JPS60126086A - Ligation promotor - Google Patents

Abstract

PURPOSE:To obtain a manifestation vector having strong transcription activity, by bonding a promoter having strong transcription activity with a promoter having transcription activity and suitable for the manifestation of exogenote to the same direction, and using the obtained ligation promoter. CONSTITUTION:The objective ligation promoter is obtained by bonding a promoter having strong transcription activity with a promoter having transcription activity and suitable for the manifestation of exogenote to the same direction. The former promoter is a Pt promoter, Ps promoter or a ligation promoter of the Pt promoter and Ps promoter of Escherichia coli phage, and the latter promoter is the promoter of the tryptophan operon of Escherichia coli or the promoter of the lactose operon of Escherichia coli.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel linked promoter, an expression vector having the promoter, and a microorganism containing the expression vector.

Expression vectors are useful substances used to express genes derived from animals, plants, or microorganisms that have been cloned using the gene 1 mulberry cultivation technique.

In recent years, with the development of recombinant DNA technology, a large number of expression vectors have been constructed.The E. coli tryptophan operon promoter (trp promoter) is most commonly used to express animal-derived proteins in E. coli. It is an expression vector containing the E. coli lactose operon promoter (hereinafter abbreviated as +ac promoter). Various properties of these promoters, including their base sequences, have been studied in detail, and somatostatin and insulin promoters have been studied in detail. .

It is used for the production of animal-derived polypeptides such as growth hormone and interferon using Escherichia coli, and is published in the following literature.

In, Itakura et al: 5science
198, 1056 (1977) D. V. Goedd.
el et al: Proc, Natl, Acad
, Sci., USA.

76, 106 (1979) 11, v. Goeddel, et al: Natur.
e 2 B 1.544 (1979) D. V. Goed
del et al: Nature 2 B 7.
411 (1980) These promoters (trp promoter and Iac promoter) are said to have relatively strong transcriptional activity, but the existence of promoters with even stronger transcriptional activity is also known. Such promoters include the P promoter and P promoter of λ phage.
t promoter, T3. T5. Examples include the promoter of T7 phage and the promoter of liposome RNA operon.

The present inventors have conducted various studies to maximize the transcriptional activity of the trp promoter and lac promoter in the production of polypeptides derived from animal cells in E. coli. As a result, we have found that the PL promoter or If you use a promoter in which P, promoter, etc. are connected in series,
They discovered that an expression vector with even stronger transcriptional activity could be obtained and completed the present invention.

The present invention will be explained in detail below.

The present invention provides a linked promoter in which a promoter with strong transcriptional activity (hereinafter referred to as promoter A) and a promoter with transcriptional activity suitable for expression of a foreign gene (hereinafter referred to as promoter B) are linked in the same direction. do.

Preferred promoters include the PL promoter, P, l promoter, and a linked promoter of the PL promoter and Pt promoter of Escherichia coli λ phage.

Although λ phage DNA itself can be used as a source of the PL promoter and the P promoter, a Pt promoter or a plasmid into which a Pt promoter has been cloned is used. Specifically 6? As a common example, plasmid pMY12-6 is used in which the P1 promoter and P, promoter have been cloned.

Plasmid pMY1.2-6, as shown in Figure 1,
A λ phage-derived DNA fragment containing the cl gene, P++ promoter, and part of the c, 11 gene, and a λ phage-derived DNA fragment containing the PL promoter were combined into plasmid pBR322 (F.

Bolivar et al.: Gene 2.9.5 (197
7)) has a structure inserted between the Pst1 site and the Bam1 site.More specifically, pMYll-6
is the entire DNA base sequence of the λ phage determined by Sanger et al. (P, Sanger et al.: J, Mo1. Bio
+, 1'62°729 (1982)), Pstl-B of positive 37,005~3B,]03 bases
gl11 fragment (including part of the 1,1 gene, P, promoter and C■ gene) and Sou 3.5,468-3.
The 5,716 base Ha'<em>~BglT1 fragment (PL
Pst [terminus and Ha e [[terminus, 1.350
It has a structure in which a DNA fragment of base pairs (hereinafter abbreviated as bp) is inserted between the PstT site of plasmid pBR322 and the BamH1 site repaired by DNA polymerase I. The cl gene of pMY12-6 is c1
Because it has the mutation 857, the c■ gene product (cl repressor) exhibits temperature sensitivity.

Promoter B includes trp promoter and I
The ac promoter is preferably used.

