtained. Further, the HB101 strain cells for transformation which had been frozen and stored at −70 ° C. were thawed, and 20 ng of p9 trp MLT was added to the suspension 1001. After 45 minutes the reaction water cooling, 4 2 ^e C with give 9 0 seconds human one preparative sucrose Uz click, SOC medium was added 9 0 cooled with 2 minutes ice, after 1 hour incubation at 37 hand, suspension 0 Transformed on a LB agar plate containing lmgZml of ampicillin and cultured overnight at 37 ° C to obtain a transformant having genes 11 and 12. DNA was isolated from this transformant, cut with the restriction enzyme EcoRI-Hindm, electrophoresed on a 1% agarose gel, and confirmed as a 629 bp fragment by EtBr staining. (Gene 11).

 (4) Expression of p9trpMLT

 Protein expression of p9trpLT under the condition of 37 ° C was confirmed.

A single colony of P9 trp MLT was added to an M9CA medium containing 2.5 g ZmI tritophan (M9 minimal medium, 1% glucose, 1 mm Mg S O-, 0.1 m MC a C l _s, 0.5% Kazami Roh acid, 0. 1 mg / ml Chia Mi down one HC 1, 0. 1 mg / in 1 Anne Pishi Li down, l OO m MH epes) Niu decorating, 3 7. C. overnight. The next day, it intends 1 5 0 amount to the new M 9 CA medium For example, OD _B. After culturing to ₀ = 0.5, 3> 5-indole acrylic acid SO ^ gZml was added, and the cells were further cultured at 37 ° C for 16 hours. After completion of the culture, 1 ml of the bacterial cell fluid was transferred to a 1.5 ml centrifuge tube, and the cells were obtained by centrifugation. The cells were lysed in 200 SDSPAGE buffer, heat-treated at 90 ° C for 5 minutes, and electrophoresed on 15% SDSPAGE. After the electrophoresis, the cells were stained with Coomassie blue and then decolorized. After decolorization, the amount of MLT expressed per total protein was measured with an LKB laser scanner. As a result, under the culture conditions of 37, expression of 10% MLT protein per total protein was confirmed. This corresponds to a protein content of 10 to 2 Omg per one under the culture condition of 37 ° C.

 (5) Activity measurement

The remaining lml of the culture, the expression of which was confirmed in (4), was separated by centrifugation. The cells were suspended in lml of PBS (-) buffer solution, sonicated for 10 minutes, and then centrifuged. By separation, a protein supernatant was obtained. After the protein supernatant through a 0.2 2 Mi black down the full I le evening one, for comparison in the same L 9 2 9 cytotoxicity test, bacteria fluid lml those Ri 1 X 1 0 ⁶ unit / ml of the active Was confirmed.

 Reference example

 (1) Isolation of a phage containing human lymphotoxin DNA:

(a) Immobilization of phage on filter Human Bok lymphoid cell DNA library one (EMBL 3 phage) the E. coli NM 5 3 8 each 3 1 0 ^lambda PFU plated on 2 0 Like LB blade preparative using, 3 7. After overnight culture in C, the phage was transferred to BA85 Nitrocellulose Fill 1 32 (5 (S & S)) and the phage on the transferred filter was added to 1.5 M sodium chloride. Then, the mixture was denatured with 0.5 M sodium hydroxide solution, and the filter was neutralized with sodium chloride and 0.5 M tris-hydrochloric acid buffer (PH 8.0). Thereafter, the phage DNA was immobilized on a filter by vacuum drying at 80 ° C for 2 hours [Maniatis T, Molecular cloning], p3 2 1 (1 9 8 2 )] ₀

 (b) Creating a probe

 It is a part of the previously reported base sequence of the lymphotoxin gene [Gray, Naturae, ρ312, 721 (11984)]

 (1) 5 '-A T GA CA C CA C C T GAAC G T C T C

 (2) 3 '-C C T C G GAA G C GAGA C A T C

 Was synthesized using a DNA synthesizer by the amidite method, and used as a probe for human genome lymphotoxin. Probe labeling was performed using these oligodeoxynucleotides at a final concentration of 1 O O pmo 1 l O O iaM Tris-HCl buffer (pH 8.0), 10 mM magnesium chloride, 5 mM DTT,

^{1.8 5 MB q (r - 32} P) ATP, 5 unit T 4 port consists Li quinuclidine Leo Chi Dokinaze solution 3 0 added during 1, 3 7, allowed to react for 45 minutes and re-phosphorylated by, further, the enzyme To inactivate, the treatment was carried out at 65 ° C for 10 minutes.

