JP2561674B2 - Method of producing protein - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a gene product using a genetic engineering technique. Specifically, it relates to a method for producing a large amount of a target gene product by arranging two or more expression units containing the target gene in series for amplification.

(Problems to be Solved by Conventional Techniques and Inventions) In recent years, many useful substances have been produced by genetic engineering techniques. However, many of them cannot be mass-produced because their products are unstable because heterologous cells are used as hosts.

In particular, when microorganisms (mainly Escherichia coli) are used, many useful proteins of higher organisms are directly expressed as they are, and most of them are decomposed by the degrading enzymes of bacterial cells. In order to overcome such a condition, conventionally, a fusion protein of a target protein and a host protein is produced in the host to stabilize it as an insoluble protein, and if the directly expressed protein becomes insoluble, it becomes insoluble (deactivated). A method for stabilizing the cells by producing them as they are), a method for secreting the cells to prevent the degradation by proteolytic enzymes in the cells and stabilizing the cells have been used. However, not all proteins can be efficiently produced by heterologous cells by these methods. For example, the method for cleaving the target protein from the fusion protein produced by the method (1) has not been successful in all cases so far because the method for cleaving the protein at a specific site is not well established. In this method, the protein solubilized in the cells is decomposed relatively quickly, so that it is not successful in many cases. In addition, the method of (1) is not successful in many cases because some proteins are not secreted depending on the type of protein.

(Means for Solving the Problem) When a protein is produced in a heterologous cell, it is degraded, but if the production is increased above the degradation, high production becomes possible.

Therefore, the present inventors have conducted extensive studies on a method for mass-producing proteins by recombinant DNA technology from such a viewpoint, developed a production method useful for such a purpose, and arrived at the present invention.

That is, the gist of the present invention is to provide an expression unit containing at least a ribosome binding site, a translation initiation codon and a structural gene.
The present invention provides a method for producing a protein, which comprises culturing a transformant transformed with an expression vector having a DNA fragment having at least one ligated to produce a protein encoded by the structural gene.

To explain the present invention below, the expression vector of the present invention,
It has a DNA fragment in which two or more expression units including at least a ribosome binding site, a translation initiation codon such as ATG and GTG, and a structural gene are linked in series.

As the ribosome binding site, a base sequence (AGGAGGT) called a so-called Shine-Dalgarno sequence (SD sequence) can be used. Of course, a part of bases may be modified within a range that does not impair the ribosome binding ability.

As structural genes, various interferons, IL-
2, genes encoding (pro) apolipoprotein A and E, TNF, cardiodilatin, cardionatrin and the like are mentioned, but not limited thereto.

The expression units can be linked in the following manner. That is, first, restriction enzyme cleavage sites are introduced at the 5'and 3'ends of the target structural gene. In this case, the restriction enzyme cleavage site to be introduced may be any, but preferably, it does not have the cleavage site in the target gene,
The one introduced at the 5'end and the one introduced at the 3'end have a common sticky end, for example, Bam HI and Bgl II, Bam
Preferred are HI and Bcl I, Bgl II and Bcl I, Sal I and Xho I and the like. The method for introducing these restriction enzyme cleavage sites into the target gene may also be carried out by using a synthetic DNA linker or the like using the restriction enzyme cleavage sites existing near the 5'end and 3'end of the target gene. Alternatively, it may be performed by an in vitro site-directed mutagenesis method using a synthetic DNA oligonucleotide mutagen. The restriction enzyme site is introduced, but preferably on the 3'side immediately after the coding region of the target structural gene. The 5'side may be at any position upstream of the coding region, but is preferably immediately before the coding region or immediately before the ribosome binding position or immediately before the promoter.

The expression unit thus introduced with the restriction enzyme cleavage site is cleaved by the restriction enzyme, and the expression unit is separated and purified by acrylamide gel or the like. The expression unit separated and purified
When DNA has the same sticky ends on the 3'side and the 5'side, it is ligated as it is with T4 DNA ligase, and when different, it is blunted with T4 DNA polymerase etc.
Ligate with 4 DNA ligase.

The expression vector of the present invention is usually a promoter region,
It has an operator region, a transcription termination factor, and optionally a repressor region.

Such promoters include trp , lac , lac UV
5, rec A or the like may be derived from Escherichia coli, P _L P _R derived from λ phage, promoter such as gene 25 or 26 derived from T5 phage, promoter of DHFR gene derived from Bacillus subtilis, etc., but tac (proceding of National Academy of Sciences USA, Proceedin
g of National Academy of Science USA, 81 , 21 (198
3)) or a strong and controllable promoter such as pac described below is preferable.

