JPH0642833B2 - Human epidermal growth factor gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to recombinant DNA technology for producing human epidermal growth factor (abbreviated as hEGF). More specifically, a synthetic gene corresponding to hEGF and DNA containing the same
Regarding

Prior Art hEGF is secreted mainly from the duodenum and the submandibular region 5
It is a polypeptide hormone consisting of 3 amino acids and has gastric acid secretion inhibitory action and epidermal cell growth promoting action. The gastric acid inhibitory action of hEGF shows its potential as a therapeutic drug for duodenal ulcer. Furthermore, it is known that hEGF binds to the EGF receptor present on the cell membrane surface to induce multifaceted biological reactions [D. G
ospodarowicz, Ann. Rev. Phys
iol. 43 251 (1981)]. The response induced by EGF is the same as the response induced by the oncogene product of oncovirus, and elucidation of the role of EGF in vivo and the mechanism of cell growth regulation is also important in order to investigate the mechanism of carcinogenesis. It is considered to be interesting. However, since naturally-occurring hEGF is extremely small, recombinant DNA
Technical production has come to the fore. Since attempts to obtain the hEGF gene from human tissues are extremely difficult due to various restrictions, the DNA sequence of hEGF has not yet been determined.

On the other hand, the previously determined amino acid sequence of hEGF [H. Gregory, Nature 257 325
An example of expressing a chemically synthesized structural gene in a microorganism based on ('75)] is known. However, since EGF is a relatively low-molecular peptide, it is recognized as a foreign substance in the bacterial cell, and is easily enzymatically decomposed,
It is expressed as a fusion peptide [J. Smith
Et al., Nucleic Acids Research
10 4467 ('82)]. Further, a method of removing an unnecessary portion from the fusion peptide has been proposed, but it is extremely uncertain. (Stephen James Brewer et al., JP-A-58-216697). An example of expressing hEGF itself is known in the yeast system [M. S. Urdea et al., Proc. Natl. Aca
d. sci. USA 80 , 7461 ('83)], its expression level is very low, and it is not suitable for mass production in combination with the slow growth rate of yeast.

Problems to be Solved by the Invention As described above, the DNA sequence of the hEGF gene has not yet been elucidated, and there have been attempts to express corresponding genes synthesized on the basis of the amino acid sequence of hEGF in various systems. In the method of expressing as a fusion peptide, there are operational complexity, difficulty in removing unnecessary parts, etc., and in the case of directly expressing the above synthetic gene without using the fusion peptide, its production amount is extremely low and practical. It wasn't what I expected.

Means for Solving the Problems As a result of intensive research conducted by the present inventors to provide a method for efficiently producing hEGF, hEG suitable for this purpose was obtained.
The present invention has been completed by finding the DNA sequence of F, establishing the method for producing the gene, the recombinant DNA containing the gene, and the method for producing the recombinant DNA.

What has been conventionally used as a DNA sequence for hEGF consists of codons suitable for expression systems such as Escherichia coli and yeast, but the present inventors have now expressed expression that is considerably different from humans. Mouse EGF (mEGF) closer to humans, completely different from codons suitable for the system
The present invention has been completed by paying attention to the above gene.

hEGF is that of mouse [J. Scott et al., Scie
, 221 236 ('83)], there is 70% homology in the amino acid sequence, and most of the differences in amino acids are one po of the codon.
It is guided by the int mutation. That is, the DNA sequence of the hEGF gene is presumed to be very similar to that of mouse. Generally, when a DNA sequence is derived from an amino acid sequence, a large number of DNA sequences are possible due to the degeneracy of codons. Therefore, as a standard for determining the sequence of the synthetic gene, it is customary to adopt the most accepted codon in the cells of the expression system.
(Morio Ikehara et al., Biotechnology Research Report 2 , 7 (198
3)].

However, recent findings show that eukaryotic genes can be expressed in prokaryotes without any problems and in some cases are more efficient than genes adapted to the expression system [M. H. C
arthurs Nucleic Acids Re
search, Symposium Series 1
1 197 ('82)]. Although there are many factors for enhancing gene expression, the stability and translation efficiency of mRNA corresponding to a structural gene are also important. In this case, the higher-order structure determined by the base sequence of mRNA is presumed to have an important meaning. Considering these points, the DNA sequence of the hEGF gene should be similar to the mouse EGF gene as long as the amino acid sequence is not changed. In fact, when the hEGF gene was designed based on this idea, it was possible to have a homology close to 90% with that of the mouse. However, in order to construct the target gene correctly, the presence of relatively long self-complementarity on the DNA strand or abnormal complementarity between double-stranded DNAs should be minimized. A slight modification was made using a computer to satisfy these conditions, and a novel DNA sequence most suitable for the production of hEGF as shown in FIG. 1 was found. FIG. 1 shows the amino acid sequence in addition to the DNA sequence.

