JPS6152284A - Rat hepatic cytochrome p-450 gene - Google Patents

Abstract

PURPOSE:A plasmid having a specified base sequence in a region coding a rat hepatic cytochrome (P-450MC) induced by 3-methylcholanthrene. CONSTITUTION:A rat hepatic cytochrome P-450 gene having a specified base sequence in a region coding a rat hepatic cytochrome (P-450MC) induced by 3- methylchloanthrene. A plasmid pAU157 having cDNA of P-450MC has been already well known, but the base sequence of a region coding the gene is elucidated to find that the region has the base numbers of the 73rd to the 1,647th base sequence shown in the figure. The P-450MC gene can be prepared by separating a plasmid DNA from Escherichia coli having the plasmid pAU157, preparing DNA having restriction enzyme sites at both ends of the P-450MC gene with the plasmid DNA, and cleaving the resultant plasmid DNA with a restriction enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a gene encoding rat liver cytochrome P-450 (hereinafter abbreviated as P-450Mo) induced by 3-methylcholanthrene (hereinafter abbreviated as MC).

P-450MC is a heme protein that binds to carbon monoxide in its reduced form and exhibits an absorption maximum at 447 nm, which is responsible for the metabolism of nutroids and fatty acids, as well as the oxidative metabolism of foreign fat-soluble organic compounds. This P-450MC, which is involved in reactions and metabolic activation of chemical mutagens, has high hydroxylation activity and is characterized by a wide range of substrate specificity.

The present inventors developed P-450MC with such characteristics.
With the aim of applying the P-450MC gene at an industrial level, we conducted various studies, determined the base sequence of the P-450MC gene, and created a DNA that contains the region encoding it in an easily usable state.
The present invention was completed by inventing a method for producing A fragment.

That is, the present invention provides a porta-1 purpuranumid containing a region encoding the P-450MC gene in an easily separable state, a method for producing the same, and P-450M.

The present invention relates to a gene, a DNA fragment containing the gene in an easily usable state, and a method for producing the same.

The present inventors have already identified cD encoding P-450MC.
An attempt was made to clone NA, and a plasmid pAU157 with a cDNA of approximately 2.7 kb was isolated.
It has been revealed that this is an almost full-length cDNA clone for -450MC mRNA. C biochemical eclipse,
925. (1988>'J However, in order to confirm that this DNA clone belonged to P-450MC,
In order to extract the coaching region of the P-450MC gene in a usable state, it was necessary to determine its base sequence and search for the coaching region. Therefore, the present inventors conducted further research and developed the above-mentioned plasmid pAU15.
We determined the entire base sequence of the cDNA insert of P.
-4,50MC consists of 523 amino acid residues, molecular weight 5
It is clear that it is a 9,300 dalton protein.
Based on the findings regarding the base sequence revealed in this way, it is possible to construct a portable planumid containing the coating region of P-450MC in an easily separable state. In this way -
By incorporating the 1°-truncated DNA into an expression vector carrying an appropriate promoter, it is possible to produce an expression vector that expresses P-450Mo in the cells of yeast, large intestine, bacteria, etc. P-450M.

The enzyme can be immobilized together with NADPH-cytochrome P-450 reductase and the oxidation reaction can be carried out.It is also possible to perform the oxidation reaction by using a bacterial cell containing this expression vector as a bioreactor.

Next, the method for producing P-450Mo gene DNA of the present invention will be explained.

The P-450MC gene of the present invention is plasmid pAU1
After isolating the plasmid DNA from Escherichia coli harboring 57, it is produced by isolating the region that coats the pP-450MC gene, that is, the region from base number 73 to base number 1647 in Figure 1. I can do it.

This P-450M.

Isolation of the gene can be advantageously carried out, for example, by the method outlined in FIG.
P-450 from E. coli harboring U157. Plasmids containing genes! ') Separate NA and use it to
A plasmid containing DNA with restriction enzyme sites at both ends of the 450MC gene! ! ! and this plasmid DN
It can be produced by cleaving A at a restriction enzyme site. This restriction enzyme site can be replaced with another restriction enzyme site, if necessary. Next, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the tore.

Note that all reagents, enzymes, etc. used in the examples are commercially available.