The pac promoter recognizes RNA polymerase −35
It consists of a region and the −10 region it joins, and −35
The region has the same nucleotide sequence as the −35 region of the phage promoter, and the −10 region has the same nucleotide sequence as the −10 region of the Escherichia coli promoter. In addition,
The “equivalent base sequence” means the same as the natural base sequence or a part of the base sequence modified by substitution, insertion or deletion to the extent that its function is not impaired. As the −35 region, a region having at least 5 to 12 base pairs including at least the base pair shown in (a) below is used.

Specifically, e.g., P of T5 phage _{25, P 26, P 28,} P
Examples include the nucleotide sequence corresponding to the −35 region of the promoter such as ₂₀₇ .

The -10 region includes at least 7 to 13 base pairs, including at least the base pair shown in (b) below, and particularly preferably the base pair shown in (c) below. use.

Specifically, for example, E. coli lac UV5, rrnD ₁ , rrnE
Examples include the nucleotide sequence corresponding to the −10 region of the promoter such as ₁ .

The pac promoter allows the −35 region to be upstream of the −10 region. At that time, both may be directly connected,
It is preferable to bind via an arbitrary base sequence of about 10 to 20 base pairs.

Examples of the operator region include those containing a base pair as shown in (d) below.

Specifically, for example, E. coli lac UV5, trp , rec A
And the base sequence corresponding to the operator region.

The operator region is not particularly limited, but is usually incorporated in the vicinity of the -35 region and the -10 region. It is particularly preferable to directly bind to the downstream of the −10 region via an arbitrary base sequence of 0 to 10 base pairs.

The -35 region, the -10 region and the operator region may each be prepared from the above-mentioned phage or E. coli or may be prepared by chemical synthesis.

Examples of the transcription terminator include the 3'region of the lipoprotein (lpp) gene, trp (operon terminator, rRNA gene terminator, λ phage to and other terminators).

The expression vector of the present invention described above is introduced into a host, for example, Escherichia coli by the CaCl ₂ method (J. Molec. Biol., 53 , 154, 1970) according to a conventional method to obtain a transformant. The desired protein can be obtained by cultivating L in broth or M9 medium.

(Effect of the invention) The expression vector of the present invention contains a plurality of target structural genes, and when a transformant transformed with this vector is cultured, the target protein can be obtained as will be apparent from Examples described later. Can be produced synergistically.

(Example) Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless the gist thereof is changed.

Example 1 A. Construction of expression plasmid a) Modification of human proapolipoprotein AI cDNA by site-specific modification method 1) N-terminal mutation Obtained according to the method described in JP-A-61-96998. Pha I-65 μg shown in FIG. 1 was added to 10 mM Tris-HCl (p
H7.5), 100mM NaCl, at 6 mM MgCl ₂ buffer 100μ in Pst
After reacting with I10u for 2 hours at 37 ° C., digested, heated at 75 ° C. for 15 minutes to inactivate the enzyme, dialyzed against water and dried. To this, 33 mM Tris-acetic acid (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM dithiothreitol buffer was added 2 mM 4-deoxytriphosphoric acid, and 40 μM was added.
3'protruding end was blunted with T4 DNA polymerase 4u, heated at 70 ° C for 10 minutes to inactivate the enzyme, dialyzed against water and dried, and then stored as an aqueous solution of 50μ. . <Fragment I> On the other hand, 20 μg of phAI-6 was mixed with 10 mM Tris-HCl (pH 7.
5), Eco R in 100 mM NaCl, 6 mM MgCl ₂ buffer 100 μ
20 μl of each of I and Sac I was reacted at 37 ° C. for 2 hours and digested. This was subjected to 5% acrylamide gel electrophoresis (89 mM Tri
s, 89 mM BoricAcid, 2 mM EDTA buffer, 10 V / cm, electrophoresis for 1.5 hours) to separate the DNA, and after staining the gel with 0.05% ethidium bromide aqueous solution, excise the larger molecular weight fragment under UV light at 340 nm. After crushing the gel with a glass rod, suspend it in 4 ml of DNA extraction buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% sodium lauryl sulfate) and leave it at 37 ° C overnight to allow the DNA from the gel. Was extracted. A large piece of gel 10000r.pm15
After removing by centrifugation for 1 minute, a smaller gel piece is removed by a glass filter and ethanol precipitation is performed 3 times to remove DNA.
Was purified and stored as a 200 μ aqueous solution. <Fragment II> A 41 base DNA fragment consisting of the following nucleotide sequence was synthesized by a DNA synthesizer (Nikka Kikai Co .; Applied Biosystem MODEL 380A).

150 pmole of this is used as a kinase buffer (50 mM Tris-HCl
(PH8.0), 10 mM MgCl ₂ , 5 mM dithiothreitol) 10μ
5 'by 20u T4 polynucleotide kinase
Phosphorylated.

