JPH082315B2 - Lymphotoxin-containing fusion proteins - Google Patents

Description

Detailed Description of the Invention [Technical Field] This invention relates to a fusion protein comprising a polypeptide containing an antibody-binding site of protein A and a lymphotoxin polypeptide, DNA encoding the fusion protein, and a method for producing the same.
the present invention also relates to a plasmid comprising the above-mentioned fusion protein, an Escherichia coli transformed with the plasmid, and a method for producing the fusion protein using the Escherichia coli.

[Background technology]

In recent years, so-called targeting therapy has been studied, in which a monoclonal antibody specific to cancer cells is linked to an anticancer substance, and the specificity of this monoclonal antibody is used to concentrate the anticancer substance in cancer tissues.

Anticancer substances used in such methods include anticancer antibiotics,
Plant toxins such as ricin and diphtheria toxin have been investigated, but no attempt has been made to use lymphotoxin as an anticancer substance in targeted therapy.

Known methods for linking a cancer-specific antibody to an anticancer substance include a method in which they are directly covalently bonded together, and a method in which a liposome encapsulating an anticancer substance is bonded to a cancer-specific antibody.

However, no attempt has been made to utilize the antibody affinity of Protein A to link a cancer-specific antibody with an anticancer substance for targeted therapy.

It is already known that a fusion protein of protein A and a physiologically active peptide can be prepared (WO 84/03103).
This is mainly for affinity purification of biologically active peptides,
and is used to prepare immunogens.

The conventional method of chemically linking anticancer substances to cancer-specific antibodies for use in targeted therapy has the drawback that the anticancer substances have difficulty entering cancer cells.
Lymphotoxin is known to selectively and directly kill cancer cells via receptors on their surface. Therefore, the present invention aims to provide a means for effective cancer therapy, such as targeted therapy, by using lymphotoxin, which has such advantageous properties, as an anticancer agent.

DISCLOSURE OF THE INVENTION

In order to achieve the above object, the present invention provides a protein A
The present invention provides a fusion protein comprising a polypeptide containing an antibody-binding site of the above-mentioned compound and a lymphotoxin polypeptide, which has both the biological activity inherent to lymphotoxin, i.e., anticancer activity, and the antibody-binding ability inherent to protein A. It also provides a method for producing the fusion protein and a vector encoding the fusion protein required for this production method.
DNA, a plasmid containing the DNA, and E. coli transformed with the plasmid are provided.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the construction of plasmid pLTM1.

FIG. 2 shows the construction of the plasmid pLTM2.

3-1 and 3-2 show the nucleotide sequence of DNA encoding native lymphotoxin and the amino acid sequence of the corresponding polypeptide in the starting plasmid pLTM2 for constructing the plasmid of the present invention.

4-1 and 4-2 show the base sequence of the DNA encoding Protein A in the plasmid pRIT2T and the amino acid sequence of the corresponding polypeptide, which are relevant to the present invention.

FIG. 5 shows the construction of the plasmid pLTM9.

FIG. 6 shows the construction of the plasmid pPRALT1 of the present invention.

FIG. 7 shows the nucleotide sequence encoding the linking portion of the fusion protein of the present invention and the corresponding amino acid sequence.

BEST MODE FOR CARRYING OUT THE INVENTION

The fusion protein of the present invention exhibits anticancer activity in its lymphotoxin peptide portion and has the ability to bind to antibodies through affinity in its protein A portion, and can thereby bind to a cancer-specific antibody, e.g., a monoclonal antibody, to form a complex. When such a complex is administered parenterally, it is expected to be concentrated in cancer cells by the action of the cancer-specific antibody, where the fusion protein is delivered to the cancer cells through the affinity between lymphotoxin and lymphotoxin receptors on the surface of the cancer cells.

A. Fusion Proteins and DNA Encoding Them The fusion peptides of the present invention comprise a polypeptide containing the antibody-binding site of protein A and a lymphotoxin polypeptide, optionally with an oligopeptide linker between the two polypeptides.

(1) Polypeptide Containing the Antibody-Binding Site of Protein A and DNA Encoding It The length of this portion is not particularly limited, as long as it has antibody-binding ability. The amino acid sequence of Protein A has already been determined, and plasmids containing DNA encoding it are commercially available. Therefore, such plasmids can be used as starting plasmids in the present invention. An example of such a plasmid is the plasmid pRIT2T available from Pharmacia (Figure 5). The portion of the nucleotide sequence encoding the amino acid sequence of Protein A in this plasmid that is relevant to the present invention is shown in Figures 4-1 and 4-2.

(2) Lymphotoxin Polypeptide and DNA Encoding It. This portion may be a polypeptide of any length, as long as it is capable of exerting the biological activity of lymphotoxin in the form of a fusion protein. In the present invention, the full-length human lymphotoxin polypeptide is used as an example.

A series of plasmids pLT1, pLTM1 and pLTM2 containing the cDNA of natural lymphotoxin have already been obtained, and their construction method is described in detail in Japanese Patent Application No. 123700/1986.
Escherichia coli x1776/pLT1 is a strain of Escherichia coli strain 87
No. 84 (FERM P-8784), Escherichia coli x177 containing the plasmid pLTM
6/pLTM2 have been deposited at the National Institute of Biotechnology, Agency of Industrial Science and Technology under Microbial Deposit No. 8785 (FERM P-8785). The methods for constructing these plasmids are described below as reference examples and are summarized in Figures 1 and 2. Therefore, the plasmids of the present invention can be constructed starting from these already obtained plasmids. The DNA encoding lymphotoxin in the starting plasmid pLTM2 of the present invention
The sequence and the corresponding amino acid sequence are shown in Figure 3-1 and
This is shown in Figure 2.

(3) Linker oligopeptide and DNA encoding it
Sequence A In the present invention, during the process of constructing an expression plasmid encoding the fusion protein of the present invention, an oligonucleotide sequence such as a consensus SD sequence may be present between the DNA encoding the polypeptide containing the antibody-binding site of Protein A and the DNA encoding the lymphotoxin polypeptide. Therefore, the fusion protein of the present invention includes not only one in which the peptide containing the antibody-binding site of Protein A and the lymphotoxin polypeptide are directly linked, but also one in which they are linked via an oligopeptide linker encoded by the above-mentioned oligonucleotide.

