JPS6363392A - Lymphotoxin-like polypeptide - Google Patents

Abstract

PURPOSE:To provide a mutant-type lymphotoxin devoid of an arbitrary series of amino acid sequence between the N-terminal of a natural lymphotoxin and the site near the 89th lysine and easily usable as a remedy for tumor. CONSTITUTION:It has been found that the cytotoxic activity of lymphotoxin is maintained even in a lymphotoxin wherein the whole amino acid sequence between the N-terminal of natural lymphotoxin to the 89th lysine is deleted. Furthermore, it has been found that the cytotoxic activity of lymphotoxin is maintained even by substituting the 89th lysine with different kind of amino acid such as a neutral amino acid (e.g. methionine). Accordingly, it is preferable to use the polypeptide of formula wherein the amino acid sequence from about 10th to about 34th sites is deleted and a non-natural amino acid sequence is inserted therein.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a mutant lymphotoxin-like polypeptide, a method for producing the same, a DNA encoding the lymphotoxin-like polypeptide, a plasmid comprising the DNA, and a method for producing the same. This relates to E. coli transformed with this plasmid.

[Conventional technology]

Lymphotoxin is an antitumor substance produced by lymphocytes. This substance inhibits the growth of tumor cells, but has little effect on normal cells, and has properties such as inhibiting cell transformation caused by chemical carcinogens and ultraviolet irradiation, so it is expected to be used as an anticancer agent. (Th mic Ho
rmones & Lm hokines (1984
), 357). In addition to antitumor activity, lymphotoxin also has various lymphokine activities.
ology Today (1985) 6-5156)
.

Aggarwal et al.
Two types of lymphotoxin were obtained, one whose terminal starts with leucine and one whose terminal starts with histidine at position 24 (Japanese Patent Application Laid-Open No. 59784827).

The present inventors also obtained cDNA of lymphotoxin in which the 26th amino acid is asparagine from the HUT-102 cell line.
A was isolated (Japanese Patent Application No. 61-61-123700) Ne
di et al., lymphotoxin is divided into four exons and the DN
It is reported that it is coded on A (Nucl
eic Ac1d Research (1985) 1
3 176361). Penn1ca and colleagues speculate that the cell-killing activity may be due to the amino acid sequence near the C-terminus of lymphotoxin (Nature (1)
984) 312724) ).

In addition, Aggarwal et al. reported that a sugar chain with a molecular weight of approximately 7000 is attached to the 62nd asparagine of naturally occurring lymphotoxin (Th
e Journal of Biolo 1csl Ch
emistr (1984) 259-1686)
, the antitumor substance TNF obtained from macrophages (
…L parallel dan 虱” L Zaitan ■ Bean Dan Ha 姐 えん え L (1985) 2
This is one of the major differences from 60-423451. Proctor et al. focused on the three-dimensional influence of this sugar chain, and speculated that this region is essential for destroying target cells (C1inical Re5e
arch 30-155A) a Aggarwal et al. demonstrated that lymphotoxin produced by genetic engineering in Escherichia coli has physiological activity, and IJ!
It became clear that a chain was not necessary, but from 1 spout to 33
It has been reported that lymphotoxin deleted up to the number 3 has no cell-killing activity (Japanese Patent Application Laid-open No. 56197-1983).
.

Aggarwal et al. conducted research on the trypsin degradation of lymphotoxin and reported that the bond between the 89th and 90th amino acids was cleaved by trypsin (The Journal of Biolog
icalChemistry (1985) 260
42334), and in order to provide resistance to trypsin degradation, which is expected to occur in vivo, an attempt was made to replace the 89th lysine with the same basic amino acid histidine (Japanese Patent Application Laid-Open No. 61-56197).

However, it is not known whether the activity is exhibited when lysine at position 89 is replaced with a different amino acid, such as a neutral amino acid or an acidic amino acid.

[Problem that the invention seeks to solve]

The present invention aims to provide a previously unknown mutant lymphotoxin that is easy to use as a tumor therapeutic drug.

[Problem that the invention seeks to solve]

Despite the above-mentioned findings of Aggarwal et al. This is a completely new finding that the cell-killing activity of lymphotoxin is maintained even when a wide range of amino acid sequences extending beyond the amino acid at position 33 and downstream of asparagine at the sugar-binding site at position 62 are deleted. I got it. Cell-killing activity was also observed for lymphotoxin in which the entire amino acid sequence from the N-terminus to lysine at position 89 was deleted. Furthermore, the inventors
Lysine at position 89 is replaced with the same basic amino acid,
It has been found that the cell-killing activity of lymphotoxin is maintained even when it is replaced with an amino acid of the opposite sex, such as a neutral amino acid such as methionine, which is not described in the specification of Japanese Patent Application No. 61-56197. The present invention was completed based on these new findings.

Therefore, the present invention provides for a natural lymphotoxin in which an arbitrary series of amino acids from the N-terminus to the vicinity of lysine at position 89 is deleted, and in some cases, a sequence of amino acids is substituted for this deleted region.
A mutant lymphotoxin-like polypeptide in which a non-natural sequence consisting of zero or less amino acids has been inserted and/or the lysine at position 89 has been replaced by an amino acid other than a basic amino acid; encoding this polypeptide The present invention attempts to achieve the above promise by providing DNA; a plasmid comprising this DNA; Escherichia coli transformed with this plasmid; and a method for producing the polypeptide using this Escherichia coli.

Blank space below [Specific explanation] (1) Polypeptide The first aspect of the polypeptide of the present invention is at position 62 from approximately the position where the α-helical region starts after the N-terminal hydrophobic region of natural lymphotoxin. An arbitrary series of amino acids downstream of and near the sugar-binding amino acid asparagine is deleted, and in some cases, a non-natural sequence consisting of 10 or less amino acids is inserted in place of this deleted region. It is something that exists.

The amino acid sequence of natural lymphotoxin and the base sequence encoding it are shown in FIG. 1, and the hydrophobicity-hydrophilicity profile of this amino acid sequence is shown in FIG. Hydrophobic-hydrophilic profiles were determined using the Kyte-Doolittle method (J
oMol, Biol, (1982) 157105). In Figure 3-1, the N-terminal hydrophobic region is shown as H, and the Chou-Fasman method (Ann
.