As a plasmid having a trp promoter, plasmid pKYP10 (Japanese Unexamined Patent Application Publication No. 1983-1971) constructed by the present inventors is used.
110600) and plasmid pKYP100 (see Reference Example 1).

As a plasmid with Iac promoter, Ful
pOP9515 (F, Ful
ler: Gene 19', 43 (1982)).

Plasmid pMY12-6 was extracted from E. coli IMY12-6 (FERM BP-409) using the method of former rnboim et al.
Nucleic Ac1ds Res, 7.1513
(1979)). If necessary, further isolated 1) N^ is subjected to cesium chloride-ethidium bromide density gradient ultracentrifugation CD, B, C1ewel
l et al.: Proc, Natl, Acad.

Sci, USA 62.1159 (1969)). This method will be used below to isolate and purify a large amount of plasmid DNA.

After digesting plasmid pMY12-6 DNA with appropriate restriction enzymes, PL promoter, Pt
The linked promoter of the present invention can be obtained by purifying a T)NA fragment containing the promoter or a promoter in which both promoters are linked, and inserting it into an appropriate restriction enzyme site upstream of the trp promoter or the Iac promoter of a plasmid having the trp promoter or the Iac promoter. can be constructed.

The reaction conditions in the above recombinant technique are generally as follows.

The digestion reaction of DNA with restriction enzymes is usually 0.1 to 20
μg of DNA from 2 to 200 mM (preferably <10',
40. mM') of Tri 5-HCj! (pH 6,
fl ~9.5 Preferably L<ha pH7.0~8. ．． 0)
, 0-200mM N a , Cj! , 2-20mM (
Preferably 5-10mM) MgCl! In a reaction solution containing t, restriction enzyme 0. ．． 1 to 100 units (preferably 1μ
1 to 3 units per g of DNA) at 20 to 70°C.
(The optimal temperature varies depending on the restriction enzyme used).
Do this for 15 minutes to 24 hours. The reaction is usually stopped at 55-7
Although heating is performed at 5° C. for 5 to 30 minutes, a method of inactivating the restriction enzyme with a reagent such as phenol or diethylpyrocarbonate can also be used.

Purification of DNA fragments generated by restriction enzyme digestion was performed using low melting point agarose gel electrophoresis (L, Wieslande
r: Analytical Biochemist
y98. 305 (1979)) and polyacrylamide gel electrophoresis (A, M, Maxam et al.: Pro
c, Natl, Acad, Scj, USA-74
, 56 (1 (1'97.7) ) etc.

DNA fragment (D-linked anti-IFdt, 2-2ooII
Tri 5-H of M (preferably lO~jOmM)
Cj! (pH 6,0-9°5, preferably pH 7,0
~8.0), 2-20mM (preferably <5-10m
M)'s MgCJ! ,,0.1~lomM (preferably 0
．． Using 0.3 to 10 units of T4 DNA ligase, 1
It is carried out at ~37°C (preferably 3-20°C) for 15 minutes to 72 hours (preferably 2-20 hours).

The recombinant plasmid DNA produced by the ligation reaction is
If necessary, the transformation method of Cohen et al. (S.

N. Cohen et al.: Proc, Natl, Acad,
Sci., USA 69.2110 (1972)) into E. coli.

Isolation of recombinant plasmid DNA from Escherichia coli containing recombinant plasmid DNA can be carried out by the method shown in Example 1 described later or by the method described in the old r.
The method of nboim et al. (11,c, Birnboim et al.:
Nuleic Ac1ds Res, 7.1513 (
1979)).

After digesting the plasmid DNA with 1-104114al restriction enzymes, the cleavage site is examined by agarose gel electrophoresis or polyacrylamide gel electrophoresis.

Furthermore, when it is necessary to determine the base sequence of DNA, the Maxano-Gilbert method (Proc, Natl, Acad,
Sci.

74, 560 (1977)).

By the above method, promoter A (Pa promoter, Pt promoter, or P, a promoter connected to a Pt promoter) is transferred to promoter B (
A promoter linked to the trp promoter or lac promoter can be constructed. T3 as promoter eight. T5゜]゛7f7faro motor,
A liposomal RNA operon promoter, a riboprotein gene (Ipp) promoter, etc. can also be used.