Fragment I 0.05pmole, Fragment II 0.0
5pmole, 5'phosphorylated primer 45pmole with 5 times concentration of polymerase and ligase buffer (0.5M NaCl, 32.5mM T
ris-HCl (pH 7.5) and 40mM MgCl _2, 5mM β- mercaptoethanol) to 12μ added meter 34.8μ system, 100 ° C., 3
Boil for 30 minutes, immediately put in a constant temperature bath at 30 ° C and let stand for 30 minutes.
It was left on ice for 10 minutes to form a heteroduplex.

This 11.6μ to 4μ 2.5mM 4 deoxynucleotide triphosphate, 2μ 10mM ATP, 2u klenow enzyme,
0.5 u of T4 DNA ligase was added, and the mixture was reacted overnight at 16 ° C. in a total system of 20 μ to circularize.

Using 2 μ of this product, the plasmid of the transformant obtained by transforming and growing Escherichia coli HB101 according to a conventional method was used to obtain a mutant plasmid which was cleaved into two DNA fragments by Pst I according to a conventional method.

Since the mutant plasmid thus obtained contains the original plasmid (wild type), it was transformed again to purify the mutant plasmid. In this way, the plasmid phA I-SP shown in FIG. 2 was obtained.

2) C-terminal modified phA I-SP 5 μg was added to 10 mM Tris-HCl (pH 7.5), 100 mM
Pst I10u and 37 ℃ in 100μM NaCl, 6mM MgCl ₂ buffer 2
After reacting for a time and digesting, heating at 75 ° C. for 15 minutes to inactivate the enzyme, it was dialyzed against water and dried. To this one
33 mM Tris-acetic acid (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate, 2 mM in 0.5 mM dithiothreitol buffer
4-deoxytriphosphoric acid is added to make 40μ system and T4DN
Blunt the 3'protruding ends with A polymerase 4u, 70 ° C
After heating for 10 minutes at 10 minutes to inactivate the enzyme, it was dialyzed against water, dried, and then stored as a 50 μ aqueous solution.
<Fragment I ′> On the other hand, 20 μg of phA I-SP was added to 10 mM Tris-HCl (pH 7.5),
Puv II Sac in 100 μM of 100 mM NaCl and 6 mM MgCl ₂ buffer
I5u was reacted at 37 ° C for 2 hours for digestion. 5% of this
Acrylamide gel electrophoresis (89mM Tris, 89mM Boric
Isolate DNA with Acid, 2mM EDTA buffer (10V / cm, 1.5 hour migration), stain the gel with 0.05% aqueous ethidium bromide solution, cut out the fragment with the higher molecular weight under UV light at 340nm, and remove the gel from the glass rod. After crushing at
Extraction, buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% sodium lauryl sulfate) 4 ml
The DNA was extracted from the gel by suspending it in the gel and leaving it at 37 ° C. overnight. After removing the large gel pieces by centrifugation at 10,000 rpm for 15 minutes, the smaller gel pieces were removed with a glass filter and ethanol precipitation was performed 3 times to purify the DNA, and the DNA was preserved as an aqueous solution of 200 μ. <Fragment II ′> A 36 base DNA fragment having the following nucleotide sequence was synthesized by a DNA synthesizer.

150 pmole of this is used as a kinase buffer (50 mM Tris-HCl
(PH8.0), 10 mM MgCl ₂ , 5 mM dithiothreitol) 10μ
5 'by 20u T4 polynucleotide kinase
Phosphorylated.

The plasmid phA I-SPB shown in FIG. 3 was obtained in the same manner as in 1) above except that the fragment I ′, fragment II ′ and 5 ′ phosphorylated primers were used.

B. Construction of human proapolipoprotein AI expression plasmid Consists of the following nucleotide sequence encoding a prosegment
DNA fragment (Arg His Phe Trp Gln Gln) 5′CGA CAT TTT TGG CAA CAA 3 ′ 3′GCT GTA AAA ACC GTT GTT 5 ′ was used as a DNA synthesizer (Nikkaki; Applied Biosystem MODEL 380).
Synthesized by A).

The above DNA fragment 45pmole was reacted at 37 ° C. for 1 hour using kinase 20u in the system of the above-mentioned kinase buffer 10μ,
5'phosphorylation was performed.

10 μg of phA I-SPB in buffer (10 mM Tris-HCl (pH 7.
5), 100mM NaCl, 6mM MgCl ₂ ) Pst I20 in 100μ system
Using u, the reaction was carried out for 2 hours to cut. This is 75 ℃ 10
After heating for a minute to deactivate the enzyme, it was desalted by dialysis against water and dried. To this, add the above-mentioned T4 DNA polymerase buffer 50μ, and add T4 DNA polymerase 10u
Was reacted at 37 ° C for 30 minutes, and the protruding 3'end was scraped off (the DNA encoding ApoA I was removed by this treatment).
The base portion up to just before was removed. After that, the enzyme was inactivated by heating at 70 ° C. for 10 minutes. Then 10-fold concentrated buffer solution H of 5.5μ (0.1M Tris-HCl, 1M NaCl, 60mM MgCl 2) was added, about a 5% acrylamide gel electrophoresis to cut at Bgl II10u the same method as described above 736bp DNA fragment (fragment MAI) was isolated and purified.