The gene can be expressed as a fusion peptide, or can be directly expressed as hEGF without being a fusion peptide.

In the former case, a DNA coding for a protein other than hEGF starting from the start codon ATG is placed at the 5′-end side of the hEGF synthetic gene, and ends with a stop codon (eg, TAG) or starts with the start codon ATG. A DNA encoding a protein other than hEGF may be placed at the 3'end of the gene and terminated with a stop codon (eg, TAG).

To use the latter for direct expression, hE
In addition to the sequence encoding the polypeptide of GF, a start codon ATG and a stop codon such as TAG are added to the 5'side and 3'side, respectively.
Side, and the 5'-end side and the 3'-end side are Eco RI and Bam HI sticky ends respectively for insertion into the vector. A recognition site for BglII is provided in the latter half of the gene. Further, a Pst I recognition site can be provided downstream of the 3'end. With the above modifications, the mouse E
It has 80% homology to the GF (mEGF) gene.

In the synthesis of the hEGF gene of the present invention, for example, the second
As shown in the figure, the hEGF gene was finally divided into 22 fragments, but care was taken here that no self-complementary sequence appeared at the 5'or 3'ends in order to avoid fragment self-association. FIG. 3 shows each DNA fragment. The method of division into fragments is not limited to the above and can be variously divided if care is taken to avoid the above self-association.

Each DNA fragment (# 1 to # 22) can be produced according to a known synthetic method. If necessary, each fragment was phosphorylated at the 5'end with a polynucleotide kinase,
It is divided into 3 groups and hybridized to form double-stranded DNA by DNA ligase. Further, complete hEGF gene was obtained by ligating each group again with DNA ligase (see FIG. 4).

This is ligated with a digestion product of pBR322 with EcoRI and BamHI to obtain a new plasmid pTB361, which is transformed into Escherichia coli DH1. The nucleotide sequence of the isolated plasmid is determined by the Sanger method using a part of the DNA fragment as a primer to confirm the presence of the target hEGF gene.

When expressing the synthetic gene of the present invention, it is preferably used as a recombinant DNA inserted into a vector such as a plasmid or bacteriophage.

The above recombinant DNA preferably has a promoter upstream of the above-mentioned initiation codon ATG, and any promoter may be used as long as it is suitable for the host used for producing the transformant.

For example, Escherichia coli;
(Eg, 294, W3110, DH1, N4830, etc.), the trp promoter, lac promoter, rec
A promoter, λP _L promoter, lpp promoter, etc., such as Bacillus subtilis
is; (eg, MI 114, etc.), yeast (Saccharomyces cerevisiae) such as SP01 promoter, SP02 promoter, penP promoter, etc.
siae; eg, AH22), PH05 promoter, PGK promoter, GAP promoter, ADH
Animal cells such as promoters (eg, monkey cells COS-
7, Chinese hamster cells CHO, etc.) SV
40-derived promoters and the like. In particular, it is preferable that the host is Escherichia coli and the promoter is the trp promoter or the λP _L promoter.

An example of the expression of the hEGF synthetic gene will be described below (see FIG. 5).

The 172 base pair DNA cut out from pTB361 with EcoRI-PstI was used as an expression vector ptrp781.
Was incorporated into the EcoRI and PstI sites of E. coli to obtain the expression vector pTB370 under the control of Ptrp.

On the other hand, a 179 base pair DNA obtained by digesting pTB361 with EcoRI-BamHI was used as an expression vector p.
The EcoRI-BamHI site of TB281 built-in _{P L}
The expression vector pTB372 under control was used.

Escherichia coli GH1 was transformed with pTB370, and growing colonies were selected using ampicillin sensitivity as an index to obtain a strain containing the target hEGF gene.

In the case of pTB372, temperature sensitive E. coli N4830
Escherichia coli D was transformed with pTB372, which was transformed by using Escherichia coli and selected with tetracycline sensitivity as an index.
H1 was transformed to express a synthetic gene.

These transformants were cultured, and the cells were quantified by a human fetal foreskin cell EGF receptor binding assay by a competitive reaction with hEGF [ ¹²⁵ I] -labeled mEGF contained in a 7 M guanidine-treated solution. DH1
/ PTB370 showed a production amount of about 2 mg / l or more (see Table 1). This expression level is hEG in E. coli.
The direct expression of F is remarkable.

