JP3078353B2 - Substantially pure cells producing Δ4-3-ketosteroid-5β-reductase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a substantially pure cells producing delta ^4-3-keto steroids -5β- reductase, delta ⁴
Substantially pure DNA and delta ^4-3-keto steroids to express 3-keto steroids -5β- reductase -5β-
The present invention relates to a method for producing a reductase. More particularly, the present invention is, for example, materials for steroid metabolism related drug development, inactivating agents steroid hormones, available delta ⁴ as such as an antigen for preparing an antibody, such as monoclonal antibodies and polyclonal antibodies as a diagnostic agent -3-
Keto steroid -5β- reductase, transformed cells efficiently produced by a genetic engineering technique, delta having the nucleotide sequence encoding the amino acid sequence of the delta ^4-3-keto steroids -5β- reductase from rat liver ⁴ 3-keto steroids -5β- reductase gene DNA and using the transformed cells, efficiently delta ^4-3-keto steroids -5
The present invention relates to a method for producing β-reductase.

[0002]

2. Description of the Related Art Cholesterol is a constituent of cells such as cell membranes and organelle membranes and is an essential substance for the survival of cells. 80-90% of the cholesterol is metabolized to bile acids in the liver, After being excreted in the juice, it is converted into steroid hormones in the adrenal glands and gonads and exerts various physiological actions, after which it is metabolized in the liver, etc.
It is known to be excreted in urine. In bile acid metabolism pathway and steroid hormone synthesis pathway from such cholesterol are involved various enzymes, among them, delta ^4-3-keto steroids -5β- reductase, 4-position of the steroid skeleton Is known to be an important enzyme that catalyzes the reaction of reducing the double bond of A to B / Cis [Journal of Biological Chemistry], Vol. 240, No. 2396 to 2401 (1965)]. For example,
Reaction formula

Embedded image (NADPH ₂ and NADP in the formula, each of nicotine adenine reduced and oxidized forms of dinucleotide phosphate) from as indicated by, delta ^4-3-keto steroids and NADPH ₂ Prefecture, 3-keto -5βH -Catalyze the reaction to produce steroids and NADP. This Δ ⁴ -3-
The reactions catalyzed by ketosteroid-5β-reductase include:

Embedded image

Embedded image Are exemplified. Delta ^4-3-keto steroids -5β- reductase having such properties,
For example, it is expected to be useful as a material for developing steroid metabolism-related drugs, a steroid hormone inactivating agent, an antigen for producing antibodies such as monoclonal antibodies and polyclonal antibodies as diagnostic agents, and the like. The diagnostic agent, e.g., by measuring the enzyme with monoclonal antibodies to the delta ^4-3-keto steroids -5β- reductase, it is possible to diagnose the enzyme deficiency.
The delta ^4-3-keto steroids -5β- reductase is known to be distributed in the liver of an animal, it has been attempted to extract purified from rat liver up to now [ "Journal of Biotechnology Logical Chemistry (J.
Biol. Chem. Volume 259, 7519-7
524 pages (1984), Vol. 260, No. 713
7 to 7141 (1985)]. However,
In the method of extracting from rat liver, for containing other similar today matters in a living body, and the amount of the resulting delta ^4-3-keto steroids -5β- reductase is extremely small, a high-purity preparation In fact, it is difficult to obtain them, and thus it is not possible to actively advance research for developing new pharmaceuticals and diagnostics using the enzyme. Therefore, the development of microbial and animal cells producing efficiently delta ^4-3-keto steroids -5β- reductase has been desired.

[0003]

[0007] The present invention is, under such circumstances, to efficiently produce useful Δ ⁴ -3- keto steroid -5β- reductase enzyme, such as the development of new drugs and diagnostic agents The purpose of the present invention is to provide a method for developing microorganisms and animal cells and mass-producing the enzyme by using them.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object, and as a result, from the cDNA library obtained based on mRNA prepared from rat hepatocytes, Screening for gene DNA that expresses the enzyme, and then constructing an expression vector using this DNA, then introducing into an appropriate cell (microorganism or animal cell) to produce a transformed cell, which is cultured in a medium. by, found that can mass-produced efficiently the delta ^4-3-keto steroids -5β- reductase, and completed the present invention based on this finding.

Namely, the present invention is characterized in that it is a cell transformed by a recombinant plasmid having a DNA expressing delta ^4-3-keto steroids -5β- reductase amino acid sequence represented by Figure 1 delta ^4-3-keto steroids and having a delta ^{4-3-substantially} pure cells producing ketosteroids -5β- reductase, nucleotide sequences encoding the amino acid sequence represented in Figure 1, cultured -5β- substantially pure DNA expressing reductase, and to the medium substantially pure cells producing delta ^4-3-keto steroids -5β- reductase is the transformed cells, then delta, and collecting the delta ^4-3-keto steroids -5β- reductase from the culture
^4-3 is intended to provide a process for the production of keto steroids -5β- reductase.

Hereinafter, the present invention will be described in detail. In the present invention, delta ^4-3-keto steroids -5β- used reductase to produce transformed cells which produce delta ⁴ -
Gene DNA expressing 3-ketosteroid-5β-reductase can be obtained by screening from a cDNA library obtained based on mRNA prepared from hepatocytes, for example, rat hepatocytes.

[0007] Then, the delta ^4-3-keto steroids -5
To illustrate a specific method for obtaining a gene DNA expressing β-reductase, first, mRN is obtained from rat hepatocytes.
Prepare A. As a method for preparing this mRNA,
For example, after crushing the excised rat liver tissue, guanidinium solution and the like are added thereto, and homogenized,
Next, the high-molecular-weight DNA is cut and subjected to an appropriate treatment to collect the total RNA. Next, this was converted to oligo (dT)
-A method of preparing a poly (A) ⁺ RNA by applying to a cellulose column and eluted only RNA having a polyA tail can be used.