Rev, Biochem, (197B) 47251-
276), the α-helical region predicted by α-her
The sugar-binding site is indicated by S. In this figure, black areas indicate the presence of amino acids, and white areas indicate amino acid deletions. The unnatural amino acid sequence consisting of 10 or less amino acids to be replaced with the deleted portion is added for convenience during the production of DNA encoding the polypeptide of the present invention, and is not suitable for the polypeptide of the present invention. It is not necessary, and if present, the number and type of amino acids are not particularly critical.

As a first specific example of the polypeptide of the present invention, the amino acid sequence from alanine at position 10 to isoleucine at position 34 of the natural amino acid sequence is deleted, and Thr -Ser -Met - Hls-＾rg-C
Mention may be made of a polypeptide in which an unnatural amino acid sequence consisting of ys has been inserted (designated D2). A portion of this arrangement is shown in FIG. 2 (A). In this figure, the numbers in the upper row indicate the amino acid ranking of natural lymphotoxin.
The numbers in parentheses indicate unnatural amino acid sequences, and therefore this figure, together with Figure 1, shows the natural sequence of amino acids 1 to 9, the unnatural amino acid sequence of 6 amino acids, and the 3 amino acid sequence.
1 shows a mutant lymphotoxin-like polypeptide of the present invention consisting of the native sequence from positions 5 to 171.

Figure 3-1B shows the hydrophobic-hydrophilic profile of this polypeptide.

As a second specific example of the polypeptide of the present invention, the amino acid sequence from alanine at position 11 to leucine at position 65 of the natural amino acid sequence is deleted, and this deleted region contains Ile.
can be mentioned (referred to as D3). A portion of this array is shown as OB in FIG. This diagram also has the same meaning as A in Figure 2 above,
Together with Figure 1, the natural sequence of amino acids 1 to 10, the non-natural sequence consisting of one amino acid, and the amino acid 66 to 1
The mutant lymphotoxin-like polypeptide of the present invention consisting of the natural amino acid sequence at position 71 is shown. Figure 3-1C shows the hydrophobic-hydrophilic profile of this polypeptide.

A third specific example of the present invention is a polypeptide (referred to as D4) in which the amino acid sequence from the N-terminus to lysine at position 89 of the natural amino acid sequence is deleted. Figure 3-2 shows the hydrophobic-hydrophilic profile of this polypeptide.

Although three types of polypeptides have been exemplified above, the polypeptide of the present invention is not limited to these, and can have any length of polypeptide region from the N-terminus to the vicinity of lysine at position 89. Within the scope of the present invention are all polypeptides that have been deleted and have inserted into this deleted region an unnatural amino acid sequence consisting of about 10 amino acids or less in some cases, and that have the cytocidal activity of lymphotoxin. belong to

A second aspect of the polypeptide of the present invention is a polypeptide in which lysine at position 89 of the natural amino acid is replaced with an amino acid other than a basic amino acid, and which still maintains the cell-killing activity of lymphotoxin. Amino acids that can replace lysine include acidic amino acids and neutral amino acids. Examples of acidic amino acids include aspartic acid and glutamic acid, and examples of neutral amino acids include methionine, glycine, alanine, serine, leucine, isoleucine, phenylalanine, tryptophan, asparagine, and glutamine.

A third aspect of the polypeptide of the present invention is a combination of the first aspect and the second aspect, that is, it has the predetermined deletion on the N-terminal side, and the lysine at position 89 is It is substituted with a predetermined amino acid.

(2) The DNA coding sequence of the present invention is not particularly limited as long as it encodes the above polypeptide and can be expressed in E. coli. One specific example is shown in the row below the amino acid sequence in FIGS. 1 and 2.

In this case, a few amino acids at the N-terminus, e.g. 9-10
It is preferable to select codons encoding these amino acids from codons that are frequently used in E. coli.

Therefore, as the coding region of the DNA of the present invention, chemically synthesized DNA is used as the DNA encoding the N-terminal hydrophobic region and the following unnatural peptide region, and the code encoding the subsequent C-terminal peptide is used. It is convenient to use the corresponding region of cDNA as the region. Furthermore, the codon encoding the amino acid at position 89 can be obtained from a codon in cDNA by introducing a point mutation using a synthetic primer.

(3) 1) The plasmid of the present invention contains the above-mentioned DNA coding region after the control B of an appropriate control sequence, and the DNA is injected into E. coli.
A plasmid that can express a coding region. Examples of promoters include PL promoter,
tac promoter, trp promoter, 1acUV
5 promoter, etc. can be used, and as the SD sequence, for example, the SD sequence of the metavirocatecase gene can be used.

Examples of plasmids capable of producing the polypeptide of the present invention include plasmid pLTD2 for polypeptide D2, plasmid pLTD3 for polypeptide D3, plasmid pLTD4 for polypeptide D4, and a polypeptide having methionine at position 89. For example, plasmid pLTM4 can be mentioned, and Escherichia coli containing these plasmids are respectively infected with Escherichia coli.
ia colt) 21776/pLTD2 m1 Laboratory Bacteria Collection No. 8896 (FERM P-8896), Escherichia colt
Z 1776/pLTD3 Microtechnical Research Institute No. 8897 (
FERM P-8897), Sherisha Colli (E,
coli) Z 1776/pLTD4
No. 8924 (FERM P-8924), and Escherichia coli
Z 1776/ pLTM4 Microtechnical Research Institute No. 8898 (
FIRM P-8898) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

(4) In the present invention, any E. coli strain that is commonly used when producing polypeptides by genetic engineering techniques can be used, such as RRI.

RB791, 5M32, HBIOI, N9
9, JM103, etc.

(5) Expression of the target polypeptide of the polypeptide is carried out by culturing E. coli transformed with the above-mentioned plasmid, for example, in LB medium according to a conventional method, and performing an induction operation depending on the promoter. Can be done. Next, the desired polypeptide can be obtained by collecting the produced polypeptide from the E. coli cells. Details will be described in later examples.

(6) Cell-killing activity of the polypeptide obtained by the above method can be measured by, for example, a microplate method. This measurement method and results will be explained in detail in Examples below.

