JPH0429352B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a recombinant plasmid incorporating the rat liver cytochrome P-450c gene and a method for producing the same. Rat liver cytochrome P-450c induced by administration of 3-methylcholanthrene is a heme protein that binds to carbon monoxide in a reduced form and whose difference spectrum shows an absorption maximum at 447 nm. This cytochrome P-
450c is involved in the metabolism of steroids and fatty acids, the oxidative metabolic reaction of foreign fat-soluble organic compounds, and the metabolic activation of chemical mutagens. The present invention aims to express cytochrome P-450c, which has high hydroxylation activity and a wide range of substrate specificity, in yeast, and to use it in oxidation reaction processes at an industrial level and in the production of organic compounds in industrial wastewater. An expression plasmid for expression in yeast that can be applied to oxidative removal, etc. is provided. In recent years, several cases have been reported in which heterologous genes were expressed in yeast using yeast alcohol dehydrogenase promoters, acid phosphatase promoters, and other promoters. Examples include expression of hepatitis B virus surface antigen up to 5×10 ⁵ molecules per bacterium, and expression of interferon. Although there are still fewer examples of expression experiments of heterologous genes in yeast than expression experiments using E. coli as a host, there has been an increasing trend in recent years. However, there has been no report on the expression of 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 its protein molecule, and controls hydroxylation activity. The present inventors conducted various studies on the expression of cytochrome P-450c in yeast and completed the present invention. The expression plasmid pAMC1 of the present invention is a cytochrome P-450c gene isolated from a recombinant plasmid pAU157 containing the entire coding region of rat liver cytochrome P-450c protein, and an alcohol dehydrogenation yeast (hereinafter abbreviated as ADH) promoter. It can be constructed by integrating it into the expression vector pAAH5 held by. - The recombinant plasmid pAMC1 obtained in the present invention and intended for expression in yeast has rat liver cytochrome P-450c, which exhibits high hydroxylation activity, located downstream of the ADH promoter, which is a strong yeast promoter.
Genes are linked. Furthermore, cytochrome P-
At the rear of the 450c gene, there is a transcription termination signal for the ADH gene, so the DNA of pAMC1
The structure makes it ideal as an expression plasmid. By introducing the pAMC1 plasmid having such a structure into a yeast host and performing large-scale expression, it is possible to isolate and purify a large amount of pure rat liver cytochrome P-450c enzyme. The obtained cytochrome P-450c enzyme is NADPH-cytochrome P-450
It is stabilized together with reductase and can perform oxidation reactions. Also productive cytochrome P-450c
It is also possible to develop the enzyme as a microsomal fraction or yeast cells that perform an oxidation reaction without separating or purifying the enzyme, and apply it as a bioreactor or together with activated sludge, etc. to wastewater treatment. Next, the present invention will be explained in more detail with reference to Examples. Example Construction of expression plasmid pAMC1 for expression of rat liver cytochrome P-450c gene in yeast: Recombinant plasmid pAU157 containing the entire coding region of rat liver cytochrome P-450c protein
(Nucleic Acids Res. 12 , p2929−2938,
It can be manufactured by the method of Yabusaki et al. 1a to 1e), cytochrome P-
Expression vector containing isolated 450c gene portion and alcohol dehydrogenase (ADH) promoter
pAAH5 (Methods in Enzymology 101 , part C
p192-201, which can be produced by the method of Ammerer et al. ), and a plasmid pAMC1 for expression in yeast was constructed as follows. Construction of recombinant plasmid pTF1 As the first step in constructing an expression plasmid, a restriction enzyme site Sal is placed at the beginning of the cytochrome P-450c gene.