Action In the present invention, the DNA sequence of the hEGF gene does not adopt the most accepted codon in the cells of the expression system, but rather has a high homology with the DNA sequence of the mEGF gene which is similar in amino acid sequence to hEGF. By so doing, the effects of mRNA stability, translation efficiency, and the higher-order structure determined by the base sequence of mRNA become favorable, and hEGF is efficiently produced. In the present invention, BglII is added to the latter half of the structural gene,
By having a Pst I recognition site near the 3'end, it is easy to confirm the success or failure of gene insertion, the insertion direction, and can be placed on several types of vectors, thus exhibiting advantages and diversity in gene manipulation. It is possible.

The hEGF gene provided by the present invention is a novel DNA
It has a sequence and has a high homology to the DNA sequence of the mouse EGF gene, which is much more human than a microorganism, and is expected to have a high expression rate of hEGF. Further, according to the present invention, hEGF is used as a host in Escherichia coli. It became possible to express directly in the system. Furthermore, by the recombinant DNA technology using a synthetic gene corresponding to hEGF of the present invention, hE
GF can be produced more efficiently, and it is useful for the production of hEGF as a therapeutic drug, elucidation of the role of hEGF in vivo and cell growth regulatory mechanism, and eventually elucidation of the carcinogenic mechanism.

Examples and Effects Next, the present invention will be described by examples.

The transformant Escherichia coli (E
scherichia coli) DH1 / pTB37
0 and Escherichia
coli) DH1 / pTB372, pRK248cIt
s is the IFO of the Fermentation Research Institute (IFO)
-14379 and IFO-14380, and FERM P-78 from the Ministry of International Trade and Industry, Institute of Industrial Science and Technology (FRI) from October 5, 1984, respectively.
83 and FERM P-7884, which have been converted to deposits under the Budapest Treaty,
Contract numbers FERM BP-843 and FER, respectively
It is stored in the same laboratory (FRI) as MBP-844.

Example 1. Synthesis of DNA Fragments DNA fragments are synthesized by solid phase synthesis by the phosphorylester method [Ito, H. et al. Nucl. Acids R
es. , 10 , 1755 (1982)]. The dimer block used as the raw material is Broka.
Et al. [Broka, C .; Nucl. Acids Re
s. , 8 , 5461 (1980)] or a fully protected dimer of a commercially available product (Wako Pure Chemical Industries, Ltd.) with pyridine (Py), triethylamine (TE).
A) and water (3: 1: 1, v / v) were dissolved in the mixture to remove the cyanoethyl group, and then pentane and ether (1: 1,
In a mixed solution of v / v), powdered one was used. DNA
The procedure for synthesizing the fragment is as follows.

25 mg of 1% polystyrene (Bachem Co.) to which dimethoxytrityl nucleoside was attached was sequentially treated with the following reagents.

(1) 3% (w / v) trichloroacetic acid (TCA) in dichloromethane [Tanaka, T, et al. Nucl. Aci
ds Res. , 10 3249 (1982)] 1 min x 2 (indicating that the same operation was performed twice) (2) Dichloromethane x 4 (3) Pyridine x 3 (4) 20 mg dinucleotide block or 30 mg monomer block Containing 0.3 ml of dry pyridine (5) Concentrating the above solution under reduced pressure (pyridine azeotrope) (6) with 0.3 ml of dry pyridine containing 25 mg of mesitylenesulfonyl nitrotriazolide (MSNT) and 5 mg of nitrotriazole. 40 ° C., 20 minutes (7) Pyridine × 2 (8) 2 minutes with 2 ml of pyridine containing 10% (v / v) acetic anhydride and 0.1M dimethylaminopyridine (DMAP) (9) Pyridine × 2 (10) Dichloromethane × 3 Using the appropriate dinucleotide or mononucleotide block, this cycle of about 40 minutes was repeated to complete the desired oligonucleotide chain. After the synthesis is completed,
0.5 M 1,1,3,3-tetramethylguanidinium-pyridine-2-aldoxime [Reese, C .; B.
Et al. Tetrahedron Lett. , 2727
(1978)] at 40 ° C. for 14 hours, the target product is taken out from the polymer carrier, and then concentrated ammonia water at 60 ° C.,
After treatment for 4 hours, all protecting groups except the dimethoxytrityl group were removed. This sample was applied to a column of reversed-phase C ₈ silica gel (Licroprep RP-8, Merck) (φ3.0 ×).
2.0 cm) and the fraction eluted with 30% acetonitrile was treated with 80% acetic acid at room temperature for 15 minutes. After washing with ether, further purification by ion exchange high performance liquid chromatography (Partisil 10 SAX, Whatman) [G
ait, M.A. J. Et al. C. S. Chem. Com
mun. , 37 (1982)] for pure DNA
A fragment was obtained. 22 kinds of D synthesized in this way
The NA fragment is as shown in FIG.