(7) A series of plasmids pLT1, pLTMl and pLT? containing the cDNA of natural lymphotoxin? '12 has already been obtained, and its production method is disclosed in Japanese Patent Application No. 12370/1986.
It is described in detail in the specification of No. 0. Plasmid pLT
Escherichia coli containing Escher 1
(ichia dish) χ1776/pLT1
No. 784 (FERMp-8784), Escherichia coli (E.
scherichia coli) χ1776/pL
TM2 is FERM P-8
785) and have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. The method for producing these plasmids will be described later as a reference example. Therefore, the plasmids of the present invention can be constructed starting from these already obtained plasmids.

To create a plasmid encoding a polypeptide with a deletion at the N-terminus, pLTMl is digested with the restriction enzyme PvuI.
Lymphotoxin cD is cleaved by digestion with [
A large fragment from which the upstream portion of NA has been removed is obtained. By digesting this fragment with exonuclease Bal 31 for various times, DNAs with the ends shortened to various degrees were obtained.
Obtain A fragment. Next, the ends of this fragment were made blunt by fill-in, a 3-mar C1al linker was added to this, and this fragment was digested with C1al and 5ail, so that the 5' end was shortened to various degrees and the end A DNA fragment is obtained in which the C1al end is the C1al end and the 3' end is the 5all end.

On the other hand, plasmid pLTM2 was cut by digesting with Pvull to give 3 % 1 % and 12a+
A C1al linker of er is added, and the resulting fragment is then digested with C1al and 5ail to obtain a linear plasmid.

These DNA fragments are ligated, used to transform E. coli, and the base sequence of the resulting plasmid is determined to select a plasmid having the desired sequence. Note that the above ligation forms a DNA 63 region encoding an unnatural amino acid sequence derived from the C1aI linker.

In this way, plasmid pLTD2 containing DNA encoding polypeptide D2 of the present invention is obtained.

Plasmid pLTD3 is also obtained by the method described above.

A plasmid containing DNA encoding the polypeptide of this invention in which lysine at position 89 has been converted to methionine can be produced as follows. That is, by digesting plasmid pLTM2 with EcoRI, an EcoRI fragment containing the site to be mutated was obtained, and this was transformed into M13
+IIplO two tJ[DNAs are inserted into the EcoR1 site, a base substitution mutation is introduced using an oligonucleotide primer according to a conventional method, and the same oligonucleotide is used to select a phage containing the desired mutation. Next, the second-terminal DNA of this mutant phage is digested with EcoRI to obtain an EcoRI 11FT fragment containing the mutation site. On the other hand, pLTM2 was digested with EcoRI to
The RI incomplete fragment is removed, and the EcoRI fragment containing the above mutation is inserted into this site. In this way, the desired plasmid pLTM4 is obtained.

A plasmid containing DNA encoding a polypeptide of the present invention lacking the amino acid sequence from the N-terminus to lysine 89 can be produced from plasmid pLTM4. That is, the lymphotoxin cDNA is cut by digesting plasmid pLTM4 at the Nco1 site that appeared due to the mutation, and the Aat n cDNA immediately after the SD sequence is
The target plasmid pLTD4 can be obtained by ligating the site using a synthetic oligonucleotide as a linker.

Next, the present invention will be explained in more detail with reference to Examples.

If! JL 7'-7, Mi)'LTD2 HiLTD
3(7) Preparation of It(1) Vector Fragment Escherichia coli (Escherichta coli)
Plasmid pLTM2 was prepared from E.coli) χ1776/pLTM2 according to a conventional method, and linearized by cutting with PvuII.

18 μg of this linearized DNA fragment was mixed with 8mer% 10vaer and 12mer, which had been phosphorylated at the 5' position in advance.
Add 500 μmof C1al linker of r, 13
0+nM Tris-HCl (pH 7,6), 2mM AT
P, 2o+M spermidine, 20mM MgCl1z
, 20μ of a solution of 300mM DTT! Inside T4DN
A ligase 100 units was added and the reaction was carried out at 22°C for 10 hours. After the reaction, DNA was recovered by performing phenol extraction and ethanol precipitation according to conventional methods.

This DNA was treated with the restriction enzyme Cla I% according to a conventional method.
and 5ail, and the larger DNA fragment was cut into 0
．． Collected by 8% agarose electrophoresis, CE 1 u
t i p-d (Schleicher & 5ch
vell). DNA was dissolved in 30 μl of pure water.

(2) Preparation of insert fragment The DNA of plasmid pLTM1 was digested with restriction enzyme PvuII, and the larger fragment was recovered using a 0.7% agarose electrophoresis gel and purified using Elutip-d.

0 g of this DNA fragment was added to 190 μl of exonuclease Ba131 reaction solution.
31 React with 2.4 units at room temperature, 1 minute, 2 minutes,
After 3 and 4 minutes, 45 μl of each reaction solution was taken out, and 4.5 μl of 0.2 M EGTA was added to each tube to stop the reaction.

This reaction solution was extracted with phenol and precipitated with ethanol to recover DNA.

The recovered DNA was diluted with 80mM) lithium-hydrochloric acid (p)l 7.4
), 10mM MgCft, '100 M
NaCl, 0.51 dATP each.

Dissolve in a solution of dCTP, dGTP, and dTTP, add 4 units of DNA polymerase large fragment, and incubate at 37°C.
The end was made blunt by reacting for 1 hour.

This reaction solution was further 5′ phosphorylated in advance.
ver C1al linker-140p*ol and 10m
MATP 1μ1140%PEG 9μ! , add 50 units of T4 ligase, bring the total volume to 25 μl, and bring to 22°C.
After 10 hours of reaction, phenol extraction and ethanol precipitation were performed to recover DNA.

This DNA was sequentially digested with restriction enzymes C1aI and 5ail, and subjected to phenol extraction and ethanol precipitation to obtain DNA.
was recovered. The DNA was dissolved in 60μi of pure water.

(3) Preparation and transformation of plasmid 1 μl of the above vector fragment and 5 μl of the inserted fragment were mixed into 40 m
M HEPES (pH 7,8), 10 M neck gclz, 10 cod DTT, 0.4mM ATP
After the total volume of the solution was made up to 30 μl by adding pure water, reaction was carried out at 12° C. overnight using 100 units of T4 ligase.

E. coli strain RRI was transformed using 10 μE of this reaction solution, and transformants resistant to 100 μg/m It of ampicillin were selected. Plasmid DNA was extracted from arbitrarily selected colonies after transformation, and the base sequence was confirmed by the dideoxy method.