B. Plasmids The plasmids of the present invention contain the DNA coding region under the control of appropriate regulatory sequences and are capable of expressing this DNA coding region in E. coli. Examples of promoters that can be used include the _PL promoter, tac promoter, trt promoter, and lacUV5 promoter. Examples of SD sequences that can be used include the SD sequence of the metapyrocatechinase gene and the consensus sequence.
SD sequences can be used.

To increase the expression level of lymphotoxin in E. coli, the inventors have synthesized three plasmids pLTM2.
Plasmids pLTM7, pLTM9, and pLTM11 were constructed.
The 3' untranslated region of lymphotoxin in the plasmid pLTM2 was deleted, and the expression level increased approximately two-fold by deleting the region that destabilizes mRNA in that region. The plasmid 2pLTM9 was created by adding an ideal consensus SD sequence to the plasmid pLTM7, and the expression level increased approximately two-fold further by improving the SD region. The plasmid pLTM11 was created by modifying the leucine and proline codons following the N-terminal methionine of the plasmid pLTM9 to favor mRNA transcription, and the expression level increased approximately two-fold further.
doubled.

(1) Construction of Plasmid pLTM7 Starting Plasmid pLTM7 Containing the Lymphotoxin Gene
2 by EcoRI to isolate the DNA containing the lymphotoxin gene.
The fragment is excised and inserted into the EcoRI site of M13 phage mp10, and specific base substitution mutations are performed using oligonucleotide primers to introduce an EcoRI site downstream of the translation termination codon of the lymphotoxin gene, resulting in mutant phage LTM 61.

The lymphotoxin gene was excised from the double-stranded DNA of this mutant phage with EcoRI and then transferred to the plasmid pLTM2
into an EcoRI vector fragment not containing the lymphotoxin gene, resulting in plasmid pLTM7. Details of this method are described in Example 1.

(2) Method for constructing plasmid pLTM9 Next, pLTM7 is cleaved with the restriction enzyme XcyI to generate a DNA fragment containing the lymphotoxin gene, to which the linker is ligated, and then further cleaved with EcoRI to obtain a 537 bp EcoRI-EcoRI DNA fragment consisting of the lymphotoxin gene and linker nucleotides.
The NA fragment was inserted into the EcoRI vector of the plasmid pLTM2, which does not contain the lymphotoxin gene, to obtain the plasmid pLT
The details of this method and the sequences of the oligonucleotides used for the linker are described in Example 2.

(3) Method for constructing plasmid pLTM11 Phage LTM81 and plasmid pLTM11 are obtained from plasmid pLTM9 by the same method as the method for constructing plasmid pLTM7. Details of this method are described in Example 3.

(4) Construction of Plasmid pPRALT1 Starting Plasmid pLTM Containing the Lymphotoxin Gene
9 by EcoRI to isolate the DNA containing the lymphotoxin gene.
The fragment is excised and inserted into the EcoRI site of the plasmid pRIT2T encoding the protein A, thereby obtaining the plasmid pP containing the gene encoding the fusion protein of the present invention.
RALT1 is obtained. Details of this method are described in Example 4.
The Escherichia coli strain x1776/pPRALT1 containing the plasmid pPRALT1 is FERM BP-1899 (original domestic deposit: FERM P-9389).
It has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology.

C. E. coli to be transformed In the present invention, any E. coli strain commonly used for producing polypeptides by genetic engineering techniques can be used, for example, N48301, RR1, RB791, SM3
2, HB101, N99, JM103, etc.

D. Expression of Target Polypeptide The target polypeptide can be expressed by culturing E. coli transformed with the above-described plasmid in, for example, LB medium according to a conventional method, and then inducing the expression using a promoter. The target polypeptide can then be obtained by recovering the produced polypeptide from the E. coli cells. Details will be described in the Examples below.

E. Cytocidal Activity The cytocidal activity of the polypeptide obtained by the above method can be measured, for example, by a microplate method. This measurement method and the results will be explained in detail in the Examples below.

The fusion protein of the present invention maintains the cell-killing activity inherent to lymphotoxin.

F. Affinity of Fusion Protein for Antibody As will be explained in detail in the Examples, the fusion protein of the present invention has affinity for antibody and can form a complex with antibody.

Next, the present invention will be described more specifically with reference to examples.

Example 1. Construction of plasmid pLTM7 (1) Introduction of an EcoRI site directly downstream of the termination codon Plasmid pLTM2 was cleaved with the restriction enzyme EcoRI, and a fragment containing the lymphotoxin gene was recovered from a 0.8% agarose gel.

0.5 pmol of this DNA fragment and double-stranded DNA of M13 phage mp10
0.5 pmol of the EcoRI digest was mixed with 66 mM Tris-HCl (pH
7.5), solution 20 containing ₅ mM MgCl2, 5 mM DTT, and 1 mM ATP
100 units of T4 DNA ligase in 1 μl at 12°C
The ligation reaction was carried out for 16 hours. After the reaction, the reaction mixture was purified by the method of Messing et al. [Messing et al., Methods in Enzymology 101 , 20-78, 198
3], and the resulting mixture was added to Escherichia coli JM103 and 0.02% X-gal
and plated with soft agar containing 1 mM IPTG at 37°C.
The phage was cultured overnight at 37°C. Single-stranded template DNA was prepared from the white plaques formed by the recombinants. Specifically, the white plaques were picked with the tip of a toothpick, and E. coli JM103 was grown. The plaques were suspended in 1.5 ml of 2xYT medium (1.6% bactotryptone, 1% yeast extract, and 0.5% NaCl) and cultured at 37°C for 5 hours. Single-stranded recombinant phage DNA was recovered from the culture supernatant by polyethylene glycol precipitation, phenol treatment, and ethanol precipitation.

Using the resulting single-stranded DNA as a template, the base sequence was determined by the dideoxy method according to the method of Messing et al. (supra), and the sequence of the cloned single-stranded DNA was confirmed.
In this way, single-stranded DNA containing the anti-coding strand of the lymphotoxin gene was obtained. This recombinant phage was used as an LTM
It was named 21.