Preparation of expression vector 2 μg of pMT I2 obtained in the reference example described later was added to a buffer solution (10 mM T
Kpn I5 with ris-HCl (pH 7.5), 6 mM MgCl ₂ ) 20μ system
It was digested by reacting with u for 2 hours at 37 ° C. To this one
Add 2μ of T4 DNA polymerase buffer at 0 times concentration and add 4 kinds of deoxynucleotide triphosphates to 1 mM, and react with T4 DNA polymerase 4u at 37 ° C for 30 minutes,
After scraping off the protruding 3'end, the enzyme was inactivated by heating at 70 ° C for 10 minutes. The thing to 37 ° C. the 10-fold concentrated buffer H at 2.5μ added Bgl II4u, D reacted for 2 hours
After cutting the NA, the protein was extracted with 25 μl of water-saturated phenol, the supernatant (aqueous layer) was separated, the phenol was removed with ether, and the mixture was dialyzed against water to desalinate and then dried. Ten
Dissolved in μ water.

Construction of pro-apolipoprotein AI expression plasmid (see FIG. 5) Prosegment 4.5pmole, fragment MAI 1 μg,
0.5 μg of the expression vector and 0.1 μM ATP of 10 μM in 10 μl of a buffer solution (10 mM Tris-HCl, 6 mM MgCl ₂ , 1 mM dithiothreitol) were added with 10 u of T4 DNA ligase, and the mixture was placed at 4 ° C. and 16 ° C.
After reacting for a period of time, each fragment was ligated to obtain pMTpAI.

C. Construction of expression plasmid containing 2 or more expression units 10 μg of pMTpAI obtained in the above B was added to 10 mM Tris-HCl.
(PH 7.5), 100 mM NaCl, using Bam H I10u, Bgl II10u a system of 6 mM MgCl ₂ buffer 100 microns, was 37 ° C. 2 hr reaction,
Disconnected. After cutting, the sample was subjected to 5% polyacrylamide gel electrophoresis (89 mM Tris, 89 mM Boric acid, 2 mM EDTA).
A 750 bp expression unit containing the SD sequence, translation initiation codon and proapoA-I was separated with a buffer solution (electrophoresis at 12 V / cm for 1.5 hours). Soak the gel in 0.05% ethidium bromide,
After about 10 minutes, it was taken out and washed with distilled water, and then the gel containing the above-mentioned expression unit was cut out under an ultraviolet ray of 340 nm. The cut gel pieces were crushed with a glass rod in a test tube, and 4 ml of DNA extraction buffer (0.5M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% sodium lauryl sulfate) was added, and the mixture was kept overnight at 37 ° C. The DNA was extracted. Then, the gel piece was centrifuged (6500 rpm, 10 minutes) to be precipitated, and the supernatant was filtered through a glass filter. To this, 12 ml of cold ethanol was added, and the mixture was allowed to stand at -70 ° C for 20 minutes and then centrifuged (12000 rpm, 20 minutes) to precipitate DNA. The precipitate was dissolved in 300 μm of 2M ammonium acetate, and ethanol precipitation was repeated twice more for purification. The finally obtained DNA precipitate was air-dried and then dissolved in 200 μl of distilled water for use. Human proapo A-obtained here
I expression unit 5 'comprises a ribosome binding site and Hitopuroapo A-I gene in side is Bam HI site that 3' Bgl the side
II site is present.

Linking of human proapoA-I expression unit Since the Bam HI site on the 5'side and the Bgl II site on the 3'side have the same sticky ends, they can be linked as they are, and the bound site is no longer Bam HI or Bgl HI . Even II is not cut.

Expression vector pMT I2 0.05 pmol was added to 10 mM Tris-HCl (p
H7.5), 100 mM NaCl, 6 mM MgCl ₂ buffer Bam with H10μ
It was cleaved by reacting with 1 u each of HI and Bgl II for 2 hours at 37 ° C. After that, the enzyme was inactivated by a phenol treatment, the phenol in the solution was removed four times with 10 μ of diethyl ether, dialyzed against water, air-dried and dissolved in 7 μ of distilled water. The above human proapoA-I expression unit was added to this in an amount of 1 pmole, and a buffer solution L containing 10 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 6 mM MgCl ₂ , and 0.2 mM ATP was added.
Then, using T4 DNA ligase 1u, the reaction was carried out at 4 ° C. overnight for ligation. Using this, JM109 competent cells (Takara Shuzo) were transformed by a conventional method to obtain transformants.