1旌■1 Boothmid LTF14 -'1(1)M1
Introduction of specific base substitution mutations using phage 3 (preparation of lymphotoxin gene with methionine at position 89) Plasmid pLTM 2 was cut with restriction enzyme EcoRI, and a fragment containing lymphotoxin cDNA was recovered from 0.8% agarose gel.

0.5 pn+ol of this DNA fragment and 0.5 pm of EcoRI-cleaved double-stranded DNA of M1313 Phage 10
66mM Tris-HCj! '(
pH7.5), 5mM MgC1z, 5mM DTT
After the ligation reaction was carried out at 12"C for 16 hours using 100 units of T4 DNA ligase in 20 μE of a solution containing 1 mM ATP, a method such as meshing [meshing, etc.] was performed using the reaction solution.・Methods in Enzymol
Escherichia coli JM103 was transformed according to 1983) and 0.02% X-ga
1 and 1 mM IPTG and cultured overnight at 37°C. Single-stranded template DNA was prepared from white plaques formed by the recombinant. That is, a white plaque was picked with the tip of a toothpick and suspended in 1.5 m/2xYT culture medium (1.6% Batato tryptone, 1% yeast extract and 0.5% NaCl) in which E. coli JM103 was growing. The suspension was incubated at 37°C for 5 hours, and the culture supernatant was purified by polyethylene glycol precipitation, phenol treatment, and ethanol precipitation.
Full-stranded recombinant phage DNA was recovered.

Using the obtained single-stranded DNA as a template, the base sequence was determined by the dideoxy method according to the method of Messing et al. (cited above), and the sequence of the cloned single-stranded DNA was confirmed. In this way, single-stranded DNA containing the anticoding strand of the lymphotoxin gene was obtained. This recombinant phage was named LTM21.

Using the single-stranded DNA of LTM21 obtained as described above as a template, a synthetic oligonucleotide: S'C
A repair reaction using DNA polymerase Klenow fragment was performed using TCTCCCATGGCCACC3' as a primer. That is, template single-stranded DNA 0.5p+*
2 pmol of 5'-terminally phosphorylated brimer was added to ol, and 7n+M Tris-HCA' (pH 7,5
), 0.1s+M EDTA, 20mM NaC
dATP, dATP,
dGTP.

Add dTTP and dCTP to 0.5mM each, make the whole 20μi, and add DNA polymerase K.
Two units of lenotm fragment were added and incubated for 20 minutes at 23°C. Subsequently, 10mM ATP
and 1 unit of T4 DNA ligase.
Incubate overnight at 2°C.

Escherichia coli JM103 was transformed using 0.1 μmol of the above double-stranded DNA according to the meshing method.

Regarding the phage plaques obtained as above,
Mutant phages were screened by plaque hybridization using the aforementioned oligonucleotide (labeled with 32P) as a probe. That is, the method of Benton-Davis et al. (W, D. Benton and R.W. Davis, Science)
) Transfer plaques from soft agar to nitrocellulose filters according to 196, 180, 1977);
Baked in vacuo at 80° C. for 2 hours. This nitrocellulose filter is 6×SS C, 10×Denh.
Hybridization was performed overnight at 23° C. in an ardt solution using a 3Zp-labeled primer oligonucleotide as a probe. Next, add this filter to 5x
Mutant phage plaques showing positive signals were isolated by washing in gSC at 46°C and autoradiography.

The base sequence was determined by the dideoxy method using the mutant phage DNA as a template, and it was confirmed that a phage containing a lymphotoxin gene with a base substitution mutation and methionine at position 89 was obtained. This mutant phage strain JM103
/LTM51.

(2) Preparation of plasmid and transformation from phage JM103/LTM51 according to the standard method.
Prepare double-stranded DNA and cut with restriction enzyme EcoRI to 8
A DNA fragment containing the lymphotoxin gene with methionine at position 9 was recovered by 0.8% agarose gel electrophoresis and purified using Elutip-d.

Plasmid pLT? I2 lymphotoxin cDNA-free EcoRI fragment 0.5 pmol and the above DNA
0.5 pmol of fragments and 40+M HEPES (p
H7,8), 10mM MgClt -10mM DT
The reaction was carried out using 100 units of T4 ligase in 0.4mM ATP at 12°C for 16 hours.

E. coli strain RRI was transformed using 10 μl of this reaction solution, and transformants resistant to 100 μg/ml ampicillin were selected. Plasmid DNA from transformant colonies
A was extracted and the base sequence was confirmed by the dideoxy method. This plasmid was named pLTM4.

1. Preparation and transformation of +1 (1) plasmid of boothmid LTD4. Plasmid pLTM4 was treated with restriction enzymes Aat II and Nco.
The larger fragment was collected on a 0.7% agarose electrophoresis gel and purified using Elutip-d.

0.1 pmol of this DNA fragment and 24 pmol of a synthetic oligonucleotide whose 5' end was phosphorylated: 5'TGCAGTAC3' were mixed and mixed with 66neM Tris-HC.
j! (pH 7,5).

5mM MgC1z, 5mM DTT and 1mM
100 units of T in 20 μl of solution containing ATP
The ligation reaction was carried out using 4DNA ligase at 16°C for 16 hours.

Escherichia coli strain JM103 was transformed using 10 μl of this reaction solution, and transformants resistant to 100 μg/ml ampicillin were selected. Plasmid DNA was extracted from arbitrarily selected colonies after transformation, and the dideoxy method was performed. The base sequence was confirmed by This plasmid is pLTD4
It was named.

Plasmid pLT prepared according to Example 1 and Example 2 of Boppeptide
D2, pLTD3, pLTD4, and pLTM4
E. coli RRI strain containing RR1/pLTD
2. RR1/pLTD3, JM103/pLTD4 and RR1/pLTM4 were incubated at 37°C in 0 mji of LB medium containing 50 Mg/ml ampicillin.
After incubating for 1 hour at -D -thiog
alactopyranos ide ; 'I PT
G) was added to 1mM and cultured for an additional 4 hours.

After the culture is completed, the culture solution is centrifuged at 600 rpm for 15 minutes to collect the bacterial cells, and the bacterial cells are injected into RPMI-
It was suspended in 1640 medium to make 330 m4. This suspension was sonicated four times for 10 minutes each to disrupt the bacterial cells, and the resultant was centrifuged at 10,000 rpm for 30 minutes to obtain a supernatant.