Next, the thus obtained poly is obtained.
(A) A cDNA library is prepared from ⁺ RNA,
First, the poly (A) ⁺ RNA is, for example, dN
After the first strand is synthesized by reacting TP _s (a mixture of equal amounts of dATP, dGTP, dCTP, and dTTP), oligo (dT) and reverse transcriptase, the second strand is synthesized with RNaseH and DNA polymerase I. Then After methylation the EcoRI site inherent in gene with EcoRI methylase, blunt and without using T ₄ DNA polymerase, then were allowed to adhere to EcoRI linkers to the both ends, was digested with restriction enzymes EcoRI, which Is electrophoresed to size fractionate the cDNA and to remove excess linker.

Next, a gene library is prepared using the cDNA thus obtained, and a target gene DNA is screened therefrom. The method is not particularly limited, and a conventionally used method is used. be able to. For example, the cD obtained based on mRNA
The NA is linked to a cloning center to create a cDNA library. On the other hand, and polyclonal antibodies delta ^4-3-keto steroids -5β- reductase, corresponding to the nucleotide sequence of a portion of the amino acid sequence of the enzyme 1
A DNA probe labeled with an isotope having a length of 5 to 25 bases or a non-radioactive compound is prepared, and the c
The target gene may be screened from the DNA library. As this cDNA library, a commercially available rat liver cDNA library can also be used.

Next, the method used in the examples of the present invention will be described. The phage vectors λgt11 and λZAP are used as cloning vectors, and cDN of the largest possible fraction from the cDNA is used.
A was ligated to these to prepare a cDNA library integrated into the λgt11 vector and a cDNA library integrated into the λZAP vector. And first, λg
The t11 library was screened using a polyclonal antibody prepared in advance to obtain about 10
After obtaining eight positive plaques λ ₂ , λ ₄ , λ ₅ , λ ₇ , λ ₉ , λ ₁₁ , λ ₁₂ , λ ₁₃ from the 0000 plaques, cloning was performed, and for each clone, Δ ⁴ -3-keto steroids -5β- Southern blot reductase enzyme preparation of lysyl endopeptidase corresponding synthetic DNA to K6 peptide obtained by the process as probes (Southern blot) was subjected to analysis, all but lambda ₄ 1.8 kbp
Since the hybridizing band was detected at a position, lambda ₄ other clones it was confirmed that at least a part contains a coding region of the enzyme. Also, this 1.
Northern blot of poly (A) ⁺ RNA derived from rat liver using an 8 kbp cDNA insert as a probe (N
The result of analysis of an Northern blot analysis revealed that the mRNA size of the enzyme was about 3.5 kbp.

Next, in comparison with the size of this mRNA,
Since the size of the resulting positive clones was small, the insert of the lambda ₁₁ were screened in λZAP library as a probe, 3 'to obtain about 100bp long positive clones Z8 to side. However, the molecular weight of the protein derived from the DNA sequence of the Z8 is 34,000 next, delta ^4-3-keto steroids -5β
-Considerably smaller than the molecular weight of the purified reductase sample;
When this cDNA clone Z8 was transfected into OS cells, the expression of the target enzyme protein was not observed.

From these results, the cDNA clone Z
8 appears to be an imperfect clone, so Primer Extended
were screened nded) library and random hexa-priming (Random Hexapriming) library λ ₁₁ insert as a probe, 8 positive clones H23-1, H2
3-2, H23-3, H241, H251, E23, E
24 and E43 were obtained. Since H241 has the longest translation region of the enzyme among them, this cDNA clone H241 was incorporated into plasmid pSVL as shown in FIG. 3, and plasmid pSV5β-241 (the plasmid was Escherichia coli MV1184 pSV5β-
241: FERM P-1221
After constructing an expression vector consisting of 9), the transformed cells were introduced into COS cells and transformed into COS-pSV5β-2.
41 out work, was cultured, the resulting protein as shown in the photograph in FIG. 4, delta ^4-3-keto steroids -
It was confirmed that it reacted with the antibody of 5β-reductase, and when the enzyme activity was examined using androstenedione as a substrate, the enzyme activity was clearly found (specific activity: 2.5 pmol / min / mg).
Protein).

Therefore, the cDNA clone H241
Was found to be a DNA that expresses a delta ^4-3-keto steroids -5β- reductase, this thing, has a nucleotide sequence shown in Figure 2 encoding the amino acid sequence represented by Figure 1 In FIG. 2, the 5 ′ upstream may have a start codon, for example, an ATG encoding Met, and the 3 ′ downstream may have a stop codon, for example, TGA. Good. SEQ ID No. 1 in the sequence listing
To show the entire nucleotide sequence of the delta ^4-3-keto steroids -5β- reductase gene DNA. It has a total length of 3189 bp
The polyA tail is missing and has a 3 'untranslated region of about 2000 bp. The derived enzyme protein has a molecular weight of 3 consisting of 327 amino acids.
7376 is of, and derived from rat liver delta ^4-3-
The six peptide fragments K6, K10, K15, K16, and K shown in FIG. 5 obtained by treating a purified sample of ketosteroid-5β-reductase with lysyl endopeptidase.
17, it was confirmed that the protein contained the entire amino acid sequence of K19.

In the present invention, DNA that express the delta ^4-3-keto steroids -5β- reductase enzyme obtained by screening from a cDNA library constructed plasmid directly integrative expression vector as shown in Example Alternatively, once it has been integrated into a suitable plasmid and introduced into a host microorganism, the transformed host microorganism can be used, for example, by means of Maniatis.
According, such as al ways, delta ^4-3-keto steroids -5
β- reductase gene DNA to prepare recombinant plasmids containing the expression vector and this recombinant plasmid was treated with the appropriate restriction enzymes, including delta ^4-3-keto steroids -5β- reductase gene, respectively After obtaining a DNA fragment and a vector fragment, an expression vector may be constructed by annealing the cohesive ends of each and ligating them using an appropriate DNA ligase.

