JPH047193B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a recombinant plasmid incorporating the rat liver cytochrome P-450c gene.

Rat liver cytochrome P-450c, which is induced by administration of 3-methylcholanthrene, is a heme protein that binds to carbon monoxide in its reduced form, and its difference spectrum shows an absorption maximum at 447 nm. It is involved in the oxidative metabolic reactions of fat-soluble organic compounds and the metabolic activation of chemical mutagens. The present invention is directed to cytochrome P-, which has high hydroxylation activity and a wide range of substrate specificity.
The present invention provides a plasmid for expressing 450c in Escherichia coli, which makes it possible to apply it to oxidation reaction processes at an industrial level, oxidative removal of organic compounds in industrial wastewater, etc.

In recent years, there have been many cases in which a plasmid constructed by connecting a promoter to a cDNA or synthetic DNA encoding a bioactive peptide derived from a higher organism was introduced into E. coli, and the bioactive peptide was expressed in E. coli. insulin, growth hormone,
Attempts have been made to produce interferon and the like using microorganisms at an industrial level. However, as for cytochrome P-450, which is localized in the microsomal membrane of cells, has a large molecular weight of 59,300 Daltons, and contains heme in its protein molecule, there is currently no report on its expression in E. coli.

The present inventors conducted various studies on the expression of rat liver cytochrome P-450c in Escherichia coli and completed the present invention.

The recombinant plasmid pMG1 of the present invention intended for expression in E. coli has a strong promoter, tac.
The rat liver cytochrome P-450c gene, which exhibits high oxidative activity, is linked downstream of the promoter. Furthermore, the SD sequence, which is a transcription factor that regulates expression in E. coli, and the distance between the SD sequence and the start codon, etc., are sufficiently satisfied.

By transforming E. coli using the obtained pMG1 plasmid, rat liver cytochrome P-450c is synthesized within the E. coli cells, and it can be isolated and purified. In this way, the obtained cytochrome P-450c specimen and rat liver NADPH
- It is possible to simultaneously immobilize cytochrome P-450c reductase and perform an oxidation reaction. In addition, the produced cytochrome P-450c is not separated or purified,
As Escherichia coli controls the oxidation reaction, it can be immobilized and applied as a bioreactor.
It is also possible to add it to activated sludge.

The recombinant plasmid pMG1 of the present invention can be constructed by connecting the recombinant plasmid pAU157 having the base sequences shown in FIGS. 1a to 1e to the tac promoter, which is a strong promoter of E. coli.

The tac promoter is “−35” of the trp promoter.
One of the strong promoters of E. coli, consisting of the “-10 region” of the lacUV ₅ promoter.
It is one.

Next, the present invention will be explained in detail with reference to Examples.

Example Construction of expression plasmid pMG1 for expression of rat liver cytochrome P-450c gene in E. coli Recombinant plasmid pAU157 containing the entire coding region of rat liver cytochrome P-450c protein
(Can be produced by the method described in Nucl. Acids. Res. 12 , p. 2929-2938).
The structural gene of 450c was transferred to the tac promoter (PL Biochemical Co., Ltd.), a strong E. coli promoter.
A plasmid pMG1 for expression in E. coli was constructed as follows. The base sequence of the cDNA contained in the pAU157 plasmid is shown in Figures 1a to 1e.

Construction of expression vector pDG1 As the first step in constructing the expression plasmid pMG1, we created an expression vector carrying the tac promoter.
A new expression vector pDG1 was constructed by modifying pDR540 (PL Biochemical). Figure 2 shows the outline. Below, we will explain how to construct it in two steps.

Step 1: Partial digestion of pDR540 plasmid Add 2 units of restriction enzyme BamHI (Takara Shuzo) to 1 μg of pDR540 plasmid DNA and digest with 10 μg of pDR540 plasmid DNA.
BamHI reaction solution [10mM Tris-HCl (PH
8.0), 7mM MgCl ₂ , 100mM NaCl, 2m
The reaction was carried out in M2-mercaptoethanol and 0.01% bovine serum albumin at 37°C for 1 hour. Next, add one unit of S1 nuclease (PL) to this reaction solution.
Biochemical) and 10μ S1 nuclease reaction solution [100mM NaOAc-HOAc (PH4.0),
300mM NaCl, 12mM ZnSO4 _] was added, and the mixture was further reacted at 18°C for 2 hours. After the reaction, an equal volume of phenol-chloroform solution [phenol:chloroform (1:1)] was added to the reaction solution, mixed, and after centrifugation at 10,000 rpm for 5 minutes, the supernatant was collected. Next, twice the volume of cold ethanol was added and the mixture was left at -80°C for 15 minutes, followed by centrifugation at 10,000 rpm for 10 minutes to collect DNA and suspend it in 10μ of distilled water.

