JPS6219085A - Gene expression plasmid parf1 aiming at rat liver nadph-cytochrome p-450 reductase gene and expression thereof in yeast and yeast strain, transformed by parf1 and expressing rat liver nadph-cytochrome p-450 reductase - Google Patents

Abstract

PURPOSE:To construct the titled yeast expression plasmid, by isolating cDNA clone capable of coding rat liver NADPH-cytochrome P-450 reductase, cutting out a region coding the above-mentioned reductase and integrating the resultant region in an expression vector. CONSTITUTION:A region coding rat liver NADPH-cytochrome P-450 reductase (hereinafter abbreviated to rat liver Fpt) is cut out of a plasmid containing cDNA coding the rat liver Fpt and integrated into an expression vector, e.g. pAAH5, holding an alcohol dehydrogenase 1 promoter to construct an expression plasmid, e.g. pARF1, which is then introduced into a yeast to afford the aimed transformant capable of producing the rat liver Fpt.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides rat liver NADPH-cytochrome P-450
The present invention relates to yeast strains that produce recombinant plus and rat liver NAI)' lo cytochrome P-450 reductases incorporating reductase genes, and methods for producing them.

Rat liver NADPH-cytochrome P-450 reduction # element (
Fpt) is a major IL113g of the liver microsomal electron transport system, and is a membrane enzyme containing flavin adenine mononucleotide and flavin mononucleotide as coenzymes] cytochrome I)-4.
50.

Only in this way can cytochrome p-450 exhibit oxidative activity towards its substrates.

The present inventors have already reported that rat liver cytochrome p-450M
We successfully isolated C (P-450MC) and expressed it in yeast (Japanese Patent Application No. 175169/1982).

P-450MC expressed in yeast exists in the yeast microsomal fraction and exhibits oxidative activity by interacting with yeast Fpt, and this yeast microsomal fraction and yeast cells producing P-450MC are It can be used for oxidation reactions of various compounds.

Furthermore, when purified rat liver Fpt was added to yeast microsomes containing this rat liver cytochrome P-450MC, cytochrome P-450MC-dependent oxidation activity increased significantly, and rat liver Fpt was expressed together with cytochrome P-450MC in yeast. Cytochrome P-4 by
The 50MC-dependent oxidation activity can be envied.

Therefore, the present inventors demonstrated that rat liver Fpt can be used as an industrially useful bioreactor. Also,
Fpt produced from yeast can also be purified and immobilized together with I)-460 for use in oxidation processes and the like.

The plasmid of the invention encodes rat liver Fpt.
A plasmid encoding rat liver Fpt is excised from a plasmid containing DNA and treated with alcohol dehydrogenase (
It can be constructed by incorporating into an expression vector carrying an A I) promoter (hereinafter abbreviated as A I). Furthermore, by introducing this plasmid into ms, a strain that produces a transformed strain that produces rat liver Fpt can be produced.

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

1) Rat liver Fp solubilized from reconstituted membrane with trypsin
Determination of the amino acid sequence of Fpt (Tanig) from rat liver.
The method of uchi et al. (Biochim, Biophys.

Acta, 51 SO, 841 (1979) M)
50mM containing 1% cholic acid, 20% glycerol, 0.1mM dithiothreitol
) Lys-hydrochloric acid buffer (pH 7,7) (1yd) was suspended to dissolve simyristoyl phosphatidylcholine corresponding to 600 times the number of moles of Fpt. Add the mixed solution to dialysate A (
0.1mM TA, 0.1mM dithios L/itol, 10mM potassium phosphate slow challenge solution, pH 7.1)
A reconstituted membrane was prepared by dialysis for 2 days.