A recombinant plasmid pTF1 was constructed which has a restriction enzyme site Hind at the end. Figure 2 shows the outline. The construction method will be described below in four steps. Step 1: Construction of recombinant plasmid pNF2 (a): Add 10 units of restriction enzyme (Takara Shuzo) Pst to 10 μg of pAU157 plasmid DNA,
Pst reaction solution [20mM Tris-HCl, (PH
7.5), 10mM _MgCl2 , 50mM ( _NH4 ) _2SO4 , _0.01
% bovine serum albumin] at 37°C for 1 hour. The reaction solution was applied to a 0.8% low melting point agarose gel (Bethesda Research) containing 0.1 μg/ml ethidium bromide (Aldrich).
Electrophoresis was performed at 100V for 90 minutes. After electrophoresis, the gel portion corresponding to the DNA fragment (approximately 330 bases) 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-HCl (PH8.0),
Phenol extraction was performed by adding 0.5 mM EDTA] and then adding an equal volume of phenol saturated with TE buffer. After centrifuging at 10,000 rpm for 5 minutes and separating the upper layer, add twice the amount of cold ethanol.
DNA was ethanol precipitated by leaving it at -80°C for 10 minutes. Thereafter, it was centrifuged at 10,000 rpm for 10 minutes, and approximately 1 μg of DNA fragments were collected and suspended in 20 μg of distilled water. (b): Next, 1 unit of restriction enzyme Sau 3A1 (Takara Shuzo) was added to 1 μg of the prepared DNA fragment.
50μ Sau 3A1 reaction solution [10mM Tris-HCl
(PH7.5), 7mM MgCl ₂ , 100mM NaCl]
After reacting at 37°C for 1 hour, perform phenol extraction and ethanol precipitation as described above to extract the DNA.
was collected and suspended in 20μ of distilled water. this
The DNA is a mixture of DNA fragments ' and ' (see Figure 2). (c): 2 μg of PUC9 cloning vector (P-
2 unit restriction enzyme (L Biochemical Co., Ltd.)
Add BamH (Takara Shuzo) and 2 units of restriction enzyme Pst, and add 50μ of BamH reaction solution [10mM Tris-HCl (PH8.0), 7mM MgCl ₂ ,
100mM NaCl, 2mM 2-mercaptoethanol, 0.01% bovine serum albumin at 37°C.
After reacting for several hours, phenol extraction and ethanol precipitation were performed, and DNA was collected and suspended in 20μ of distilled water. In addition, restriction enzyme BamH and restriction enzyme
Sau3A1 has a common recognition site, so they can be cloned together. (d): Mix 20μ of the DNA fragments obtained in (b) and (c),
Add 5 units of _T4 DNA ligase (Takara Shuzo) to 60μ of _T4 DNA ligase reaction solution [66mM Tris-HCl (PH7.6), 6.6mM _MgCl2 ,
16 in 10mM dithiothreitol, 1.0mM ATP]
The reaction was carried out at ℃ for 3 hours. After the reaction, the reaction solution was processed using the method of Cohen et al. (Proc. Natl. Acad. Sci. USA 69 , P2110
~2114) using Escherichia coli
JM103 strain [△ (lac-pro), thi, sfr A, sup
E, end A, sbc B, hsd R ^- , F tra D36,
proAB, lac ^q , ZΔ45, obtained from PL Biochemical Co.]. For culturing E. coli, LB medium (10g polypeptone, 5g yeast extract,
The LB plate medium used was LB medium 1 to which 12 g of agar was added. The same applies thereafter. To clone the desired DNA fragment, we used the method of Ruther et al. (Mol.Gen.Genetics,
178p475-477), 200 μg/ml of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (Hani Chemical), 2 μmole/ml of isopropylthiogalactoside (Tokyo Kasei) and
Contains 25 μg/ml ampicillin (Sigma)
The transformants were spread on LB plates, and white colonies appearing among the blue colonies were isolated. Next, Birnboim et al.'s method (NuCl.Acids.Res 7
Plasmid DNA was isolated from the white colony according to the method (p1513-1523), and 1 μg of plasmid was
1 unit of restriction enzyme EcoR per DNA
and 1 unit of restriction enzyme Pst,
After reacting for 1 hour at 37℃ in the Pst reaction solution, 0.8
% was analyzed by agarose electrophoresis to select recombinant plasmids incorporating the DNA fragment.