The type of plasmid used for constructing the expression vector is not particularly limited, and is appropriately selected depending on the type of host cell used. For example, as a host cell, Escherichia coli belonging to the genus Escherichia (E.
coli), for example, E. coli. coli DHI, HB10
1, MV1184, MV1304, W3110,
When using the same C600 strain, plasmid pINI
In the case where Bacillus subtilis is used in, for example, plasmids pTUB218 and pTUB2,
When yeast such as Saccharomyces cerevisiae is used, for example, plasmid pAM82 is preferably used.

On the other hand, monkey kidney (COS) is used as a host cell.
When animal cells such as cells, Chinese hamster overly (CHO) cells, and CHO-dhfr (folate reductase deficient) cell lines are used, viral vectors expressing the foreign gene with the late promoter of the SV40 virus, such as plasmids pSVL and pSV2 -Dhfr and the like can be preferably used.

With respect to the method of introducing the expression vector into the above-mentioned host, for example, when the host cell is a microorganism belonging to the genus Escherichia, the expression vector may be introduced in the presence of, for example, calcium ions, or by using the competent cell method. It may be introduced, and in the case of a microorganism belonging to the genus Bacillus, a competent cell method, a protoplast method, or a microinjection method is used. Selection for the presence or absence of the expression vector introduced into a host microorganism selected microorganisms grown by culturing the microorganism in a selective medium based on the drug resistance marker of the vector, delta ^4-3-keto steroids -5β
Obtaining a substantially pure cell producing reductase.

On the other hand, when the host cell is an animal cell such as COS or CHO, for example, a calcium phosphate method or an electroporation method is used. In addition, drug resistance and the like are used to select animal cells into which the expression vector has been introduced. It drug resistance gene may be incorporated into an expression vector, separately to construct a drug-resistance gene expression vector, performs cotransfection by both vectors mixture substantially to produce delta ^4-3-keto steroids -5β- reductase Get pure cells.

When culturing the cells transformed as described above, when the cells are microorganisms, the cells are cultured in a medium containing nutrients such as carbon sources and nitrogen sources necessary for the growth of the microorganisms and inorganic components. Can be performed. As the carbon source, for example, glucose, starch, sucrose, molasses, dextrin, etc., as the nitrogen source, for example, peptone, meat extract, casein hydrolyzate, corn steep liquor, nitrate, ammonium salt, etc., as inorganic components Examples thereof include salts containing cations such as sodium, potassium, calcium, magnesium, cobalt, zinc, manganese, and iron, and anions such as chlorine, sulfuric acid, and phosphoric acid.

The form of culture is preferably liquid culture, and industrially, it is advantageous to carry out deep aeration stirring culture. The culture temperature, microorganisms are grown, Δ ⁴ -3- keto steroid -5β
-It is appropriately selected within a range in which a reductase is produced. In the case of a microorganism belonging to the genus Escherichia, it is usually selected at a temperature in the range of 20 to 40 ° C. The cultivation time depends on the conditions, but the cultivation may be terminated at an appropriate time in consideration of the time required for the enzyme to reach the maximum yield, and is usually about 12 to 27 hours.

[0021] After culturing in this manner, delta ^4-3-
Keto steroids -5β- reductase separated by directly collecting a liquid culture containing cells may be purified, also, delta ⁴ -
When 3-ketosteroid-5β-reductase is present in the cells, the obtained culture is recovered by filtration or centrifugation, and then the cells are collected by a ball mill or a machine using ultrasonic waves. disruption method and disrupted by enzymatic disruption method such as lysozyme, as necessary to separate collected solubilize delta ^4-3-keto steroids -5β- reductase by adding a chelating agent or a surfactant, It may be purified. For purification methods, for example, delta ^4-3-keto steroids -5β
-Reducing enzyme-containing solution is concentrated under reduced pressure, membrane concentrated, salted out with ammonium sulfate or sodium sulfate, methanol, ethanol,
After means such as fractional precipitation with a hydrophilic organic solvent such as appropriately selected from acetone, to obtain a combined precipitation, the further precipitate containing the delta ^4-3-keto steroids -5β- reductase, needs Accordingly, it may be dissolved in water or a buffer solution and dialyzed with a dialysis membrane to remove impurities of lower molecular weight.In addition, ion exchange chromatography with an adsorbent or a gel filtration agent, adsorption chromatography, It may be purified by a gel filtration method or the like. Thus on substantially obtained by pure delta ^4-3-keto steroids -5β- reductase may be lyophilized if necessary.

On the other hand, when the transformed cells are animal cells such as COS cells and CHO cells, the cells may be cultured using a conventional means for tissue culture of animal cells, for example, the modified Dulbecco's Eagle method. A transformant containing an MEM medium and about 10% by weight of fetal bovine serum is conveniently seeded on a Petri dish at about 10 ⁴ cells / cm ² , usually at a temperature of 30 to 37 ° C. for 2 to 6 days. After culturing to the extent,
Using known enzyme separation and recovery means, the desired Δ ⁴ -3
A solution containing ketosteroid-5β-reductase may be recovered. Thus delta ^4-3-keto steroids -5β- reductase-containing solution thus obtained, by purification in the same manner as in the culture of the transformed microorganism of, substantially pure delta ^4-3- Ketosteroid-5β-reductase is obtained.