In the Examples of this application, we will construct a 3-linked promoter of PaPtPtrp and 2-linked promoters of p and 'PLrp, and construct expression vectors containing them (respectively pPtT
2 and p, Ptr4) were described. pP
In LT2, the intrinsic blow motor of the teI-lacycline (T,) resistance gene has lost its function, and the gene is under the control of expression by the P'+t PtPt□3-linked promoter. Therefore, when a foreign gene is inserted downstream of the 3-linked promoter in p P t T Z, TC resistance is not expressed, and the only selection marker in the plasmid DNA is immunity to λ phage (derived from the properties of the cl gene). becomes. Since this selectable marker of immunity to λ phage is difficult to use, it is desirable to introduce a marker for resistance to antibiotics. Also Pa P
cl! downstream of the P8 promoter in the 3-linked promoter of tPtr-! Because a portion of the gene (including the translation initiation site) is present, translation begins from the translation initiation site of the middle cII gene, but this is not a desirable property for an expression vector. On the other hand, in pPLT4,
3 linked promoter to PR promoter and CI+
pPtT4 contains the PtPtrp2-linked promoter from which part of the gene has been removed, and carries the ampicillin (Ap) resistance gene and the Tc resistance gene.
can be used more advantageously.

Examples of the present invention will be shown below.

Voice Example 1 Construction of a PR'P+-PLrp3 linked promoter in which P promoter, Pt promoter and trp promoter were linked in the same direction: Plasmid pPLT2 having a PRPL P trp3 linked promoter was obtained as follows (Fig. 1). reference).

First, pMY12-6 plasmid DNA was transferred to E. coli IMYI.
It was isolated and purified from I-6 by the method described above.

pMY12-6 plasmid DNA (approximately 4.6 Kb
) Approximately 4 pg of 10 mM Tris-HCj! (pH
7,5), 100mM NaCj! , 7 m M M
g CI! , and 8 units of restriction enzyme Bam II dissolved in 40 μβ of a buffer containing 6 mM 2-mercaptoethanol (hereinafter abbreviated as “Y-100 buffer”).
1 (manufactured by Takara Shuzo Co., Ltd.; hereinafter all restriction enzymes are manufactured by Takara Shuzo Co., Ltd. unless otherwise specified), and a digestion reaction was carried out at 37°C for 2 hours.

Subsequently, after phenol and chloroform extraction and ethanol precipitation, the DNA fragments were purified with total [40 μβ of 50 mM T
ris-HCj! (p147.6), 7mM MgC
fz, 6mM 2-mercaptoethanol, 0.25
mM dA'TP, 0.25mM dCTP.

0.25mM dGTP and 0.25mM dT'r
Buffer containing P*<hereinafter referred to as “DNA polymerase I buffer”
6 units of Escherichia coli DNA polymerase K1enow fragment (manufactured by Pethesda Research Laboratory, hereinafter the same) was added, and the mixture was reacted for 2 hours at 15°C. The protruding end was changed to a flat end. The reaction was stopped by phenol extraction, and after chloroform extraction and ethanol precipitation, the DNA
A total amount of 40μβ of 10mM Tris-11C!
! , (pH 7,6), 50mM NaC7! ,7'
mM MgCff1. and dissolved in a buffer containing 6mM 2-mercaptoethanol (hereinafter referred to as "Y-50 buffer"), 8 units of pstT were added, and the mixture was heated at 37℃ for 2 hours.
A time digestion reaction was performed. After heat treatment at 65℃ for 10 minutes,
Low melting point agarose gel @ pneumophoresis [1, Wiasla
nder:AnalyLic++ IBiochemi
98 + 305 (1979)) C) A DNA fragment of about 1.35 Kb containing the cl gene, ■) 8 and ! +'l made.

Next, pKYP1 with the trp portal promoter
Approximately 5 μg of 00 plasmid DNA (approximately 4.45 Kb, see Reference Example 1) was digested with 10 units of Xhol in 40 pH trrY-1001i solution for 2 hours at 37°C. Subsequently, after phenol and chloroporum extraction and ethanol precipitation, the DNA fragments were mixed with DNA polymerase Ig! in a total volume of 40 μl. Dissolve the injection solution and add 6 units of E. coli DNA polymerase I/Klenoi I + fragment.
After incubation for 2 hours at 15°C, the 5'-protruding ends generated by Xhol digestion were converted to flat ends. The reaction was stopped by phenol extraction, and after chloroform extraction and ethanol precipitation, the entire DNA fragment (iL4 o/7I! ys
Dissolve in O buffer, add 10 units of Pstr, 37
The digestion reaction was carried out at ℃ for 2 hours. After heat treatment at 65°C for 10 minutes, tr
Larger plasmid DNA Ig containing p promoter
The L piece (approximately 3.7 Kb) was purified.