Example 2 Phosphorylation of oligo DNA 25 μl of phosphorylation reaction solution of each DNA fragment [2.5 μg of oligo DNA, 50 mM Tris-HC
1, pH 7.6, 10 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 1 mM ATP, 2.5 units T4
Polynucleotide kinase (Takara Shuzo)] at 37 ° C,
After reacting for 1 hour, the 5'end was phosphorylated. This reaction solution was frozen as it was, thawed, and used in the next reaction.

Example 3 Ligation of DNA Fragments The hEGF gene has a double-stranded structure in a single series of steps as shown in FIG. 4 (-in the figure indicates that the 5'-terminal hydroxyl group is phosphorylated). For example, block I was connected as follows. 12 species (DNA fragments 1 to 12
5 μl each of the phosphorylation reaction solution of the DNA fragment obtained by the operation of Example 2) was added to make 60 μl. To this, 1.4 units of T4 DNA ligase (Takara Shuzo) was added, incubated at 14 ° C for 25 hours, and then treated at 65 ° C for 10 minutes to stop the reaction. In order to digest the block I dimer, which is the main product here, with the restriction endonuclease EcoRI (Takara Shuzo), the reaction solution was mixed with the following three components: 50 mM NaCl, 0.01% bovine serum albumin (BSA). ), 7 mM MgCl ₂ and 120 units of Eco RI at 37 ° C., 1.
After reacting for 5 hours, using a 6% acrylamide gel, a buffer solution (pH 8.3) [100 mM Tris-HC was used.
1, 100 mM boric acid, 2 mM EDTA] at 25 mA for 1.5 hours. After electrophoresis, stain the gel with 0.6 mg / l ethidium bromide (EtBr) and
A gel piece containing a DNA fragment of 01 bp was encapsulated in a dialysis tube, immersed in a running buffer, and the DNA fragment was electroeluted from the gel [J. Mol. Biol. , 110 , 1
19 (1977)]. This dialysis tube solution is 0.01
M Tris-HCl, (pH 7.6), 0.1M NaC
l and 0.001M EDTA saturated phenol extracted 3 times, then ether extracted and then NaCl was added to 0.2M. Subsequently, 2 volumes of cold ethanol was added to precipitate the DNA at -20 ° C. By the same operation as above, block II (# 13 to # 2
2) was prepared.

Example 4 Cloning of hEGF Gene (FIG. 5) The Escherichia coli plasmid pBR3 was used as a cloning vector.
22 was used. pBR322 DNA was added to 20 μl of a reaction solution [10 mM Tris-HCl, pH 8.0, 7 mM MgCl 2.
₂ , 100 mM NaCl, 2 mM 2-mercaptoethanol, 0.01% bovine serum albumin (BSA), 19 units EcoRI (Takara Shuzo), 5 units BamH
I (Takara Shuzo)] at 37 ° C. for 1 hour, diluted 3 times with water, and treated at 65 ° C. for 10 minutes to inactivate the enzyme. 0.5 μl of this reaction solution was mixed with about 20 equivalents of DNA fragment blocks I and II to obtain 66 mM Tris.
-HCl (pH 7.5), 6.6 mM MgCl ₂ , 10 mM dithiothreitol (DTT) and 1 mM ATP,
As a 10 μl reaction solution, T4 DNA ligase (New England Biolabs) was allowed to act at 14 ° C. for 2 hours to bind the hEGF gene to the plasmid.

E. coli DH1 strain [Selson, M. et al. E. Nature, 217 ,
1110-1114 (1968)] was transformed.
That is, 50 μl of competent cells stored at −70 ° C. [Hanahan, D. et al. J. Mol. Bio
l. , 166 , 557 (1983)] was incubated at 0 ° C. for 15 minutes, and then 4 μl of the reaction solution was added. After further incubation at 0 ° C for 30 minutes, 42
Every 1.5 minutes at 0 ° C., another 5 minutes at 0 ° C. 200 μl of LB medium (10 g of bactotryptone, 11 g of bacto yeast extract, 8 g of NaCl per 11 g) was added to the reaction solution.
Was added) and incubated at 37 ° C. for 50 minutes. This E. coli was added with L containing 35 μg / ml of ampicillin.
The cells were spread on B agar medium and cultured overnight at 37 ° C. Select 60 strains from the ampicillin resistant colonies, and add 7μ
LB agar medium containing g / ml of tetracycline was inoculated, but 59 strains were not obtained. Next, 16 of these 59 strains were selected, and the plasmid DNA of this converted strain was subjected to the alkaline method [Maniatis, T. et al. Et Molecular C
longing (Cold Spring Harbou
r), 368-369 (1982)],
Digested with EcoRI and BamHI, digested with EcoRI and BglII, and digested with PstI. From the electrophoretic pattern of these digests on a 2% agarose gel, 14 strains were found to be the transformants in which the hEGF gene was correctly inserted. The cloning vector thus obtained was named pTB361.