It was named pNF1. Note that agarose was purchased from Wako Pure Chemical Industries. (e): 5 μg according to the method of Birnboim et al.
Prepare pNF1 DNA and use 5 units of restriction enzyme.
Add Sma (Takara Shuzo) and 50μ Sma
(Takara reaction [10mM This-HCl, (PH8.0), 7mM
37 in _MgCl2 , 20mM KCl, 7mM 2-mercaptoethanol, 0.01% bovine serum albumin).
After reacting for 1 hour at ℃, DNA was collected and suspended in 20μ of distilled water. Approximately 2 μg of this DNA solution is
Add 2 μg of Sal linker (Takara Shuzo) and 10 units of T ₄ DNA ligase,
The reaction was carried out in a T ₄ DNA ligase reaction solution at 16°C for 3 hours. Next, the reaction solution was mixed with Escherichia coli DH1 [F ^- , recA ₁ ,
end A ₁ , gyr A96, thi−1, hsd R17, Sup
E44, λ ^- strain stored at Kyushu University School of Medicine Gene Information Facility, also stored at Sumitomo Chemical Co., Ltd. Takarazuka Research Institute) and spread on LB plates containing 25 μg/ml ampicillin. Plasmid DNA was prepared from the obtained colonies, 1 unit of restriction enzyme Sal (Takara Shuzo) was added to 1 μg of plasmid DNA, and 50 μ of the reaction solution [10 mM Tris-HCl, (PH7.5), 7 mM MgC12,
175mM NaCl, 0.2mM EDTA, 7mM 2-mercaptoethal] at 37°C for 1 hour, analyzed by 0.8% agarose electrophoresis as usual, and selected plasmids that can be cleaved with the restriction enzyme Sal.
The obtained plasmid was named pNF2. Step 2 Construction of recombinant plasmid pCF2 (a): Add 10 units of restriction enzyme Pst and 10 units of restriction enzyme Pvu (Takara Shuzo) to 10 μg of pAU157 plasmid DNA, add 50 μg of reaction solution [20 mM Tris-HCl, (PH7) .5), 10mM _MgCl2 ,
The reaction was carried out at 37° C. for 1 hour in 50 mM (NH ₄ ) ₂ SO ₄ , 0.01% bovine serum albumin. reaction solution
Analyze the DNA sections of interest by 0.8% low melting point agarose gel electrophoresis according to the method described above.
isolated. (b): Approximately 1 μg of DNA fragment was added with 1 unit of restriction enzyme Sau3A1 and reacted in 50 μg of Sau3A1 reaction solution at 37°C for 1 hour. The DNA was collected and suspended in 20 μg of distilled water. . (c): Perform the same procedure as step 1 (c) and use the restriction enzyme.
A cloning vector pUC9DNA cut with BamH and Pst was prepared. (d): Mix 20μ of the DNA fragments obtained in (b) and (c),
Add 5 units of T ₄ DNA ligase to 60μ
The mixture was reacted for 3 hours at 16°C in the T ₄ DNA ligase reaction solution. After the reaction, the reaction solution was transformed into Escherichia coli JM103 strain and white colonies were selected in the same manner as in step 1(d). Isolate plasmid DNA from this white colony and 1 μg
of plasmid DNA, 1 unit of restriction enzyme Pst and 1 unit of restriction enzyme Sma
was added, reacted at 37°C for 1 hour, and analyzed by 0.8% agarose gel electrophoresis.
Select a plasmid that has integrated the fragment ′, and use it as pCF1.
And so. (e): Next, according to the method of Birnboim et al., 5 μg of
Prepare pCF1 DNA and use 5 units of restriction enzyme.
Add Sma to 37% in 50μ Sma reaction solution.