Example P-450mRNA Partial MiM SD male and female rats (4 weeks old, 100-12Of) were treated with MC dissolved in corn oil at a rate of 25 W/kp.
It was administered intraperitoneally, and 14-15 hours after administration, the rats were sacrificed and the liver was removed. After cutting the liver into small pieces,
5mM) Lis-HCl (pH 7,5), 25mM
NaCl, 5mM MgCl2, 0.2M sucrose.

Add 5% Triton
Centrifuge for 10 minutes at R and add 20% 2MM to the supernatant obtained.
0 polysomes were precipitated by adding an equal volume of the above buffer containing 12 to 26,400Xf, 10
Collected by centrifugation for 50 A 260/mJ
5mM M? The yield of 0 polysomes suspended in CI2 was approximately 15OA260 per 2 livers.020m7! Add an equal volume of extraction buffer (0.2M sodium acetate (pH 5.0) e1% S
DS) was added, and phenol/l/40ml saturated with 2-fold diluted extraction buffer was added and shaken.

After standing for 2-8 minutes, 40ml of chloroform was added and further shaken. The water layer and organic layer were separated by centrifugation at 15,000Xf for 1 minute, and 80ml of chloroform was added to the aqueous layer and extracted again. Repeated extraction with chloroform until the middle layer disappeared, and the final product was obtained. Add twice the volume of cold ethanol to the aqueous layer and add to -20°C.
Then, the RNA was precipitated overnight. Recovered RNA
The RNA was washed twice with 3M sodium acetate (pH 6,0), then dissolved in 0.1M sodium acetate (pI (7,0)) and subjected to econol precipitation again.
The cells were collected, washed with 75% ethanol, vacuum dried, and incubated at -80°C. RNA from polysomes
The extraction rate was 83-87%.

Next, the obtained RNA was passed through an oligo(rlT) cellulose column, and mRNA containing poly(rlT) was fractionated.

Dissolve the RNA in a small volume of sterile water and equilibrate it to FJJ 87.
MCO, 5M NaCl,] OmM) Lis-HCl (pH 7,5), 1mM EDTA, (1,1
%SDS), 1 (1-15 A 260/rr+
After heat treatment at 065° C. for 5 minutes, the sample was diluted to a concentration of 71%, applied to a Shitaoligo (dT) cellulose column previously equilibrated with an equilibration buffer, and thoroughly washed with the equilibration buffer. Collect both eluted fractions with sterile water and make a final concentration of 2%.
of aqueous potassium vinegar solution and 2 volumes of cold ethanol were added to precipitate RNA. 75% RNA with +1 fold
After washing with ethanol, it was dissolved in sterile water and passed through an oligo(dT) cellulose column again. Approximately 0.12 vv of mRNA was obtained from 12 livers by the above procedure.

Next, extract the mRNA from 1. (Size fractionated by 1-80% sucrose density gradient centrifugation. 10-80%
0.1 M NaCl with a sucrose linear density gradient of .

05% SDS, ]mM E
DTA, 10 mM) Lis monohydrochloric acid (
RNA heat-treated at 65°C for 5 minutes to pT(7,5), 2
~4A260 layered, TV 865 type vertiquat)
v rotor (DuPont Sor va/A),
Centrifugation was performed at 265°onox, t' for 1 hour. After centrifugation,
The mixture was fractionated into two fractions of 0.8+t+J, and mRNA was recovered from each fraction by ethanol precipitation. The mRNA of each fraction was added to a rabbit reticulocyte in vitro translation system at a rate of OB〃A260/mJ, and 1 mci/mJ of
The reaction was carried out at 30° C. for 1 hour in the presence of S-methionine.

Total translation activity was determined by collecting a TCA-insoluble fraction of a portion of the reaction solution and measuring its radioactivity. On the other hand, 25 μl of the reaction solution was added with PT buffer (0.1 M potassium phosphate (T) H7,4), 150 mM Na (Jl,
2mM methionine, 2% l-lydone X-100) 75μ
4, add 10 μ2 of anti-P-450 antibody, and incubate at 4°C.
I left it there overnight. To this, 50 μ4 of Protein A-Sepha 0-2CL-4B equilibrated with PT buffer was added,
The reaction was allowed to proceed at 4° C. for 1 hour while stirring for 10 minutes. The gel was collected by centrifugation at 12.00 rpm for 5 minutes, washed twice with 1 ml of PT buffer, and then
10 nJ of Aquasol was added, and the radioactivity adsorbed to the gel was measured. This is called P-450 synthesis activity.