Step 2: Construction of pDG1 Add 10μ of DNA solution prepared in Step 1.
Add 1 μg of Bgl linker (Takara Shuzo) and 4 units of T ₄ DNA ligase (Takara Shuzo),
20μ T ₄ DNA ligase reaction solution [66mM
Tris-HCl (PH7.6), 6.6mM _MgCl2 , 10mM
dithiothreitol, 1.0mM ATP] at 16℃15
Allowed time to react. After the reaction, the reaction solution was prepared using the method of Cohen et al. (Proc. Natl. Acad. Sci. USA 69 , p2110-
Escheric hia coli
DH1 strain (F- ^, recA1, endA1, gyrA96, thi-
1. Transformed into hsdR17, supE44, λ ^- (also preserved at Kyushu University School of Medicine Genetic Information Facility / Sumitomo Chemical Co., Ltd. Takarazuka Research Institute),
LB plates containing 100 μg/ml ampicillin (Sigma) (10 g polypeptone per plate,
5g yeast extract, 5g NaCl,
The transformants were spread on 12 g of agar (containing 12 g of agar), and the colonies that appeared were isolated. Next, Birnboim
method (Nucl, Acids, Res. 7 , p1513-
1523), plasmids from colonies according to
Prepare DNA, add 1 unit of restriction enzyme Bgl to 1 μg of plasmid DNA,
Bgl reaction solution [10mM Tris-HCl (PH7.5), 7
The reaction was carried out at 37° C. for 1 hour in mM MgCl ₂ , 100 mM NaCl, 7 mM 2-mercaptoethanol. After the reaction, the reaction solution was applied to a 1.0% agarose gel (Sigma) containing 0.1 μg/ml ethidium bromide (Aldrich) and electrophoresed at 100 V for 60 minutes. The migration pattern was observed under an ultraviolet lamp, and a plasmid that had been cleaved at one site with the restriction enzyme Bgl was selected and named pDG1 plasmid. The constructed expression vector retains the tac promoter and has 7 bases downstream of the SD sequence,
It has a recognition site for the restriction enzyme Bgl, and by incorporating the desired DNA fragment into this site,
A target protein can be expressed.

Binding of DNA containing the tac promoter and the initiation codon of cytochrome P-450c (construction of pDGN101) As the second step in constructing the expression plasmid pMG1, from the expression vector pDG1 constructed in
EcoR-BglDNA containing tac promoter
A fragment was isolated containing approximately 300 base pairs of Sau3A containing the start codon of the cytochrome P-450 gene.
- Connect the Pst fragments and create a recombinant plasmid.
pDGN101 was obtained. The outline of the construction is shown in Figure 3.
Below, we will explain how to construct it, dividing it into four steps.

Step 1: Preparation of 3600 base pair EcoR-Pst fragment Add 1 unit of restriction enzyme EcoR (Takara Shuzo) and 1 unit of restriction enzyme Pst (Takara Shuzo) to 1 μg of pBR322 DNA (Wako Pure Chemical Industries), and add 20 μg of EcoR reaction solution. [100mM Tris
-HCl, (PH7.5), 7mM _MgCl2 , 50mM
NaCl, 7mM 2-mercaptoethanol,
0.01% bovine serum albumin] at 37°C for 1 hour. After the reaction, the reaction solution was applied to a 1.0% low melting point agarose gel (Bethesda Research) containing 0.1 μg/ml ethidium bromide for electrophoresis. After electrophoresis, the gel portion corresponding to the 3.6 Kb EcoR-Pst DNA fragment was cut out under an ultraviolet lamp, placed in an Eppendorf tube, and heated at 65° C. for 5 minutes. Add twice the volume of TE buffer [10mM Tris] to the melted gel.
- Add HCl (PH8.0), 0.5mM EDTA],
Next, phenol saturated with TE buffer was added to perform phenol extraction. 10000r.pm
After centrifuging for 5 minutes at
was precipitated with ethanol. then at 10000rpm
Centrifuge for 10 minutes and collect approximately 0.2μg of 3.6Kb EcoR-
Pst DNA fragments were collected and suspended in 10μ of distilled water.