A plasmid was prepared from these transformants by a conventional method to obtain a plasmid pMT (pAI) ₁ into which 1 unit of the proapo AI gene was introduced.

Bgl I The pMT (pAI) ₁ 0.05pmole at buffer H10μ
Cleavage was performed by reacting with I1u for 2 hours at 37 ° C. After that, the enzyme was inactivated by a phenol treatment, the phenol in the solution was removed 4 times with 10 μ diethyl ether, dialyzed against water, air-dried and dissolved in 7 μ of distilled water. To this, 1 pmole of the above human proapoA-I expression unit was added, and T4 DNA ligase 1u was used in buffer L10μ at 4 ° C.
The reaction was carried out overnight and ligated.

Using this, JM109 competent cells (Takara Shuzo) were transformed by a conventional method to obtain a transformant.

A plasmid was prepared from these transformants according to a conventional method to obtain a plasmid pMT (pAI) ₂ into which 2 units of the proapo AI gene had been introduced.

Using this pMT (pAI) ₂ , a plasmid pM in which three proapoA-I expression units were ligated by the same method as above.
T (pAI) ₃ was obtained.

D. Cultivation of a bacterium in which 1 to 3 human proapo A-I expression units are linked and the productivity of the human proapo A-I i Proapo A-I expressing bacterium was L broth (10 g bactopeptone, 5 g yeast extract, 10 g salt /) Ampicillin was added so that the concentration became 50 mg /, and the mixture was cultured at 30 ° C. overnight. 1/50 volume of this culture solution was inoculated into fresh L broth, and after culturing at 30 ° C for 2 hours, isopropyl-β-D-galactopyranomide (IPTG) was added to a final concentration of 2 mM, and further at 30 ° C. Three
The culture was shaken for a period of time. After that, centrifuge the cells (6500r.pm,
The cells were collected for 10 minutes). The cells were treated with 10 mM Tris-HCl (pH 7.
5), suspend in 0.1 mM EDTA solution and add 0.1 ml culture to 62.5 mM Tri
s-HCl (pH6.8), 2% sodium lauryl sulfate, 5%
2-mercaptoethanol, 10% glycerol, 0.002
1% at 100 ℃ in buffer solution containing% bromophenol blue
The reaction was carried out for 0 minutes, and the mixture was subjected to SDS-12.5% acrylamide gel electrophoresis (10 V / cm, electrophoresis for 1.5 hours). After running the gel
After staining with an aqueous solution of 25% methanol and 8% acetic acid (V / V / V) containing 0.2% Coomassie Brilliant Blue R250 (manufactured by Sigma), it was decolorized with the same aqueous solution, dried, and stored.

The rogel was subjected to a densidometer (manufactured by Shimadzu Corporation), and the productivity of human proapoA-I was confirmed by the concentration ratio with standard human apoA-I 1 μg. The results are shown in Table-1.

Reference Example 1 Construction of plasmid pMT I2 (see FIG. 4) (1) Construction of hybrid promoter DNA fragment having the same sequence as the lac UV5 promoter 10 region, operator region and transcription start site, and the same sequence as the -35 region of T5P25 promoter. DNA fragments (from below) were prepared using Model 308A, DNA synthesizer manufactured by Nikkaki Co., Ltd.

Eco RI 37 bases: Equivalent to DNA fragment containing −35 region of T5 phage P25 promoter 37 bases: DNA fragment complementary to 43 bases: lac UV5 Equivalent to DNA fragment containing region and operator region 43 bases: Complementary to: DNA fragment 100 μg of each synthetic DNA fragment 5 in 2 ml of 28% aqueous ammonia solution
The reaction was carried out at 5 ° C. for 6 hours to perform deprotection, and purification was carried out using a 17% acrylamide-7M urea gel. As a result, 10 μg of each fragment was obtained. Then, 1 μg of these DNA fragments was added to 1u.
T4 polynucleotide kinase and 10 mM Tris-HCl (pH 7.
6), and reacted at 37 ° C. for 1 hour in 10 μm of a buffer containing 10 mM MgCl ₂ to phosphorylate the 5 ′ end. Next, NaCl was added to 50 mM, and the above DNA fragment and were mixed, heated at 100 ° C. for 3 minutes, gradually cooled, and annealed to obtain a double-stranded DNA fragment of 37 bp and 43 bp. 2 μg of each was obtained.

0.5 μg of each of these DNA fragments was added to 1 μm of plasmid pBR322 treated with Eco RI and Hin d III, and T4 DNA ligase 1 was added.
u, 10 mM Tris-HCl (pH 7.5), 6 mM MgCl ₂ , 10 m
Insertion was carried out by reacting for 16 hours at 4 ° C. in 10 μm of a buffer containing M ATP and 1 mM DTT.