105,000 Xl The reconstituted membrane was collected before ultracentrifugation for 1 hour, suspended in dialysate A, and treated with trypsin 100
J# (1sf/sg, 2mMII Ct) was added and solubilized with stirring at 0°C overnight. Again, 105.
000 xf/, by ultracentrifugation for 1 hour.
After removing the solubilized Fpt fragment (Fpt-L) from the reconstituted membrane, it was lyophilized, and about 17'17 mol was applied to Applied Biosystems' 47oA peptide sequence number -, followed by automatic Edoman degradation: 't7j The PT) (-amino acids obtained in each cycle were analyzed and identified using high performance liquid chromatography under the following conditions. Condition ■: Column; 413ondapak C+a (4
X250wm), solvent; CHaCN: 10 mM
Vinegar d sodium buffer (pH 4,5) = 42:58,
Flow 4; 0.8 aJ/fI)in detection; 269 nm
, Condition ■: Column; 20 rba) < CN (4,6X
25011111, solvent; 15mM sodium acetate buffer (pH 6,8), acetonitrile-methanol (
4:1) 12-16% discontinuous gradient end, flow rate;
1.0 in, detection; 269 nm 2) Preparation of synthetic NA probe OC/rTT deduced from the amino acid sequence of Fpt-T.
C/TTC8'16 mixed DNA) was synthesized using Abrite Noquiosystems 880A DNA synthesis machine. The protective group was removed by incubating the obtained oligonucleotide (17 mar) in ammonia water at 50° C. for 4 hours. high performance liquid chromatograph,
(-#Bondapalc Cts, 0.1 M
The oligonucleotide fraction was separated with triethylamine-acetate buffer (pH 7.0, niacetonitrile = 95:5 to 60:40), lyophilized, 80% acetic acid was added, and the mixture was left for 20 minutes in Murogakure. The trityl group was removed. The oligonucleotide fraction was collected by high performance liquid chromatography again. Approximately 100 oligonucleotides obtained
pmol to 100 nci(7) Cr-82P]
ATP (~5800 Ci/mol, ~2Q prno
l), containing 15° units of T4 polynucleotide kinase.

8) Partial purification of Fpt mRNA Phenobarbital was intraperitoneally administered to SD pigeon rats (4 weeks old, 100-120 fl) at a rate of 80q/Kp. 14-15 hours after administration, the rats were The liver was removed and the liver was removed. After cutting the liver into small pieces, add 10 volumes of 25mM)
Hydrochloric acid (pH 7.5), 25 mM NaCl, amM
MgCr2゜0.2M sucrose, 5% Triton X-10
Add 0.1 scoop/ml heparin sodium, homogenize, and centrifuge at 17.27.000×I for 10 minutes. To the resulting supernatant, add the above buffer containing 20% 2 M MyCtx isometrically to the polysomes. precipitated. Polysomes were collected by centrifugation at 26.400×II for 10 min and then incubated at 5 m
One M MyCtx was suspended. The yield of polysomes was approximately 15OA260 per f liver. 20-Part 1] Add an isometric extraction buffer (0.2M sodium acetate (pH 5.0), 1% 5DS) to the soybean suspension, and
The mixture was saturated with twice diluted extraction buffer, added with 40% of t-cotenol, and shaken.

After leaving it for 2-8 minutes, add 40% chloroform and shake it again. The aqueous layer and the organic layer were separated by centrifugation at 15,000×f for 1 minute, and the aqueous layer was extracted again by adding chloroform 80−. Repeat the extraction with chloroform until the intermediate layer disappears, and add twice the volume of cold ethanol to the final aqueous layer, and add -2
RNA was precipitated by standing at 0°C overnight.

The recovered RNA was treated with 8M sodium acetate I]rum (pH 16,
After washing twice with 0.0, 0.1M sodium acetate (p
H7,Q) and subjected to ethanol precipitation again. The precipitated RNA was collected, washed with 75% ethanol, vacuum dried, and stored at -80°C. The extraction rate of RNA from polysomes was 88-87%.

Next, the obtained RNA was passed through an oligo(dT) cellulose column, and mRNA having poly(~) was fractionated.
Dissolve NA in a small amount of sterile water and add equilibration buffer [0.5M
NaCl, 10mM) Lis-HCl (pTT7.5)
, 1mM EDTA, 0.1% SDS 1, 10~
It was diluted to 15A260/-. After heat treatment at 65° C. for 5 minutes, it was applied to an oligo(dt) cellulose column equilibrated with an equilibration buffer and thoroughly washed with the equilibration buffer. Fractions eluted with sterile water were collected, and an aqueous potassium acetate solution with a final concentration of 2% and 2 volumes of cold ethanol were added to precipitate RNA. Recovery [Nota RNA
was washed with 75% ethanol and then dissolved in sterile water.