As a control, E. coli RRI and JM103, which had not been transformed with the plasmid of the present invention, were treated in the same manner to obtain supernatants.

The above sample was diluted 10 times with RPM I medium to prepare an analysis stock solution. Each of these was diluted in eight 2-fold series.

Lymphotoxin activity was determined by E., A., Carswell et al. [Proceedings of the National Academy of Sciences].
Of Science of the United Stake (Proc-Natl-Acad-Sci”-USA
, 72+3666.1975) by a microplate method. 5% fetal bovine serum, 0
．． Mouse LM cells were cultured in RPMI-1640 medium containing 5% penicillin, 0.5% streptomycin, and 0.2% glucose, and 1 x 104 cells/1
00 μl into the wells and add the above diluted sample 1.
00μE and 72℃ at 37℃ in 5% CO□ atmosphere.
Cultured for hours. After washing the plate, add 0.05% crystal violet formalin ethanol solution 1
00 μi was added to stain the cells for 30 minutes. Next, 0.
05M NaHtPOa・100 μl of ethanol solution
The dye was eluted, and the absorbance at 570 nm was measured using a photometer (Mini Reader ■, manufactured by Guinatec).

The absorbance (normal scale) and the dilution factor (logarithmic scale) were plotted on a semi-logarithmic graph, and the number of units of cell killing activity in the sample was calculated, with the activity required for 50% cell killing as one unit. The calculated lymphotoxin activity was as follows.

RRI/pLTM2 700RRI/pL
TD2 200RRI/pLTD3
100RRI/pLTM4 3
0RR110 μM103/pLTD4 10μM103
0 (1) Preparation of mRNA - H) T lymphocytic leukemia cell line HUT-102 was suspended in 20 ml of RPMI-1640 medium containing 5% fetal bovine serum, and incubated at 37°C in a 5% CO□ incubator for 2 to 3 hours.
Culture was performed for 1 day. Increase the medium by 2 times,
The cells were cultured for 2 to 3 days each time, and 11 culture solutions were finally obtained. The resulting culture solution was centrifuged at 4°C, 3000 rpm for 10 minutes, the cells were collected, and buffered with phosphate buffer (1 omM sodium phosphate, pH 7, 2, 0, 15M NaCl).
After suspending in 50 ml and washing, centrifugation was performed to obtain a T cell pellet.

T, Maniatis etc.”Mo1ecul
ar Cloning”Col 1d Spring t(
arbor Laboratory 1982 P, 1
It was isolated using the guanidium/CsC1 method according to 96. The T cell pellet was diluted with 5 times the volume of guanidium isothiocyanate solution (6M guanidium isothiocyanate, 5M
M sodium citrate, pH 7, 0, 0, 1 M β-mercaptoethanol, 0.5% sarcosyl), placed in a glass homogenizer container, homogenized 10 times with Teflon, and then transferred the homogenate to an SW55 centrifugal tube ( 1.51!111 containing 5.7 M CsC4 and 0.1 M EDTA (pH 7.5) in Llltra C1ear™ and incubated at 15° C. for 35. Ultracentrifugation was performed at OOOrpm for 20 hours.

After removing the guanidium solution, the wall was coated with 3 guanidium solution.
After washing twice, the centrifuge tube was cut at the top of the C5Cj2 solution. After removing the CsCl solution, the precipitate was washed twice with 80% ethanol and treated with 10mM Tris-11cj! pH
It was dissolved in 500 μl of buffer (TBS) containing 7, 4, 5 mM EDTA and 10% SDS. Add this to 500μ of a chloroform-N-butanol mixture (4:1)! After re-extracting the organic layer with 3500 μl of TE, the aqueous layers were combined and subjected to ethanol precipitation to obtain mRNA.

Molecular Cloning of mRNA
” by passing it through an oligo(dT) cellulose column according to the method described in
NA was purified.

(2) Synthesis of cDNA Synthesis of cDNA second strand is performed using Gubler-Hoffman
It was done in accordance with the law. Water 8.4 μm, IMT
ris-HC1! (pH 8,3) 1μl' (50m
M), 0.6M MCI! 1μl (30wM)
, 0.16M MgC1! t 1μil (8mM),
20mMDT71 μm (1mM), 25o+M
dNTP (dATP, dCTP, dGTP,
dTTP (mixture of 25mM each)) 1.6 μl (2mM each)
, RNasin (biotic 30U/μN) 1μ
l, oligo(dT) 12-18 (manufactured by PL) 1
μI! (2ujri, and HUT-102poly (A
)" RNA (1Mg/μf) 3μl, total volume 1
After making 9 μl and incubating at 37°C for 5 minutes,
Reverse transcriptase (Life 5science+ 12.5U
/ /112) 1μβ was added and reacted at 37°C for 1 hour. The numbers in parentheses of the salt solution indicate the final concentration. 0 after reaction.
5 1 μl of MEDTA and 0.5 μl of lO% SDS
After stopping the reaction by adding β, phenol/chloroform extraction and ammonium acetate/ethanol precipitation were performed twice.
After washing with 80% ethanol and drying under reduced pressure,
Dissolved in 50 μl of water. Take 5 μl of this and
After adding μg of sel and leaving the mixture at room temperature for 10 minutes, phenol extraction was performed, and the aqueous layer was subjected to 0.7% agarose gel electrophoresis to examine the extent of first strand elongation.

The synthesis of the second strand was also carried out according to the conditions for the second strand synthesis of the Gubler-Hoffman method. cDNA first strand aqueous solution 45. IJA', Wed 33.5111, I M
Tris-HCI (pH 7,5) 2 pl (2
0mM), 0.5M MgC1zlμ1(5d)
, I M (NH4)zsOtl pl (10m
M), IM KCI 1 0 pl (100+
(mM), 10mM β-NAD 1pl (0,
1+M), 5mg/ml B SA 1 pi (
50Mg/ml! ), 4mMdNTP1111!
(40 μM), E.

coli DNA ligase (PL, 0.5μg/μm) 1μ/
, E. coli DNA polymerase I (PL.