The total translation activity showed a peak in the 18-20S fraction, and the P-
450 synthesis activity showed a peak around 188. p-
In the fraction with the highest 450 synthesis activity, P-450mRN
A accounted for about 4.7% of the total mRNA. Therefore, the three parts before and after the two parts with the highest P-4,50 synthesis activity (
16-288) was used to synthesize c'DNA.
This was done using the g method. First, plasmid pSV7186
(P-T.

Piochemical) and psV1982 (P-T, t
Approximately 60 dTs were added to the primer and 5 to 10 dGs were added to the linker using primer DNA and linker DNA prepared from Iocomical Co., Ltd. and terminal transferase.

Approximately 1.4 μ2 of this primer and heat treatment (6
Approximately 2 μ2 of mRNA subjected to 5 minutes at 5°C was
mM) Lis-hydrochloric acid (pH 8,8),
8mM MPCI2.80mMMCI,
5.5 units of reverse transcriptase in 0.8 mM DTT, 2 mM dN TPs were added and incubated for 20 minutes at 45°C. After treatment with Phenoluk DQ form, DNA was recovered by ethanol precipitation.
Next, this DNA was subjected to dC addition using terminal transferase, 10 to 20 dC additions were confirmed, and the 20 reaction product was recovered by ethanol precipitation and then digested with Bindlff.

0.02 pmol of DNA thus obtained and 0.04 pmo7I of linker DNA were added to 10 ttl of 10
mM) Rinu hydrochloric acid (pH 7,5), 1 mM ED
TA, 0. After incubation in IMNaCJ at 65°C for 2 minutes and then at 42°C for 30 minutes, the mixture was cooled to 0°C, followed by 20mM (pH 7.5), 4mM MyCl.
2゜10mM (NH4), SO2,0.1M
MCI, 0.1 mM β-NAD.

50 μf/ml BSA and DNA ligase at position 101 were added to bring the total volume to 100 μl. After overnight incubation at 12°C, 40 aM dNTPs, 0
．． Add 15mM β-NAD, 2 units of DNA ligase, 5 units of DNA polymerase T, and 1.25 units of ridanuclease H, take a total volume of 104μ, and incubate at 12°C.
The mixture was incubated for 1 hour and further incubated at 25°C for 1 hour. This T')NA warm solution was used to transform E. coli DI (1 strain), and ampicillin-resistant colonies were selected.

Selection of P-450MCcDNA clones P-450MCc
In order to select clones containing DNA, positive,
Hybridization, translation, assay and immunoprecipitation assays were performed.

After culturing E. coli containing the plasmid and amplifying the plasmid DNA by adding chloramphenicol,
Plasmid DNA was prepared by the method of irnbojm-Doly.
was prepared. 20 μ2 of plasmid DNA is BamHI
After cutting it into a linear shape, it was boiled for 10 minutes and rapidly cooled. Then, an equal volume of IN NaOH was added, and after standing at room temperature for 20 minutes, 1/2 volume of neutralization buffer (IN NaCJ) was added.
, 0.8M sodium citrate, 0.5
M) Lis-magazine acid (pT(8,0>,
The mixture was neutralized by adding IN HCJ) and cooled with water. This denatured DNA was transferred to an 8-nitrocellulone filter, and after air-drying the filter, 2 ml of 6xSSC was added at 50 mJ.
Washed. After air drying, the filter was heated at 80° C. for 2 hours. Next, add 1 ml of filter FC sterilized water and
Boil for a minute and wash again with 1 mJ of sterile water. 7
Hybridization G solution containing mRNA (100-500 pf/ml poly(A) RNA, 6
5% deionized form 7 MiF, 20 mM PI
FES (pH 6,4) -0,2% SDS, 0.4M
NaCl, 100lit/ml tRNA) 80
μl was added and incubated at 50° C. for 3 hours. Then, the filter was injected with 10mM
Lis-hydrochloric acid (pH7,6), 0.15M Na
Cl, 1 mM EDTA, 0.5% SDS in 1 mJ;
It was washed 10 times while keeping the temperature at 65°C. Finally, SDR
Wash the filter twice with the above buffer without
It was added to 800 μm sterile water containing μ9 tRNA, boiled for 1 minute, and then rapidly cooled with dry ice-ethanol. The eluted mRNA was treated with pheno-yv-poo*lum, and the mRNA was recovered by ethanol precipitation.