CaCl ₂ was added to the reaction solution to 100 mM, and E. coli HB10
The mixture was mixed with competent cells of 1 and treated at 0 ° C. for 10 minutes and 37 ° C. for 5 minutes for transformation. The transformant acquired tetracycline resistance, and tetracycline was added to the medium at 20 μg / ml.
You can easily select by inserting.

In this way, a pac promoter plasmid pBRpac having a promoter activity was obtained. The base sequence of the pac promoter was determined by the dideoxy method according to a conventional method. The dideoxy method is described in detail in the literature Anal. Biochem. Vol. 152,232. (Competent cells were collected in a logarithmic growth phase by a standard method, suspended in 1/3 volume of 0.1MCaCl ₂ at 0 ° C, left standing for 30 minutes, and collected again to 1/100 volume. obtained by suspension of the 0.1MCaCl _{2.) (2)} the plasmid obtained as the introduction of the transcription termination factor to hybrid promoters pBRpac (4.4Kb
p) 1 μg of Bam HI and Pvu II 1 u each for 10 mM Tris-HCl
(PH 7.5), buffer containing 100 mM NaCl, 6 mM MgCl ₂ 10 μ
Digested by reacting at 37 ° C for 2 hours. The reaction product was evaporated to dryness after ethanol precipitation and stored.

Meanwhile, the plasmid pIN III Al (The EMBOJ.Vol 3,10,2
By the 437,1984) 5μg Kpn I5u, 10mM Tris -HCl (p
H7.5), 6 mM MgCl ₂ in 50 μl of a buffer solution, reacted at 37 ° C. for 2 hours and digested, and 330 mM Tris-acetic acid (pH 7.9), 660 mM
Add 5.5 μl of buffer containing KAc and 100 mM Mg (Ac) ₂
The DNA was treated with 10u of 4 DNA polymerase at 37 ° C for 15 minutes, the protruding 3'end was scraped off, and the mixture was heated at 70 ° C for 10 minutes to deactivate the oxygen. Next, 100 mM Tris-HCl (p
H7.5), 1M NaCl, 60mM MgCl ₂ buffer 6.0μ was added and digested with Bam H I5u for 2 hours at 37 ° C, 60μ of water-saturated phenol was added, and after deproteinization, ethanol precipitation was performed 3 times for purification. . As a result, lpp containing transcription termination factor
0.5 μg of ^{3 ′} region (˜500 bp) fragment was obtained. This DNA
The fragment was digested with the above-mentioned digestion plasmid (pBRpac) using T4 DNA ligase 1u, 10 mM Tris-HCl (pH 7.5), 100 mM MgC
l _2, 10mM ATP, 4 ℃ in a buffer 10μ containing 1 mM DTT, 1
After reacting for 6 hours and inserting, a plasmid pPac was obtained. This plasmid can be stably maintained in JM105 strain or JM109 strain which is a lac Ig strain. That is, using pPac 0.1 μg, (1)
The JM105 strain was transformed according to the method described to obtain a transformant, which was used as the minimum medium 1 containing 20 mg / ml of ampicillin.
When pPac
1 mg was obtained.

(3) Introduction of multilinker Next, a multilinker having the SD sequence and the start Met sequence shown in FIG. 4 was synthesized with a model 308A manufactured by Nikkaki Co., Ltd.
As a result of purification according to the method of (1), 10 μg of DNA was obtained. 1 μg of the above plasmid pPac was added to 10 mM Tris-HCl (p
H7.5), 100 mM NaCl, 6 mM MgCl ₂ in 10 μm of a buffer solution, and cut with Bam HI 1u at 37 ° C. for 2 hours, followed by ethanol precipitation and evaporation to dryness for storage.

This cleaved plasmid pPac and 1 μg of the above multilinker
10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM ATP, 1
Using T4 DNA ligase 1u in buffer 10μ containing mDTT,
The reaction was carried out at 4 ° C for 16 hours to obtain a plasmid pMT2 having a multilinker inserted therein.

(4) Introduction of lac repressor gene Further, plasmid pMC9 (proc.Natl.Acad.Sci.USA, 80 ,
3015 (1983)) 10 μg to 100 mM Tris-HCl (pH 7.5), 10
Eco R I10 in 100 μM buffer containing mM NaCl and 6 mM MgCl ₂
Using u, the reaction was carried out at 37 ° C. for 2 hours for cleavage, precipitation with ethanol, evaporation to dryness and storage. As a result, 2.5 μg of a 1.7 Kbp DNA fragment encoding the repressor gene lac I was obtained. Then, 1 μg of the above plasmid pMT2 was added to 100 mM Tr
Buffer containing is-HCl (pH 7.5), 10 mM NaCl, 6 mM MgCl ₂
The reaction was performed at 37 ° C. for 2 hours using Eco R I1u in 10 μm, cleaved, precipitated with ethanol, evaporated to dryness and stored.