 (c) Detection of target phage

The probe labeled according to the above (b) was individually associated with the filter to which the DNA of the above (a) was fixed. This association reaction was performed using a 6-fold concentration of SSC (0.15 M sodium chloride-0.015 M sodium citrate) containing a 277.5 KB q-labeled probe, and a 5-fold residue. Degree of den A solution consisting of a heart solution (Denhardt's), 0.1% SDS and 200 μg / ml denatured salmon sperm DNA. After immersion for 6 hours, after the reaction, the filter was immersed in 6 XSSC-0.1% SDS solution three times at 40 ° C for 15 minutes, washed, and further washed with 0.2 XSSC-0.1% SDS solution. Washed by immersion 3 times at 0 ° C.

Radioautograms were taken from the washed filters, and plaques that responded to the probe were searched for by overlaying a set of two Replicap filters with radioautograms. Ri by this method, Ri O 6 X 1 0 ⁵ plaques to afford two EMBL 3 full Aji to respond to the probe.

 (2) Isolation of the human lymphotoxin DNA fragment:

 From these phages, the phage DNA was extracted and purified by a grise mouth-step gradient method [ManiattsT, Molecuulararcloning, p83 (1992)].

 The obtained DNA was cleaved with restriction enzyme EcoRI (manufactured by Toyobo), separated by 0.5% agarose gel electrophoresis, and the DNA fragment was separated from the agarose gel by a Southern blot method using a BA852-torose cell mouth. Transcribed on a surface filter (S & S). The probe used in (1) was associated with this filter to obtain a reacted 2.4 kb DNA fragment. This DNA fragment completely contained the signal peptide portion of the human photophosphotoxin to the termination codon.

 (3) Construction of a human lymphotoxin structural gene:

 (a) Preparation of gene a

The DNA fragment recovered as described above is digested with the restriction enzyme Rsll (NEB), and a DNA fragment of 528 bp containing a part of exon 4 of the lymphotoxin gene (hereinafter referred to as “DNA fragment”). The gene a) was isolated. This gene is shown below. 5 '-GACCGTGCCT TCCT CCAG

 3'— GCACGGAAGGAGGTC

CCTCAGCCCTAGTACTGTCTTCTTTGGAGCCTTCGCTCTGTAGA GGAGTCGGGATCATGACAGAAGAAACCT CGGAAGCGAGACATCT CCGACGTGAGCTACATGGTGCCCCGACGCAAGGT CGAGTGGGT C

CCTCAGCCCTAGTACTGTCTTCTTTGGAGCCTTCGCTCTGTAGA GGAGTCGGGATCATGACAGAAGAAACCT CGGAAGCGAGACATCT

ACTT GGAAAAAT C CAGAAAGAAAAAATAAT TGATTT CAAGAC C T TGAACCTTTTTAGGTCTTTCTTTTTTATTAACTAAAGTTCTGGA

TCTCCCCATTCTGCCTCCATTCTGACCATTTCAGGGGTCGTCAC AGAGGGGTAAGACGGAGGTAAGACTGGTAAAGTCCCCAGCAGTG

CACCTCTCCTTTGGCCATTCCAACAGCTCAAGTCTTCCCTGATC GTGGAGAGGAAACCGGTAAGGTTGTCGAGTTCAGAAGGGACTAG AAGTCACCGGAGCTTTCAAAGAAGG

 TTCAGTGGCCTCGAAAGTTTCTTCCTTAA

(b) Preparation of gene b

 The rest of exon 3 and exon 4 of the lymphotoxin gene were synthesized using one or two oligodoxynucleotides (1) to (12) below, which were synthesized organically using a DNA synthesizer. And synthesized.