Step 2 Preparation of tac promoter fragment 1 unit of restriction enzyme EcoR and 1 unit of restriction enzyme Bgl were added to 1 μg of expression vector pDG1, and the mixture was reacted in a 20 μg EcoR reaction solution at 37° C. for 1 hour. After reaction, step 1
Perform low melting point agarose electrophoresis using the same procedure as above to perform EcoR-Bgl of approximately 420 bases.
DNA fragments were excised from the gel, the DNA was collected, and suspended in 10μ of distilled water.

Step 3 Preparation of Sau3A1-Pst fragment containing the start codon of cytochrome P-450c gene Add 5 units of restriction enzyme Pst to 5 μg of pAU157 DNA, add 20 μg of Pst reaction solution [20 mM Tris-HCl (PH7.5), 10 mM
MgCl ₂ , 50mM (NH ₄ ) ₂ SO ₄ , 0.01% bovine serum albumin] at 37°C for 1 hour, and after electrophoresis on low melting point agarose, approximately 300 base pair Pst
After cutting out the DNA fragment and collecting the DNA, 10μ
was suspended in distilled water. It has been found that the initiation codon of the cytochrome P-450c gene is present 4 bases downstream of the Sau3A1 cleavage site in this DNA fragment. So, this
Add 3 units of restriction enzyme Sau3A1 (Takara Shuzo) to the solution containing the PstDNA fragment, and
Sau3A1 reaction solution [10mM Tris-HCl (PH
7.5), 7mM MgCl ₂ , 100mM NaCl] at 37°C for 1 hour, and an equal amount of phenol
After treatment with chloroform solution, DNA was collected and
Suspended in 10μ of distilled water. As is clear from Figure 3, this DNA solution contains two types of
DNA fragments are mixed.

Step 4 Construction of recombinant plasmid pDGN101 The 3600 base pair EcoR-PstDNA fragment prepared in Steps 1, 2, and 3, and the 400 base pair
Mix 0.2 μg each of the EcoR-Bgl DNA fragment and the Sau3A1-Pst DNA fragment mixture containing the start codon, and make 7.2 units.
T ₄ DNA ligase was added, and the mixture was reacted for 2 hours at 16°C in a 45μ T ₄ DNA ligase reaction solution. Then, according to the method of Cohen et al., E. coli was grown in the reaction solution.
The DH1 strain was transformed. Transformants
Ruther et al.'s method (Mol.Gen.Genetics 178,
200 μg/ml of 5-bromo-4-chloro-3-indolyl-β-D-
Galactoside (Hani Chemical), and 15μg/
Contains ml of tetracycline (Sigma)
It was spread on an LB plate, and the blue colonies that appeared were isolated. Plasmid DNA was prepared from the colony according to the method of Birnboim et al.
2 units of restriction enzyme Hind (Takara Shuzo) and 2 units of restriction enzyme Bgl (Nippon Gene) were added to 2 μg of plasmid DNA, and the mixture was reacted in a 20 μg Hind reaction solution at 37° C. for 1 hour. The reaction solution was analyzed by 1.0% agarose gel electrophoresis, and 2300 base pairs, 900 base pairs,
5 of 450 base pairs, 350 base pairs and 250 base pairs
A plasmid in which two DNA fragments were detected was selected and named pDGN101.

Construction of expression plasmid pMG1 Recombinant plasmid pDGN101 constructed by
An expression plasmid pMG1 was constructed from the pAU157 plasmid and pBR322 plasmid.
An outline of the construction is shown in Figure 4. Below, 4
We will explain the construction method in two steps.

Step 1 Preparation of 300 base Hind-Pst fragment Add 2 units of restriction enzyme Hind to 2 μg of pDGN101 plasmid, and add 20 μg of Hind-Pst fragment.
After reacting in the reaction solution for 1 hour at 37°C, electrophoresis was performed on 1.0% low melting point agarose, and a 3900 base pair
HindDNA fragment was isolated. Next this
Two units of the restriction enzyme Pst were added to the DNA solution, and the mixture was reacted for 1 hour at 37°C in a 20μ Pst reaction solution, and a 300 base pair Hind-Pst DNA fragment containing the tac promoter was prepared by the same procedure. This DNA is hereinafter referred to as DNA fragment a.