After reacting at ℃ for 1 hour, DNA was collected and suspended in 20μ of distilled water. Approximately 2 μg of this DNA solution is
Add 2 μg of Hind linker (Takara Shuzo) and 10 units of T ₄ DNA ligase, and add 60 μg of Hind linker (Takara Shuzo) and
The reaction was carried out in a T ₄ DNA ligase reaction solution at 16°C for 3 hours. Next, the reaction solution was mixed with Escherichia coli
coli) DH1 in the same manner as described above,
Plasmid DNA was prepared from the obtained colonies. Add 1 unit of restriction enzyme Sal to 1 μg of the obtained plasmid DNA, and add 50 μg of plasmid DNA.
Hind reaction solution [10mM Tris-HCl, (PH
7.5), 7mM MgCl ₂ , 60mM NaCl] at 37°C.
The mixture was reacted for 1 hour, analyzed by 0.8% agarose electrophoresis, and a plasmid that could be cleaved with the restriction enzyme Hind was selected, and the resulting plasmid was named pCF2. Step 3 Construction of recombinant plasmid pNC1 (a) Add 5 units of restriction enzyme to 5 μg of pNF2 DNA
Pst and 5 units of the restriction enzyme Hind were added, and the mixture was reacted for 1 hour at 37°C in a 50μ Hind reaction solution, and the DNA was collected and suspended in 20μ distilled water. (b) Perform the same operation on 5 μg of pCF2DNA,
After collecting the DNA, it was analyzed by 0.8% low melting point agarose gel electrophoresis, the smaller DNA fragment of Pst-Hind was excised from the gel, and after phenol extraction and ethanol precipitation, the DNA fragment was collected.
Suspended in 20μ of distilled water. (c) Mix 20μ of the DNA solutions prepared in (a) and (b), add 5 units of T ₄ DNA ligase,
The reaction was carried out at 16°C for 3 hours in a 60μ T ₄ DNA ligase reaction solution. The resulting reaction solution was transformed into Escherichia coli DH1 using a conventional method, and plasmid DNA was prepared from the colonies that appeared. To 1 μg of the obtained plasmid DNA, 1 unit of restriction enzyme Sal and 1 unit of restriction enzyme Hind were added, reacted in a 50 μHind reaction solution at 37° C. for 1 hour, and analyzed by 0.8% agarose gel electrophoresis. A plasmid that retains the desired structure was selected and designated pNC1. Step 4 Construction of recombinant plasmid pTF1 (a) Add 1 unit of restriction enzyme Pst to 1 μg of pNC1 DNA and incubate at 37 μg in a 50 μl Pst reaction solution.
After reacting for 1 hour at ℃, DNA was collected and suspended in 20μ of distilled water. Add 1.5 units of alkaline phosphatase (Takara Shuzo) to this DNA solution, and add it to the 100μ alkaline phosphatase reaction solution [50mM Tris-HCl, (PH8.0)] at 60℃60.
It was allowed to react for a minute. After the reaction, phenol extraction was performed twice, and after ethanol precipitation, the DNA was collected and diluted with 20μ
was suspended in distilled water. (b) 10 units of restriction enzyme Pst was added to 10 μg of pAU157 plasmid DNA, and the mixture was reacted in a 50 μg Pst reaction solution at 37° C. for 1 hour to recover DNA.
