JP2518862B2 - New plasmid vector - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for efficiently producing Escherichia coli protease III. Furthermore, the present invention relates to a novel plasmid vector that enables secretion of a gene product in Escherichia coli or intracellular production.

E. coli protease III is also called protease Pi (Swamy, KH & Goldberg, AL (1981) Nature,
292, 652-654), by Cheng and Zipser, first isolated from the periplasm of E. coli, purified (Cheng, Y
-SE & Zipser, D. (1979) J. Biol. Chem, 254 , 4698-470.
6). Then, the gene (P'tr) of this enzyme was cloned (Dykstra, CC & Kushner, SR (1985), J. Bacte.
riol., 163 , 1055-1059), and its entire nucleotide sequence was determined (Finch, PW et al (1986) NAR, 14 , 7695-7703). As a result, this enzyme is a protein with a molecular weight of 110K consisting of a single-chain polypeptide of 939 amino acid residues,
It was revealed that it was synthesized as a precursor having a signal peptide consisting of 23 amino acid residues. This signal peptide is considered to play an essential role in the secretion of protease III from the cytoplasmic membrane. Further, Protease III exists in the microbial cells in a very stable form although its amount is small, and it is an enzyme that can be easily purified from the microbial cells by utilizing this property.

As a substrate specificity of protease III, this enzyme is 6500
Although it has been reported that only the front and rear peptides are degraded (Cheng, Y-SE & Zipser, D. (1979) J. Biol. Chem., 25
4, 4698 to 4706) the cleavage site, Ty insulin B chain
It was unknown except that it cleaved the r-Leu (16-17) and Phe-Tyr (25-26) bonds. The present inventors have used various peptide hormones as substrates to
As a result of investigating the cleavage site by III, this enzyme showed that human β-endorphin Lys-Asn (19-20), human rheumorphin Arg-Arg (6-7) and Thr-Arg (13-14),
Arg-Lys (14-15) of h-VIP, His of human calcitonin
-Thr (20-21), Phe-Gly of substance P (8-
9) and Gly-Leu (9-10), Phe of human glucagon
-Thr (6-7) and Lys-Tyr (12-13), h-ACTH
It was found that the Tyr-Ser (2-3) bond of E. coli was specifically degraded. Moreover, the present inventors were able to clarify that this enzyme decomposes a peptide having a molecular weight of about 3000 at a specific site by using various protease digests of β-casein as a substrate. The substrate specificity of protease III, which recognizes the size of the substrate, is extremely useful industrially as a tool (reagent) for investigating the structure-function relationship of peptides / proteins. But,
The production amount of protease III in E. coli was very low, which made it extremely difficult to isolate and purify it.

The present inventors have conducted various studies on a plasmid containing the protease III gene (ptr) for the purpose of making Escherichia coli produce a large amount of E. coli protease III and facilitating its isolation and purification. It was found that a large amount of protease III was expressed by substituting for tac promoter with, and completed the present invention.

That is, the present invention provides a plasmid derived from E. coli with ptr
Consisting of inserting a fragment containing the gene and then transforming E. coli with the plasmid vector obtained by replacing the ptr promoter with the tac promoter and culturing this transformant in the presence or absence of inducer A method for producing Protease III is provided.

 Hereinafter, the present invention will be described in detail.

Construction of plasmid vector pDR42S The ptr gene has been cloned from the E. coli chromosome as a 19Kb BamHI fragment (Dykstra, CC & Kush.
ner, SR (1985) J. Bacteriol., 163 , 1055-1059). This cloned plasmid is called pCDK3,
This is used as the ptr gene source (in the present invention, pG185, which is completely equivalent to pCDK3, was used). Construction of pDR42S, a plasmid originating from protease III, is carried out as follows according to the chart shown in FIG.