Next, the obtained mRNA was size-fractionated by 10-80% sucrose density gradient centrifugation.

0,1 M with 10-80% sucrose linear density gradient
NaCl, 0.5% SDS, tmM ET
)'FA.

10mM) Lis-hydrochloric acid (pt(7,5) di, RNA heat-treated at 65℃ for 5 minutes, 2.4A260 was layered, and TV8
65 type particle rotor (DuPont 5or
vatl) at 265.000Xf for 1 hour. After centrifugation, the mixture was fractionated into two fractions of 0.8-m, and mRNA was recovered from each fraction by ethanol precipitation.

Both mRNAs were subjected to electrophoresis on a denaturing agarose gel containing 2.2 M formaldehyde, blotted onto a nitrocellulose filter, and analyzed by Northern hybridization using a synthetic NA probe. Ta. As a result, bands were detected on both sides of 22S, and CI')NA synthesis was performed using these bands.

4) CI) NA cloning CDNA cloning was performed by the Okayama-Berg method. First, -8'-oligo (dt') addition ps
V7186-derived plasmid pricemer (purchased from Pharmacia P-L Biochemicals) approximately 1.8μ
f and m that was previously heat treated (65°C, 5 minutes)
About 2af of RNA was added to 50mM) Lis-HCl (pH 8,8
)8mM MgCz2, 80mM KCt, 0.8
mMDTT, 5.51 in 2 m MdNTPa
Reverse transcriptase at position 1 was added and incubated at 45°C for 20 minutes.

- After treatment with pure alcohol-chloroform, DNA was recovered by ethanol precipitation. Next, this DNA
was subjected to dC addition using terminal transferase, and approximately 10 dC additions were confirmed. The reaction product was recovered by ethanol precipitation and then digested with Hindm.

The thus obtained DNA (0.021) mO6 and 8'-oligo (dG)-added psV19B2-derived p(indux linker (purchased from Pharmacia Pl-L Biochemicals) 0.04 pmoz) were added to 10 bL (DlomM
) Lis-HCl (pH 7,5), 1mM EDTA, 0
．． in 1M NaC6 at 65°C for 2 minutes, then at 42°C at 80°C.
The molecular ftl ink was incubated and then cooled to 0°C.

To this, 20mM) Lis-HCl (pH 7,5), 4mM
MIC1g, 10mM (NH4) $1804
, o, 1MK, czO, 1mM β-NAD, 50
tt I /m#BsA.

Add 10 units of 1'3NA ligase and reduce the total volume to 100 μt.
And so. After incubating overnight at 12℃, 40μ
M dNTPs, 0.15τ■β-NAD.

2 units DNA ligase, 5 units DNA polymerase I,
Add 1.25 units of ribonuclease H to make a total volume of 104 μt, and incubate at 12°C for 1 hour, then at 25°C for 1 hour.
Time ink ikibe.

-I hit it. Using this DNA solution, E. bacterium DH1 strain was transformed, and ampicillin-resistant colonies were selected.

5) F p t $r) NA Rion (D?
From the ambirelin-resistant colony extracted from Ij, F
In order to select clones containing ptcI)NA, colony hybridization was performed using a synthetic probe.

E. coli carrying the plasmid was plated to form colonies, and then replicated onto a nitrocellulose filter. 0.2 colonies per filter
The DNA was bound to the filter by soaking it in NaOH and lysing it. Filter 0゜5M Tris -
After neutralization by soaking in HCL (pH 8,0), 80℃
The filter was dried in an airtight oven for 2 hours to fix the DNA on the filter. 4xSSC (tamM sodium citrate A-150mM NaCz), 10X Den
harclt (0,02% Ficoll, o, og%B
SA% 0.02% polyvinylpyrrolidone).

A mixed solution of 100 mg/d salmon testis DNA and the filter were packed in a plastic bag and incubated at 60 L for 8 hours. A synthesized NA probe was added to the above mixture, which was again packed in a plastic bag together with the filter and incubated at 40°C overnight. The filter was taken out, kept at 40°C, washed four times with 4x SSC, and an autoradiogram was taken to isolate colonies giving positive regnal. Four positive probes were obtained, one of which was named pRFl.