Step 2 Pvu-HincDNA fragment and
Preparation of Pst-HincDNA fragment 5 units of restriction enzyme Pvu (Bio-Labo) was added to 5 μg of pAU157 plasmid DNA, and after 20 μg of Pvu reaction [7mM Tris-
HCl (PH7.4), 150mM NaCl, 6mM
_MgCl2 , 6mM2-mercaptoethanol,
100μg/ml bovine serum albumin] at 37℃
After reacting for 1 hour, DNA was collected. Next, add 5 units of restriction enzyme to this DNA solution.
Add Hinc (Takara Shuzo) and 20μ Hinc
Reaction solution [10mM Tris-HCl, (PH8.0), 7
After reaction for 1 hour at 37°C in 1.0% low melting _point agarose gel, a 3200 base pair Pvu-Hinc DNA fragment was isolated (DNA fragment b and ) At the same time, a 2100 base pair HincDNA fragment was prepared in the same manner, and this
Three units of restriction enzyme Pst were added to the DNA solution, and the mixture was reacted for 1 hour at 37°C in a 20μ Pst reaction solution. The resulting reaction products were separated on a 1.0% low melting point agarose gel, and the 1000 base pair
Pst-HincDNA fragment was isolated (DNA
Let it be fragment c. ) Step 3 Hind-Pvu of 700 base pairs
Preparation of DNA Fragment Two units of restriction enzyme Pvu were added to 2 μg of pBR322 DNA, and the mixture was reacted in a 20 μg Pvu reaction solution at 37° C. for 1 hour to recover DNA. Next, add 2 units of restriction enzyme to this DNA solution.
Add Hind and dilute in 20μ Hind reaction solution.
The reaction was carried out at 37°C for 1 hour. Reaction product, 1.0%
Separation was performed using a low melting point agarose gel, and a 700 base pair Pvu-Hind DNA fragment was isolated (referred to as DNA fragment d). Step 4: Mix approximately 0.2 μg of each of the DNAs prepared in Steps 1, 2, and 3. unit's
T ₄ DNA ligase was added, and the mixture was reacted at 16° C. for 15 hours in a 30μ T ₄ DNA ligase reaction solution. After the reaction, transform E. coli strain DH1 with the reaction solution,
200μg/ml 5-bromo-4-chloro-3
-indolyl-β-D-galactoside and
It was spread on an LB plate containing 10 μg/ml ampicillin (Sigma), and the blue colonies that appeared were isolated. Plasmid from colony
Prepare DNA, add 1 unit of restriction enzyme Hinc to 1 μg of plasmid DNA,
DNA fragments of 3300 base pairs, 500 base pairs, and 1300 base pairs were detected among the plasmids, which were analyzed on a 1.0% agarose gel after reacting for 1 hour at 37°C in a 20μ Hind reaction solution. A plasmid was selected and named pMG1.

Expression of rat liver cytochrome P-450c using expression plasmid pMG1 Rat liver cytochrome P-450c was expressed in Escherichia coli using the constructed expression plasmid pMG1.
The expression was carried out. The detailed method will be described below.

