JPH0523743B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing rat liver cytochrome P-450c.

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 utilizes 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 encoding a bioactive peptide derived from a higher organism or a synthetic DNA 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-450c, which is localized in the microsomal membrane of cells, has a large molecular weight of 59,300 daltons, and contains heme in the protein molecule, there is currently no report on its expression in E. coli.

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, and it can also be added to activated sludge.

The recombinant plasmid pMG1 of the present invention is a recombinant plasmid pAU157 (Nucl. Acids. Res. 12 , p2929-
29 Can be produced by the method described in 38. ) can be constructed by connecting it to the tac promoter, a strong E. coli promoter.

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 , p2929-2938).
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.

I Construction of expression vector pDG1 As the first step in constructing expression plasmid pMG1, an expression vector carrying the tac promoter was constructed.
A new expression vector pDG1 was constructed by modifying pDR540 (PL Biochemical). 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 1μg of pDR540 plasmid DNA, and digest with 10μg of pDR540 plasmid DNA.
BamHI reaction solution [10mMTris-HC (pH8.0),
The reaction was carried out at 37°C for 1 hour in 7mM MgC ₂ , 100mM NaC, 2mM mercaptoethanol, 0.01% bovine serum albumin. Next, add 1 unit of S1 nuclease (PL Biochemical Co., Ltd.) to this reaction solution.
and 10μ S1 nuclease reaction solution [100mM NaOAc-HOAc (PH4.0), 300mM
NaC, 12mMZnSO ₄ ] and then heated to 18℃ for 2 hours.
Allowed time to react. After the reaction, an equal amount of phenol-chloroform solution [phenol:chloroform (1:1)] was added to the reaction solution, mixed, and heated at 10,000 rpm for 5 minutes.
After centrifugation for a minute, the supernatant was collected. Next, add twice the volume of cold ethanol, leave at -80°C for 15 minutes, and centrifuge at 10,000 rpm for 10 minutes to collect DNA.
Suspended in 10μ of distilled water.

Step 2: Construction of pDG1 Add 1 μg to the 10 μ DNA solution prepared in Step 1.
of Bg linker (Takara Shuzo) and 4 nits of T ₄ DNA ligase (Takara Shuzo), and
T ₄ DNA ligase reaction solution [66mMTris-HC
(pH7.6), 6.6mMMgC ₂ .
10mMdithiothreito, 1.0mMATP] at 16℃
The reaction was allowed to proceed for 15 hours. After the reaction, the reaction solution was processed using the method of Cohen et al. (Proc. Nat. Acad. Sci. USA 69 , p2110).
−2114) using Escherichia coli (Escherichia coli).
DH1 strain ( ^F- , recA1, endA1, gyrA96, thi-
1, hsdR17, supE44, λ ^- , strain stored at Kyushu University School of Medicine Genetic Information Facility, also stored at Takarazuka Research Institute, Sumitomo Chemical Co., Ltd.) and transformed into 100 μg/ml.
LB plates containing ampicillin (Sigma) (each containing 10 g polypeptone, 5 g yeast extract, 5 g NaC, 12 g agar)
The transformants were spread and the colonies that appeared were isolated. Next, Birnboim et al.'s method (Nucl, Acids,
Res, 7 , p-1513-1523), plasmid DNA was prepared from the colony, 1 unit of restriction enzyme Bg was added to 1 μg of plasmid DNA, and Bg reaction solution [10mM Tris-HC
(PH7.5), 7mMgC ₂ , 100mM NaC,
7mM mercaptoethanol] at 37°C for 1
Time reacted. After the reaction, the reaction solution was applied to a 1.0% agarose gel (Sigma) containing 0.1 μg/ml ethidium bromide (Aldrich) and incubated at 100 V for 60 min.
Electrophoresis was performed for minutes. The migration pattern was observed under an ultraviolet lamp, and a plasmid cleaved at one site with restriction enzyme Bg was selected and named pDG1 plasmid. The constructed expression vector retains the tac promoter and has a recognition site for the restriction enzyme Bg at 7 bases downstream of the SD sequence, and by integrating the target DNA fragment into this site, the target protein can be expressed. I can do it.