Approximately 1.35K derived from pMY]2-6 thus obtained
b DNA fragment (approximately 0.1 Mg) and p KYP100-derived approximately 3.7 K b DNA fragment (approximately 0.177 g)
, 20mM Tris-HCj! (pH7,6)
, 10mM MgC11z, l'omM dithiothreitol, and 0.5mM ATP (hereinafter, this dilute solution will be abbreviated as "T4 DNA ligase buffer") 20μ! The binding reaction was carried out at 4° C. for 18 hours using 2 units of 74 DNA ligase (manufactured by Takara Shuzo Co., Ltd., the same applies hereinafter).

Using the reaction solution containing the recombinant plasmid DNA thus obtained, Escherichia coli MP347 strain [F-1-acZam
YI4 ±rpam8 galstrA (λ mutual ±o25
2 c1857 ΔH1)) (FF, RM BP-40
8) using the method of Cohen et al. (S, N, Cohen
et al.: Proc, Natl, Acad, Sci, U
SA 69.2110 (1972)) (hereinafter, this method will be used for the transformation of E. coli) to obtain a tetracycline (Tc) resistant strain. Plasmid DNA was isolated from these transformed strains using the method shown below.

That is, the transformed strain was 50. 8 ml LG medium containing μl/l 111 ampicillin [Batato tryptone 1.0
9i, #'lN: tkiss 0.5%, NaCj'0.5%
, glucose 0.1%, pH 7,2).
Cultured at 0”c for 15-18 hours.
orpn+, collect bacteria by centrifugation for 5 minutes, and transfer the bacterial cells to 400μ
l of 50mM Tri s-H'CI (pH 7,5)
The cells were suspended in a buffer containing 10 mM EDTA and frozen in dry ice and ethanol. After thawing at 20'C, add 40μl of 2.5M KCj? and 5w/m of 40μf
l lJ zozyme (suspended in solution H) was added and left in water for 15 minutes. After freezing and thawing as described above, centrifugation was performed at 15.00 rpm for 20 minutes to remove the supernatant. After extracting this supernatant with a mixture of phenol and chloroborum, twice the amount of ethanol was added to precipitate the DNA. The resulting DNA fragments were collected by centrifugation, and
RNase A (Sigma
50 to IOoμβ of 10 mM Tris containing
-11Cj! A partially purified plasmid DNA was prepared by suspending the DNA in 1 mM EDTA (pH 7.5) (hereinafter, this method will be used for plasmid isolation). When we analyzed its structure, we found that it was IP.

It was confirmed that the plasmid I) P+-T'2 possessed by the T2 strain had the desired structure.

p l(Y
Escherichia coli strain MP347 carrying the P I 00 plasmid was grown on LG agar plates containing 20 μg/ml of teilacycline (10 g/A Bactodrypton, 5 g/Il Yeast Equiy, 5 g/1 NaCj!, Ig/#Glucose).

E. coli strain MP347 (+p, strain 72) carrying the pPLT2 plasmid containing the P,I-PL Ptrp 3-linked promoter could not grow on 15 g/6 agar, pH 7.2>, but was grown on tetracycline 20 μg/ml.
1, G agar plates containing 37-42°C. In the case of pKYPloo, the transcription of the Tc resistance gene is controlled by the trp promoter alone, but in the case of pPLT2, the transcription of the Tc resistance gene is controlled by the trp promoter alone.
It is controlled by the rp3-linked promoter.

The above differences in tetracycline resistance reflect differences in transcriptional activity of promoters, and PR-P, -Pt
The rp3-linked promoter was shown to have stronger transcriptional activity than the trp promoter alone.

E. coli strains containing pMY12-6 and pPLT2 are
Escherichia coroni IMY12 respectively
-6 (FERM BP-409), Escheri
-chiacoli I Pt T2 (FERM B
P-407) has been deposited with the Institute of Microbial Technology (Kikoken), Agency of Industrial Science and Technology.

Example 2 Plasmid vector with PL-Ptrp 2-linked promoter 1) Construction of PLT4: Approximately 5 μg of pPLT2 plasmid DNA was added to 50 μl of pPLT2 plasmid DNA.
by lO units of Bg/II in l-100 buffer,
Digestion reaction was carried out at 37°C for 2 hours.