One platinum loop of E. coli DH1 recombinant having this plasmid pTB361 was inoculated into 1.5 ml of LB medium containing 35 μg / ml of ampicillin, and cultured at 37 ° C. overnight with shaking. 0.3 ml of this culture solution was added to 25 ml of the same medium dispensed in a 200 ml flask, and the mixture was shake-cultured at 37 ° C. for 6.5 hours. Then, this culture solution was added to 125 ml of the same medium dispensed in a 500 ml flask, and further 45 The culture was shaken for a minute. Next, chloramphenicol was added at 170 μg / ml, and the culture was continued overnight to amplify the plasmid DNA. 150 ml of this culture broth, 6000 rpm,
After centrifugation at 4 ° C for 9 minutes, the obtained bacterial cells were washed with physiological saline, and 4 ml of the reaction solution [25 mM Tris-HCl, pH was used.
8.0, 50 mM glucose, 10 mM EDTA, 1 mg / ml
Lysozyme] was added and suspended. After 20 minutes in ice, 8 ml of alkaline solution [1% (w / v) SDS, 0.
2N NaOH] and added in ice for 5 minutes, then 6ml
5M potassium acetate buffer (pH 4.8) was added and the mixture was kept in ice for 10 minutes and centrifuged at 10,000 rpm at 4 ° C. for 20 minutes. To the obtained supernatant, twice the amount of ethanol was added, and after shaking, the mixture was kept at -20 ° C for 10 minutes and then 10,000
It was centrifuged at 0 rpm at 4 ° C. for 20 minutes. After air-drying the precipitate, 4 ml of a buffer solution [1 mM Na ₂ EDTA (pH 8.
0), 10 mM Tris-HCl (pH 8.0)] and dissolved in 3.9 g of cesium chloride (CsCl) and EtBr.
3mg with Beckman 50Ti rotor
5,000 rpm, 15 ° C, 64 hours CsCl-EtBr
Equilibrium density gradient centrifugation was performed.

Collect the bands of plasmid DNA and double the volume of buffer [1
mM Na ₂ EDTA, pH 8.0, 10 mM Tris-H
Cl, pH 8.0], an equal amount of chloroform-phenol (1: 1, v / v) was added, and the mixture was washed twice with EtBr.
After removal of ethanol, ethanol precipitation was performed. The precipitate was added to 0.6 ml of a buffer solution [1 mM EDTA, 10 mM TriS.
-HCl, pH 8.0, 0.3M NaCl],
Another ethanol precipitation was performed.

The nucleotide sequence of the hEGF gene incorporated in the plasmid pTB361 isolated here is the method of Wallace et al. [Wallace, R. et al. B. Gene, 16 , 2
1-26 (1981)]. That is, pTB3
61 DNA was added to 10 μl of reaction solution [7 mM Tris-HC
1, pH 7.5, 7 mM MgCl ₂ , 50 mM NaCl,
4 units of PvuII (Takara Shuzo)] were reacted at 37 ° C. for 1 hour. 1 μl of an aqueous solution of DNA fragment # 7 (1.0 A260 / ml) was added to this reaction solution as a primer, heated at 100 ° C. for 5 minutes, and then rapidly cooled in an ice bath. Subsequent operations were carried out according to the general dideoxy method. Similarly, DNA fragments # 14 and # 18 were used as primers.
Was used to confirm that the nucleotide sequence of the hEGF gene was correct.

Example 5 Construction of plasmid for expression of hEGF and production of transformant (FIG. 5) i) 10 μg of pTB361 obtained in Example 4 above was added to a reaction solution [50 mM NaCl, 6 mM TriS-HCl.
(PH 7.6), 6 mM MgCl ₂ , 6 mM 2-mercaptoethanol, 0.01% BSA, 50 units Eco
RI, 10 units PstI (Takara Shuzo)], 37 ° C,
After reacting for 1.5 hours, a 172 bp DNA fragment was purified by 2% agarose gel electrophoresis according to a conventional method (described above). On the other hand, the expression vector contains ptrp781 [Ku
rokawa, T .; Nucl. Acids Re
s. , 11 , 3077-3085 (1983)]. ptrp781 DNA was transformed into Ec
After digestion with orRI and PstI, double the amount of water was added to the reaction solution, and the enzyme was inactivated at 65 ° C. for 10 minutes.