Step 1 Labeling of expressed protein by maxi-cell method The constructed pMG1 plasmid was labeled with E. coli strain CSR603 (uvrA6, rec
A1, phr, thr, 1eu, pro, his, arg, lac,
gal, ara, xy1, mt1, stf) (strain kept at Kyushu University Department of Molecular Genetics; also kept at Sumitomo Chemical Co., Ltd. and Takarazuka Research Institute), and the transformant CSR603 (pMG1) strain was transformed into Obtained. The resulting colonies were cultured using the MaxiCell method of Sancar et al. (JMB 148) .
The expressed protein was labeled according to p45-62). First, E. coli CSR603 (pMG1) strain was
Medium [1% casamino acid, 0.1 μg/ml thiamine + M9 medium, however, M9 medium contains Na ₂ HPO ₄ 6
g, KH ₂ PO ₄ 3 g, NaCl 0.5 g, NH ₄ Cl
Add distilled water to 1g to make 1, and after autoclaving, add 10ml of 0.01M CaCl ₂ and 1M MgSO ₄
Add 1 ml of ] and cultured overnight at 37°C. Subplant 0.1ml of that into 10ml of K medium.
Incubate at 37°C and grow to an OD ₆₆₀ of 0.2. Transfer 10ml of the culture solution to a sterilized watch glass and place it under a 15W ultraviolet lamp for about 90 minutes.
Irradiation was performed for 5 to 20 seconds at a distance of cm. After irradiation, the culture solution was transferred to a 100 ml Erlenmeyer flask, incubated at 37°C for 1 hour, and cycloserine was added at 100 μg/
ml and incubated at 37°C for 8 to 12 hours. Centrifuge at 3000 rpm for 15 minutes to collect bacteria, and add Hershey salt (5.4 g NaCl, 3.0
g KCl, 1.1g NH ₄ Cl, 15mg CaCl ₂ .
2H ₂ O, 0.2g MgCl ₂・6H ₂ O, 0.2mg
_FeCl3・_6H2O , _{87mg KH2PO4} , 12.1g
Washed twice with Trizmabase/1). Next, add the bacteria to 5 ml of Hershey medium [Hershey salt
Per 100ml, 0.5ml threonine (2%), 1
ml leucine (1%), 1ml proline (2%),
The suspension was suspended in 1 ml arginine (2%) and 0.1 ml thiamine (0.1%) and incubated at 37°C for 1 hour. Furthermore, ³⁵ S-methionine (1000 Ci/mmole) was added to the solution at a concentration of 50 μCi/ml, and the mixture was incubated at 37° C. for 1 hour. Bacteria were collected by centrifugation, suspended in 0.1 ml of 2% SDS (sodium dodecyl sulfate), and then heat-treated at 100°C for 4 minutes to dissolve the protein. As described above, 75μ of sedimentation buffer [25% TritonX-
100, 190mM NaCl, 6m MEDTA, 50m
M Tris HCl (PH7.4)] and anti-P-
10 μg of 450cIgG was added and left at 4° C. overnight. To this, add T buffer [2% TritonX-
100, 2mM Met, 150mM NaCl, 5m
Protein A-SepharoseCL-4B equilibrated with MEDTA, 50mM Tris-HCl (PH7.4)]
(Pharmacia) was added thereto, and the mixture was allowed to react at room temperature for 1 hour while stirring every 10 minutes. The gel was centrifuged at 12,000 rpm for 5 minutes, and the precipitated gel was washed twice with 1 ml of T buffer to remove non-specifically adsorbed substances. The resulting gel was boiled for 1 minute in 50μ sample buffer (described above) to elute the immunoprecipitate adsorbed to the gel, and the eluate was then purified using the method of Laemmli et al.
227 p680-685), SDS-polyacrylamide gel electrophoresis and fluorography were performed. The results are shown in Figure 5. A
In case of pAU157 used as control, no immunoprecipitate was detected, but in case of B using pMG1, an immunoprecipitate corresponding to rat liver cytochrome P-450c was confirmed. In addition, the expression level is estimated from the detected amount per E. coli cell,
It is thought that there are 10 ² to 10 ³ molecules.

From the above, it was confirmed that rat liver cytochrome P-450c protein was expressed by introducing the recombinant plasmid pMG1 into E. coli strain CSR603.

[Brief explanation of drawings]

Figures 1a to 1e show the base sequence of cDNA contained in the pAU157 plasmid. Figure 2 shows
An overview of expression vector pDG1 construction is shown. FIG. 3 shows an overview of plasmid pDGN101 construction. FIG. 4 shows an outline of the construction of the expression plasmid pMG1. FIG. 5 shows fluorography results showing the expression level of rat liver cytochrome P-450c protein in E. coli. Figure 6 shows the expression plasmid
A restriction enzyme map of pMG1 is shown. 〓 is promoter, 〓 is P-450c gene, 〓 is Ori gene,
〓 represents ampicillin resistance gene. Also,
Ah is the Aha site, Ec is the EcoR site, and Hc is the
Hinc site, Hd represents Hind site, Ps represents Pst site, Pv represents Pvu site, and Pv represents Pvu site.

Claims (1)

[Claims] 1 Plasmid pMG1 consisting of approximately 5200 base pairs, characterized by the restriction enzyme map shown below, and having the base sequence AGGAAACAGCAGATCATG at the junction between the SD sequence of the promoter and the P-450c gene start codon ATG. . 2. Transformed with plasmid pMG1, which consists of approximately 5,200 base pairs and is characterized by the restriction enzyme map shown below, and whose base sequence at the junction between the SD sequence of the promoter and the P-450c gene start codon ATG is AGGAAACAGCAGATCATG. E. coli.