Subsequently, after phenol and chloroform extraction and ethanol precipitation, the DNA fragments were isolated from the total (i 40111 DNA
Polymerase ■ Slow (dissolved in K solution, 6 units of E. coli DN)
Add A polymerase I-Kleno++ fragment and 1
The reaction was carried out at 5° C. for 2 hours to convert the 5'-protruding ends generated by X hol digestion into flat ends.

The reaction was stopped by phenol extraction, and after chloroporum extraction and ethanol precipitation, the DNA fragments were dissolved in a total volume of 40 μl2 of Y-100 buffer, IO units of BamHI was added, and the digestion reaction was carried out at 37° C. for 2 hours. 65℃, 10
After heat treatment for 1 min, low melting point agarose gel electrophoresis was used to analyze approximately 0.0 min containing the P, -Ptrp-linked promoter.
A 7'Kb plasmid DNA fragment was purified. Next, approximately 5 μg of pK Y +)100 plasmid DNA was injected into 10 units of Xho in Y-100 buffer at 50°C.
Digestion reaction was carried out for 2 hours at 37°C. Subsequently, after phenol and chloroform extraction and ethanol precipitation, the DNA fragments were dissolved in 40 μl of total N DNA polymerase 1 buffer, 6 units of E. coli DNA polymerase [-KIeno+q fragment was added, reacted at 15°C for 2 hours, and Xhol The 5'-overhangs generated by the digestion were converted to flat ends. The reaction was stopped by phenol extraction, and after chloroform extraction and ethanol precipitation, the DNA fragment was dissolved in a total volume of 40 μl of Y-100 buffer, 10 units of BamHl was added, and the digestion reaction was performed at 37°C for 2 hours. . After heat treatment at 65'C for 10 minutes, the larger plasmid DNA was analyzed using low melting point agarose gel electrophoresis.
The fragment (4,0 Kb) was purified. p obtained in this way
An approximately 7 Kb DNA fragment (approximately 0.0
5°C g) and approximately 4.5°C from pKYP 100. The OK b DNA fragment (0°l/7g) was subjected to a ligation reaction at 4°C for 18 hours using 2 units of T4 DNA ligase in 20 μl of T4 DNA ligase mild 1#i solution.

Escherichia coli strain MP347 was transformed with the recombinant plasmid DNA thus obtained to obtain an Ap shochu-resistant strain. A Tc-resistant strain was selected from among them, plasmid DNA was isolated, and its structure was analyzed. As a result, it was confirmed that plasmids pP and T4 possessed by the IPLTd strain had the desired structure.

E. coli MP347 strain carrying pKYP100 plasmid containing only trp promoter was treated with 20μ of tetracycline.
pP containing the PL-Ptrp2 linked promoter could not grow on LG agar plates containing g/ml.
Escherichia coli MP347 strain carrying the L T 4 plasmid ([
PUT4 strain) grew when cultured at 37-42°C on LG agar plates containing 20 μg/ml of tetracycline. As described in Example 1, the difference in tetracycline resistance reflects the difference in the transcriptional activity of the promoter, and it was shown that the PL-Ptrp2-linked promoter has stronger transcriptional activity than the trp promoter alone.

In addition, the E. coli strain containing pPLT4 is l1scheric.
l+1acoli IPL T4 (FERM BP
406) and has been deposited with the Kikoken.

Reference Example 1 Preparation of pKYP-100: 50°C
) was added with 50 units of Hh a l and 10 mM Tr
is-HCffi (pH 7,5), 7mM MgC
L, total gL containing 6mM 2-mercaptoethanol
The reaction was carried out in 100 μl of reaction solution at 37° C. for 2 hours. Hh
Digestion with aI 1 & 5% polyacrylamide gel electrophoresis (A, M, Maxam et al.: Proc, Na
tl, Acad, Sci.

74, p. 560 (1977), hereinafter abbreviated as PAGE], a DNA fragment of approximately 180 bp containing the trp promoter was purified. At this time, 1) the two DNA fragments other than the NA fragment cannot be separated well by PAGE and are therefore purified together. The three purified DNA fragments (approximately 4 μg) were dissolved in 50 mM Tris-HCl (pH
7,6), 7mM MgC6z, 10mM 2-mercaptoethanol, 0.25mM dATP, 0.
25mM dCTP.