15U/μJ) 2μm1. E. coli RNaseH (Takara Shuzo 1.3U/μl) 0.5μl, 32p-dcT
P1μ1 (10μl) was mixed to give a total volume of 100μl, and the mixture was reacted at 12°C for 1 hour and then at 22°C for 1 hour.

A 1 μl sample was taken before and after the reaction, and the amount of radioactivity incorporated was measured by TCA precipitation. After stopping the reaction by adding 4 μl of 0.5 M EDTA and 5 μl of 10% 5DS to the reaction solution, phenol-chloroform extraction was performed, and ethanol precipitation was performed twice to reduce the precipitate to 80%.
After washing with ethanol and drying under reduced pressure, the precipitate was dissolved in water 1
It was dissolved in 0 μl.

(3) Preparation of cDNA library cDNA aqueous solution 10 pl, water 2 All % I MT
ris-acetate (pH 7,9) 0.7μIl
(35!IM), 1M potassium acetate, 1.3 μm
(65mM), 0.1M magnesium acetate 2μj
! (10mM), 10mM DTT1 ttl
(1mM), 5mg/mf BSA 1 pit
(250 pg/ml, 2mMd NTP 1
PI (0.1mM) and 1μl of T4 DNA polymerase (manufactured by Takara Shuzo, 1.5U/μJ) were mixed, and the total amount was 2.
The reaction was carried out at 37° C. for 10 minutes at 0 μE. 0.5M
After stopping the reaction by adding 1 μl of EDTA and 5 μl of 10% SD SO, phenol-chloroform extraction was performed, ethanol precipitation was performed twice, the precipitate was washed with 80% ethanol, dried under reduced pressure, and 10 μl of water was added. dissolved in.

EcoRI linker (manufactured by Takara Shuzo, GGAATTCC, 1
pg/μJ) to 2μl of water, 115x linker kinase buffer (0,33M Tris-HCI pH 7,6
, 5mM ATP, 5mM spermidine, 50+++M
MgC1z, 75mMDTT, 1mg/mj! B.S.A.
)2All, T4 DNA kinase (Takara Shuzo, 6U/
1μl) was added to make total tloμE, and the mixture was reacted at 37°C for 1 hour. To this, cDNA blunt-ended as described above was added.
Solution 5μ! , 2 μl of 5× linker-kinase buffer, 1 portion of water
．． 5μ/, T4 DNA ligase (Takara Shuzo, 175U/
μl) 1 μN, T4 RNA ligase (PL, 0.8 p
g/pi) 0.5 ul'c processing, ig=2o
The reaction was carried out overnight at 12°C or for 6 hours at 22°C.

After heating the reaction solution at 65°C for 10 minutes to inactivate the ligase, add 65 ttl of water and 10 pl of 10X EcoRIQ buffer.
, EcoRI (manufactured by Takara Shuzo?, 5U/μI 5μ
1 was added to make a total of ff1looμl, and the mixture was reacted at 37°C for 3 hours. A 1/10 volume sample was collected before and after EcoRI digestion and subjected to 10% polyacrylamide gel electrophoresis to check the ligation of the EcoRI linker and the success or failure of the EcoRI digestion reaction. The reaction solution was extracted with phenol, and 5 M NaCl was added to the aqueous layer to give 0.3 M
The EcoRI linker monomer was removed by applying it to a Sepharose CL-4B column (2 mJ). It was developed with TE (pH 7,6), 0.3 M NaCl, and after collecting 4 to 5 drops, counting was performed. Certain fractions were ethanol precipitated and each fraction was dissolved in 10 μl of water.

Plasmid pUC9 was cleaved with EcoRl and treated with calf intestinal alkaline phosphatase (CIP) according to a standard method. 1 μf (0.4 μg) of this preparation, 10 μl of cDNA prepared by the above method, 20 μl of linker-kinase buffer, and 64 μl of water were mixed and heated at 68°C for 10 minutes, and then 5 μl of T4 DNA ligase was added to make a total volume of 100 μ2. The reaction solution reacted overnight at ℃ 65
After heating for 10 min at °C, 1 ottll of E. coli 11
The mixture was mixed with 210 μl of competent cell suspension of strain 776 to perform transformation. The transformed bacterial solution prepared in this way was heated to 300mj! x 1776 medium (containing 50 μg/ml ampicillin) and cultured at 37° C. overnight. A portion was frozen and stored at -80°C as a 15% glycerin solution.

(4) Screening cDNA of cDNA library
20 μl of the frozen glycerin preservation solution of the transformed bacteria containing
Diluted 5.000 times with 1776 medium and diluted solution 100 times.
μ2 was spread on nitrocellulose placed on an ampicillin-containing χ1776 agar plate and cultured overnight at 37°C. Colonies on nitrocellulose were transferred to two sheets of nitrocellulose. This replica, χ1776
After culturing on a medium for 3 hours, the cells were transferred to a medium containing 10 Mg/ml chloramphenicol and further cultured overnight.

The filter was soaked in 10% SDS, denaturing solution (
0,5N NaOH, 1,5M NaCl), neutralization solution (1,5M NaCl, 0,5M Tris-MCI)
, pH 8,0), then 5 m per filter.
Solution [Proteinase K (Me
rk) 1 mg/mi! ,0,5 MNaCl,
10 Tris-HCI, pH 7, 4, 5mM E
The reaction was carried out at 37°C for 1 hour in DMAO01%5DS).

After sandwiching the filter between filter paper and rubbing it vigorously with a rower,
It was washed twice with 2XSSC and heat treated at 80°C for 3 hours.

The above nitrocellulose filter, 6XSsc, 1
Hybridization was performed overnight at 42° C. with P-labeled oligonucleotide probe #71 in 0×Denhardt solution. Next, add this filter 6x
After washing in SSC at 55°C and autoradiography for 2 days, 3 colonies showed positive signals.

Next, this filter was heated to 65'C30 in 1xSSCi solution.
After washing for several minutes to wash away the positive signal, it was dried and used again for hybridization with oligonucleotide probe #72. 6 x S S C, 10 x Den
Hybridization was performed overnight at 42° C. with oligonucleotide probe #72 labeled with 32p in hardt solution. This filter was then placed in 6XSSC for 5
After washing at 0°C and performing autoradiography for 2 days, the same three colonies as when #71 probe was used showed positive signals.