The plasmid pRFl was isolated from Escherichia coli, cut with various restriction enzymes, and the fragment was 0.8%.
A restriction enzyme map was created by analysis using low gel electrophoresis. Figure 8 shows the pRFl cDNA insert (length 2.6
kb) is shown.

7) Determination of base sequence of pRFl cDNA insert Using the dideoxy method, pRFlc
The entire base sequence of the DNA insert was determined.

After cutting the plasmid DNA with restriction enzymes and cloning into M18 γ-di mp8 and mp9, single-stranded D
NA was isolated and analyzed using the M18 sequencing kit (Takara Shuzo C strain). FIG. 4 shows the entire base sequence of the pRf'l cDNA insert. The amino acid sequence of Fpt deduced from the base sequence is also shown.

It is known that the terminal amino acid of rat liver Fpt is acetylated (Black & Co.
on, J. Biol, Cham, , 257592
9 (1982) Although the amino acid sequence of the terminal amino group has not been clarified, Figure 4 shows the Suiko-Mota start codon A'1.
If Fpt is read from G, then rat liver Fpt is estimated to be a protein consisting of 678 amino acid residues and a molecular weight of 76.900. The underlined amino acid sequence in FIG. 4 indicates the amino acid sequence determined for the Fpt specimen solubilized from the reconstituted membrane with trypsin, and matched the amino acid sequence read from the base sequence. Furthermore, the amino acid sequence near the terminal end of the amino group shows high homology with the previously reported amino acid sequence of rabbit liver Fpt.

Based on these facts, the pRF 1 cDNA insert is a full-length cDNA containing the entire coding region of Fpt.
It turned out to be.

The recombinant plasmid pRF1, which contains the entire coding region of Fpt, has a Fpt gene portion of about 2.8
kb was isolated and the expression vector pAAH5□ carrying the ADH promoter (Methods in E
zymolofy 101, part Cp19
2-201. , Ammerer et al. ), and as the first step in constructing a plasmid for self-expression in yeast, a recombinant plasmid 1) RFCC5 having restriction enzyme sites and 1 ndiu at the beginning and end of the Fpt gene was constructed. Figure 1 shows the outline.

Step 1: Construction of recombinant plasmid pRF”R111 (a)1 #f(ripRF1 plus tFDNAIc
25-L-1--t) 17) Restriction enzyme PvuII
Add 250 #t of restriction enzyme reaction solution M.
[10mM) Lis-HCl, (pll 7.5), 10
mM 8 (gctz, 1mM dithiothreitol, 50
The reaction was carried out at 87°C for 1 hour in mM NaCz). The reaction solution was applied to a 0.7% low melting point arose gel (Bethesda Research) containing 0.5Af/- ethidium bromide (Aldrich), electrophoresed at 70V for 120 minutes, and then electrophoresed with ultraviolet lamp F. l) NA fragment l (approximately l, Qkb
) was cut out, placed in an Eppendorf tube, and heated at 65°C for 5 minutes. Add TE buffer [10mM] Lis-HCl (pH 8,0), 1mM to the melted gel.
Phenol extraction was performed by adding an equal volume of 7x/-JLT saturated with ED'l'Al.

After centrifuging at 12.00 rpm for 5 minutes and separating the upper layer, 2 volumes of cold ethanol were added and the mixture was left at 80°C for 15 minutes to precipitate 13NA with ethanol. Then, centrifuge at 12.00 rpm for 10 minutes.
Approximately 0.2 SF of DNA fragment 1 was collected and suspended in 10 μL of distilled water.

(b) Next, the prepared 0.2 μf DNA fragment 1 2 5 units / ) (D restriction # element Rsax (NEB) was added, and the mixture was heated at 87°C in 20 μt restriction enzyme reaction solution M.
After incubation for an hour, the I') NA fragment 1' (700
The gel portion corresponding to bl)) was cut out and similarly precipitated with ethanol to recover a 0.15 af DNA fragment 1', which was suspended in 10 μm of distilled water.