In a reaction solution containing 0.25mM dGTP and 0.25mM dTTP with a total volume of 30 μp, 8 units of Escherichia coli DNA polymerase 1-Klenow fragment were added and allowed to react at 5° C. for 2 hours. Through this reaction, the 3'-overhanging end generated by Hha I digestion is treated with DNA polymerase I-Kl.
The eno-fragment's 3'-5' exonuclease activity and 5'->3' repair synthesis activity convert it into a flat end. D was then heat treated at 72°C for 30 minutes.
After inactivating the NA polymerase I Klenow fragment, IM NaCl! The NaCρ concentration was set to 50mM, and 8
One unit of Hindl was added and reacted at 37°C for 2 hours. After digestion with Hindl, a DNA fragment of about 100 bp containing the trp promoter was separated and purified by PAGE.

On the other hand, 8 units of Ec were added to 5 μg of plasmid pBR322.
oR+ was added, 10mM Tris-HCJ (pH
7,5), 50mM Na'CI! , 7mM MgCj
! The reaction mixture was reacted at 37° C. for 2 hours in a reaction solution containing 6 mM 2-J captoethanol in a total volume of 20 μ2. After the reaction, after phenol and chloroform extraction and ethanol precipitation, the DNA fragments were added to a total volume of 20 pl of 50 mM Tri.
s-HCl (pH 7,6), 7rr+M MgClz
, 6mM 2-mercaptoethanol, 0.25mM
dATP: 0.25mM dCTP, 0.25m
M dGTP, dissolved in 0.25 mM dTTP, followed by 8 units of E. coli DNA polymerase l -Kleno-
The fragments were added and reacted at 15°C for 2 hours. E C' O
The 5'-overhanging ends generated by RI digestion were converted to flat ends by the repair synthesis activity of the DNA polymerase I-Klenow fragment.

After inactivating the DNA polymerase Ienow fragment by heat treatment at 72°C for 30 minutes, IMNaC
NaCj with l! The concentration was 50mM and 8 units of Hind
U was added and reacted at 37°C for 2 hours.

After digestion with H'1ndllll, large plasmid DNA fragments (approximately 4
．． 33Kb) was purified.

Approximately 1 containing the trp promoter thus obtained
00bp DNA fragment (approximately 50ng) and pBR322 Yamaki approximately 4.33Kb DNA fragment (approximately 0.2.17g)
) with 50 ng of 5'-phosphorylated X h'o I
Linkal (pCCTCGAGG, manufactured by Collaborative Research) was added, and 20mM-Tris-HC was added.
j! (pH 7,6), 10mM MgC#z, 10
In a reaction solution containing mM dithiothreitol and 0.5 mM ATP in a total volume of 20 μl, 1 unit of T4 DNA ligase was added, and the binding reaction was carried out at 4° C. for 40 hours.

Using the recombinant plasmid DNA thus obtained, Escherichia coli strain 88101 was transformed, and the obtained Ap''
The plasmid DNA of the transformed strain of Tc'' was isolated and purified.
coRI Xho I, Hin d m, l(a e
m, C1aIXTaqT.

A plasmid in which an approximately 100 bp DNA fragment containing a trp promoter and two Xhol linkers were cloned by digestion with Rsal (manufactured by New England Biolabs) was selected and named pKYP-100.

[Brief explanation of drawings]

FIG. 1 shows the construction process of pPLT2. FIG. 2 shows the construction process of pPLT4. FIG. 3 shows the construction process of pKYP-100. Patent Applicant (Representative Applicant) (102) Hiroshi Anzai, Chairman of the Cancer Research Association, Kyowa Hakko Kogyo Co., Ltd.

Claims (1)

[Scope of Claims] (1) A promoter with strong transcriptional activity (hereinafter abbreviated as promoter A) and a promoter with transcriptional activity suitable for expression of a foreign gene (hereinafter abbreviated as promoter B) are arranged in the same direction. A linked promoter bound to. (2) The linked promoter according to claim 1, wherein promoter A is a PL promoter, a PR promoter, or a linked promoter of a PL promoter and a PR promoter of Escherichia coli λ phage. (31) Iff motor B is the promoter of the E. coli tryptophan operon (hereinafter abbreviated as trp promoter) or the promonitor of the E. coli lactose operon (
The linked promoter according to claim 1, which is a 1ac promoter (hereinafter abbreviated as 1ac promoter). (4) An expression vector having a linked promoter according to any one of claims 1 to 3. Expression vector according to claim 4 characterized by +51' pPL T 2 or pPtT4. (6) A microorganism comprising the expression vector according to claim 4 or 5. (7) Esocielichia coli Ipt T, 2° (
8) Esocielichia coli IPLT4゜