Using the single-stranded DNA of LTM21 obtained as described above as a template, and the synthetic oligonucleotide: CGC TCT GTA GAA TTC GGA AAA ATC CAG as a primer, a repair reaction was carried out using DNA polymerase Klenow fragment.
Two pmol of a 5'-terminally phosphorylated primer was added to the reaction mixture, which was then incubated in 10 μl of a solution containing ₇ mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 20 mM NaCl, and 7 mM MgCl2 at 60° C. for 20 minutes, followed by incubation at 23° C. for 20 minutes. dATP, dGTP, dTTP, and dCTP were also added to the reaction mixture.
The mixture was added to a concentration of 0.5 mM, and 2 units of DNA polymerase Klenow fragment were added to a total volume of 20 μl. The mixture was then incubated at 23° C. for 20 minutes.
The mixture was incubated for 1 minute.
and 1 unit of T4 DNA ligase was added, and the mixture was incubated at 12°C overnight.

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

The phage plaques obtained as described above were subjected to the ^same procedure as above.
The mutant phages were screened by plaque hybridization using as a probe.
That is, the method of Benton Davis et al. [WD Benton and RW Davis, Science 196 , 180, 1
Plaques were transferred from the soft agar medium to a nitrocellulose filter according to the method described in [977] and baked in a vacuum at 80°C for 2 hours. The nitrocellulose filter was then washed with 6x SSC, 1
Hybridization was carried out overnight at 23°C in 0x Denhardt's solution using a ^32P -labeled primer oligonucleotide as a probe.
The mutant phage plaques were isolated by washing in PBS at 59°C and autoradiography to show a positive signal.

A double-stranded phage was isolated from this phage plaque by rapid isolation, digested with EcoRI to obtain a 715-bp fragment, and sequenced using this as a template by the dideoxy method. This mutant phage was confirmed to have a base substitution mutation and an EcoRI site immediately downstream of the termination codon of the lymphotoxin gene. This mutant phage was designated JM 103/LTM 61.

(2) Plasmid Preparation and Transformation Double-stranded DNA was prepared from phage JM 103/LTM 61 according to standard methods, digested with the restriction enzyme EcoRI, and the DNA fragment containing the lymphotoxin gene was recovered by 0.8% agarose gel electrophoresis and purified with Elutip-d.

The lymphotoxin gene-free E of plasmid pLTM2
0.5 pmol of the coRI fragment and 0.5 pmol of the above DNA fragment were dissolved in 40 mM HEPES
T4 in S (pH 7.8), ₁₀ mM MgCl2, 10 mM DTT, 0.4 mM ATP
The reaction was carried out using 100 units of ligase at 12°C for 16 hours.

10 μl of this reaction mixture was used to transform E. coli strain RR1, and transformants resistant to 100 μg/ml ampicillin were selected. Plasmid DNA was extracted from transformant colonies and the base sequence was confirmed by the dideoxy method. This plasmid was named pLTM7.

Example 2. Construction of Plasmid pLTM9 (Figure 5) 24 μg of plasmid pLTM7 (156 μl) was incubated with 60 units of restriction enzyme XcyI (24 μl) and 20 μl of XcyI buffer at 37°C for 4 hours, and then 9 μg (20 μl) of each of the following 5'-phosphorylated oligonucleotides was added to the reaction mixture: The mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation and dried.
The reaction was carried out using 100 units of T4 ligase in _gcl2 , 10 mM DTT, and 0.4 mM ATP for 16 hours at 12° C. The reaction mixture was extracted with phenol and chloroform, and the DNA was then precipitated by ethanol, and the precipitate was dried.

This precipitate was dissolved in 160 μl of distilled water and diluted with 300 units of
The mixture was mixed with EcoRI (20 μl) and 20 μl of EcoRI buffer (20 μl), incubated at 37° C. for 2 hours, and the reaction mixture was frozen.

The reaction mixture was separated by electrophoresis using a 2% agarose gel. The gel portion containing the 537 bp DNA fragment was excised and eluted by electrodialysis in a dialysis membrane. The eluate was applied to an Elutip-D column to adsorb the DNA. The column was then washed, and the DNA was eluted and recovered by increasing the ionic concentration of the eluent. The ethanol precipitate was dried, and the resulting DNA was dissolved in 20 μl of distilled water.

The lymphotoxin gene-free E of plasmid pLTM2
0.5 pmol of the coRI fragment and 0.5 pmol of the above DNA fragment were dissolved in 40 mM HEPES
S (pH7.8), 10mM MgCl2, _10mM DTT, 0.4mM MgCl2, _10m
100 units of T4 ligase in 0.4 mM DTT and 0.4 mM ATP
The reaction was carried out at 2°C for 16 hours.

10 μl of this reaction mixture was used to transform E. coli strain RR1, and transformants resistant to 100 μg/ml ampicillin were selected. Plasmid DNA was extracted from the transformant colonies and the base sequence was confirmed by the dideoxy method. This plasmid was named pLTM9.

Example 3. Construction of Plasmid pLTM11 (1) Plasmid pLTM9, in which the codons for the second leucine and third proline amino acids were converted, was cleaved with the restriction enzyme Eco
The fragment containing the lymphotoxin gene was isolated by 0.8% RI digestion.
It was recovered from an agarose gel.

0.5 pmol of this DNA fragment and double-stranded DNA of M13 phage mp10
0.5 pmol of EcoRI digestion product was mixed with 66 mM Tris-HCl (pH
7.5), solution 20 containing ₅ mM MgCl2, 5 mM DTT, and 1 mM ATP
100 units of T4 DNA ligase in 1 μl at 12°C
The ligation reaction was carried out for 16 hours. After the reaction, the reaction mixture was purified by the method of Messing et al. [Messing et al., Methods in Enzymology 101 , 20-78, 198
3], and E. coli JM103 was transformed with 0.02% X-gal
and plated with soft agar containing 1 mM IPTG at 37°C.
The phage was cultured overnight at 37°C. Single-stranded template DNA was prepared from the white plaques formed by the recombinants. Specifically, the white plaques were picked with the tip of a toothpick, and E. coli JM103 was grown. The plaques were suspended in 1.5 ml of 2xYT medium (1.6% bactotryptone, 1% yeast extract, and 0.5% NaCl) and cultured at 37°C for 5 hours. Single-stranded recombinant phage DNA was recovered from the culture supernatant by polyethylene glycol precipitation, phenol treatment, and ethanol precipitation.