172 bp DNA and plasmid DN thus obtained
Each A has a single chain cohesive end generated by EcoRI digestion and PstI digestion at both ends.

These two are mixed, and 66 mM TriS-HCl, pH is added.
In the presence of 7.5, 6.6 mM MgCl ₂ , 10 mM DTT and 1 mM ATP, T4 DNA ligase (NEB) was allowed to act for 5.5 hours at 14 ° C. to bind the DNA, and the Escherichia coli DH1 strain was prepared in the same manner as described above. Was transformed. Next, this E. coli was mixed with LB containing 7 μg / ml tetracycline.
It was spread on an agar medium and cultured at 37 ° C. for 1 day. The resulting tetracycline-resistant colonies were then inoculated on LB agar medium containing 35 μg / ml of ampicillin, and non-erasable transformants were selected. Further, in the same manner as described above, the plasmid DNA of the transformant was digested with EcoRI and PstI,
Further digested with Bg1II and HindIII, hEG
A transformant in which the F gene was correctly inserted was selected. The expression plasmid thus obtained was designated as pTB370, and the transformant was designated as Escherichia coli DH1 / pTB370.
I named it.

ii) The expression vector having the λP _L promoter gene was constructed as follows (Fig. 6). The plasmids ptrp601 [Tsuro Kurokawa, Thesis of The University of Tokyo (1983)] were digested with restriction enzymes EcoRI and Cla.
After cutting with I, the cohesive ends of the resulting single chain were filled in with DNA polymerase I (Klenowfragment), treated with phenol, and precipitated with ethanol. This linear DNA was treated with T4 DNA ligase at 14 ° C. to make circular DNA, and Escherichia coli was transformed by the same method as described above. From this, a plasmid in which the trp promoter downstream was EcoRI was isolated and named pTB56. It was

Next, this plasmid pTB56 was digested with PvuII to obtain a linear DNA, and then a synthetic oligonucleotide (EcoR
(I linker) and T4 DNA ligase reaction was performed. After digesting this reaction product with EcoRI, about 0.28 kbp DNA fragment containing the trp promoter gene was purified by 2% agarose gel electrophoresis according to a standard method.

On the other hand, pBR322 DNA was digested with EcoRI to give a linear DNA, and the phosphate group at the 5'end was removed by treatment with alkaline phosphatase to give 0.28 kbp DN.
It was mixed with the AEcoRI fragment, reacted with T4 DNA ligase at 14 ° C., ligated with DNA, and transformed into Escherichia coli. From this, the trp promoter was Ec of pBR322.
isolation of the plasmid cloned into the oRI site,
It was named pTB57.

Next, a linear DNA obtained by partially digesting this plasmid pTB57 with EcoRI was treated in the same manner as described above to crush one EcoRI recognition site to form a circular DNA, which was then transformed into Escherichia coli. A plasmid was obtained from the selected colony, and tr was obtained from the pattern of restriction enzyme cleavage.
The plasmid lacking the EcoRI recognition site on the upstream side of the p promoter was designated as pTB91.

Furthermore, after digesting the plasmid pTB91 with EcoRI, the cohesive end of the single chain was filled in with DNA polymerase I, mixed with a synthetic oligonucleotide (BglII linker),
It was ligated with ligase and a BglII recognition site was introduced downstream of the trp promoter gene, and this plasmid was named pTB334.

Using pTB57 and pTB334 thus obtained, t
A plasmid having an EcoRI recognition site upstream of the rp promoter and a BglII recognition site downstream thereof was constructed. First, pTB344 was treated with restriction enzymes Hpa I and Ps.
After cutting with t I, about 0.78 kbp DNA fragment was eluted and purified by 2% agarose gel electrophoresis.

Also, pTB57 was cleaved with the same restriction enzyme, and then 3.85 kbp DNA fragment was eluted and purified by 1% agarose gel electrophoresis. After mixing both of them and ligating them with T4 DNA ligase, Escherichia coli was transformed, a plasmid was obtained from the obtained colonies, and a transformant having the desired plasmid was selected based on the pattern of restriction enzyme cleavage.
The plasmid isolated from this was named pTB340.

Next, a plasmid pAD329 having a λP _L promoter
[Adhya, S. Cell, 29 , 939-94.
4 (1982)], a 0.35 kbp DNA fragment having a λP _L promoter gene was isolated. First, the plasmid pAD329 was digested with restriction enzymes BglII and HpaI and then subjected to 2% agarose gel electrophoresis to give about 0.45.
The kbp DNA fragment was eluted and purified. Then this 0.45k
After partially digesting the bp DNA fragment with Hinf I,
By subjecting to 2% agarose gel electrophoresis, a DNA fragment of about 0.35 kbp was eluted and purified. 0.35 thus obtained
The kbp DNA fragment was digested with BglII and Hinf at both ends.
It has a sticky end generated by I digestion.