The delta ^4-3-keto steroids -5β- reductase obtained by the method of the present invention is the presence of NADPH _2, the property of catalyzing the reactions that reduce 4-position of a double bond in the A / BCIS Steroid Backbone For example, it is useful as a material for developing steroid metabolism-related drugs, a steroid hormone inactivating agent, and an antigen for preparing antibodies such as monoclonal antibodies and polyclonal antibodies as diagnostic agents. Especially monoclonal antibodies to delta ^4-3-keto steroids -5β- reductase can be expected as a diagnostic agent of the enzyme deficiency.

The symbols of the nucleotide sequence and the amino acid sequence described in the specification of the present invention are based on conventional abbreviations in the art, and examples thereof are listed below. In addition, all amino acids are assumed to be in the L form. DNA: deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine Ala: alanine Arg: arginine Asn: asparagine Asp: aspartic acid Cys: cysteine Gln: glutamine Glu: glutamic acid Gly: glycine His: leucine lysine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Val: Valine

[0025]

[Sequence list]

SEQ ID NO: 1 Sequence length: 3189 base pairs Sequence type: nucleic acid Number of strands: double stranded Topology: linear Sequence type: cDNA for mRNA Origin: Synthetic based on rat liver derived Sequence name: delta ^4-3-keto steroids -5β- reductase gene

[0026]

[Table 1]

[0027]

[Table 2]

[0028]

[Table 3]

[0029]

[Table 4]

[0030]

Next, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention. Preparation Example 1 Preparation of poly (A) ⁺ RNA 2 g of isolated rat liver tissue was crushed in liquid nitrogen,
After homogenization in 0 ml of 6M guanidine isothiocyanate, the high molecular weight DNA was cut through a 18.5 gauge needle to reduce the viscosity, and then the homogenate was diluted with 1/3 volume of 5.7M cesium chloride and 0.5%. 1M EDT
A (pH 7.5) overlaid on a solution containing 35,000 rp
and centrifuged at 25 ° C. for 18 hours. After centrifugation,
The precipitate was dissolved in 3 ml of water, treated with an equal volume of phenol / chloroform, the aqueous layer was transferred to another test tube, and 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added thereto, followed by centrifugation. Separation treatment, collection of the precipitate, drying under reduced pressure
200 mg of NA were obtained. After dissolving this crude RNA in 1.5 ml of water, the mixture is kept at 65 ° C. for 5 minutes, quenched, and cooled to 1.5 ml of 40 m
M Tris-HCl (pH 7.6), 1.0 M NaCl, 2 mM
EDTA, 0.2% SDS solution was added. Then, the whole amount of this solution was added to 20 mM Tris-hydrochloric acid (pH 7.6), 0.5 M
After adsorbing on a 75 mg oligo (dT) -cellulose (Pharmacia) column equilibrated with NaCl, 1 mM EDTA and 0.1% SDS, washing with 10 ml of the same solution, 5 ml of 20 mM Tris-HCl (pH 7.1). 6), 0.1M
Po with NaCl, 1 mM EDTA and 0.1% SDS solution
RNA other than ly (A) ⁺ RNA was eluted. Then 10
mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.05%
Poly (A) ⁺ RNA was eluted with the SDS solution, and 1 ml of the first eluted fraction was fractionated. To this, 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added, and the mixture was allowed to stand at −20 ° C. overnight, centrifuged at 15,000 rpm for 30 minutes, and the precipitate was collected and dried under reduced pressure. Thus, 200 μg of poly (A) ⁺ RNA was obtained.

Preparation Example 2 Oligo (dT) prime λgt
Preparation of 11 Library Poly (A) ⁺ RNA obtained in Preparation Example 1 was dissolved in water to 2 μg / μl, and 2.5 μl thereof was transferred to a microtube, heated at 65 ° C. for 5 minutes, and quenched. Later
50 mM Tris-hydrochloric acid (pH 8.3), 10 mM MgC
l ₂ , 140 mM KCl, 10 mM dithiothreitol and 2 mM dNTPs (dATP, dGTP, dCTP, d
A mixture of equal amounts of TTP), 5 μg of oligo (dT) (Pharmacia) and 1.5 units of reverse transcriptase (Takara Shuzo) were added to make a total volume of 20 μl and reacted at 42 ° C. for 1 hour. 80 mM Tris-hydrochloric acid (pH 7.
5), 200 mM KCl, 10 mM MgCl ₂ , 25 μg / m
lBSA, RNase H (Takara Shuzo) 60 units and D
NA polymerase I (Boehringer Mannheim) 5
After adding the unit to make the total volume 150 μl, the reaction was carried out at 12 ° C. for 1 hour, and then the reaction was carried out at 22 ° C. for 1 hour. Then 2
0 μl of 0.25 M EDTA solution and 10 μl of 10%
After the reaction was stopped by adding an SDS solution, an equal amount of phenol / chloroform was added, and the mixture was centrifuged at 10,000 rpm for 5 minutes to separate the aqueous layer. Next, an equal volume of 4M ammonium acetate and 2 volumes of ethanol were added to the aqueous layer, and the mixture was centrifuged at 15,000 rpm for 15 minutes. The precipitate was collected and dried under reduced pressure. Hydrochloric acid (pH 8.0), 10 mM EDTA, 80 μMS-adenosylmethionine, 100 μg / ml BSA and 2 units of EcoRI methylase (Promega Biotech)
Was added to make 10 μl and reacted at 37 ° C. for 1 hour. After the reaction, 40 μl of water was added to the reaction solution, the mixture was treated with an equal amount of phenol / chloroform, and the aqueous layer separated by centrifugation was added with an equal amount of 4M ammonium acetate and 2 times the amount of ethanol to obtain a mixture of -70. After standing at ℃ for 15 minutes,
After centrifugation at 000 rpm for 15 minutes, 67 mM Tris-hydrochloric acid (pH 8.8), 6.7 mM MgCl ₂ , 16.6 mM ammonium sulfate, 10 mM _2- mercaptoethanol,
6.7 μM EDTA, 0.167% BSA, 750 μ each
M dATP, dGTP, dCTP, dTTP and T4
4 units of DNA polymerase (manufactured by Takara Shuzo) was added to make a total volume of 12 μl, and reacted at 37 ° C. for 1 hour. Then, the mixture was treated with an equal amount of a phenol / chloroform solution, precipitated with ethanol, and the precipitate was recovered by centrifugation and dried under reduced pressure.