Perform 0.8% agarose gel electrophoresis to remove DNA
Cut out the gel part corresponding to the fragment and insert the DNA
was collected and suspended in 20μ of distilled water. (c) Mix 20μ of the DNA solutions prepared in (a) and (b), add 5 units of T ₄ DNA ligase,
Reaction was performed at 16°C for 3 hours with 60μ of T ₄ DNA ligase reaction solution. After the reaction, the reaction solution was transformed into Escherichia coli strain DH1. (d) Prepare plasmid DNA from the obtained colonies, add 1 unit of restriction enzyme Sal and 1 unit of restriction enzyme Hind to 1 μg of plasmid DNA, and react in 50 μg of Hind reaction solution at 37°C for 1 hour. , analyzed by 0.8% agarose electrophoresis. Of the plasmids analyzed,
We first selected one that incorporated a DNA fragment of approximately 1.8 Kb. Since there are two possible ways of integration into the DNA fragment depending on its orientation, we further analyzed the structure of the plasmid DNA. 1 unit of restriction enzyme for 1 μg of the above plasmid
Add Hind and 1 unit of restriction enzyme Stu (Nippon Gene), and add 50μ of Stu reaction solution [10mM Tris-HCl, (PH7.5), 100mM NaCl,
10mM MgCl ₂ , 6mM 2-mercaptoethanol, 0.1mg/ml bovine serum albumin] at 37°C.
The mixture was reacted for 1 hour and analyzed by 0.8% agarose gel electrophoresis. Of the plasmids analyzed,
A DNA structure whose DNA fragments were connected in the forward direction of ``→→'' was selected and used as a pTF1 plasmid. Construction of expression plasmid pAMC1 From the pTF1 plasmid constructed in step 1, the rat liver cytochrome P-450c gene portion was extracted.
Yeast expression vector carrying the ADH promoter
pAAH5 (obtained from Washington Research Foundation, Methcds in Enzymology, 101part
Cp192-201 can be produced by the method of Ammerer et al. ) to construct an expression plasmid pAMC1. Figure 3 shows the outline. The construction method will be explained in three steps below. Step 1: Contain rat liver cytochrome P-450c gene
Isolation of 1.8Kb Hind fragment (a): Add 5 units of restriction enzyme Sal to 1μg of pTF1 plasmid and incubate with 37
After reacting for 1 hour at ℃, DNA was collected and suspended in 20μ of distilled water. Next, add to this DNA solution,
2 units of E. coli polymerase. Add the fragment and add 50μ of polymerase reaction solution [40mM potassium phosphate (PH7.5),
The reaction was carried out at 37°C for 1 hour in 6.6mM MgCl ₂ , 1.0mM 2-mercaptoethanol, 33μM dNTP].
After the reaction, DNA was collected and suspended in 20μ of distilled water. Approximately 1μg of DNA was added to 20μ of the resulting DNA solution.
A Hind linker and 3 units of T ₄ DNA ligase were added, and the mixture was reacted for 3 hours at 16°C in a 60μ T ₄ DNA ligase reaction solution. After the reaction, the reaction solution was transformed into E. coli strain DH1, and plasmid DNA was prepared from the obtained colonies. 1 unit of restriction enzyme per 1 μg of plasmid DNA
Add Hind and 37 min in 50μ Hind reaction solution.
The reaction was carried out for 1 hour at ℃, and after the reaction, it was analyzed by 0.8% agarose gel electrophoresis. Among the plasmids examined, one incorporating an approximately 1.8 Kb Hind fragment was selected and designated pTF2. (b): 2 units of restriction enzyme for 2 μg of pTF2 DNA
Add Hind and 37 min in 50μ Hind reaction solution.
After reacting at ℃ for 2 hours, DNA was collected. The obtained DNA solution was subjected to 0.8% low melting point agarose gel, and after electrophoresis, 1.8Kb HindDNA
Cut out the fragment from the gel, collect the DNA,
Suspended in 20μ of distilled water. (c): Add 1 unit of restriction enzyme Hind to yeast expression vector pAAH5 carrying 0.5 μg of ADH promoter, and add it in 50 μg of Hind reaction solution.