On the other hand, the plasmid pTB340 was digested with restriction enzymes BglII and EcoRI and then subjected to 1% agarose gel electrophoresis to elute and purify about 4.35 kbp DNA.
The DNA obtained here was digested with BglII and Ec at both ends.
It has sticky ends generated by oRI digestion. 0.35 kbp containing the λP _L promoter gene thus obtained
Mix DNA fragment and DNA of about 4.35 kbp, and
After circular DNA was formed with NA ligase, Escherichia coli was transformed, and a plasmid having a λP _L promoter, a Bgl II recognition site upstream and an EcoRI recognition site downstream was isolated therefrom, and named pTB281.

Using this, a plasmid for expressing hEGF was constructed (Fig. 5). First, the plasmid pTB described above in Example 4
361 10 μg was added to the reaction solution [100 mM NaCl, 10 mM
TriS-HCl, pH 8.0, 7 mM MgCl ₂ , 2
mM 2-mercaptoethanol, 0.01% BSA, 5
0 unit EcoRI, 20 unit BamHI (Takara Shuzo)], and after reacting at 37 ° C. for 1.5 hours, 2% agarose gel electrophoresis was performed to contain the hEGF gene.
The 9 bp DNA fragment was eluted and purified. On the other hand, the plasmid pTB281 was also treated with EcoRI and B in the same manner as above.
After digestion with amHI, double the amount of water was added and the mixture was kept at 65 ° C. for 10 minutes to inactivate the enzyme. Mix both of these, 14 ℃
Then, T4 DNA ligase was allowed to act to bind the DNA.

Transformation of E. coli was performed as follows. E. coli N483
LB medium was added to an overnight culture of strain 0 (commercially available from Pharmacia Japan) and diluted 100 times. After shaking culture at 37 ° C. for 2 hours, centrifugation was performed at 3,300 rpm, 4 ° C. for 8 minutes, and the obtained bacterial cells were washed with 10 mM NaCl. A 50 mM CaCl ₂ solution was added thereto, the mixture was kept in ice for 15 minutes, centrifuged at 3,300 rpm at 4 ° C. for 4 minutes, and suspended again in 50 mM CaCl ₂ . 7 μl of the reaction solution obtained above was added to E. coli N4830 suspended in 100 μl, and the mixture was incubated at 0 ° C. for 45 minutes. Then 37
After incubating at 0 ° C. for 2 minutes, 900 μl of LB medium was added, and then incubated at 30 ° C. for 1 hour. This Escherichia coli was spread on LB agar medium containing 35 μg / ml of ampicillin and cultured at 30 ° C. overnight. The resulting ampicillin resistant colonies all lacked resistance to 7 μg / ml tetracycline. Next, plasmid DNA was taken from a part of this transformant, and EcoRI and Ba were added.
By digesting with mHI and further with BglII, hEG
A transformant in which the F gene was correctly inserted was selected. The plasmid thus obtained was designated as pTB372.

The pTB372 obtained above was then treated with pRK248cIts (repressor) [Berna by the same procedure as described above.
rd, H.D. Et al. Methods in Enzymol
, 68 , 482-492 (1979)], and the obtained transformant was spread on LB agar medium containing 35 μg / ml ampicillin and 7 μg / ml tetracycline. , 30
Cultured overnight at ° C. From the resulting colonies, the plasmid DNA obtained in the same manner as described above was digested with a restriction enzyme, and a transformant containing the hEGF gene was selected from the pattern, and this was selected as Escherichia coli DH1 / pTB372, pRK248.
It was named cIts.

Reference Example 1 hEGF Production Method i) Escherichia coli DH1 / pTB370 7 μm
The cells were cultured in LB medium containing g / ml tetracycline at 37 ° C with shaking overnight. To 0.5 ml of this culture medium, 10 ml of M9 medium containing 0.4 μg / ml of tetracycline [containing 0.4% casamino acid and 1% glucose] was added, and the mixture was incubated at 37 ° C. for 4 hours.
After culturing with shaking for 3 hours, 3β-indole acrylic acid (I
AA) was added to adjust the concentration to 30 μg / ml. After culturing for another 4 hours, the culture solution 10.5
ml was centrifuged at 7,000 rpm at 4 ° C for 10 minutes, and the obtained bacterial cells were frozen at -70 ° C. After dissolving this, 1 ml of the reaction solution [7 M guanidine hydrochloride, 2 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 M Tri]
Incubated in S-HCl, pH 7.0] at 0 ° C. for 1 hour. This reaction solution was centrifuged at 20,000 rpm at 4 ° C for 30 minutes, and the obtained supernatant was treated with TEN [20 mM Tr.
iS-HCl, pH 8.0, 1 mM EDTA, 0.2M
It was dialyzed against 4 liters of BaCl] at 4 ° C. twice, and the precipitated insoluble matter was removed by centrifugation at 20,000 rpm, 4 ° C. for 30 minutes. The solution thus obtained was stored at -20 ° C.