The plasmid pV185 is cut with the restriction enzyme HindIII to obtain a 6.7 Kb HindIII fragment containing the ptr gene, which is inserted into the commercially available plasmid pUC19. Hind pUC19 for that
III digestion, E. coli alkaline phosphatase (BA
P) and dephosphorylated, and use this for plasmid p
Mix with 6.7 Kb HindIII fragment excised from V185 and ligate. Here, there are two insertion directions of the ptr gene, but one in which the lac promoter of pUC19 and the direction of the ptr gene are the same is selected.
Named). Then, the 2.5 Kb Sal I-Hpa I region (probably containing all or part of the promoter region) derived from Escherichia coli located on the 5'side of the ptr gene from pUC67 is deleted. To do this, first add pUC67 to Sal.
After digestion with I, fill up with Klenow fragment to make Sal I site as blunt end (blunt end). It is then digested with Hpa I and the digest is subjected to agarose gel electrophoresis. Isolation and purification of the 7.1 Kb DNA fragment by electroelution of T4 DNA
After ligation with ligase, E.coli JM109
Are transformed. Isolate a plasmid from the ampicillin-resistant transformant and confirm that it is the desired plasmid by a restriction enzyme digestion mode (this plasmid is called pUC4
Named 2). The resulting plasmid has a unique Hind III site 3'downstream of the ptr gene, which is converted to a Sal I site using a Sal I linker (this plasmid is designated pUC42S). Then, about 20 bp of pUC42S
The EcoR I-BamH I DNA fragment consisting of was excised from the expression vector pDR540 containing the tac promoter by 400b.
pDR42S is constructed by replacing the p EcoR I-BamH I DNA fragment.

Production of Protease III The plasmid vector pDR42S obtained by the above method was introduced into Escherichia coli by a conventional method, and the obtained transformant was cultured in the presence or absence of isopropyl β-D-thiogalactoside (IPTG). Protease III is expressed, and protease III is recovered from the culture medium by a conventional method.

 Hereinafter, the present invention will be described in more detail with reference to Examples.

Example Construction of plasmid pDR42S Plasmid pV185, which is completely equivalent to pCDK3 known as the ptr gene source, was obtained by Researcher Itaya, Mitsubishi Kasei Life Research Institute. 10 μg of this plasmid pV185 was digested with 50 units of Hind III in 100 μl reaction solution at 37 ° C. for 1 hour. The digest was then subjected to 0.7% agarose electrophoresis. A 6.7 Kb HindIII fragment containing the ptr gene was extracted from agarose gel by electroelution and then purified by DE-52 column chromatography. The amount of 6.7 Kb Hind III fragment recovered by ethanol precipitation was about 2 μg. On the other hand, vector plasmid
pUC19 (manufactured by TOYOBO) (2 μg) in 50 μl reaction solution, 1
It was completely digested with 0 unit of Hind III at 37 ° C. for 1 hour, and then 1 unit of Escherichia coli alkaline phosphatase was added and the reaction was carried out at 37 ° C. for another 30 minutes. After stopping the reaction by the phenol-chloroform extraction method, DN
A was recovered by ethanol precipitation. The recovered amount was 2 μg. The 6.7 Kb Hind III fragment obtained as described above and 0.1 μg each of pUC19 treated with E. coli alkaline phosphatase after Hind III digestion were mixed, and 5 units of T4 DNA ligase (manufactured by TOYOBO) were mixed in a 20 μ reaction solution. The reaction was carried out at 16 ° C for 30 minutes to circularize the plasmid. Next, Escherichia coli JM109 was transformed with the circularized plasmid, and pUC67 was obtained from the transformant.

Then, 10 μg of this pUC67 was added to
It was completely digested with 00 units of Sal I for 1 hour at 37 ° C. After recovering the DNA by ethanol precipitation,
5 units of DNA polymerase (Klenow) was added to the reaction solution of, and the mixture was reacted at 37 ° C for 30 minutes, and then treated at 65 ° C for 10 minutes. After recovering the DNA by ethanol precipitation, 50 units of Hpa I in 100 μl reaction solution
After complete digestion for 1 hour at 0 ° C, the digest was subjected to 0.7% agarose gel electrophoresis. Cut out a 7.1 Kb DNA fragment and 6.7 Kb H
Elution purification was performed as for the ind III fragment. The amount of DNA recovered was 5 μg. 0.1 μg of this 7.1 Kb DNA fragment
Cyclization was performed with 5 units of T4 DNA ligase in a 20μ reaction solution at 16 ° C for 30 minutes. Escherichia coli JM109 was transformed with the cyclized plasmid, and pUC42 was obtained from the transformed strain.