Binding of DNA containing the tac promoter and the start codon of cytochrome P-450c (construction of pDGN101) As the second step in constructing the expression plasmid pMG1, tac
An EcoRI-BeDNA fragment containing the promoter was isolated, and this was combined with an approximately 300 base pair Sau3A-Pst fragment containing the start codon of the cytochrome P-450 gene.
The fragments were ligated to obtain a recombinant plasmid pDGN101. 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 pBR322DNA (Wako Pure Chemical Industries),
EcoR reaction solution [100mM Tris-HC, (PH7.5),
7mMgC ₂ , 50mM NaC, 7mM 2-mercaptoethanol, 0.01% bovine serum albumin] at 37°C for 1 hour. After reaction. reaction solution
Electrophoresis was performed using a 1.0% low melting point agarose gel (Bethesda Research) containing 0.1 μg/ml ethidium bromide. 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 to the melted gel [10mM Tris-HC (PH8.0),
Phenol extraction was performed by adding 0.5mM MEDTA] and then adding phenol saturated with TE buffer. Centrifuge at 10,000 rpm for 5 minutes, separate the upper layer, add 1/40 volume of 4M NaC and 2 volumes of ethanol, and leave at -80℃ for 10 minutes.
DNA was ethanol precipitated. then at 10000rpm
Centrifuge for 10 minutes and collect approximately 0.2μg of 3.6Kb EcoR-P st
DNA fragments were collected and suspended in 10μ of distilled water.

Step 2 Preparation of tac promoter fragment Add 1 unit of restriction enzyme EcoR and 1 unit of restriction enzyme Bg to 1 μg of expression vector pDG1.
was added and reacted for 1 hour at 37°C in a 20μ EcoR reaction solution. After the reaction, perform low melting point agarose electrophoresis using the same procedure as in step 1.
The base EcoR-Bg DNA fragment was 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 to 5 μg of pAU157 DNA.
Add Pst and add 20μ of Pst reaction solution [20mM Tris
−HC (PH7.5), 10mMgC ₂ , 50mM
(NH ₄ ) ₂ SO ₄ , 0.01% bovine serum albumin]
After reacting at 37°C for 1 hour and performing electrophoresis on low melting point agarose, a PstDNA fragment of approximately 300 base pairs was excised.
After DNA collection, it was suspended in 10μ of distilled water. In addition,
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. Therefore, we added 3 units of the restriction enzyme Sau3A1 (Takara Shuzo) to the solution containing this PstDNA fragment, and added 20μ of the restriction enzyme Sau3A1 (Takara Shuzo).
Sau3A1 reaction solution [10mMTris-HC (pH7.5),
After reacting for 1 hour at 37°C in 7mM MgC ₂ , 100mM NaC] and treating with an equal volume of phenol-chloroform solution, the DNA was collected and suspended in 10μ of distilled water.

Step 4 Construction of recombinant plasmid pDGN101 The 3600 base pair plasmid prepared in Steps 1, 2, and 3
EcoR−PstDNA fragment, 400 base pairs of EcoR
- Retains BgDNA fragment and start codon
0.2 μg of each Sau3A1-Pst DNA fragment mixture was mixed, 7.2 units of T ₄ DNA ligase was added, and the mixture was reacted in a 45 μg T ₄ DNA ligase reaction solution at 16° C. for 2 hours. Thereafter, E. coli strain DH1 was transformed with the reaction solution according to the method of Cohen et al. Transformants were prepared using the method of Ruther et al. (Mo. Gen. Genetics
178, P475-477), spread on LB plates containing 200 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside (Hani Chemical) and 15 μg/ml tetracycline (Sigma). , the blue colonies that appeared were isolated. mentioned above
Plasmid DNA was prepared from colonies according to the method of Birnboim et al., and 2 units of restriction enzyme Hind (Takara Shuzo) and 2 units of restriction enzyme Bg (Nippon Gene) were added to 2 μg of plasmid DNA and added to 20 μg of Hind reaction solution. at 37℃1
Time reacted. The reaction solution was analyzed by 1.0% agarose gel electrophoresis, and five DNAs of 2300 base pairs, 900 base pairs, 450 base pairs, 350 base pairs, and 250 base pairs were detected.
Select the plasmid for which the fragment is detected,
It was named pDGN101.