Plasmid DNA was extracted from these three colonies,
The base sequence was determined using the dideoxy method according to a conventional method. As a result, one of these clones contained the entire coding sequence of the lymphotoxin peptide (shown in Figure 5).
It contained. This plasmid was named pLTI (
Figure 6).

1 Consideration 1 The above plasmids pt and rt of Bootmid LMTI were transformed into Escherichia coli RRI strain, the transformant was cultured according to a conventional method, and plasmid DNA was extracted from the cultured cells.

40 μm (20 pg) of pLT1 plasmid DNA.

20ttl EcoRI buffer (X 10), 140u
6 of distilled water and 4 μl of EcoRI enzyme solution (40 units).

The mixture was mixed and reacted at 37°C for 3 hours. This reaction mixture was extracted with 100 μβ of phenol and chloroform, washed with 200 μl of ether, and then subjected to ethanol precipitation. After drying, the precipitate was dissolved in 100 μl of a dye solution for electrophoresis, and 1×TBE (Tris-B
AGE separation by electrophoresis on a 1.2% agarose gel in (orate-EDTAg solution), and the desired approximately 0.
The gel containing 9 kb of DNA was excised. This gel piece was placed in a dialysis tube, electrophoretically eluted in 1x TBE for 2 hours, the contents of the dialysis tube were collected, and Eluti
After adsorbing DNA, the column is washed and the ion concentration of the eluent is increased to remove DNA.
A was eluted and collected. After ethanol precipitation, the precipitate was dried for 3 minutes, and the resulting DNA was dissolved in 20 μl of distilled water.

10μtl of solution containing plasmid pKK223-3 (10
Atg), 5μβ EcoR1 buffer (XIO), 36
μβ distilled water and 3 μl EcoRI (30 units)
were mixed and reacted at 37°C for 3 hours. This reaction mixture was then supplemented with 50 μl of 50 iM Tris-H (J
(pH 8.0) and 10 μl of bacterial alkaline phosphatase (BAP) C-75i (1/10 dilution) were added and reacted at 65° C. for 1 hour. The reaction mixture was diluted with 100 μβ
Extract with 200 μl of phenol and chloroform.
After washing with 1 liter of ether, ethanol precipitation was performed, and the precipitate was dried for 3 minutes. This precipitate was dissolved in 20 μl of distilled water.

5 μl of the pKK223-3 digest, the pt, t
I Digestive proboscis 5. cl, 101! 100mM HEPES
(pH 7,8), 10μ2 of 30mM MgC1z,
1μβ of 300mM DTT.

A solution (20 units) of 1 μβ of 10 mM ATP and 2 μE of T4 DNA ligase was mixed and reacted at 12° C. overnight.

Transformation was performed by mixing 10 μl of this reaction mixture with 200 μl of competent cells of Escherichia coli strain JM103, spread on H plates, and cultured at 37° C. overnight. Twelve of the many colonies formed were analyzed by rapid isolation, and three colonies were confirmed to contain the desired plasmid. This plasmid was named pLTM1.

1 Thought ■1 Boothmi LTM2 (7) -”, 7
:z) 100 μN (10 pg) of plasmid pLTl
'l l solution, 40, cl PvuII buffer (X 10
), 260 pl of distilled water, and 4 .mu.l of PvuII enzyme solution (40 units) were mixed and reacted at 37.degree. C. for one day. To this reaction mixture was added 360 μE of distilled water, 4012
Pvul buffer solution and 4 μl of Pvul enzyme solution (40 units) were added, and the reaction was carried out at 37° C. for 1 day. This reaction mixture was extracted with 400 .mu..beta. of phenol and chloroform, washed with 800 .mu.l of ether, precipitated with ethanol, and the resulting precipitate was dried.

This precipitate was dissolved in 100 μl of agarose electrophoresis dye solution and electrophoresed on a 1.2% agarose gel in IXTBE. Gels containing DNA approximately 1.6 kb and 1.7 kb in length were excised.

This gel piece was placed in a dialysis tube and incubated in lxTBE for 2 hours.
After time electrophoresis elution, the content of the dialysis tube was collected and applied to an Elutip-d column to adsorb DNA, the column was washed, and the DNA was eluted and collected by increasing the concentration of the eluent. The DNA was precipitated with ethanol from the eluate, dried for 3 minutes, and the precipitate was washed with 20μ
1 of distilled water. Add 380/J l to this solution.
IM Tris-HCI (pH 8,0) and 4 μE
BAPC-75 enzyme solution (1/10 dilution) was added,
The reaction was carried out at 65°C for 1 hour.

The reaction mixture was extracted with 300 μβ of phenol and chloroform, and then washed with 600 μl of ether.

Escherichia coli strain JM103 containing plasmid pCTM 4 was cultured according to a conventional method, and plasmid DNA was extracted from the cultured cells according to a conventional method. This pCTM 4 plasmid DNA solution 100μl (DNAIOμg), 401
Pvul buffer (XIO), 260μ4' distilled water,
and 4 μl of PvuI enzyme solution were mixed and reacted at 37° C. for 1 day. The reaction mixture was extracted with 200 μl of phenol and chloroform, and after washing with 200 μl of ether, the DNA was precipitated with ethanol and dried. This precipitate was dissolved in 360 μl of distilled water. To this DNA solution, add 40 μl of AatII buffer (xlO) and 4 μl of
AatlI enzyme solution (40 units) was added and heated at 37°C.
It reacted for one day. This reaction solution was extracted with 200 .mu..beta. of phenol and chloroform, washed with 200 .mu.2 of ether, then DNA was precipitated with ethanol, and the precipitate was dried. This precipitate was dissolved in 100 μl of agarose electrophoresis dye solution and 1.3%
Agarose gel electrophoresis was performed and a gel containing approximately 1.9 kb of DNA was excised. This gel piece was placed in a dialysis tube and electrophoretically eluted in 1× TBE for 3 hours. Collect the contents of the dialysis tube and use Eluti
It was applied to a p-d column to adsorb DNA, the column was washed, and the ion concentration of the eluent was increased to elute and collect the DNA. DNA was precipitated from this eluate with ethanol, and the precipitate was dried for 3 minutes and then dissolved in 400 μE of distilled water.

The solution was extracted with 300 μβ portions of phenol and chloroform and washed with 600 μl of ether.