Using the resulting single-stranded DNA as a template, the base sequence was determined by the dideoxy method according to the method of Messing et al. (supra), and the sequence of the cloned single-stranded DNA was confirmed.
In this way, single-stranded DNA containing the anti-coding strand of the lymphotoxin gene was obtained. This recombinant phage was used as an LTM
It was named 71.

Using the single-stranded DNA of LTM71 obtained as described above as a template, a repair reaction was carried out using DNA polymerase Klenow fragment with the synthetic oligonucleotide: T TTA AAT ATG TTA CCT GGT GTT GG as a primer.
Two pmol of a 5'-terminally phosphorylated primer was added to the reaction mixture, which was then incubated in 10 μl of a solution containing ₇ mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 20 mM NaCl, and 7 mM MgCl2 at 60° C. for 20 minutes, followed by incubation at 23° C. for 20 minutes. dATP, dGTP, dTTP, and dCTP were also added to the reaction mixture.
The mixture was added to a concentration of 0.5 mM, and 2 units of DNA polymerase Klenow fragment were added to a total volume of 20 μl. The mixture was then incubated at 23° C. for 20 minutes.
Subsequently, 1 μl of 10 mM ATP and 1 unit of T4 DNA ligase were added, and the mixture was incubated at 12° C. overnight.

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

The phage plaques obtained as described above were subjected to the ^same procedure as above.
The mutant phages were screened by plaque hybridization using as a probe.
That is, the method of Benton Davis et al. [WD Benton and RW Davis, Science 196 , 180, 1
Plaques were transferred from the soft agar medium to a nitrocellulose filter according to the method described in [977] and baked in a vacuum at 80°C for 2 hours. The nitrocellulose filter was then washed with 6x SSC, 1
Hybridization was carried out overnight at 23°C in 0x Denhardt's solution using a ^32P -labeled primer oligonucleotide as a probe.
The mixture was washed in PBS at 59°C and autoradiographed to isolate mutant phage plaques that showed a positive signal.

A double-stranded phage was isolated from this phage plaque by rapid isolation and digested with EcoRI to obtain a 537-bp fragment. The nucleotide sequence of this fragment was determined by the dideoxy method using the template. It was confirmed that base substitution mutations had occurred, altering the codons for the second leucine and third proline amino acids in the lymphotoxin gene. This mutant phage strain was designated JM 103/LTM 81.

Double-stranded DNA was prepared from phage JM103/LTM81 in the usual manner, digested with the restriction enzyme EcoRI, and the DNA fragment containing the lymphotoxin gene was recovered by 0.8% agarose gel electrophoresis and purified with Elutip-d.

The lymphotoxin gene-free E of plasmid pLTM2
0.5 pmol of the coRI fragment and 0.5 pmol of the above DNA fragment were dissolved in 40 mM HEPES
T4 in S (pH 7.8), ₁₀ mM MgCl2, 10 mM DTT, 0.4 mM ATP
The reaction was carried out using 100 units of ligase at 12°C for 16 hours.

10 μl of this reaction mixture was used to transform E. coli strain RR1, and transformants resistant to 100 μg/ml ampicillin were selected. Plasmid DNA was extracted from transformant colonies and the base sequence was confirmed by the dideoxy method. This plasmid was named pLTM11.

Example 4. Construction of plasmid pPRALT1 (Figure 6) Plasmid pRIT2T (obtained from Pharmacia) 5 μg
The digestion was carried out by incubating 10 μl of the DNA fragment with 15 units of EcoRI (1 μl) at 37° C. for 5 hours. The reaction mixture was then diluted with 40 μl of 25 mM Tris-HCl (pH 8.0) buffer and 1 μl of 100 μl of 15 μl of EcoRI.
0 μl alkaline phosphatase (E. coli C75)
(1/10 diluted) was added and reacted at 65°C for 40 minutes.

The reaction mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation and dried.

0.5 pmol of this DNA precipitate and 0.5 pmol of the EcoRI fragment containing the lymphotoxin gene of the plasmid pLTM9 were diluted in 40 mM HEPES
The reaction was carried out at 12° C. for 16 hours using 100 units of T4 ligase in 10 mM MgCl ₂ , 10 mM DTT, and 0.4 mM ATP (pH 7.8).

10 μl of this reaction mixture was used to transform Escherichia coli strain RR1, and transformants resistant to 100 μg/ml ampicillin were selected. Plasmid DNA was extracted from the transformant colonies and the base sequence was confirmed by the dideoxy method. This plasmid was named pPRALT1.

Example 5. Confirmation of expression product by in vitro translation. Plasmid pPRALT1 of the present invention encoding a fusion protein of lymphotoxin and protein A, and protein A
The expression product of the in vitro translation of the plasmid pRIT2T encoding the nucleotide sequence was tested using a DNA expression kit (Amersham).
The apparent molecular weights of the expression products were determined by SDS-PAGE and comparison of the band positions of the expression product and molecular weight standard bands. The expression product of pPRALT1 had a molecular weight of approximately 48 K, and the expression product of pPIT2T had a molecular weight of approximately 30 K. This confirmed that the fusion protein of the present invention was expressed by the plasmid pPRALT1.

Example 6. Expression and purification of polypeptide The E. coli HB101 strain containing the above-mentioned plasmid pPRALT1 was incubated in 30 ml of LB medium at 37°C overnight, and then inoculated into 3 L of LB medium and cultured at 37°C for 7 hours.
A jar fermenter (manufactured by Iwashiya) was used for the cultivation. After the cultivation was completed, the culture medium was centrifuged to collect the bacterial cells. The collected bacterial cells were then soaked in phosphate buffer (10 mM sodium phosphate (pH
7.2) After adding 1 mM EDTA and suspending thoroughly, the cells were disrupted by passing through a Manton-Gaulin homogenizer three times at 8000 psi. The mixture was centrifuged at 10,000 rpm for 30 minutes to obtain the supernatant.