Next, pUC42 (2 μg) was completely digested with 10 units of Hind III in a 50 μ reaction solution at 37 ° C. for 1 hour. After recovering the DNA by ethanol precipitation, 2 units of DNA polymerase (Klenow) was added to 50 μl of the reaction solution, reacted at 37 ° C. for 30 minutes, and then incubated at 65 ° C. for 10 minutes. DNA recovered by ethanol precipitation
Was added with 1 unit of Escherichia coli alkaline phosphatase in a 50 μl reaction solution, treated at 37 ° C. for 30 minutes, and then extracted with phenol-chloroform. About 1.5 μg of DNA was recovered by ethanol precipitation. Obtained DNA
0.1 μg was mixed with 0.01 μg Sal I linker (Takara Shuzo) (molar ratio 1: 100), and 5 units of T in a 20 μ reaction solution were mixed.
4 DNA ligase was added and treated at 16 ° C for 30 minutes. Escherichia coli JM109 was transformed with the thus circularized plasmid, and pUC42S was obtained from the transformed strain.

Finally, the tac promoter and ptr gene were ligated as follows. First, pUC42S (4μg) is 100μ
20 units of EcoR I and 20 units of BamH in the reaction solution of
It was completely digested with I for 1 hour at 37 ° C. and subjected to 0.7% agarose electrophoresis. The 7 Kb EcoR I-BamH I fragment was excised and eluted and purified in the same manner as in the case of the 6.7 Kb Hind III fragment. The amount of DNA recovered by ethanol precipitation is about 3
It was μg.

On the other hand, pDR540 (Pharmacia) 10 μg, 100 μg
50 units EcoR I and 50 units BamH I in the reaction solution
Was digested for 1 hour at 37 ° C. and subjected to 1.5% agarose electrophoresis. 400 bp EcoR I-Bam containing tac promoter
The HI fragment was excised and elution-purified as in the case of the 6.7 Kb Hind III fragment. The amount of DNA recovered by ethanol precipitation was about 0.5 μg.

7Kb EcoR I-BamHI fragment obtained as described above.
Mix 1 µg with 0.1 µg of 400bp EcoR I-BamHI fragment,
16 with 5 units of T4 DNA ligase in μ reaction solution
The plasmid was circularized by treating at 30 ° C. for 30 minutes. Escherichia coli JM109 was transformed with the circularized plasmid, and pDR42S was obtained from the transformed strain.

The desired plasmid pDR42S obtained as described above.
The detailed restriction enzyme sites of are shown in FIG. In the figure, the underlined restriction sites are shown to exist independently on this plasmid. Transformant, Escherichia coli JM109 /
pDR42S has been deposited with the Institute of Microbial Engineering, Institute of Industrial Technology (FERM P-9520, date of deposit: August 14, 1987).

In this plasmid pDR42S, the lac promoter derived from pUC19 and the tac promoter derived from pDR540 are located in tandem in this order and control the ptr gene located 3'-downstream thereof. Since the E. coli chromosome-derived DNA region remaining 5'upstream of the ptr gene is only 48 bp, which is too short to contain the ptr gene promoter, pDR42S
In, the promoter of the ptr gene seems to be partially or completely deleted.