(C) Next, prepared 0 and 15 μf I) NA fragment 1
'N1a & (7) Hindm'J linker (Takarazosha) and 8 units of T4 DNA ligase were added, and 20aL of T4 ligase reaction solution [66mM] Lis-HCl, (pH 7,5), 66mM MgCr2.
16°C in 10mM DTT, 1mM ATP]
, and reacted for 200 hours. After the reaction, add 1aL of 1.5M NaCl and 5Q:L2.yt restriction enzyme Hindm.
(Takara Shuzo Co., Ltd.) and reacted at 87°C for 8 hours.
The enzyme was inactivated by incubation at 65°C for 5 minutes.

(d) Add 8 units of restriction enzyme dm to 1#f pBR822 and 87°C in 20μt restriction enzyme reaction solution M.
After reacting for 1 hour, phenol extraction and ethanol precipitation were performed, and the DNA was recovered and suspended in 20 μt of distilled water. Add 199mM Tris-HCl (I)H8,0) to the suspension.
26μt and E. coli alkaline phosphmatase 0.5
The unit was added and reacted at 60°C for 1 hour. After the reaction,
Extract 8 times with a 1:1 mixture of phenol and chloroform, centrifuge at 12.00 rpm for 5 minutes, separate the upper layer, and collect 1) NA by ethanol precipitation.
It was suspended in 0 μt of distilled water.

(e) 5 DNA fragments each obtained in (c) and (d)
Each microliter was mixed, 8 units of Ta DNA ligase was added, and E. coli DH1 strain was transformed using 201t of T4. For culturing E. coli, use L medium (109 polypeptone per 11, 6f yeast extract, 5f Na
149 agar was added to the plate medium. The same applies thereafter.

In order to clone the desired DNA fragment 1' with a Hindm linker, the transformant was spread on a trL plate containing 100 μg/- of ampicillin (Sigma), and colonies were isolated. Next, Birnbo
im et al.'s method (Nucl, Ac1ds, Rea
, 7, p151B-152111), plasmid L)NA was isolated from the colony, and 0.1/4
5 units of restriction I## for 1 plasmid DNA
Add dul for Sokawa 1 and add 87 in 4 to the restriction enzyme reaction solution.
After reacting at 1 hour at 0.8°C, the mixture was analyzed by 0.8% agarose gel electrophoresis to select a recombinant plasmid incorporating DNA fragment 1' that can be excised with Ilindm. 1 mHf (Takara Shuzo Co., Ltd.) of the obtained Chronidzol was added to limit W# elementary reaction solution H (10 mM) in lithium-hydrochloric acid (pH 7,5), 10 m M MpCzg, 1 mM dithiothreitol, 100 mM NaCz). °C,
After reacting for 1 hour, it was analyzed by 0.8% agarose gel electrophoresis to select a plasmid in which the FptON end was connected in a direction close to the HI cleavage site of pBR822.
One of them is pRFRl 11 and (la)SJlf
I's pkF1 plus i-do DNA with 26 units @l
# Add elementary Pvull and limit 260 μt a # elementary reaction e
The reaction was carried out in M at 87°C for 1 hour. The reaction solution was fractionated by 0.8% low melting point agarose gel electrophoresis, and the target 1) NA
Fragment 2 (approximately 1.8 kkl was reacted according to the spinning speed method. After the reaction, 1.5 M NaCz 1 im L and 60
:x, Ni7) restriction enzyme H1ndlll was added and reacted at 87C for 8 hours, followed by incubation at 66°C for 6 minutes.

(c) 1) N obtained in (b) and step 1(d)
Mix 6 μt each of A fragments and make 8 uniq TonoT41)
NA Q case was added and reacted in 20 sL of T4 DNA ligase reaction solution at 15C for 4 hours. After the reaction, Escherichia coli DHI strain was transformed with the reaction solution and colonies were isolated in the same manner as in step 1(e). Plasmid DNA was isolated from the obtained colony, and 0.1 μg of plasmid DNA was
The DNA was cut by adding 5 units of the restriction enzyme Hindul or 5 units each of the restriction enzymes Neo I and BamHI to A, and analyzed by agarose gel electrophoresis. Select a clone in which DNA fragment 2 has been inserted so that the end is T) BR822 (7) close to the EcoRI site and insert p
It was designated as RFP2.