ii) Escherichia coli DH1 / pTB372, pR
29 K248cIts in M9 medium containing 35 μg / ml ampicillin and 7 μg / ml tetracycline
Culture was performed overnight at 0 ° C with shaking. 35 μg in 0.5 ml of this culture
Add 10 ml M9 medium containing / ml ampicillin,
After shaking culture at 9 ° C for 4 hours and shaking culture at 42 ° C for 2 hours, the same treatment as described above was performed, and the obtained solution was stored at -20 ° C.

The products obtained in i) and ii) above were subjected to radioreceptor assay (RRA method) [Cohen, S. et al. Et Pro
c. Natl. Acad. Sci. USA, 72 , 13
17-1321 (1975)].

EGF activity was expressed by weight of purified mouse EGF standard showing the same activity. First, human fetal foreskin cell Flow700
0 (flow Laboratories, Inc. commercially available) in a Dulbecco's Minimal Essential (DMEM) medium containing 10% fetal bovine serum to a diameter of 1.6.
cm cell culture dish (Linbro, Flow
Laboratories, Inc. Cultured (commercially available). After discarding this medium and washing the cells with a DNA medium containing 0.1% BSA, 0.2 ml of the medium and mouse EGF (Collab labeled with ¹²⁵ I by the chloramine T method were used.
orative Research, Inc. Commercially available) 5
ng, and an appropriate amount of each product obtained above, add 37 ° C
The cells were cultured for 1 hour. Next, after washing with the same medium, 0.2N N
Treat with aOH, transfer to a tube, and gamma-ray counter
The incorporated I was measured. By the same operation, the amount of human EGF in the product was calculated from the calibration curve obtained by the competitive reaction with mouse EGF of known weight. The results are shown in Table 1.

Further, Escherichia coli DH1 / pTB370 strain was cultured, and after induction with IAA, the EGF activity in the lysate was measured together with the growth by the method already described. The results are shown in FIG. In the figure, the broken line shows the growth of the strain and the solid line shows the EG.
F activity is shown.

[Brief description of drawings]

FIG. 1 shows DN of the synthetic gene of the present invention corresponding to hEGF.
FIG. 2 is a diagram showing an A sequence and an amino acid sequence, FIG. 2 is a diagram showing an example of division into DNA fragments during hEGF gene synthesis of the present invention, and FIG. 3 is a diagram showing h of the present invention.
It is a figure which shows an example of the DNA fragment for EGF corresponding synthetic gene manufacture, and FIG. 4 is a schematic diagram which ligates each DNA fragment of FIG. 3 and manufactures an hEGF synthetic gene. FIG. 5 is a construction diagram of an expression plasmid incorporating the hEGF-corresponding gene of the present invention, and FIG. 6 is a plasmid p.
It is a construction drawing of TB281. FIG. 7 is a graph showing cell growth and EGF activity when EGF was produced using the synthetic gene of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1:19) (56) Reference US Patent 4395486 (US, A) Proc, Natl, Acads, Sc i, USA, 1983 [80] P.I. 7461-7465 Nucleic Acids Research, 1982 [10] P. 4467-4482 Nature, 1983 [303] P. 722-725

Claims (2)

[Claims] 1. DNA sequence AACAGTGATTCAGAATGTCCTCTCT
CACACAGTGGATACTGCCTCCATGA
CGGCGTGTGTATGTATATTGAAGCA
CTAGACAAAATACGCATGCAACTGTG
TAGTTGGCTATATTTGGTGAACGATG
CCAGTACCGAGATCTGAAATGGTGGG
A DNA having a synthetic gene for expressing human epidermal growth factor represented by GAACTGCGA. 2. A DNA sequence AACAGTGATTCAGAATGTCCTCTCT characterized in that a plurality of oligodeoxynucleotides are enzymatically linked and inserted into a vector if desired.
CACACAGTGGATACTGCCTCCATGA
CGGCGTGTGTATGTATATTGAAGCA
CTAGACAAAATACGCATGCAACTGTG
TAGTTGGCTATATTTGGTGAACGATG
CCAGTACCGAGATCTGAAATGGTGGG
A method for producing a DNA having a synthetic gene for expressing human epidermal growth factor represented by GAACTGCGA.