After washing the mRNA twice with 70% ethanol, it was dissolved in 5 μm of sterile water and incubated in vitro according to the method described above.
It was added to the ro translation system to perform protein synthesis. The reaction product and the immunoprecipitate with the anti-P-450MC antibody were analyzed by 5DS-polyacrylamide gel/l/electrophoresis and hybridized with mRNA giving a band that migrated at the same migration position as P-450MC. Five clones gave positive results, one of which was named pAU157.

To create a restriction enzyme map of the pAU157 eDNA insert, plasmid DNA was prepared and cut with various restriction enzymes, and the size of the cut fragment was determined to be 0.8-
The restriction enzyme map shown in FIG. 8 was obtained from the cleavage patterns of various restriction enzymes analyzed by 1.0% agarose gel and 5% polyacrylamide gel electrophoresis. This restriction enzyme map was completely different from the restriction enzyme map of P-450 cDNA cloned from the liver of a rat treated with MC. Note that the restriction enzyme that does not have a recognition site in the eDNA insert of pAU157 is AccI +
A, v a T + B a m HI + B W
J IT T E c o RI @ Ht n d
llr *Nar I, 5phI, 5tuI.

Determining the base sequence of pAU157 cDNA insert Using the Mayam-Gjlbert method, pAU157
The entire base sequence of the cDNA insert was determined. After cutting the plasmid DNA with a restriction enzyme and dephosphorylating the 5' end of the resulting fragment, it was injected with polynucleotide kinase and γ-
” Phosphorylate again using P-ATP to convert the 5′ end to 7
Labeled with 2P. After treating the sample with another restriction enzyme and separating the cleavage fragment, base-specific chemical modification was performed, limited digestion was performed, and separation was performed by paternal acrylamide toge/L' electrophoresis. Autoradiography was performed to determine the base sequence. I read it. In Figure 1, pAU157 cDNA-r
The complete base sequence of the insert is shown. In this figure 1, 5'
The C following the dG homopolymer, which is the binding site with the nil vector DNA, is shown as base number 1.

Translation start codon ATG is found at base number 73, and stop codon T is found at base numbers 1645-1647.
AG was seen. Therefore, from base number 788 to 1
The reading frame up to position 644 was considered to be the reading frame encoding the protein. There are two other possible reading frames for proteins with one base shifted, but in the other two reading frames, P-4, which is estimated to have 856,000 molecules
It is not possible to code 50MC. When converting the base sequence corresponding to this longest reading frame into amino acids, a total of 5
It was found to encode 24 amino acids. The obtained amino acid sequence is shown in FIG. P-450 in Figure 1
2 to 2 of the amino acid sequence of the part encoding MC,
The 22 amino acid sequence up to B-th is purified P-450
M. It was confirmed that the obtained cDNA clone corresponded to P450 y cm RNA. Since the amino terminal of the purified enzyme is a glolin residue, the methionine residue corresponding to the translation initiation codon ATG is likely to have been modified and removed after translation. P- calculated from 528 amino acid residues excluding methionine
The molecular weight of 450MC was 59,800.

On the other hand, regarding the untranslated region, other experimental results showed that the 5' end is missing 8 obp.
At the 8' end, from base number 2115 and 25
From the 99th position, the po! of eukaryotic mRNA! J (A)
Sequence thought to be chain addition signal, AATAAA
, was found, and also the 8'-terminal Niha, 61-62bp
A poly(A) structure was present. Therefore, pAU
157 eDNA insert is P-450MCmRN
It was revealed that this was almost full-length eDNA of A.

Construction of Votaple Plasmid pTF1 Plasmid pTF'l containing only the coding region of P-450MC was constructed from pAU157.

10μ? (7) Completely cleave pAU 157 DNA with 10 units of restriction enzyme PstJ and create p-450MC.
Ps of approximately 5 soz group pairs located at the 5' end of the cDNA
The fragments were separated by 0.8% low melting point agarose gel/l' electrophoresis (100 V, 90 minutes) and recovered from the gel.