To this supernatant, 5% w/v ammonium sulfate was added to effect ammonium sulfate precipitation, and the supernatant was purified on a Butyl Toyopearl (manufactured by Toso) column.

The supernatant was added to 20 ml of Butyl Toyopearl 650M equilibrated with phosphate buffer (10 mM sodium phosphate (pH 7.2), 0.02% Tween 20) containing 5% w/v ammonium sulfate, and immediately washed with 100 ml of the equilibration solution. The protein adsorbed to the column was eluted with phosphate buffer containing no ammonium sulfate. The eluted fraction was the target polypeptide.

Example 7: Confirmation of antibody binding ability of target polypeptide It was confirmed that the target polypeptide binds to the antibody, and that this binding is due to the affinity of the protein A moiety. Specifically, the target polypeptide obtained in Example 6 was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane using an electroblotting device (Biometra).
After blocking with phosphate buffer containing A, the antibody was bound to peroxidase-labeled bovine anti-rabbit IgG antibody (Betchil Laboratories), washed, and then 4-chloro-1
The color was developed using a 100-naphthol solution and hydrogen peroxide solution. As a result, a dark purple band was observed at a molecular weight of 44,000. The target polypeptide bound to a peroxidase-labeled bovine antibody.

Cell extracts (supernatants) obtained by the method described in Example 2 from E. coli N48301 containing the plasmid pPRALT1 and E. coli RR1 containing the plasmid pLTM7, as well as culture supernatants from HUT-102 cells, were diluted with phosphate buffer to 1 x ¹⁰⁴ U/ml for the E. coli extract and 1 x ¹⁰³ U/ml for the HUT-102 supernatant. 10 µl of column carrier IgG Sepharose 6 Fast Flow (Pharmacia) was added to 300 µl of each solution and gently shaken at room temperature for 1 hour. These were centrifuged at 6000 rpm to obtain supernatant A. The precipitated carrier was washed three times with 200 µl of phosphate buffer. After separating the carrier from the wash by centrifugation at 6000 rpm, 300 µl of phosphate buffer containing 1 mg/ml of Protein A (Sigma) was added to the carrier and gently shaken at room temperature for 1 hour. This mixture was then washed.
After centrifugation at 6000 rpm, supernatant B was obtained.

The lymphotoxin activity of the thus prepared supernatants A and B was measured by the above-mentioned method, and the following results were obtained.

From the above results, lymphotoxin activity was observed in supernatant B of the extract containing the product from E. coli containing the plasmid pPRALT1 of the present invention, i.e., the fusion protein of lymphotoxin and protein A. This indicates that
This means that the fusion protein was adsorbed to the carrier via the antibody (IgG) immobilized on the carrier, and then displaced and eluted by adding protein A.

Therefore, this experiment demonstrated that the polypeptide of the present invention, which is a fusion protein of protein A and lymphotoxin, forms a complex with the antibody.

Example 8. Cytocidal activity The cytocidal activity of the polypeptide of interest was evaluated by BBA Aggarwal et al.
( J. Biological Chemistry , 260 , 2345-2354). Mouse L-cells were cultured in Eagle's MEM medium containing 5% calf serum, 0.5% penicillin, 0.5% streptomycin, and 1 μg/ml actinomycin D.
M cells were cultured, and 3 x 10 ⁴ cells/100 μl were placed in a well. 100 μl of the diluted ampoule was added, and the mixture was incubated at 5% C.
The cells were cultured for 24 hours at 37°C under _{an O2} atmosphere. After washing the plate, 100 μl of a 0.05% crystal violet-formalin-ethanol solution was added and the cells were stained for 30 minutes. The dye was then eluted with 100 μl of a _0.05 % M _NaH2PO4 -ethanol solution, and the absorbance at 570 nm was measured using a photometer (MiniReader II, Dynatech).

The absorbance (normal scale) versus dilution factor (logarithmic scale) was plotted on a semi-logarithmic graph, and the number of units of cytocidal activity in the sample was calculated, with the activity required for 50% cell killing being 1 unit. The cytocidal activity value was corrected using the reagent TNF (Genzyme) as an internal standard.

The cytocidal activity of the target polypeptide obtained in Example 6 was 8.8 x 10 ⁵ units (per mg of protein).
Therefore, the fusion protein of the present invention expressed by the plasmid pPRALT1 has lymphotoxin activity.

Reference Example 1. Preparation and screening of cDNA library (1) Preparation of mRNA Human T lymphocytic leukemia cell line HUT-102 was suspended in 20 ml of RPMI-1640 medium containing 5% fetal bovine serum and cultured at 37°C in a 5% _CO2 incubator for 2 to 3 days.
The medium was doubled in volume and cultured for 2-3 days each time, and finally culture solution 1 was obtained.
Centrifuge at 3000 rpm for 10 minutes at ℃ to collect the cells, and then transfer them to 50 ml of phosphate buffer (10 mM sodium phosphate, pH 7.2, 0.15 M NaCl).
The cells were washed by suspending in PBS, and then centrifuged to obtain a T cell pellet.

T. Maniatis et al. “Molecular Cloning” Cold Sprin mRNA
g Guanidium/C according to Harbor Laboratory 1982 p.196
The T cell pellet was isolated using the sCl method. The T cell pellet was diluted with 5 volumes of guanidinium isothiocyanate solution (6 M guanidinium isothiocyanate, 5 mM sodium citrate, pH 7.0, 0.1 M
β-mercaptoethanol, 0.5% sarkosyl), and place in a glass homogenizer container.
After homogenization, the homogenate was gently placed on 1.5 ml of a solution containing 5.7 M CsCl and 0.1 M EDTA (pH 7.5) in a SW55 centrifuge tube (Ultra Clear, trademark), and the homogenate was then transferred to a centrifuge tube.
Ultracentrifugation was carried out at 35,000 rpm for 20 hours at 5°C. After removing the guanidinium solution, the wall was washed three times with guanidinium solution, and the centrifuge tube was cut at the top of the CsCl solution. After removing the CsCl solution, the precipitate was washed twice with 80% ethanol and resuspended in 10 mM
The fragment was dissolved in 500 μl of a buffer solution (TES) containing Tris-HCl pH 7.4, 5 mM EDTA, and 10% SDS.
After extraction with 500 μl of N-butanol mixture (4:1), the organic layer was extracted again with 500 μl of TES, and the aqueous layers were combined and subjected to ethanol precipitation to obtain mRNA.