Construction of expression plasmid pMG1 Expression plasmid pMG1 was constructed from the recombinant plasmids pDGN101 and pAU157 plasmids and pBR322 plasmid constructed in the above. below,
The construction method will be explained in four steps.

Step 1 Preparation of 300 base Hind-Pst fragment Add 2 units of restriction enzyme Hind to 2 μg of pDGN101 plasmid, react in 20 μg of Hind reaction solution at 37°C for 1 hour, perform electrophoresis on 1.0% low melting point agarose, A 3900 base pair HindDNA fragment was isolated. Next, 2 units of restriction enzyme Pst were added to this DNA solution, and the reaction was carried out for 1 hour at 37°C in a 20μ Pst reaction solution.
300 base pair Hind-Pst containing promoter
DNA fragments were prepared. From now on, this DNA
It is called DNA fragment a.

Step 2 Pvu-HincDNA fragment and
Preparation of Pst-HincDNA fragment Add 5 units of restriction enzyme Pvu (Bio-Lab) to 5 μg of pAU157 plasmid DNA,
Pvu reaction solution [7mMTris-HC (PH7.4),
150mM NaC, 6mMgC ₂ , 6mM 2-mercaptoethanol, 100μg/ml bovine serum albumin]
The mixture was reacted for 1 hour at 37°C, and the DNA was collected. Next, add 5 units of restriction enzyme to this DNA solution.
Add Hinc (Takara Shuzo) and add 20μ of Hinc reaction solution [10mM Tris-HC (PH8.0), 7mMgC
₂ , 60mM NaC, 7mM2-mercaptoethanol] at 37℃ for 1 hour, and after electrophoresis on a 1.0% low melting point agarose gel, a 3200 base pair Pvu-
A HincDNA fragment was isolated (referred to as DNA fragment b). At the same time, a 2100 base pair HincDNA fragment was prepared in the same manner, and 3 units of restriction enzyme Pst were added to this DNA solution, and 20μ
The reaction was carried out in the Pst reaction solution at 37°C for 1 hour. The obtained reaction product was separated on a 1.0% low melting point agarose gel, and a 1000 base pair Pst-Hinc DNA fragment was isolated (referred to as DNA fragment c) Step 3: 700 base pair Hind-Pvu
Preparation of DNA fragment: Add 2 units of restriction enzyme to 2 μg of pBR322 DNA.
Add Pvu and incubate at 37°C in 20μ Pvu reaction solution.
After a time reaction, DNA was collected. Then this DNA
Add 2 units of restriction enzyme Hind to the solution and add 20μ
The mixture was reacted for 1 hour at 37°C in the Hind reaction solution. The reaction products were separated on a 1.0% low melting point agarose gel, and a 700 base pair Pvu-Hind DNA fragment was isolated (referred to as DNA fragment d). Step 4 The DNA prepared in Steps 1, 2, and 3 was separated into approximately
Mix 0.2 μg each, add 3 units of T ₄ DNA ligase, and mix in 30 μg of T ₄ DNA ligase reaction solution.
The reaction was carried out at 16°C for 15 hours. After the reaction, E. coli is collected in the reaction solution.
DH1 strain was transformed and 200μg/ml of 5-brome-
4-chloro-3-indolyl-β-D-galactoside and 10 μg/ml ampicillin (Sigma)
The blue colonies that appeared were isolated. Prepare plasmid DNA from colonies, add 1 unit of restriction enzyme Hinc to 1 μg of plasmid DNA, and add 20 μg of Hind.
After reacting in the reaction solution at 37°C for 1 hour, the reaction product was analyzed on a 1.0% agarose gel. Among the analyzed plasmids, DNA fragments of 3300 base pairs, 500 base pairs, and 1300 base pairs were detected, and the 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 detailed method is described below. Step 1: Labeling of expressed protein using the maxi-cell method.