Culture of transformed Escherichia coli JM109 / pDR42S Escherichia coli K-12 strain JM10 transformed with plasmid pDR42S
LB medium containing 40 μM ampicillin (or M9-0.2%
The cells were cultivated with shaking in casamino acid medium) at 37 ° C. The time when the production of protease III was the highest was examined by changing the time of induction with IPTG and the time of collecting the cells after induction. As a result, 0.4 mM IPTG was added at the time when the turbidity of the culture solution grew to a Klett value of 50 to 100, and further culturing was continued.
Protease III is most prominent when cells are collected 4 to 6 hours after TG addition.
It was found that the production of The cells thus obtained were frozen and stored at -20 ° C.

Crushing of bacteria and purification of protease III After thawing frozen cells at room temperature,
The cells were suspended in 50 volumes of 10 mM Tris-HCl buffer (pH 8) and sonicated to disrupt the bacteria. Protease III is extremely stable and loses almost no activity when left at 37 ° C overnight, so it is better to perform sonication as carefully as possible. After the sonication treatment, the mixture was centrifuged at 15000 rpm for 30 minutes at 4 ° C., and the centrifuged supernatant was dialyzed against 10 mM Tris-hydrochloric acid buffer solution (pH 8). Then, it was applied to a DE-52 column equilibrated with the same buffer. Protease III adsorbs to DE-52 under these conditions. A suitable column volume is about 1/50 of the volume of the bacterial culture solution. After washing the column with 10 mM Tris-HCl buffer (pH 8) in an amount about 10 times the column volume, protease III was eluted with the same buffer containing 0.1 M NaCl. PM of elution fraction
Sephacryl S-300 after concentration by ultrafiltration using -10
It was purified by Superfine column chromatography until Protease III gave a single band on SDS-PAGE. The purification yield of the protease III thus purified was about 30%. The amount of Protease III produced was SDS-PA
It was estimated to be about 60 mg / by performing GE and comparing the band of protease III with the degree of staining of the band of a marker of known protein concentration. The yield of the purified standard is about 15 mg /
Which is the reported yield of 0.5 mg /
(Dykstra, CC & Kushner, SK (1985), J. Bacteriol.,
163 , 1055-1059), about 30 times. Also,
Escherichia coli JM109 transformed with pDR42S and subjected to IPTG induction during culture does not have pDR42S.
It is about 1000 times higher than JM109. The above results are shown in Table 1 below.
Shown in

As is clear from the above examples, according to the method of the present invention,
Protease III can be produced extremely efficiently. However, the plasmid pDR42S used in the method of the present invention
Can be used not only for protease III but also for expression of other proteins. Therefore, the present invention also provides an expression vector, pDR42S, which enables efficient intracellular expression and secretion of a gene product.

The expression vector according to the present invention is useful as a secretory vector that can be controlled and has high expression efficiency. This expression vector contains a unique BamHI, Xb downstream of the tac promoter.
Since it has a I and Sal I sites, it is possible to insert a gene encoding a heterologous protein as a BamHI-SalI fragment or an XbaI-SalI fragment. Also, protease III
In order to secrete and express a heterologous protein in Escherichia coli using the gene encoding the signal peptide of Escherichia coli, as shown in FIG.
You can use the fl II site. In this case, the amino terminus of the heterologous protein and the signal peptide of protease III can be linked by a chemically synthesized oligo DNA linker. In this way, the present inventors have already succeeded in expressing ribonuclease H.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of the construction process of the novel plasmid vector pDR42S. FIG. 2 is a restriction enzyme digestion map of pDR42S. FIG. 3 is a schematic diagram showing a construction example of a plasmid vector for secretory expression of a heterologous protein using pDR42S.

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Claims (4)

(57) [Claims] 1. A pUC-type plasmid vector capable of replication and expression in Escherichia coli, which contains a tac promoter region, a gene encoding a signal peptide of Escherichia coli protease III, and a structural gene of Escherichia coli protease III in this order. 2. The plasmid vector according to item (1), which contains an origin of replication derived from plasmid pUC18 or pUC19. 3. The plasmid vector according to item (1) or (2), which contains an ampicillin resistance gene as a selectable marker. 4. The plasmid vector according to item (1), which is characterized by the following restriction enzyme cleavage map.