The mRNA was passed through an oligo(dT) cellulose column according to the method described in "Molecular Cloning," to purify poly(A) ₊ RNA.

(2) Synthesis of cDNA The synthesis of the first strand of cDNA was carried out according to the Gubler-Hoffman method.
M), 0.6M KCl 1μl (30mM), 0.16M MgCl ₂ 1μl (8m
M), 20mM DTT 1μl (1mM), 25mM dNTP (dATP, dCT
1.6 μl (2 mM each) of a 25 mM mixture of dTP, dGTP, and dTTP, RNase A
asin (Biotech 30U μl) 1 μl, oligo (dT) 12
-18 (PL Co., Ltd.) 1 μl (2 μl), and HUT-102poly
(A) ₊ 3 μl of RNA (1 μg/μl) were mixed to make a total volume of 19 μl.
The mixture was pre-incubated at 37°C for 5 minutes, and then 1 μl of reverse transcriptase (Life Science, 12.5 U/μl) was added.
The reaction was allowed to proceed at 80°C for 1 hour. The final concentration of the salt solution is shown in parentheses. After the reaction, 1 μl of 0.5 M EDTA and 0.5 μl of 10% SDS were added to terminate the reaction, followed by phenol-chloroform extraction, ammonium acetate-ethanol precipitation twice, and precipitation at 80°C.
After washing with 50μ% ethanol and drying under reduced pressure,
5 μl of this was taken, to which 1 μg of RNase was added, and the mixture was left at room temperature for 10 minutes, followed by phenol extraction. The aqueous layer was subjected to 0.7% agarose gel electrophoresis to examine the degree of first strand elongation.

The second strand was synthesized under the same conditions as those for the Gubler-Hoffman method.
33.5μl of water, 2μl of 1M Tris-HCl (pH7.5) (20mM),
0.5M MgCl2 _1μl (5mM), 1M ( _NH4 ) _{2SO4 1μl} (10mM
M), 10μl of 1M KCl (100mM), 1μl of 10mM β-NAD (0.1
5mg/ml BSA 1μl (50μg/ml), 4mMdNTP 1μl
(40 μM), E. coli DNA ligase (PL, 0.5 μg/μl) 1
μl, E. coli DNA polymerase I (PL, 15 U/μl) 2 μl
l, E. coli RNaseH (Takara Shuzo 1.3U/μl) 0.5μl, 10 ³²
1 μl (10 μl) of P-dCTP was mixed to make a total volume of 100 μl, and the mixture was allowed to react at 12°C for 1 hour, followed by 22°C for 1 hour. 1 μl of sample was taken before and after the reaction, and the amount of radioactivity taken up was measured by the TCA precipitation method. 0.5 MEK was added to the reaction solution.
After stopping the reaction by adding 4 μl of DTA and 5 μl of 10% SDS,
Phenol-chloroform extraction was carried out, followed by ethanol precipitation twice. The precipitate was washed with 80% ethanol and dried under reduced pressure, and then dissolved in 10 μl of water.

(3) Preparation of cDNA library: 10 μl of cDNA solution, 2 μl of water, 0.7 μl (35 mM) of 1M Tris-acetate (pH 7.9), 1.3 μl of 1M potassium acetate
(65 mM), 0.1 M magnesium acetate 2 μl (10 mM), 10 mM
DTT 1μl (1mM), 5mg/ml BSA 1μl (250μg/ml), 2m
1 μl (0.1 mM) of mdNTP, T4 DNA polymerase (Takara Shuzo 1.
The mixture was mixed with 1 μl of 10% SDS (5 U/μl) to a total volume of 20 μl, and incubated at 37° C. for 10 minutes. The reaction was stopped by adding 1 μl of 0.5 M EDTA and 0.5 μl of 10% SDS, followed by phenol-chloroform extraction and ethanol precipitation twice. The precipitate was washed with 80% ethanol, dried under reduced pressure, and then dissolved in 10 μl of water.

EcoRI linker (Takara Shuzo, GGAATTCC, 1μg/μl)
To 2 μl of the solution, add 5 μl of water, 5× linker kinase buffer (0.
33M Tris-HCl pH 7.6, 5mM ATP, 5mM spermidine, 50mM
Add 2 μl of 30 mM MgCl ₂ , 75 mM DTT, 1 mg/ml BSA, and 1 μl of T4 DNA kinase (Takara Shuzo, 6 U/μl) to make a total volume of 10 μl.
The reaction was carried out at 7°C for 1 hour. 5 µl of the blunt-ended cDNA solution, 2 µl of 5x linker kinase buffer,
l, water 1.5μl, T4 RNA ligase (Takara Shuzo, 175U/μl
(1μl, T4 RNA ligase (PL, 0.8μg/μl) 0.5μl
The reaction mixture was heated to 65°C for 10 minutes to inactivate the ligase, and then 65 μl of water, 10 μl of 10x EcoRI buffer, and EcoRI were added.
(Takara Shuzo 7.5U/μl) 5 μl was added to make a total volume of 100 μl.
The reaction was allowed to proceed at 37°C for 3 hours. One-tenth-volume samples were taken before and after EcoRI digestion and subjected to 10% polyacrylamide gel electrophoresis to check the success of EcoRI linker ligation and EcoRI digestion. The reaction mixture was extracted with phenol, and the aqueous layer was adjusted to 0.3M NaCl by adding 5M NaCl. The EcoRI linker monomer was then removed by loading onto a 2ml Sepharose CL-4B column. The mixture was developed with TE (pH 7.6) and 0.3M NaCl. After collecting 4-5 drops each, the counted fractions were ethanol precipitated and dissolved in 10µl of water.