 (1) 5 '-A A T T C A T G C T C C C T G G T G T T G G

(2) 3 '-G T A C G A G G G A C C A C AA C C G G A G T G

 T G

 (3) 5 '-C C T C A C A C C T T C A G C T G C C C A G A C

 T

 (4) 3 '-G AA G T C G A C G G G T C T G A C G G G C A G

 T

 (5) 5 '-G C C C G T C A G C A C C C C AA G A T G C A T

 C T T G C C

 (6) 3 '-C G T G G G G T T C T A C G T A G AA C G G G T

 G T C G T G

 (7) 5 '-C A C A G C A C C C T C A AA C C T G C T G C T

 C

 (8) 3 '-G G A G T T T G G A C G A C G A G T G G A G T A

 A

 (9) 5 '-A C C T C A T T G G A G A C C C C A G C A A G C

 A G

 (10) 3 '-C C T C T G G G G T C G T T C G T C T T G A G T

 G A

 (11) 5 '-A A C T C A C T G C T C T G G A G A G C A A A C

 A C G

(12) 3 '-CGAGACCTCTCGTTTGTGCCT G That is, 800 pmo1 of each of the above oligopeptides was dissolved in 15 ^ 1 of sterile water, and T4 polynucleotide kinase, 400 mM ATP, 10 times the volume of kinase buffer The solution was added to adjust the solution to 501, and reacted at 37 ° C for 2 hours. After that, the enzyme was destroyed by treatment at 65 ° C for 10 minutes, the 12 oligodeoxynucleotides were combined into one tube, and after concentration, Sephadex G50 gel Desalting and unreacted phosphoric acid were treated using a filtration column (Pharmacia LKB). After desalting and recovery, the solution was further concentrated, dissolved in 50 il of annealing buffer, reacted at 90 ° C for 3 minutes, and cooled slowly. To this was added a ligation buffer solution equivalent to 2000 units of T4 DNA ligase (Takara Shuzo), and the mixture was allowed to react overnight at 16 ° C.

 After completion of the reaction, the mixture was subjected to 5% acrylamide gel electrophoresis. After staining with ethidium bromide, a 158 bp DNA fragment (hereinafter referred to as “gene b”) was cut out along with the gel and dissolved in an elution buffer. Elution was performed, and the Sephadex G25 gel filtration column (Bulmasia LKB) was desalted and concentrated.

 This gene b is shown below.

Five'-.

3'- GTACGAGGGACCACAACCGGAGTGTGGAAGTCGACGG

ACTCACTGCTCT GGAGAG CAAACAC G TGAGTGACGAGACCTCTCGTTTGTGCCTG

(c) Binding of gene a and gene b and their amplification

Bluescript M1 3 KS (+) Restrict vector vector (Toyobo) After digestion with the enzyme EcoRI, the phosphoric acid at the 5 'end is removed by treatment with alkaline phosphatase, and the mixture of this and the gene a and gene b prepared as described above is added to the mixture. By reacting with T4 DNA ligase, plasmid 1 (blue script M13 KS (+) — gene a—gene b) having a structural gene for human lymphotoxin was obtained. Was. Further, E. coli XL1 blue strain cells for transformation, which had been frozen and stored at 170 ° C., were thawed, and 20 ng of plasmid 1 was added to the suspension 1001. After reacting for 45 minutes under water cooling, a heat shock was given at 42 for 90 seconds, and the mixture was further ice-cooled for 2 minutes. To this, add SOC Medium 900-1 and incubate at 37 ° C for 1 hour, then place the suspension on an LB agar plate containing 100 S / ml ampicillin. Then, the cells were cultured overnight at 37 ° C. to obtain transformants having the genes a and b (transformants ab). Next, the blues script M13KS (+)-genea-geneb was separated from this transformant, cut with the restriction enzyme EcoRII, and then subjected to low melting point agar electrophoresis. A 687 bp human structural gene for binding to the gene a and b was separated and recovered.

 () Construction of modified rephotoxin:

 (a) Preparation of structural gene fragments

 The gene a-b obtained in the above (3) was cut with a restriction enzyme PVuII (Toyobo) to obtain a DNA fragment of 6553 bp (hereinafter, referred to as gene c).

 The gene c is shown below.

5 '-CTGCC 3'—GACGG DVlOVOODVVOVOiXDVOOiOiiDDVViODOOVVVDOVDVDOID

z ε 00 / ∑6df / JOd £ 0 l / £ 6 OM AAGTCACCGGAGCTTTCAAAGAAGG

Next, the following two oligodeoxynucleotides, which were designed to have an EcoRI site at the head and ATG as a start codon, were synthesized, and T4polynucleotides were synthesized. The 5 'terminus was phosphorylated using leotide kinase.

 (1) 5 '-AA T T C A T G AA T T A C G A G T T C C

(2) 3 '-G T A C T T AA T G C T C AA G G

 Thereafter, the oligonucleotide was desalted and concentrated, dissolved in an annealing buffer, treated at 90 ° C. for 3 minutes, then slowly cooled and annealed to obtain gene d.

 The gene d is shown below.