Next, add 1 μ2 of this fragment to 1 unit of restriction enzyme 5au8A.
〇Meanwhile, cloning vector pUC9
(PL Biochemical Co., Ltd.) (2μ2) was digested with 2 units of the restriction enzyme Bam) (I. Since the restriction enzymes BamHI and 5au8AI have a common recognition site, both were
NA! It was possible to recombine with J gauze and transform. 20 OAt/ml (') 5'-Fum4-
LB (11 per D1 op polypeptone, 52
A colony growing as a white colony on an agar plate (containing yeast extract and 5fNaCJ) was isolated, and Shibu7 Numi FDNA was prepared from this. ◇ Confirm that the plasmid DNA contains the desired DNA fragment. After that, after O reaction, which was cut with restriction enzyme SmaI,
After the phenol-chloroform treatment, the DNA was recovered by ethanol precipitation. Next, this DNA2/j
For f, SaJ Nilinker 2 μ2 and 10 units of 7
After adding 4DNA') gauze and reacting at 16°C for 3 hours, the mixture was transformed into E. coli DHI strain. An ampicillin-resistant colony was isolated, plasmid DNA was prepared from it, and it was confirmed that it had an SaA⋅ cleavage site.

10 μ2 of pAU157 DNA was mixed with 10 units of restriction enzyme P.
A PvulT-PstI fragment of about 12,001 base pairs located at the 3' end of the P450MC cDNA was completely digested with stI and 10 units of the restriction enzyme PvuTr, and was then similarly prepared and digested with 5au8AI. and mixed with the C9 cloning vector (P-L Biochemical) cut with restriction enzymes BamHI and PstI, religated with T4 DNA ligase, and the reaction mixture was used to transform Escherichia coli JM108 strain. Isolate white colonies in the same manner as
Target PstI-8au8AI to plasmid DNA
The resulting plasmid D was confirmed to contain the fragment.
After cutting NA5μ2 with 5 units of restriction enzyme SmaI, adding a Hind linker and performing a DNA ligase reaction, E. coli DT (1 strain was transformed). From the obtained colony, plasmid DNA was transformed into iAI The plasmid DNA was digested with restriction enzymes Ps tI and T
(cut with indlff, smaller PstT-)Ti
The ndlI fragment was recovered from low melting point agarose and mixed with the previously prepared PstT and Hindllr digested plasmid DNA containing the 5' end.
Rebinding was performed using J gauze. After the reaction, E. coli D
H1 strain was transformed, plasmid DNA was isolated from the obtained colony, cut with 5aJI and HIndTff, and the desired plasmid DNA was selected.The plasmid DNA thus obtained was cut with PstI, and treated with alkaline phosphatase. provided. On the other hand, pA
Cut U 157 DNA with PstI and create T4DNA!
The mixture was religated with J gauze and transformed into E. coli strain DHI.

Plasmid DNA was prepared from the obtained colonies, and one having the desired DNA structure was selected and named pTF1.

By cutting pTFl with Sal I and HlndTff, a DNA fragment containing only the P-450MC coding region can be easily recovered. In addition, the 5alI and Hindllr sites at both ends can be repaired with DNA polymerase I or digested with nuclease S1 to make blunt ends. 31 If a linker with an appropriate restriction enzyme site is added, , it is possible to convert to other restriction enzyme sites. From pTF 1
, 1) P-450,,c can be synthesized within microbial cells by removing a fragment containing the coding region of -450MC, connecting it to an appropriate promoter, and introducing it into microorganisms.

Next, construction of an expression plasmid expressing p-450MC and an example of expression of P-450MC will be explained. Construction of expression plasmid pAMC1 pTF1 plasmid, rat liver P-450M (.

The gene part was extracted and the yeast expression vector pAAH5 (WashingtonRe
Search Foundation, get it, Met
hods InEnzymology+ 101 pn
rtcy192-201 can be produced by the method of Amm@rsr et al. ) to construct the expression plasmid pAMC1 Step 1: 1.8 kb H1ndu containing the rat liver P-450MC gene
Isolation of fragment (a): Add 5 units of restriction enzyme-hari to 1 μ2 of pTF1 plasmid, add 50 μ! 8 in the industrial reaction solution of
React at 7°C for 1 hour, collect DNA, and collect 20μ! Next, 2 units of E. coli polymerase Kluno fragment were added to this DNA solution, and 50 μl of polymerase reaction solution [40 mM KPO4 (
pH7,5), 6.6mMMfcl, 1.
0mM 2-mercaptoethanol, 33μM dNTPs
] for 1 hour at 37°C. After the reaction, the DNA was collected and suspended in 20 μl of distilled water. Obtained DNA solution 2
0μ, about 1μ2 Windier linker and 3
Unit T, DNA! Add J gauze and 60μj T
DNA! The reaction was carried out in J gauze reaction solution at 16°C for 3 hours. After the reaction, the reaction solution was transformed into Escherichia coli strain DH1, and plasmid DNA was prepared from the obtained colonies. 1 unit of restriction enzyme Hind per 1 μ2 of plasmid DNA
Tff was added, and the mixture was reacted for 1 hour at 37° C. in 50 μjl of HindTIr reaction solution. After the reaction, it was analyzed by 0.8% agarose gel/L' electrophoresis. Among the plasmids examined, one incorporating a HindllT fragment of approximately 1.8 kb was selected, and pTF2 and 1~Ta0(b):2μ2T fragment were selected.
ITF2 DNA contains 2 units of restriction enzyme Hindlr.
Add 50μ! Hindlff reaction solution at 37℃2
After a period of time, the DNA was collected.