On the other hand, 1 μg of EcoRI linker
mM Tris-HCl (pH 7.6), 10 mM MgCl ₂ , 10 mM
Dithiothreitol, 0.1 mM spermidine, 0.1 mM E
DTA, 1 mM ATP and 3 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) were added to make a total volume of 10 μl, and reacted at 37 ° C. for 30 minutes. Then, this is T4
Adding the whole amount to the sample after the DNA polymerase treatment,
Further, 60 units of T4 ligase (manufactured by Pharmacia) was added and reacted at 14 ° C. overnight.
0 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM
MMgCl ₂ , 7 mM _2- mercaptoethanol, 100μ
g / ml BSA and 250 units of EcoRI were added and reacted in a total volume of 40 μl at 37 ° C. for 2 hours. This reaction solution was fractionated on a 1% low melting point agarose gel, and
After collecting the gel containing the DNA of the base No. 00,
After the gel was melted, an equal amount of phenol was added thereto, and the mixture was ice-cooled for 10 minutes.
Centrifugation was performed at 4 ° C. for 10 minutes. An equivalent amount of phenol was added to the obtained aqueous layer, and after repeating this operation, the aqueous layer was treated twice with chloroform, and 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added. −70
After centrifugation at 15,000 rpm for 15 minutes, the precipitate was washed twice with 75% ethanol,
It was dried under reduced pressure. Next, 1 μm of a lambda phage vector λgt11 (manufactured by Stratogene) arm was added to the dried precipitate.
g, and the mixture was reacted at 26 ° C. for 10 minutes using a ligation kit (Takara Shuzo), and further reacted using an in vitro packaging kit (Stratogene). The lambda phage vector λgt11 library thus obtained was infected with Escherichia coli Y1090 (manufactured by Stratogene), and the total number was examined. As a result, 1 × 10 ⁶ pfu (plaque form) was obtained.
ing unit).

Preparation Example 3 Preparation of λZAP Library A λZAP library was prepared in the same manner as in Preparation Example 2 except that Lambda phage vector λZAP was used instead of Lambda phage vector λgt11 in Preparation Example 2.

Preparation Example 4 Random prime λgt11 la
Preparation of Ibrary A random prime λgt11 library was prepared in the same manner as in Preparation Example 2, except that a random primer (manufactured by Takara Shuzo) was used instead of oligo (dT) in Preparation Example 2.

Example 1 For the λgt11 library of Preparation Example 2, Δ ⁴ -3
In order to select the cDNA of ketosteroid-5β-reductase, screening was first performed using the enzyme-specific polyclonal antibody. That is, 1 × 10 ⁵ clones of the oligo (dT) prime λgt11 library obtained in Preparation Example 2 were cloned into Escherichia coli Y1090.
Was spread on a 1.5% agar medium (containing 10 g of bactotryptone, 5 g of bactoeast extract, and 10 g of NaCl per liter), and LB
A nitrocellulose membrane soaked in 10 mM IPTG was placed on the lysed plaque on the plate, and cultured for another 3 hours to transfer the protein onto the nitrocellulose membrane. The membrane was then washed with TBST [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% Tween.
20], followed by 20% fetal bovine serum-TBS
Incubation was carried out at room temperature for 30 minutes in solution T.

Next, the membrane was coated with a polyclonal antibody for 20 minutes.
After reacting for 12 hours in a solution diluted 50-fold with a 50% fetal bovine serum-TBST solution, washing with a TBST solution three times for 10 minutes, " ¹²⁵ I" -anti-mouse antibody was added with a 20% fetal bovine serum-TBST solution. The reaction was performed for 2 hours with the liquid diluted 500 times. Next, the membrane was washed 5 times with a TBST solution for 10 minutes, dried by ventilation, and subjected to autoradiogram overnight. The medium at the position where the signal appeared was cut out, and the SM solution [0.1 M NaCl, 8 mM MgSO ₄ , 50 mM Tris-
Hydrochloric acid (pH 7.5), 0.01% gelatin]
After dilution and re-opening on an LB plate, the screening was repeated in the same procedure to purify the plaques, and the eight clones λ ₂ , λ ₄ , λ ₅ , λ ₇ , λ ₉ , λ ₁₁ , λ ₁₂ , λ ₁₃
I got Each of these clones was re-introduced to an LB plate, the lysed plaques on the LB plate were transferred onto a nitrocellulose membrane, and the membrane was 0.5 M N
aOH and 1.5M NaCl solution for 5 minutes, 3M sodium acetate solution (pH 5.5) for 5 minutes,
Dried under reduced pressure at 2 ° C. for 2 hours. Next, this membrane was put into a plastic bag, and 6 times concentration SSC (1 time: 150 mM NaCl,
15 mM sodium citrate), 1x Denhardt's solution (0.1
02% ficoll, 0.02% polyvinylpyrrolidone,
The mixture was kept at 28 ° C. for 4 hours in 10 ml of a solution containing 0.02% BSA) and 0.5% SDS. Next, after removing the solution, 50 mg of a synthetic DNA consisting of 20 bases whose 5 ′ end was labeled with ³² P shown in FIG. 6 was added, and the mixture was hybridized at 28 ° C. overnight. 1% SD
After washing twice with the S solution at 28 ° C. and then drying with air, the autoradiogram was performed overnight, and the seven clones λ ₂ , λ ₅ , λ ₇ , λ ₉ , λ ₁₁ and λ ₁₂ which reacted positively were obtained. , Λ ₁₃
I got