phr, thr, 1eu, pro, his, arg, lac, gal, ara,
xyl, mtl, str) (Kyushu University Department of Molecular Genetics Department; also stored at Sumitomo Chemical Co., Ltd. and Takarazuka Research Institute).
CSR603 (pMG1) strain was obtained. The obtained colonies were cultured, and the expressed protein was labeled according to the Maxixel method of Sancar et al. (JMB 148 p45-62). First, E. coli CSR603 (pMG1) strain was grown in K medium [1% casamino acids, 0.1 μg/ml thiamine + M9 medium, however, M9 medium contained 6 g of Na ₂ HPO ₄ , 3 g of KH ₂ PO ₄ ,
Add distilled water to 0.5g of NaC and 1g of NH ₄ C to make 1
After autoclaving, add 10ml of _0.01MCaC2 ,
Add _1ml of 1MMgSO4. ] 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 at a distance of about 90cm directly under a 15W ultraviolet lamp.
Irradiation was performed for 5 to 20 seconds. After irradiation, the culture solution was transferred to a 100 ml Erlenmeyer flask, cultured at 37°C for 1 hour, cycloserine was added at 100 μg/ml, and incubated at 37°C for 8 to 12 hours. Collect bacteria by centrifugation at 3000 rpm for 15 minutes, and add Hershey salt (5.4 g NaC
, 3.0gKC, 1.1gNH ₄ C, 15mg CaC ₂・
2H ₂ O, 0.2gMgC ₂・6H ₂ O, 0.2mg FeC ₃・
_6H2O , _87mgKH2PO4 , _{12.1gTrizmabase} /1
) was washed twice. Next, the bacteria were grown in 5 ml of Hershey medium [per 100 ml of Hershey salt, 0.5 ml threonine (2%), 1 ml leucine (1%), 1 ml proline (2%), 1 ml arginine (2%), 0.1 ml thiamine (0.1%)]. and incubated at 37°C for 1 hour. Furthermore, ³⁵ S-methionine (1000Ci/
mmole) to 50μCi/ml, and at 37℃
Incubated for hours. Collect bacteria by centrifugation, 0.1
After suspending the protein in 2% SDS (sodium dodecyl sulfate), it was heat-treated at 100°C for 4 minutes to dissolve the protein. The crude extract prepared as above
25μ to 75μ sedimentation buffer [25%
Triton X-100, 190mM NaC,
6mMEDTA, 50mM TrisHC (pH 7.4)], 10μg of anti-P-450cgG, and incubated at 4℃ for 1 hour.
I left it for the night. To this, add T buffer [2% Triton
−100, 2mM Met, 150mM NaC,
50μ of Protein A-Sepharose CL-4B (Pharmacia) equilibrated with 5mMEDTA, 50mM Tris-HC (pH 7.4) was added, and the mixture was allowed to react at room temperature for 1 hour while stirring every 10 minutes. 12000rpm
The precipitated gel was washed twice with 1 ml of T buffer to remove non-specifically adsorbed substances. The resulting gel was incubated in 50μ sample buffer (above) for 1 hour.
After boiling for minutes to elute the immunoprecipitate adsorbed to the gel, the eluate was subjected to SDS-polyacrylamide gel electrophoresis and fluorography according to the method of Laemmli et al. (Nature 227p680-685). The results are shown in Figure 1. A is an example of pAU157 used as a control, and no immunoprecipitate was detected, but in B, using pMG1, an immunoprecipitate corresponding to rat liver cytochrome P-450c was confirmed. Furthermore, the expression level is estimated to be 10 ² to 10 ³ molecules per E. coli cell, estimated from the detected amount.

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 the drawing]

FIG. 1 shows fluorography results showing the expression level of rat liver cytochrome P-450c protein in E. coli.

Claims (1)

[Claims] 1. Rat liver cytochrome P-, which is characterized by culturing E. coli transformed with plasmid pMG1.
How to make 450c.