The obtained DNA solution was applied to a 08% low melting point agarose gel, and after electrophoresis, 1.8 kb Hindff DNA
Cut out the fragment from the gel, collect the DNA, suspend it in 20 μl of distilled water, and add 90(c): 0.5 tty of A.
Yeast expression vector p carrying the DII promoter
A A) T 5 ("via s old gton Re5
I got it from the search Foundation),
Add 1 unit of restriction enzyme T (IndW) and react for 1 hour at 37°C in 50 μl of ndlff reaction solution.
After collecting A, it was suspended in 20 μl of distilled water. this DNA
220 .mu.l K 2.0 units of alkaline phosphatase was added, and the mixture was reacted for 60 minutes at 60.degree. C. in a 100 .mu.l alkaline phosphatase reaction solution. After the reaction, phenol extraction was performed twice, and after ethanol precipitation, the DNA was collected and suspended in 20 μl of distilled water. (d): Mix 20 μl of the DNA solutions prepared in (b) and (c), T4DNA on the 7th floor! Add J gauze and T4DNA! J4DNA! The mixture was reacted with J gauze at 6°C for 3 hours. After the reaction, the reaction mixture was transformed into Escherichia coli DH1, and plasmid DNA was prepared from the obtained colonies. 1 unit of restriction enzyme HI n d■ was added to 01 μl of plasmid DNA, and 50 μl of H ndllr was added.
The mixture was reacted in the reaction solution at 37° C. for 1 hour, and after the reaction, it was analyzed by 0.8% agaron gel gel electrophoresis. Among the plasmids analyzed, those incorporating a DNA fragment of approximately 1.8 kb were selected. Some of the obtained plasmids contained ADT (
1.8kb HindlI in the opposite direction from the promoter
Some DNA fragments are connected. Therefore, 1 unit of restriction enzyme 5tuI and 1 unit of restriction enzyme BamH were added to 1μ2 of the selected plasmid DNA, and the mixture was reacted for 1 hour at 37°C in 50μl of Bam) II reaction solution. After the reaction, the reaction solution was analyzed by 0.8% agarone electrophoresis, and the DNA generated from the expression vector pAAH5 was analyzed.
In addition to the A fragment, there is a 2.7 kb DNA fragment and a 1.1 kb D
Plasmids in which NA fragments were detected were selected. This plasmid contains the ADH promoter and P-450M in the forward direction.
The C gene was connected and it was named pAMCl.

Expression of P-450MC Rat liver P-450,,c was expressed in yeast using the constructed expression plasmid pAMC1.The detailed method is described below.Step 1: Isolation of yeast transformants Rat liver P-450□, in yeast. In order to express
The method of Beggs et al. (Nature, 275.p10
4-109 (1978)), the recombinant plasmid pAMC1 was cultured in Saccharomyces yeast (Saccharomyces yeast).
anycescerevisiae ) S HY 8
Strain (MATa, 5te-vc9.ura8, t,
rpl, hisLleu2-8, 1eu2-112.
adel, Can1 [Cir+] (ATCC44771
), and colonies exhibiting the "Leu" phenotype were selected to obtain the transformant SHY8 (pAMC1).Step 2: Preparation of crude extract The yeast sIy carrying the expression plasmid pAMC1
8 (pAMC 1 ) strain from which leucine was removed
jt SD synthetic medium [0,67% Bacto-yeAs
tnitrogenbase Wlo amino
acids (Difco), 2% Dextrone
.

in 20 W/mA of tridi1-fan-2Q μm/ml histidine, 20 μS′/mJ adenine sulfate],
1 ml of the 0 culture solution cultured to 2 x 10' cells/ml was centrifuged at 7,000 rpm for 8 minutes, and after harvesting, the 0 culture solution was suspended in 1 mJ of 1.2 M sorbitol again.
Centrifuge, add 0.2+nJ of Zymolyase solution [1.2M
Tulpitou #, 50mM potassium phosphate (pH 7,5
), 14mM 2-mercapto: r-p/-)V.