Next, these clones were spread on an LB plate, and 15 ml of each clone was placed on each LB plate.
Phage was infiltrated into SM, transferred to a test tube together with the upper layer agar, centrifuged at 8,000 rpm for 10 minutes, and then 60 units of DNase I (Takara Shuzo) and RNase A (Sigma) were added to the supernatant. 100 μg
Was added and kept at 37 ° C. for 30 minutes. 20 equivalents of this
% Polyethylene glycol and 2.5 M NaCl were added, and the mixture was ice-cooled for 1 hour and centrifuged at 15,000 rpm for 20 minutes to obtain a precipitate. This precipitate was suspended in 0.5 ml of SM solution, and treated by adding an equal volume of chloroform.
Cesium chloride was added to the aqueous layer after centrifugation so that the density was 1.15, and this was added to 1.6 (2 ml).
1.5 (3 ml), layered on a solution of SM to which cesium chloride was added to a volume of 1.4 (3 ml),
Centrifuged for hours. After centrifugation, densities of 1.5 and 1.4
The band containing the phage appearing during the
The precipitate was dissolved in hydrochloric acid (pH 7.5) and centrifuged at 40,000 rpm for 1 hour to obtain a precipitate. This precipitate is
The mixture was treated with mM EDTA, 0.5% SDS, and 50 μg / ml protease K (Sigma) at 65 ° C. for 1 hour, and then an equal amount of phenol was added to the reaction solution. After treating with phenol / chloroform and centrifuging again, the aqueous layer is treated with chloroform. One-tenth volume of 3M sodium acetate and 2.5 volumes of ethanol are added to the aqueous layer and left at -70 ° C for 15 minutes. did. Then 15,
The precipitate was collected by centrifugation at 000 rpm for 15 minutes, washed twice with 75% ethanol, dried under reduced pressure, dissolved in 50 μl of water containing 5 μg / ml RNase A, and 10 μl of H Buffer [Man
iatis et al., Molecular Cloning,
104, Cold SpringHarbor (198)
2) Examination of EcoRI digested to insert during, about the longest fragments delta ^4-3-keto steroids -5β- reductase gene clone with the 3.0kbp were obtained, approximately obtained after digestion 3 The 0.0 kbp insert was recovered by low melting point agarose gel electrophoresis. Next, this insert was completely digested with EcoRI to obtain 1.8 kbp and 1.2 kbp inserts. Using the 1.8 kbp inserted fragment as a probe, Northern blot analysis of the poly (A) ⁺ RNA obtained in Preparation Example 1 revealed that the mRNA size of the enzyme was about 3.5 kbp. , This mRNA
The clone is smaller in size compared to the size of
It was found to be an incomplete clone.

Example 2 Since the clone obtained in Example 1 was an incomplete clone, the λZAP library obtained in Preparation Example 3 and the random primed λgt11 library obtained in Preparation Example 4 were subjected to Δ ⁴ − In order to select a complete cDNA of 3-ketosteroid-5β-reductase, screening was performed using the cDNA clone obtained in Example 1. That is, about 2 × 10 ⁵ clones of the λZAP library and about 2 × 10 ⁵ clones of the random primed λgt11 library were respectively used for Escherichia coli XL1-Blue and Y10
90 was used as an indicator bacterium, spread on a 1.5% LB agar medium (containing 10 g of bactotryptone, 5 g of bactoeast extract, and 10 g of NaCl per liter), and the lysed plaque on the LB plate was transferred to a nylon membrane. , This membrane was treated with 0.5M NaOH, 1.5M NaC.
1M solution for 5 minutes, 3M sodium acetate solution (pH 5.5)
And dried at 80 ° C. under reduced pressure for 2 hours.

Next, the membrane was placed in a plastic bag, and 5 times-concentrated SSC (1 time: 150 mM NaCl, 15 mM sodium citrate), 5 times Denhardt's solution (1 time: 0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02%
BSA), 50 mM sodium phosphate (pH 6.5), 0.1
% SDS (sodium lauryl sulfate), 250 μg / ml
10 ml of a solution containing salmon sperm DNA and 20% formamide
Incubated at 37 ° C for 4 hours. After removing the solution,
To this 5 'about 1.8kbp and 1-labeled end at ³² P.
2 kbp DNA insert probe (10 ⁸ cpm / μg; 1.8 kbp and 1.2 kbp D obtained by completely digesting the phage clone obtained in Example 1 with EcoRI restriction enzyme
After adding 20 ng (using an NA fragment) and hybridizing at 37 ° C. overnight, the membrane was subjected to 2 times SSC, 0.1% SSC.
Washing with a DS solution three times at room temperature and at 37 ° C. for 10 minutes,
Autoradiogram was performed overnight after drying with ventilation. next,
The medium at the position where the signal appeared was cut out, and the SM solution [0.1
M NaCl, 8 mM MgSO ₄ , 50 mM Tris-hydrochloric acid (p
H7.5), 0.01% gelatin], 1 ml, diluted and reapplied on an LB plate, and repeated screening with the same probe to purify plaques to obtain 20 clones. The clone was further infected with Escherichia coli XL1-Blue, and LB agar medium plates (13 cm × 9 cm) were spread on two plates.
Phage was infiltrated into SM by adding 5 ml of SM solution, transferred to a test tube together with the upper layer agar, and centrifuged at 8,000 rpm for 10 minutes. 100 μg of DNase I (Takara Shuzo) and RNase A (Sigma) 60 units in the supernatant
Was added and kept at 37 ° C. for 30 minutes. 20 equivalents of this
% Polyethylene glycol and 2.5 M NaCl were added, and the mixture was ice-cooled for 1 hour and centrifuged at 15,000 rpm for 20 minutes to obtain a precipitate. This precipitate was suspended in 0.5 ml of SM solution, and treated by adding an equal volume of chloroform.
Cesium chloride was added to the aqueous layer after centrifugation so that the density was 1.15, and this was added to 1.6 (2 ml).
1.5 (3 ml), layered on a solution of SM to which cesium chloride was added to a volume of 1.4 (3 ml),
Centrifuged for hours. After centrifugation, densities of 1.5 and 1.4
The band containing the phage appearing during the
The precipitate was dissolved in hydrochloric acid (pH 7.5) and centrifuged at 40,000 rpm for 1 hour to obtain a precipitate.