This cut plasmid pMT2 and 1 μg of the above 1.7 Kbp DNA fragment were used.
10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM ATP, 1
Ligation was performed using T4 DNA ligase 1u in a buffer solution containing 20 μm mM DTT to obtain a plasmid pMT I2.

Example 2 A <Modification of Chloralphenicol Acetyltransferase (CAT) Structural Gene> pCM4 (Gene Gene, 20 , 305)
(1982)) 1 μg (0.3 p mol) of 10 mM Tris-HCl (pH 7.
5), 100mM NaCl and 6mM MgCl ₂ buffer (H) 10μ
The reaction was carried out with 0.1 u of Bam HI for about 10 minutes at 37 ° C, and partial digestion was performed with Bam HI. Bam HI was inactivated by heating this product at 70 ° C. for 10 minutes, then dialyzed to desalt, and lyophilized by a vacuum pump.

To this, add 17μ of water and add 10 times more T4 DNA.
Polymerase buffer (33 mM Tris-acetate, 6 mM MgOAC, 2.
5mM 4-deoxynucleotide) 2µ and T4 DNA polymerase 1µ were added and reacted at 37 ° C for 15 minutes. Then 70
℃ was heated for 10 minutes, after which the enzyme was inactivated, Bgl II linker; reaction's instructions which are attached by (GCAGATCT Takara Shuzo Co., Ltd.) was added, ligase Chillon kit (Takara Shuzo Co.) ( The reaction was carried out at 12 ° C. for 2 hours, and the products were linked. E. coli HB101 competent cell (Takara Shuzo Co., Ltd.)
Made in accordance with the attached instructions,
A BglII linker was introduced into the 3'side of the CAT structural gene.

As a result of this operation, the CAT structural genes are 2 of Bam HI and Bgl II.
It can be separated as an expression unit of about 750 bp by double digestion. And since the Bam HI site on the 5'side and the Bgl II site on the 3'side have the same sticky ends, they can be ligated as they are, and the bonded site is no longer Bam HI or Bg H I.
l II even cut not be (Figure 7) B CAT ligated expression vector expression unit pMT I2 0.05p mol of 10 mM Tris-HCl (p
H7.5), 100m NaCl and 6mM MgCl ₂ buffer system 10μ Ba
m H I, was cut reacted Bgl II each 1u and 37 ° C. 2 hours. Then, the enzyme was inactivated by a phenol treatment, the phenol in the solution was removed 4 times with 10 μ of diethyl ether, dialyzed against water, air-dried and dissolved in 7 μ of distilled water. 1 pmol of the above CAT expression unit was added to this, and a ligation kit (Takara Shuzo Co., Ltd.) was used to perform a ligation reaction according to the attached instructions. Using this, JM109 competent cells (manufactured by Takara Shuzo Co., Ltd.) were transformed by a conventional method to obtain a transformant.

Plasmids were prepared according to a conventional method from these transformants, CAT gene was cleaved with Bam H I and Bgl II to obtain a one to four ligated plasmid. (Eighth
Fig.) Cultivation of a bacterium in which one to four C CAT expression units are linked and the productivity of the CAT a) The CAT-expressing bacteria obtained in the above B are respectively L broth (10 g bactotryptone, 5 g yeast extract, 10 g salt / 5 in liters)
Ampicillin was added to a concentration of 0 mg /, and the mixture was cultured at 30 ° C overnight. 1/50 volume of this culture solution was added to fresh L broth, and the mixture was shake-cultured at 30 ° C. for 2 hours, and then isopropyl-β-D-
Galactopyranoside (IPTG) was added so that the final concentration would be 2 mH, and the mixture was further shake-cultured at 30 ° C. for 3 hours. 10mM T cells
Suspend in ris-HCl (PH7.5), 0.1 mM EDTA solution, and add 0.1 ml culture to 62.5 mM Tris-HCl (PH6.8) 2% sodium lauryl sulfate, 5% 2-mercaptoethanol, 10% glycerol, 0.002 The mixture was reacted at 100 ° C. for 10 minutes in a buffer system containing% bromophenol blue, and subjected to SDS-12.5% acrylamide gel electrophoresis.

After electrophoresis gel is 0.2% Coomassie Brilliant Blue R
25% methanol containing 250 (manufactured by Sigma), 8% acetic acid (V
/ V / V) aqueous solution, then decolorized with the same aqueous solution, dried and stored.

b) The gel was subjected to a densitometer (manufactured by Shimadzu Corporation) to measure the production amount. The results are shown in Table-2.