After reacting at 37°C for 1 hour, the DNA was collected and suspended in 20μ of distilled water. Add 2.0 units of alkaline phosphatase to 20μ of this DNA,
The reaction was carried out at 60°C for 60 minutes in a 100μ alkaline phosphatase reaction solution. After the reaction, phenol extraction was performed twice, and after ethanol precipitation, the DNA was collected and suspended in 20μ of distilled water. (d): Mix 20μ of the DNA solutions prepared in (b) and (c), add 2 units of T ₄ DNA ligase,
Reaction was carried out at 16°C for 3 hours in a T ₄ DNA ligase reaction solution. After the reaction, the reaction solution was transformed into E. coli DH1, and plasmid DNA was extracted from the obtained colonies.
was prepared. Add 1 unit of restriction enzyme Hind to 1 μg of plasmid DNA, and add 50 μg of restriction enzyme Hind.
The mixture was reacted in a Hind reaction solution at 37°C for 1 hour, and after the reaction, it was analyzed by 0.8% agarose gel electrophoresis. Of the plasmids analyzed, approximately 1.8Kb
We selected one that incorporated a DNA fragment. Some of the obtained plasmids had a 1.8 Kb HindDNA fragment connected in the opposite direction to the ADH promoter. Therefore, the selected plasmid
1 unit of restriction enzyme Stu per 1μg of DNA
and 1 unit of restriction enzyme BamH1 were added, and the mixture was reacted for 1 hour at 37°C in a 50μ BamH1 reaction solution. After the reaction, the reaction solution was analyzed by 0.8% agarose gel electrophoresis, and the expression vector pAAH5
In addition to the DNA fragments generated from
A plasmid in which a 1.1 Kb DNA fragment was detected was selected. This plasmid had the cytochrome P-450c gene connected to the ADH promoter in the forward direction, and was named pAMC1. Expression of rat liver cytochrome P-450c using expression plasmid pAMC1 Rat liver cytochrome P-450c was expressed in yeast using the constructed expression plasmid pAMC1. The detailed method will be described below. Step 1: Isolation of yeast transformants To express rat liver cytochrome P-450c in yeast, the method of Beggs et al. (Nature, 275p104-
109 (1978)) to create a recombinant plasmid.
pAMC1 was transferred to Saccharomyses cerevisiae strain SHY3 (MATa, ste-vc9, uta3, trp1, his1, leu2-
3, leu2−112, ade1, can1 [Cir ⁺ ]
(ATCC44771), select colonies showing Leu ⁺ phenotype, and transform SHY3
(pAMC1) strain was obtained. Step 2 Preparation of crude extract Yeast carrying expression plasmid pAMC1
SHY3 (pAMC1) strain was grown in 10 ml of synthetic medium without leucine [0.67% Bacto-yeast nitrogen base].
W/O amino acids (Difco), 2% Dextrose,
20 mg/ml tryptophan, 20 μg/ml histidine, 20 μg/ml adenine sulfate].
The cells were cultured until the concentration reached 10 ⁷ cells/ml. 1 ml of culture solution,
The cells were centrifuged at 7000 rpm for 3 minutes to collect the bacteria, and then suspended in 1 ml of 1.2M sorbitol. Centrifuge again, and 0.2 ml of Zymolyase solution [1.2M sorbitol, 50mM calcium phosphate (PH7.5), 14mM 2-mercaptoethanol, 400μg/ml Zymolyase 60000]
and incubated at 30°C for 30 minutes.