This precipitate was washed with 20 mM EDTA, 0.5%
SDS, 50 μg / ml protease K (manufactured by Sigma)
At 65 ° C. for 1 hour, then add an equal amount of phenol to the reaction solution, treat the aqueous layer after centrifugation with an equal amount of phenol / chloroform, and again treat the aqueous layer after centrifugation with chloroform. Then, 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added to the aqueous layer, and the mixture was heated at -70 ° C.
Left for 5 minutes. Subsequently, the precipitate was collected by centrifugation at 15,000 rpm for 15 minutes.
2% ethanol, dried under reduced pressure, and then
/ Ml RNase A in 50 μl of water, and 10 μl of the precipitate is dissolved in H buffer [Maniatis et al., Mole.
color Cloning, 104, Cold Sp
ring Harbor (1982)]
Examination of RI digested to insert 'and clones with long fragments of 100bp in the end, 5' 3 than the clones obtained in Example 1 delta has a 100bp long fragment on the end ^4-3-
Each of the ketosteroid-5β-reductase gene clones was obtained, and the respective inserts obtained after digestion were recovered by low melting point agarose gel electrophoresis. These clones were then used for pBluescript p
hagemid to create a restriction map. Both of the two clones obtained had the same restriction enzyme map, and were named Z8 and H241, respectively. Of these two clones, H241 was codin
It was a complete clone containing all the g regions. However, when Z8 was transfected into COS cells, the expression of the target enzyme protein was not observed.

Example 3 The clone p5β-241 (H24) obtained in Example 2
After complete digestion with XbaI using 1), 0.5 units of bacterial alkaline phosphatase (manufactured by Toyobo Co., Ltd.) was added to 50 mM Tris-HCl (pH 8.0), and the mixture was reacted at 65 ° C. for 1 hour (hereinafter, referred to as “1”). Abbreviated as BAP processing). After treating the reaction solution twice with a phenol / chloroform solution, 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added to the aqueous layer, followed by centrifugation to collect the vector. Insert fragment recovered from gel and vector p digested with XhoI
SVL (Pharmacia) was ligated using a ligation kit (Takara Shuzo), and plasmid pSV5
It was named β-241. Exponentially growing Escherichia coli MV1184 (purchased from Takara Shuzo) cultured in 100 ml of LB medium (containing 10 g of bactotryptone, 5 g of bactoeast extract, and 10 g of NaCl per liter) was collected, and 40 ml of ice-cold 30 mM. Potassium acetate, 100 mM RbCl, 10 mM CaCl ₂ , 50 mM Mn
After suspending in Cl ₂ and 15% glycerin (pH 5.8), the cells were left at 0 ° C. for 5 minutes, centrifuged, and the cells were collected.
4 ml of 10 mM MOPS, 75 mM CaCl ₂ , 10 mM Rb
The cells were suspended in Cl and 15% glycerin (pH 6.5) to obtain competent cells. DN of plasmid pSV5β-241 ligated to 200 μl of this E. coli suspension
A solution (20 μl) was added and left at 0 ° C. for 30 minutes.
800 μl of LB medium was added thereto, the mixture was incubated at 37 ° C. for 1 hour, spread on an LB agar plate containing 50 ng / ml of ampicillin, and cultured overnight to obtain a transformant Escherichia coli MV1184pSV5β-241. The Escherichia coli MV1184 pSV5β-241 transformed with the plasmid pSV5β-241 was sent to the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology.
FERM P-12219 ".

Next, the transformed single white colony was cultured overnight in 2 ml of LB medium containing 50 μg / ml ampicillin, and collected by centrifugation. 1mg / ml
After adding 50 mM Tris-hydrochloric acid (pH 8.0), lysozyme (manufactured by Sigma), 50 mM EDTA (pH 8.0), and 0.6 ml of 15% sucrose, the mixture was reacted at 37 ° C. for 15 minutes.
12 μl of 10% SDS was added and mixed, then 60 μl of 5M potassium acetate was added, and the mixture was allowed to stand at 0 ° C. for 30 minutes.
Thereafter, the mixture was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was treated with an equal amount of phenol / chloroform.
The aqueous layer was treated twice with ether, twice the volume of ethanol was added, and the mixture was allowed to stand at -70 ° C for 15 minutes.
The precipitate is collected by centrifugation at 00 rpm for 15 minutes,
After washing twice with 75% ethanol, it was dried under reduced pressure. next,
This precipitate is dissolved in water containing 5 μg / ml of RNase A,
Clones containing the insert were selected by digestion with XbaI. A single colony containing this clone was
After culturing overnight at 37 ° C. in 6 ml of LB medium containing 0 μg / ml, 5 ml thereof was inoculated into 500 ml of LB solution and cultured at 37 ° C. Chloramphenicol (20 μg / ml) was added to the bacterial solution in the logarithmic growth phase, and the mixture was further cultured overnight.
The cells were collected by centrifugation at 0 rpm for 10 minutes, and then
8% sucrose, 10% Triton X, 25 mM EDT
A, 10 mM dissolved in 15 ml of a solution containing 50 mM Tris-HCl (pH 8.0) and 0.25 M Tris-HCl (pH 8.0)
1.5 ml of g / ml lysozyme was added and lysed by heating. Furthermore, the supernatant after centrifugation at 14,000 rpm for 30 minutes was treated by adding an equal amount of phenol / chloroform, and 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added to the aqueous layer. After leaving at -70 ° C for 15 minutes, the precipitate was recovered by centrifugation at 3,000 rpm for 15 minutes.