Plasmid pU9 was cleaved with EcoRI and treated with calf intestinal alkaline phosphatase (CIP) in the usual manner. 1 μl (0.4 μg) of this preparation was mixed with cDNA 1 prepared by the above method.
0 μl, 20 μl of 5x linker kinase buffer and 64 μl of water
The reaction mixture was mixed and heated at 68°C for 10 minutes, and then 5 μl of T4 RNA ligase was added to make a total volume of 100 μl. This reaction mixture was incubated overnight at 12°C and heated at 65°C for 10 minutes. 10 μl of each mixture was then added to E. coli.
The resulting solution was mixed with 210 μl of competent cells of the x1776 strain, and transformation was carried out.
The cells were added to 300 ml of x1776 medium (containing 50 μg/ml ampicillin) and cultured overnight at 37°C. A portion of the culture was made into a 15% glycerol solution and stored frozen at -80°C.

(4) Screening of the cDNA library 20 μl of frozen glycerol stock solution of cDNA-containing transformants
Dilute 5,000 times with x1776 medium and add 100 μl of the diluted solution.
The mixture was spread onto nitrocellulose placed on an ampicillin-containing x1776 agar medium plate and cultured overnight at 37°C.
The colonies on the nitrocellulose were transferred to two sheets of nitrocellulose. The replicas were cultured on x1776 medium for 3 hours, then transferred to medium containing 10 μg/ml chloramphenicol and cultured overnight. The filters were then successively immersed in 10% SDS, denaturing solution (0.5 N NaOH, 1.5 M NaCl), and 10% SDS for 5 minutes each.
After placing the filter on a neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0), 5 ml of proteinase K solution (proteinase K (Merk) 1 mg/ml, 0.5 M NaCl, 10 T
The filter was placed between filter paper and rubbed vigorously with a roller, then washed twice with 2x SSC and heated at 80°C for 3 hours.

The nitrocellulose filter was washed with 6×SSC, 10
Hybridization was performed overnight at 42°C with ^32P -labeled oligonucleotide probe #71 in 6x Denhardt's solution. The filters were then washed in 6x SSC at 55°C and autoradiographed for 2 days. Three colonies showed positive signals.

The filter was then washed in 1x SSC solution at 65°C for 30 minutes to wash away the positive signal, then dried and used again for hybridization with oligonucleotide probe #72. Hybridization with ^32P -labeled oligonucleotide probe #72 in 6x SSC, 10x Denhardt's solution was performed overnight at 42°C. The filter was then washed in 6x SSC at 50°C and autoradiographed for 2 days. The same three colonies as when the #71 probe was used showed positive signals.

Plasmid DNA was extracted from these three colonies.
The nucleotide sequences were determined by the dideoxy method in the usual manner, and one of these clones was found to contain the entire coding sequence for the lymphotoxin peptide.
This plasmid was designated pLT1 (FIG. 1).

Reference Example 2. Construction of Plasmid pLMT1 (FIG. 1) The plasmid pLT1 was transformed into Escherichia coli RR1 strain, the transformant was cultured in a conventional manner, and plasmid DNA was extracted from the cultured cells.

40 μl (20 μg) pLT1 plasmid DNA, 20 μl Eco
RI buffer (x10), 140 μl of distilled water and 4 μl of EcoRI
The enzyme solution (40 units) was mixed and reacted at 37°C for 3 hours. This reaction mixture was extracted with 100 μl each of phenol and chloroform, washed with 200 μl of ether, and then precipitated with ethanol. After drying, the precipitate was dissolved in 100 μl of electrophoresis dye solution and diluted with 1x TBE (Tris-Borate-E
The gel was electrophoresed on a 1.2% agarose gel in 1x TBE buffer (AGE separation), and the gel containing the target DNA of approximately 0.9 kb was excised. This gel piece was placed in a dialysis tube and quenched with 1x TBE.
The contents of the dialysis tube were collected and applied to an Elutip-d column to adsorb the DNA.
The column was washed, and the DNA was eluted and recovered by increasing the ion concentration of the eluent. After ethanol precipitation, the precipitate was dried for 3 minutes, and the resulting DNA was dissolved in 20 μl of distilled water.

Plasmid pKK223-3 containing solution 10 μl (10 μl), 5 μ
l of EcoRI buffer (x10), 36 μl of distilled water and 3 μl
The reaction mixture was mixed with 50 μl of 50 mM Tris-HCl (pH 7.5) and incubated at 37° C. for 3 hours.
The mixture was added with 10 μl of PBS (pH 8.0) and 10 μl of bacterial alkaline phosphatase (BAP) C-75 solution (1/10 dilution) and incubated at 65°C for 1 hour. The reaction mixture was extracted with 100 μl each of phenol and chloroform, washed with 200 μl of ether, and then precipitated with ethanol. The precipitate was dried for 3 minutes and dissolved in 20 μl of distilled water.

5 μl of the pKK223-3 digest, 5 μl of the pLTI digest
μl, 10 μl of 100 mM HEPES (pH 7.8), 10 μl of 30 mM
gCl ₂ , 1 μl of 300 mM MDTT, 1 μl of 10 mM ATP, and 2
Mix 12 μl of T4 DNA ligase solution (20 units)
The reaction was carried out at 0°C overnight.

10 μl of this reaction mixture was mixed with 200 μl of competent cells of E. coli JM103 strain to carry out transformation, which was then spread on an H plate and cultured overnight at 37°C. Of the many colonies that formed, 12 were analyzed by rapid isolation, and it was confirmed that 3 colonies contained the desired plasmid. This plasmid was then cloned into pLTM1.
It was named.