400μ? /ml Zymolyase 60,000] and incubated at 30°C for 30 minutes.

Spheroplasts were collected by centrifugation at 7.00 Or, n, m for 8 min, and 0.4 mj! Buffer A [1,
2M Trupitot IV, 50mM Trls-HC
After washing with J (pH 7,5)), centrifuge again and add 50μ of SDS solution [2% dodecyl/I/sodium sulfate-50m
MTris-HC71 (pH 7,5)) was then heat treated at 100°C for 5 minutes to give a 10.00 Or, p
After centrifugation for 5 min at
pH 6,8), 2% (W/V) sodium dodecyl sulfate, 5% (V/V) 2-mer'captoethanol, 1
0% (W/V) glycero-yv, 0.001% bromophenol blue] were added.

Step 8: Identification of expressed protein Out of approximately 100 μl of the crude extract of the yeast sHy8 (pAMCl) strain extracted in Step 2, 20 μl! was subjected to 5DS-polyacrylamide gel, and the method of Laemma et al.
Nature 227. Electrophoresis was performed according to p680-685). After electrophoresis, acrylamide gel and nitrocellulose filter (5ehleicher &
scn'lI'11) and plotting buffer [25mM Trls-HCJ (pH 8,8),
3 in 192 mM glycine, 20% methanol].
A voltage of 0 V was applied for about 10 hours to transfer the white matter to the nitrocellulose filter. After 0 electrophoresis, the nitrocellulose filter was washed with blocking solution [3% gelatin, 50 m
M Tris-MCI (pH 7,5), 200m
MNaCJ, 0.05% Tween 20) and stirred for 30 minutes. Next, 1μ2/ml of anti-P-
Buffer containing 450 MCIgG [1% gelatin, 50
mM Tris-T (CJ (pH 75), 200 m
M NaCl, 0.05% Tween 20) and further stirred for 2 hours. The nitrocellulose membrane was then soaked in TBS solution C containing 0.05% Tween 20.
50mM Tri 5-T (CJ (pH 7,5),
The cells were washed four times for 40 minutes each with 200mM NaCJ) and immersed in blocking solution again. Next, remove the blocking solution, soak in 50 mJ of 125I-Protein A solution (7 μc +) for 1 hour, and add 0.05% Tween.
It was washed four times for 30 minutes each with a TBS solution containing 20, and finally washed with a TBS solution. The treated nitrocellulose filter was dried with tear paper and subjected to autoradiography.

When calculated based on p-450, a CM preparation, approximately 4 x 10' molecules of rat liver P-450Mo protein were produced per cell.

[Brief explanation of the drawing]

Figure 1 (Figures 1a to 1e) shows plasmid pAU1
The base sequence of 57 cDNA inserts is shown. In the figure, ring group number 73 to 1644 is P-450M
Corresponds to the region encoding C protein. FIG. 2 (Parts 1 to 4) is a schematic diagram showing the method for constructing the plasmid and P-450MC gene DNA fragment of the present invention. FIG. 8 shows a restriction enzyme map of the eDNA insert portion of plasmid pAU157. In the figure, the thick line indicates the P-450, c coding region. (N) 0(JL 0(In 0(JOO<* 01-4
12 -- ε scream 0

Claims (6)

[Claims] (1) A plasmid that retains the region encoding the rat liver cytochrome P-450_M_C gene in an easily separable state. (2) Rat liver cytochrome P-450_ having the base sequence from base number 73 to base number 1647 in Figure 1
The plasmid according to claim 1, which retains a DNA fragment in which restriction enzyme recognition sites are connected to both ends of M_C gene DNA. (3) Claim 2 having the structure shown in FIG.
plasmid pTF1. (4) Rat liver cytochrome P-450_M_C gene and a DNA fragment containing this in an easily separable state. (5) The DNA fragment according to claim 4, which has restriction enzyme recognition sites at both ends of the region encoding the rat liver cytochrome P-450_M_C gene. (6) Restriction enzyme recognition site is Sal I or Hind
The DNA fragment according to claim 5 which is III.