5 '-AATTCATGAATTACGAGTTCC

3 '-GTACTTAATGCTCAAGG

(b) Preparation and amplification of structural gene

 Blues script M13KS (+) vector (Toyobo) was cut with the restriction enzyme EcoRI, and the 5'-terminal phosphoric acid was removed by treatment with alkaline phosphatase. Then, this is mixed with gene c and gene d, and T4 DNA ligase is allowed to act thereon to produce plasmid 2 having gene c and gene d (Blues Ml 3 KS (+) — Gene c—gene d) was obtained.

Furthermore, to melt the rough or Jimeichi 7 O ^e C transforming E. coli XL 1 Blue a share cells which had been frozen at, brass Mi de 2 2 0 in the virtual cloudy solution 1 0 0 1 ng was added. After reacting for 45 minutes under ice-cooling, a heat shock was applied at 42 ° C for 90 seconds, followed by further ice-cooling for 2 minutes. After adding SOC Medium 900 ^ 1 to this and incubating at 37 ° C for 1 hour, the suspension was placed on an LB agar plate containing lOO ^ gZml of ampicillin. And then cultured overnight at 37. As a result, a transformant having the gene c and the gene d (transformant c-d) was obtained. Next, the blue script M13KS (+) — gene c-gene d was isolated from the transformant c-d, cut with the restriction enzyme EcoRI, and then subjected to low melting. at point agar electrophoresis, structural gene 6 7 3 b _P gene c and gene d are bonded (modified Li lymphotoxin butoxy down structural gene; gene c - d) were separated and recovered.

 The genes cd were as shown in FIG.

 5 '-AATTCATGAATTACGAGTTCCCTGCC 3 "-GTACTTAATGCTCAAGGGACGG

GGATGAGAGGGTTCCGGTGGAGGAGGGGTGAGATGGACCGGGTA

CTCCAGGTCGAGAAGAGGAGGGTCATGGGGAAGGTACACGGAGA

AAGTCACCGGAGCTTTCAAAGAAGG

TTCAGTGGCCTCGAAAGTTTCTTCCTTAA

 (5) Preparation of modified lymphotoxin expression vector:

 The expression vector pkk2 2 3 — 3 vector (Pharmacia LKB) was digested with the restriction enzyme EcoRI, and the 5′-terminal phosphoric acid was removed by treatment with alkaline phosphatase. Of the gene c-d and T4 DNA ligase to produce plasmid 3 (pkk22-3 — 3 — MLTDN

A) was obtained.

 Test example 1

In MEM medium containing 1 0% FCS, L 9 2 9 cells were suspended to be 2 X 1 0 ⁵ cells / ml of镍度, which one 0 0 ^ 1 to 9 6-well plates dispensing Then, the cells were cultured at 37 ° C for 5 hours. Then, the peptide of the present invention 101 and serially diluted 10-fold and actinomycin D (2.5 g- / m 1) l O jl were added, and cultured at 37 for 18 hours. did. Furthermore, add MTT (5 mg / m1) 201 After culturing at 37 ° C for 5 hours, 10% SDS-0.01N hydrochloric acid IOOI was added, and the mixture was further incubated at 37 ° C for 18 hours to lyse the cells. . After lysis, the absorbance at 540 to 690 nm was measured using a Thai Yuichi Check Multiscan (Fuchiichi Laboratories) to confirm the survival of the L920 cells.

The amount of biological activity required to kill 50% of L929 cells was defined as 1 unit, and the antitumor activity of the peptide of the present invention was determined. 9.48 was X 1 0 ⁷ units / mg.

 Test example 2

 The stability of the lymphotoxin (recombinant lymphotoxin) purified from the body of Escherichia coli and the peptide of the present invention at 4 ° C. was tested as follows.

 That is, two kinds of samples were prepared at a concentration of 550 g Zml of the recombinant lymphotoxin and 310 g / ml of the peptide of the present invention. Immediately after the preparation and two months later, each sample was electrophoresed with 15% SDS-PAGE, stained and decolored, and the purity was measured with an LKB laser-scanner.

 The purity of the lymphotoxin and the peptide of the present invention in the electrophoresis immediately after the preparation was 95% or more for the lymphotoxin and 95% for the peptide of the present invention. The lymphotoxin stored for months and that of the peptide of the present invention had a purity of 0% lymphotoxin and 60% of the peptide of the present invention.