Collect spheroplasts by centrifugation at 7000 rpm for 3 minutes, wash with 0.4 ml of buffer A [1.2 M sorbitol, 50 mM Tris-HCl (PH7.5)], centrifuge again, and add 50 μ of SDS solution [2 % sodium dodecyl sulfate-50mM Tris-HCl (PH7.5)]. Next, heat treated at 100℃ for 5 minutes,
After centrifuging at 10,000 rpm for 5 minutes, collect the supernatant and
50μ sample buffer [62.5mM Tris−HCl
(PH6.8), 2% (W/V) sodium dodecyl sulfate, 5% (V/V) 2-mercaptoethanol,
10% (w/v) glycerol, 0.001% bromophenol blue] was added. Step 3: Identification of expressed protein Out of approximately 100μ of the crude extract of the yeast SHY3 (pAMC1) strain prepared in Step 2, 20μ was subjected to SDS-polyacrylamide gel and subjected to the method of Laemmli et al. (Nature 227 , p680-685). Therefore, electrophoresis was performed. After electrophoresis, layer the acrylamide gel with a nitrocellulose filter (Shleicher & Scnuli), add blotting buffer [25mM Tris-
HCl (PH8.3), 192 mM glycine, 20% methanol], a voltage of 30 V was applied for about 10 hours, and the protein was transferred to a nitrocellulose filter. After electrophoresis, filter the nitrocellulose filter with blocking solution [3% gelatin, 50mM Tris-HCl (PH
7.5), 200mM NaCl, 0.05% Tween20]
Stir for 30 minutes. Next, 1 μg/ml anti-P-450c
Buffer containing IgG [1% gelatin, 50mM Tris
−HCl (PH7.5), 200mM NaCl, 0.05%
Tween 20] and stirred for an additional 2 hours. Subsequently, the nitrocellulose membrane was coated with 0.05% Tween20
TBS solution containing [50mM Tris-HCl (PH7.5),
The cells were washed four times for 40 minutes each with 200mM NaCl and immersed in blocking solution again. Next, remove the blocking solution and soak for 1 hour in 50 ml of ¹²⁵ -Protein A solution (7 μCi) containing 0.05% Tween20.
Washed 4 times for 30 minutes each with TBS solution, and finally washed with TBS solution. The treated nitrocellulose filter was dried on filter paper and autoradiography was performed. Figure 4 shows the analysis of the expressed protein of SHY3 (pAMC1) using the above method. A shows the results for the pAAH5 vector used as a control, in which no protein recognized by anti-P-450c IgG was detected. On the other hand, B shows the case where the expression plasmid pAMC1 was used, and anti-P-
450c The protein molecule that reacts with IgG is cytochrome P.
It was detected at a position corresponding to the molecular weight of -450c. When calculated based on the purified cytochrome P-450c sample, approximately 4 x 10 ⁵ molecules of rat liver cytochrome P-450c protein were produced per cell. In the above, SHY3 strain was used as a yeast host, but Satucharomyces strain AH22 (a, leu2-3, leu2-
112, his4-519, can1 [cir ⁺ ]; ATCC38626] and NA87-11A strain (α, pho3-1, pho5-1,
trp1, his3, leu2, [cir ⁺ ]; Obtained from the 4th Laboratory, Department of Fermentation Engineering, Faculty of Engineering, Osaka University; stored at Sumitomo Chemical Co., Ltd., Takarazuka Research Institute) as hosts, Leu ⁺ transformation, AH22 (pAMC1) strain, respectively. and
The NA87-11A (pAMC1) strain was obtained, and the expression of rat liver cytochrome P-450c protein was similarly confirmed.

[Brief explanation of drawings]

Figures 1a to 1e show the base sequence of the rat liver cytochrome P-450c gene cloned into the pAU157 plasmid. Figure 2 shows an overview of the construction of recombinant plasmid pTF1. FIG. 3 shows an overview of the construction of recombinant plasmid pAMC1. Figure 4 shows rat liver cytochrome P-450c expressed in yeast.
This is the result of scanning with a densitometer. A is SHY3 (pAAH5) used as a control and B is
SHY3 (pAMC1).

Claims (1)

[Scope of Claims] 1. Constructed by integrating the cytochrome P-450c gene isolated from recombinant plasmid pAU157, which has the entire coating region of rat liver cytochrome P-450c protein, into expression vector pAHH5 carrying an alcohol dehydrogenase promoter. A plasmid that expresses cytochrome P-450c in yeast. 2. Claim 1, which is pMAC1, which consists of about 14,200 base pairs and is represented by the restriction enzyme map below.
Expression plasmids as described in Section.