Step 8 Construction of recombinant plasmid pRFC2 (a) pRFRl of lnf 11 DNA 20 uni.

The restriction enzyme NcoI, 20 units of restriction enzyme, and 1 were added, and after reacting in 100 μt of restriction enzyme reaction solution H at 87C for 1 hour, the longer one was subjected to 0.8% low melting point agarose gel electrophoresis. Excise the NA fragment from the gel 1
DN was extracted by phenol extraction and ethanol precipitation.
The A fragment was collected and suspended in 20ttl of distilled water.

(b) Perform the same operation as in (a) on 1bfl of pRFP2 DNA and apply it on 0.8% low melting point agarose gel 11E.
Extract the shorter DNA fragment by gK electrophoresis, collect it, and transfer it to 20μ.
It was suspended in distilled water.

(c) (a) and (b) M prepared DNA solution 20
8 units of T4 DNA ligase were added, and the mixture was reacted at 16° C. for 200 hours in a 5 Q a L T4 DNA ligase reaction solution. E. coli strain DH1 was transformed using the obtained reaction solution, and plasmid DNA was prepared from the colonies that appeared.

5 units of -indm for restriction enzymes were added to the obtained 0.1 μf plasmid DNA, reacted in 20AL restriction enzyme reaction solution M at 87°C for 1 hour, and subjected to 0.8% agarose gel electrophoresis. It was analyzed in A plasmid retaining the target structure was selected and named pRFc2.

Ste Nobu 4 Construction of recombinant plasmid pRFC5 (a) Add 20 units of restriction enzyme I to 1#N pRFC2 DNA and react in 100 μt of restriction enzyme reaction solution H at 87°C for 1 hour. Later, phenol,
DNA was extracted with chloroform and precipitated with ethanol.
was collected and suspended in 20#t of distilled water. Ko (7) D
Add 0.5 units of alkaline phosphatase to N A In l & 0.1M) Lith-HCl (pHg
, 0) at 60°C for 1 hour. After the reaction, extraction with 1:l of phenol:chloroform was performed eight times, and after precipitation with ethanol, the DNA was collected and suspended in 20 μt of distilled water.

(b) A 20 uni-lid portion was excised from pRFI DNA of 1##, and the DNA was collected and suspended in 20 μt of distilled water.

(c) Shiri DNA solution 2 prepared in (a) and (b)
Mix 0 μt, add 8 units of 141) NA ligase, and add 141) of 50 #t of Ta DNA ligase to
The reaction was carried out at 5°C for 8 hours. After the reaction, E. coli D was added to the reaction mixture.
The H1 strain was transformed.

(d) Plasmid DNA was prepared from the obtained colonies, and 0. 5 units of restriction enzyme 5caI and 5 units of restriction enzyme S-a for 1 μl of plasmid DNA.
c I was added and reacted for 1 hour at 87°C in restriction enzyme reaction solution II (1"); analyzed by 0.8% agarose gel electrophoresis. Of the analyzed pluses, the target structure was retained. A plasmid was selected and named pRFCC5.

2) Construction of pRFCC5 plasmid DNA constructed in 1, rat liver Fpt
The gene part was extracted and the yeast expression vector pAAH5 (Washington
Obtained from Re5search Found-ation,
Methods in Enzymology.

The method will be described in detail.

(a) 1 a II (7) pRFcc5DNAjc
20: Add L neat restriction@enzyme-Uundu+ and incubate at 87°C in 100 aA(7) restriction enzyme reaction solution M.
After reacting for 1 hour, it was subjected to 0.8% low melting point agarose gel electrophoresis.
The fragment was excised from the gel, the DNA was collected, and suspended in 2 μl of distilled water.

(b) 1 μl of yeast expression vector pAAH5 (Wash
Inton Research Foundation
20 units of restriction enzyme XHi
ndu+ was added and reacted in 100 nA restriction enzyme reaction solution M at 87°C for 1 hour. After collecting the DNA, 20#L
was suspended in distilled water.