[Brief description of drawings]

FIG. 1 shows the preproapo AI cDNA plasmid. Figure 2 shows the 5'of the apoA-I gene by site-directed mutagenesis.
The plasmid with Pst I and Sal I sites introduced is shown on the side. Figure 3 shows the 5'of the apo AI gene by site-directed mutagenesis.
A plasmid having Pst I and Sal I sites introduced on the side and a Bgl II site introduced on the 3'side is shown. FIG. 4 shows the construction of an expression vector using a hybrid promoter (pac). FIG. 5 shows the construction of a proapolipoprotein AI expression plasmid. FIG. 6 shows the construction of a plasmid in which a plurality of proapolipoprotein AI expression units are linked. Fig. 7 shows B on the 3'side of the CAT gene by site-directed mutagenesis.
The plasmid into which the gl II site was introduced is shown. Figure 8 shows CAT
The construction of the expression plasmid is shown.

Claims (26)

(57) [Claims] 1. A promoter region and a transcription terminator, wherein two or more expression units containing a ribosome binding site, a translation initiation codon and a structural gene are linked between the promoter region and the transcription terminator. A method for producing a protein, which comprises culturing a transformant transformed with an expression vector having the DNA fragment to produce a protein encoded by the structural gene. 2. The promoter is a trp promoter, a lac promoter, a lacUV5 promoter, a recA promoter,
P _L P _R promoter, Gene 25 or 26 promoter, DHFR gene promoter, tac promoter, and methods according to claim 1, wherein the promoter is selected from the pac promoter. 3. The transcription termination factor is lipoprotein (lpp)
3'region of gene, terminator of trp operon, rRN
The method according to claim 1, which is a transcription termination factor selected from a terminator of A gene and a to terminator of λ phage. 4. The method according to claim 1, wherein the ribosome binding site is a Shine-Dalgarno sequence (SD sequence). 5. A structural gene, a gene encoding interferon, a gene encoding IL-2, a gene encoding proapolipoprotein A or E, a gene encoding TNF, a gene encoding cardiodilatin, and cardio. The method according to claim 1, which is a structural gene selected from genes encoding Natrin. 6. The method according to claim 1, wherein the expression vector is an expression vector having 2 to 4 expression units. 7. The method according to claim 1, wherein the expression vector is an expression vector having an expression unit containing a structural gene in proapolipoprotein AI. 8. The method according to claim 7, wherein the expression vector is pMT (pAI) ₂ . 9. The method according to claim 7, wherein the expression vector is pMT (pAI) ₃ . 10. The method according to claim 1, wherein the expression vector is an expression vector having an expression unit containing a chloramphenicol acetyltransferase structural gene. 11. The method according to claim 10, wherein the expression vector is pMT (CAT) ₂ . 12. The method according to claim 10, wherein the expression vector is pMT (CAT) ₃ . 13. The method according to claim 10, wherein the expression vector is pMT (CAT) ₄ . 14. A promoter region and a transcription termination factor, wherein two or more expression units containing a ribosome binding site, a translation initiation codon and a structural gene are linked between the promoter region and the transcription termination factor. An expression vector having the following DNA fragment. 15. The promoter is trp promoter, lac
Promoter, lacUV5 promoter, recA promoter, P _L P _R promoter, gene25 or 26 promoter, DH
The expression vector according to claim 14, which is a promoter selected from a FR gene promoter, a tac promoter, and a pac promoter. 16. The transcription termination factor is lipoprotein (lp
p) 3'region of the gene, terminator of trp operon,
15. The expression vector according to claim 14, which is a transcription termination factor selected from the terminator of rRNA gene and the to terminator of λ phage. 17. The expression vector according to claim 14, wherein the ribosome binding site is a Shine-Dalgarno sequence (SD sequence). 18. A structural gene, a gene encoding interferon, a gene encoding IL-2, a gene encoding proapolipoprotein A or E, a gene encoding TNF, a gene encoding cardiodilatin, and a cardio. 15. The expression vector according to claim 14, which is a structural gene selected from genes encoding Natrin. 19. The expression vector according to claim 14, which is an expression vector having 2 to 4 expression units. 20. The expression vector according to claim 14, which is an expression vector having an expression unit containing a structural gene in proapolipoprotein AI. 21. The expression vector according to claim 20, wherein the expression vector is pMT (pAI) ₂ . 22. The expression vector according to claim 20, wherein the expression vector is pMT (pAI) ₃ . 23. The expression vector according to claim 14, which is an expression vector having an expression unit containing a chloramphenicol acetyltransferase structural gene. 24. The expression vector according to claim 23, wherein the expression vector is pMT (CAT) ₂ . 25. The expression vector according to claim 23, wherein the expression vector is pMT (CAT) ₃ . 26. The expression vector according to claim 23, wherein the expression vector is pMT (CAT) ₄ .