Lymphotoxin is originally a protein with a molecular weight of 18,800, but analysis by swimming after storage for two months shows that it is a protein with a molecular weight of 17,000 and 16,000. Degraded, there was no lymphotoxin protein with a molecular weight of 18,800. On the other hand, the peptide of the present invention is a protein having a molecular weight of 180,000, and after storage for 2 months, has a molecular weight of 17,000 and a molecular weight of 16,000, similar to lymphotoxin. A decomposition product of 0 was confirmed, but the peptide of the present invention having an original molecular weight of 18,000 Protein was also present at 60%.

 From this, it was confirmed that the peptide of the present invention was excellent in storage stability as compared with that of linhotoxin.

 Test example 3

 An SD female rat (body weight: 197 to 21.3 g) was anesthetized by intraperitoneal administration of 5 ml of 25% urethane and fixed in the dorsal position. The experiment was performed after about 10 minutes with the jugular vein visible. Administration of each protein and blood collection were performed from the right jugular vein. The dose of each protein was 250 g rat. Immediately after administration to the rat (0 minute), 5, 10, 15, 20, 40, 60, and 90 minutes later, approximately 0.15 ml of blood is collected, and the blood is promptly collected at each blood collection. Suction into two capillaries (made by DRUMMOND, length for micro-macro-clit: 75 mm) and centrifugation (Kubota-macro-climate KH-120) Using 0 S and centrifuged at 12 000 rpm for 3 minutes). This serum was serially diluted, and its blood activity was examined by an L929 cytotoxicity test.

 As a result, both the recombinant lymphotoxin and the peptide of the present invention were 12 minutes, and the peptide of the present invention could maintain almost the same blood activity as the lymphotoxin. confirmed.

Incidentally, the side effects of antihypertensive such as in the above experiments is not observed, also, LD _5D value of the present invention Bae butyrate de was separately by measurement, 1 X 1 0 ^B ukg more (mouse, intravenous) der is, It was also confirmed that the safety was high.

 Industrial availability

 According to the present invention, it has become possible to express 10% or more of natural LT, which conventionally could only be expressed at about 1 to 2%.

In particular, when the gene encoding the peptide obtained in Example 1 (the peptide of the present invention) having high homology to natural lymphotoxin (the peptide of the present invention) is used as the LT structural gene, a high concentration of 37% is obtained. It was possible to express. Therefore, the present invention paves the way for the industrially advantageous production of LT and its clinical use, and is extremely useful.

Claims

The scope of the claims  1. It has an SD region 13 bases upstream of the start codon, the base sequence of the 10 region is TT AACT, the base sequence of the 135 region is TTGACA, and Lymphotoxin expression vector with a size of 2.7 to 3.1 kb excluding the gene to be read.  2. It has an SD region 13 bases upstream of the start codon, the base sequence of the 10 region is TT AACT, the base sequence of the 135 region is TTGACA, and is a lymphotoxin. Transformation of host microorganisms with a lymphotoxin expression vector of 2.7 to 3.1 kb excluding the gene encoding Characteristic method for producing lymphotoxins.  3. The method for producing lymphotoxins according to claim 2, wherein the culturing is performed in the presence of 3β-indoleacrylic acid.  4. The following amino acid sequence A sn-T yr-G lu-P he-P ro-A la-G ln-T hr — A la— A rg— G ln— H is — P ro — L ys — M et-H is — L eu — A la-H is — Ser — T hr — L eu — Lys-Pro-A la-A la-H is — L eu — I le — G ly — A sp — P ro — S er — L ys — G in— A sn-Ser-L eu-L eu-T rp-A rg ^, -A la-A sn-T hr-A sp-A rg-A la-P he-L eu-G ln -A sp -G ly -P he -Ser -L eu -S er -A sn—A sn—S er —L eu -L eu—V al —P ro —T hr —S er —G ly—I le-T yr-P he-V al-T yr — S er — G in— V al-V al — P he — S er — G ly-Lys-A la-T yr-S er-P ro- L ys-A la — T hr — S er — S er — P ro —: L eu — T yr — L eu-A la-H is-G lu-V al-G ln-L eu-P he — S er — S er — G in — T yr — P ro — P he — H is-V al-P ro-L eu-L eu-S er-S er-G ln-Lys-Met-V al-T yr-Pro-G ly-L eu-G ln-G lu -P ro-T rp-L eu-H is-Ser-M et — T yr-H is-G ly-A la-A la-P he-G ln-L eu-T r-G ln-G ly-Asp-Gln-Leu-Ser-Thr-His-Thr-Asp-G1y-Ile-Pro-His-Leu-Val-Leu-Ser -P ro-Ser-T hr-Val-P he-P he-G ly-A la-P he-A la-Le A peptide represented by