0.5 unit of alkaline phosphatase was added to this DNA solution, and the mixture was reacted at 60° C. for 1 hour in 25 aL of 0.1 M Tris-HCl (H8.0). After the reaction, extraction with a mixture of phenol and chloroform was performed eight times, and after precipitation with ethanol, the DNA was collected and suspended in 20#t of distilled water.

(C) DNA solution 20 prepared in (a) and (b)
μt was mixed, 8 units of T4 DNA ligase was added, and the mixture was reacted with T4 DNA4 DNA ligase at 16° C. for 4 hours. After the reaction, transform E. coli DHI strain with the reaction solution,
Plasmid DNA was prepared from the resulting colonies. 0
．． Add 5 units of the restriction enzyme NcoI and 6 units of the restriction enzyme Dansei to 1 al of plasmid DNA, and after reacting in restriction enzyme reaction solution H at 87°C for 1 hour,
It was analyzed by 0.8% agarose gel electrophoresis. A plasmid retaining the desired structure was selected and named pARF1. In pARF1, between the ADH promoter of pAAH5 and the Al)H terminator, the Lalit liver Fpt and gene are connected to the ADH promoter in the forward direction.

Expression of P-450 reductase was performed. The method is described below.

Step 1 Isolation of Yeast Transformants Bacteria were collected using YPD medium (1% yeast extract, 2% lipeptone, 2% glucose). After washing the obtained bacterial cells with 0.2 M LiCz, the obtained pellet was injected with 20 μt of I M LiCL and pARFl.
1 μg (10 μt) of plasmid DNA, plus 70%
80 sL of polyethylene glycol 4000 was added and the mixture was thoroughly suspended and incubated at 80°C for 1 hour. Thereafter, 140 μt of sterilized water was added to the mixed solution, and 100 μt each was prepared on an SD plate (0.67% yeast nitrogen source, 2% glucose, 2% agar, 24-” (pARFl) strain).

Step 2 Preparation of yeast total protein 24 afi/-histidine).

Add 2M1NaO to 0.9-mL yeast culture in logarithmic growth phase.
After adding 0,1-H, 9% 2-mercaptoethanol and incubating for 10 min in water, 80% 'I''C
Add 0.2 - of A to the mixture and leave it on ice for 10 minutes. 12
．． The precipitate was collected by centrifugation at 00 rpm for 2 minutes, washed with ice-cold A7T, and then dried.

STERIP 8 Retrieval of expressed protein The total protein of the yeast AH22 (pARFl) strain prepared in step 2 was mixed with sample buffer [4% sodium dodecyl sulfate, 0.16M], lithium-hydrochloric acid (4% sodium dodecyl sulfate, 0.16M), which had been boiled at 100°C for 5 minutes. p) I6.8), 0.88M 2-mercaptoethanol, 20% glycerol, 0.01% bromophenol blue] were placed in 8 mouths and dissolved while incubating at 100°C. Solution 7.5%S
The cells were applied to a DS-polyacrylamide gel, and electrophoresis was performed according to the method (-685). After electrophoresis, acrylamide gel and nitrocellulose 1), 174 JLtter (Schleicher & 5ch
Niil1) in one layer and blotting buffer [2
5mM) Lis-HCl (1)88.8), 192mM glycine, 20% methanol) at a voltage of 80V for approximately 10
Over time, the proteins were transferred to the nitrocellulose filter. After electrophoresis, the nitrocellulose filter was diluted with blocking solution [8% gelatin, 50mM] lithium-hydrochloric acid (p
H7,5) 206mM NaCt, 0.05%Tw
een2G) and stirred for 80 minutes. next,
Buffer solution containing 20 afi/- anti-ppt-gG [1
% gelatin, 50mM Tris-HCl (pH 7.5), 2
00mM NaCz, 0.05% Tween 20) and further stirred for 2 hours. The nitrocellulose membrane was then soaked in a TBS solution containing 0.05% Tween 20.
50mM) Lis-HCl (PH7,5), 26QmM
NaCz) F Washed 4 times for 80 minutes each and immersed in blocking solution again. Remove blocking solution and remove 20
After soaking in +J-I-Protein A solution P& (2 μCi) for 1 hour, the cells were washed four times for 80 minutes each with a TBS solution containing 0.05% cD Tween 20, and finally washed with a TBS solution.