Reference Example 3. Preparation of plasmid pLTM2 (Figure 2) 100 μl (10 μg) of plasmid pLTM1 solution, 40 μl of Pv
uII buffer (x10), 260 μl distilled water, 4 μl PvuII
The enzyme solution (40 units) was mixed and reacted at 37°C for 1 day. To this reaction mixture, 360 μl of distilled water, 40 μl of Pvu
PvuI buffer and 4 μl of PvuI enzyme solution (40 units) were added and reacted at 37° C. for 1 day. The reaction mixture was extracted with 400 μl each of phenol and chloroform.
After washing with 100 μl of ether, the DNA was precipitated with ethanol and the resulting precipitate was dried. The precipitate was dissolved in 100 μl of agarose electrophoresis dye solution and electrophoresed on a 1.2% agarose gel in 1x TBE. The gel containing DNA fragments of approximately 1.6 kb and 1.7 kb in length was excised. The gel fragments were placed in a dialysis tube and electrophoresed in 1x TBE for 2 hours. The contents of the dialysis tube were recovered and applied to an Elutip-d column to adsorb the DNA. The column was then washed, and the DNA was eluted and recovered by increasing the concentration of the eluent. The DNA was ethanol precipitated from the eluate, dried for 3 minutes, and the precipitate was dissolved in 20 μl of distilled water. To this solution, 380 μl of 1 M Tris-HCl (pH 8.0) and 4 μl of BAP were added.
C-75 enzyme solution (1/10 diluted) was added and reacted for 1 hour at 65° C. The reaction mixture was extracted with 300 μl each of phenol and chloroform, and then washed with 600 μl of ether.

The E. coli JM 103 strain containing the plasmid pCTM4 was cultured in a conventional manner, and the plasmid DNA was extracted from the cultured cells in a conventional manner.
A was extracted from this pCTM4 plasmid DNA solution (100 μl (DNA
The ion concentration was increased to elute and recover DNA. DNA was precipitated from the eluate with ethanol, and the precipitate was collected for 3 min.
After drying for 3 minutes, the solution was dissolved in 400 μl of distilled water.
The mixture was extracted with 100 μl each of phenol and chloroform and washed with 600 μl of ether.

100 μl of the solution containing the approximately 1.6 kb and 1.7 kb DNAs, 50 μl of the solution containing the approximately 1.9 kb DNA, 20 μl (75 μg) each of 5' phosphorylated oligonucleotides #73 and #74: CATGCTCCCGGGTGTTGGTCTTACTCCATCAG #73 TGCAGTACGAGGGCCCACAACCAGAATGAGGTAGTC #74 10 μg of tRNA, and 50 μl of 3 M sodium acetate were mixed, and 1 ml of ethanol was added to the mixture, which was then left at -80°C for 20 minutes and centrifuged at 16,000 rpm at 4°C for 10 minutes to separate the DNAs.
The precipitate was precipitated with RNA, the supernatant was removed, the precipitate was dried, and the mixture was diluted with 8 μl of 30 mM MgCl ₂ , 4 μl of distilled water, and 8 μl of
The DNA was dissolved in a mixture of 100 mM HEPES (pH 7.5) and annealed at 65°C for 20 minutes, 42°C for 30 minutes, room temperature for 5 minutes, and then on ice for 5 minutes.
P, 1 μl of 300 m DTT, 9 μl of 40% PEG, and 2 μl of
A T4 DNA ligase solution (20 units) was added, and the reaction was carried out at 20°C overnight.

10 μl of this ligation reaction mixture was used to transform Escherichia coli RR1 strain according to the Hanahan method, and the resulting transformant was spread on an LB ampicillin plate and cultured overnight at 37° C.
Fifty-five colonies emerged, of which nine were analyzed by rapid isolation, and two were confirmed to contain the desired plasmid, which was designated pLTM2.

This plasmid was used to transform Escherichia coli JM103 and x1776 strains, respectively.
coli ) JM 103/pLTM2, and Escherichia coli (
E. coli ) x1776/pLTM2 was obtained.

Continued from the front page (51) Int.Cl. ⁶ Identification symbol Internal reference number FI Technical marking location // (C12P 21/02 C12R 1:19) (71) Applicant 999999999 Nissan Chemical Industries, Ltd. 3-7-1 Kanda Nishikicho, Chiyoda-ku, Tokyo (71) Applicant 999999999 Tosoh Corporation 4560 Tomita, Shinnanyo City, Yamaguchi Prefecture (72) Inventor Tetsuzo Miki 2-1 Shinshinden, Abiko City, Chiba Prefecture A-908 (72) Inventor Seiji Kato 1-9-2 Minamidai, Sagamihara City, Kanagawa Prefecture (72) Inventor Hiroshi Nagata 4-19-12 Tsukushino, Machida City, Tokyo

Claims (9)

[Claims] 1. A fusion protein comprising a polypeptide containing the antibody-binding site of protein A and a lymphotoxin polypeptide, said fusion protein having the biological activity of lymphotoxin and the ability to bind to an antibody. 2. The fusion protein according to claim 1, wherein the polypeptide containing the antibody-binding site of protein A and the lymphotoxin polypeptide are linked directly or via a linker peptide. Claim 3: The following amino acid sequence: The fusion protein according to claim 2, comprising: 4. DNA encoding a fusion protein comprising a polypeptide containing the antibody-binding site of protein A and a lymphotoxin polypeptide, said fusion protein having the biological activity of lymphotoxin and the ability to bind to an antibody. Claim 5: The following base sequence: The DNA according to claim 4, having the following structure: 6. A plasmid comprising DNA encoding a fusion protein comprising a polypeptide containing the antibody-binding site of protein A and a lymphotoxin polypeptide, said fusion protein having the biological activity of lymphotoxin and the ability to bind to an antibody. 7. The plasmid of claim 6 designated pPRALT1. [Claim 8] Escherichia coli transformed with a plasmid containing DNA encoding a fusion protein comprising a polypeptide containing the antibody binding site of protein A and a lymphotoxin polypeptide, said fusion protein having the biological activity of lymphotoxin and the ability to bind to an antibody. [Claim 9] A method for producing a fusion protein comprising a polypeptide comprising the antibody binding site of protein A and a lymphotoxin polypeptide, said fusion protein having the biological activity of lymphotoxin and the ability to bind to an antibody, said method comprising culturing Escherichia coli transformed with a plasmid comprising DNA encoding said protein, and recovering said fusion protein from the culture.