D of lengths of approximately 1.5 k b and 1.7 k b as described above.
100 μl of solution containing NA, about 1.9
50μ112 of a solution containing DNA of k b length
0 pH (75 ng) each of 5' phosphorylated oligonucleotides #73 and #74 (Figure 8), 10 μg of tRNA
and 50 μl of 3M sodium acetate, and add 1
Add ml of ethanol and place at -80°C for 20 minutes.
DN by centrifugation at 16,000 rpm for 10 minutes at °C.
Precipitate Class A with tRNA, remove the supernatant, dry the precipitate, add 8 μl of 30oM MgCl, 4 pl of distilled water, and 8110 100 mM HEPES (pH 7).
, 5) and at 65°C for 20 minutes.
30 minutes at 42°C, 5 minutes at room temperature, followed by 5 minutes on ice.
Annealed for minutes. Add 1μl of 10mM ATP to this
, 1μl 300mM DTT, 9μE040% PE
G, and 2 μl of T4 DNA ligase solution (20 units) were added, and the reaction was carried out at 20° C. overnight.

Using 10 μl of this ligation reaction mixture, Hanahan
Transform E. coli RRI strain according to the method and transform it into LB
The cells were spread on thiampicillin plates and cultured overnight at 37°C. In this way, 55 colonies appeared. Of these, 9 colonies were analyzed by rapid isolation method, and 2
It was confirmed that the colony contained the desired plasmid. This plasmid was named pLTM2.

This plasmid allows E. coli strains JM103 and 117
76 strains were transformed, and each Escherichia coli Es
cherichia coli) JM 103/pLT
M2, and Escherichia coli
a colt) 21776/pLTM 2 was obtained.

[Brief explanation of drawings]

FIG. 1 shows the nucleotide sequence encoding natural lymphotoxin and the corresponding amino acid sequence in the starting plasmid pLTMZ for constructing the plasmid of the present invention. FIG. 2 shows the lymphotoxin-like polypeptide D2 of the present invention.
(A in the figure) and the sequence of the connection site between the N-terminal sequence and the C-terminal sequence of D3 (B in the figure) are shown. Figures 3-1 and 3-2 show natural lymphotoxin and lymphotoxin-like polypeptides D2 and D3 of the present invention.
, and the hydrophobicity-hydrophilicity profile of D4. Figures 4-1 and 4-2 are diagrams of the production system of the plasmid of the present invention. Figure 5 shows the sequence of the cDNA insert in plasmid pLTl. Figure 6 shows the construction of plasmid pLTM1. Figure 7 shows the construction of plasmid pLTM2. Figure 8 shows the sequences of synthetic oligonucleotides #73 and #74. 〈ω　　Q: The 5th Country Met Leu Pro Gly Val Gly L
euCATG CTCCCG GGT GTT GGT
CTTTGCAG TACGAG GGCCCA C
M CCA GMN Trie Jimie Figure 8 Thr Pro Ser Ala ACT CCT TCA G $73 Synthetic oligonucleotide TGA GGA AGT C174
Synthetic oligonucleotide Pvu

Claims (1)

[Claims] 1. An arbitrary series of amino acid sequences from the N-terminus of natural lymphotoxin to a site near lysine at position 89 is deleted, and in some cases, this deleted region is replaced by 10 amino acids. A non-natural sequence of up to 3 amino acids has been inserted,
And/or a mutant lymphotoxin-like polypeptide in which lysine at position 89 is replaced with an amino acid other than a basic amino acid. 2. The polypeptide according to claim 1, wherein a region consisting of amino acids approximately at positions 10 to 34 is deleted. 3. The polypeptide according to claim 2, consisting of the following amino acid sequence: [There is an amino acid sequence]. 4. The polypeptide according to claim 1, wherein a region consisting of amino acids approximately 11 to 65 is deleted. 5. The polypeptide according to claim 4, which has the following amino acid sequence: [There is an amino acid sequence]. 6. The polypeptide according to claim 1, wherein the entire region from the N-terminus to the vicinity of lysine at position 89 is deleted. 7. The polypeptide according to claim 6, which has the following amino acid sequence: [There is an amino acid sequence]. 8. The polypeptide according to claim 1, wherein lysine at position 89 is replaced with methionine. 9. An arbitrary series of amino acids from the N-terminus of natural lymphotoxin to the vicinity of lysine at position 89 is deleted, and in some cases, a non-natural sequence consisting of 10 or less amino acids replaces this deleted region. is inserted, and/or a DNA encoding a mutant lymphotoxin-like polypeptide in which lysine at position 89 is replaced with an amino acid other than a basic amino acid. 10. An arbitrary series of amino acids from the N-terminus of natural lymphotoxin to the vicinity of lysine at position 89 is deleted, and in some cases, a non-natural sequence consisting of 10 or less amino acids replaces this deleted region. is inserted and/or the lysine at position 89 is replaced with an amino acid other than a basic amino acid. 11. Claim 10 contains a tac promoter/SD sequence of the metapyrocatechase gene upstream of the DNA, and the polypeptide can be expressed in E. coli under the control thereof. Plasmids listed. 12. An arbitrary series of amino acids from the N-terminus of natural lymphotoxin to the vicinity of lysine at position 89 is deleted, and in some cases, a non-natural sequence consisting of 10 or less amino acids replaces this deleted region. is inserted, and /
Alternatively, E. coli transformed with a plasmid containing DNA encoding a mutant lymphotoxin-like polypeptide in which lysine at position 89 is replaced with an amino acid other than a basic amino acid. 13. Escherichia coli
coli) RRI/pLTD2, Escherichia coli R
RI/pLTD3, Escherichia coli RRI/pLT
The E. coli according to claim 12, which is Escherichia coli RRI/pLTD4. 14. An arbitrary series of amino acids from the N-terminus of natural lymphotoxin to the vicinity of lysine at position 89 is deleted, and in some cases, this deleted region is replaced by a non-natural sequence consisting of 10 or less amino acids. is inserted, and /
Alternatively, E. coli transformed with a plasmid containing DNA encoding a mutant lymphotoxin-like polypeptide in which lysine at position 89 is replaced with an amino acid other than a basic amino acid is cultured, and the polypeptide is extracted from this culture. A method for producing a mutant lymphotoxin-like polypeptide, the method comprising collecting a mutant lymphotoxin-like polypeptide.