In the strain, a band thought to be iFp rainbow expressed at the same position as the purified Fpt sample was confirmed. On the other hand, AH22
(pAAH5) When all proteins of the strain were migrated, F
No band was observed at the pt position, A) 122(I)A
It is thought that the Fpt band detected in the RFI strain was expressed by pARF1. When the expression density was calculated for all Fpti preparations, it was found that approximately 1×10” molecules of rat liver NADPf (-cytochrome P-4fiO reductase protein) were produced per yeast cell.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an outline of the construction of plasmid 1) RFCC5 containing the Fpt coding region. Figure 2 shows plasmid pARF1 for yeast self-expression of Fpt.
FIG. 2 is a diagram showing an outline of the construction. FIG. 8 shows the ffl# elementary map of the CDNA insert portion of plasmid pRF1. In the figure, the thick line indicates the coding region of Fpt. Figure 4 (Parts 1 to 5) shows c of plasmid pRF1.
The complete base sequence of the DNA insert is shown. Procedural amendment (spontaneous) Ushiguchi, Commissioner of the Japan Patent Office, Showa [ / p month, Black 1) Yu Akira 1, Indication of the case 1985 Patent side No. 146653 2, Name of the invention Rat liver NADP) I-Cytochrome P Transformation using the -450iW original enzyme gene and expression plasmid pARF1 and pARF1 for the purpose of expressing it in yeast,
Yeast strain expressing f-cytochrome P-450 reductase (rat liver NADP) 3.Relationship with the person making the amendment Patent applicant postal code 100 4. Detailed description of the invention in the specification to be amended 5. Contents of amendment (1) On page 5, line 13 of the specification, the phrase "expression vector holding a promoter" has been replaced with [promoter (yeast ADH gene promoter is Wa
shington Research Foundation
ion's U.S. Patent Application Box 299733,
In the United States, when using for industrial or commercial purposes, permission from the right holder is required. ). (2) On page 4, lines 1 and 2, “Patent Application 1984-1
75159J is corrected to JP-A-6L-56072J. (3) Expression vector pAAH5 (tosoe
t,hodsinEnzymology101. par
tCP192-201. , Amlerer et al. ) into [
Yeast expression vector carrying yeast ADH gene promoter and terminator 1) AAH5 (formerly ngton Research Foundation
Available from Methods in Enzymolo
gy 101. part CP192-201゜Am
It can be produced by the method of Merer et al. ),
” he corrected. (4) r p A A on page 29, lines 6 to 9
H5 (Washington Research F
Obtained from Foundation, Methods in
Enzymology, 101 part CP19
2-201, Ammerer et al. , )" should be corrected to read rpAAH5J. (5) r p A A H5 (klashington Re
5earch Foundation)" is corrected to rpAAH5 as J. that's all

Claims (10)

[Claims] (1) A plasmid containing the entire coding region of the rat liver NADPH-cytochrome P-450 reductase gene. (2) Plasmid pRF1 according to claim 1, which has the structure shown in FIG. (3) An expression plasmid for expressing the rat liver NADPH-cytochrome P-450 reductase gene in yeast. (4) The expression plasmid according to claim 3, which is constructed by incorporating the rat liver NADPH-cytochrome P-450 reductase gene into the expression vector pAAH5 carrying an alcohol dehydrogenase I promoter. (5) The yeast expression plasmid pARF1 according to claim 3, which has the structure shown in FIG. (6) Rat liver NADPH-cytochrome P possessing the rat liver NAOPH-cytochrome P-450 reductase gene
Yeast that produces -450 reductase. (7) The yeast according to claim 6, which belongs to the genus Saccharomyces. (8) Saccharomyces cerebice AH22 (pAEF1
) strain according to claim 6. (9) A method for constructing plasmid pARF1 for yeast expression, which comprises isolating the rat liver NADPH-cytochrome P-450 reductase gene from plasmid pRF1, and incorporating it into expression vector pAAH5 carrying an alcohol dehydrogenase I promoter. . (10) A method for producing yeast for producing rat liver NADPH-cytochrome P-450 reductase, which comprises transforming yeast using yeast expression plasmid pARF1.