21 ml of Tris-hydrochloric acid (pH
7.4) was added and dissolved, and 20 g of cesium chloride and 10 m
g / ml of ethidium bromide (1 ml) was added, and 50,000 was added.
After separating the closed circular plasmid DNA by centrifugation at 4 ° C. overnight at 4 ° C., an equal amount of butanol was added thereto to remove ethidium bromide, followed by centrifugation.
The aqueous layer was further treated twice with butanol. The sample was washed with Tris-HCl containing 0.2 M NaCl (pH
After gel filtration to remove impurities by CL4B Sepharose gel (manufactured by Pharmacia) washed in 7.4), 2 volumes of ethanol was added, and the mixture was allowed to stand at -70 ° C for 15 minutes.
Then, the mixture was centrifuged at 3,000 rpm for 30 minutes, and the precipitate was washed twice with 75% ethanol and dried, and the concentration was 1 μg.
/ Μl. In determining the nucleotide sequence over the entire region from the obtained plasmid, the inside of this gene was digested with each restriction enzyme, and each plasmid pBl
euscript and linked to Escherichia coli XL-
Blue was used as a host bacterium for transformation by a conventional method. The single clone confirmed to be inserted was inoculated into 10 ml of a 2 × YT medium (containing 16 g of bactotryptone, 10 g of bactoeast extract, and 5 g of NaCl per liter) containing 50 μg / ml ampicillin and 0.01% thiamine. After culturing at 37 ° C. for 1 hour, helper phage M1
3KO7 (Takara Shuzo) was infected with 6 × 10 ⁸ pfu and cultured overnight. After centrifugation at 10,000 rpm for 10 minutes, a 1/5 volume PEG-NaCl solution (20% polyethylene glycol) was added. 6000, 2.5 M NaCl)
Was added and left at room temperature for 15 minutes. Then 15,000
5 μg / ml was added to the precipitate after centrifugation at 0 rpm for 10 minutes.
Water containing RNase A was added to dissolve, left at room temperature for 15 minutes, and then treated with an equal amount of phenol. The aqueous layer after centrifugation was treated with phenol / chloroform, and 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added to the aqueous layer, and the mixture was left at -70 ° C for 15 minutes.
The precipitate after centrifugation at 00 rpm for 15 minutes was washed twice with 75% ethanol and dried under reduced pressure. The DNA thus obtained was subjected to ExoIII / Mung-beannucl.
Ease deletion system [manufactured by Takara: Gene, 28 , 351-359 (1984)] and Sequence Kit (United Stat)
es Biochemical Co., Ltd.) using a, Δ ⁴ -
The nucleotide sequence near the region encoding 3-ketosteroid-5β-reductase was determined. Figure 1 shows the amino acid sequence,
FIG. 2 shows the nucleotide sequence encoding the amino acid sequence.

[0044] From this result, delta ^4-3-keto steroids -5β- reductase gene consists 978 bases, 326
A gene encoding the amino acid of the residue, starting from ATG encoding the start codon Met, to Met-Asn
-5'- encoding Leu-Ser-Thr-Ala
ATG AAC CTC AGC ACT GCA-
It has a 3 ′ base sequence and the C-terminal amino acid sequence is −
5'-CCA TTTCAT GACGAA encoding Pro-Phe-His-Asp-Glu-Tyr
Have nucleotide sequences of TAC-3 ', DNA fragment containing the nucleotide sequence that encodes a polypeptide that is delta ^4-3-keto steroids -5β- reductase gene from rat having a nucleotide sequence consisting of the stop codon of TGA Was confirmed. The entire nucleotide sequence of DNA containing the delta ^4-3-keto steroids -5β- reductase gene obtained in this manner is shown in SEQ ID NO: 1.

[0045] Example 4 Example 3 was obtained in delta ^4-3-keto steroids -5β-
In order to confirm that the vector into which the reductase gene was inserted expresses the desired enzyme activity, a vector to be expressed in animal cells was constructed. First, clone containing delta ^4-3-keto steroids -5β- reductase gene p5β-2
The XbaI-XbaI fragment (1.9 kbp) of No. 41 was synthesized from "Science" Vol. 234, Nos. 1258-12.
According to page 61 (1986), the plasmid pSVL
Recombined into expression vector. For this recombination, 10 μg of clone p5β-241 DNA was first digested with 15 units of XbaI in H buffer (supra) at 37 ° C. for 2 hours and cut, and then subjected to low-melting point agarose gel electrophoresis. The extract was purified and dried. Separately, plasmid pSVL (Pharmacia) DNA
3 μg of M buffer [Maniatis et al., Molecu
lar Cloning, 104, Cold Spri
ng Harbor (1982)].
aI was added and reacted at 30 ° C. for 2 hours. After the reaction, BA
The DNA was treated with P (described above) and ligated to a DNA fragment of about 1.9 kbp using a ligation kit. This was transfected into COS7 cells by the electroporation method.

The expression was confirmed delta ^4-3-keto steroids -5β- reductase according to Example 5 COS cells. 25 mM Tris-HCl (pH 7.
4) 137 mM NaCl, 5 mM KCl, 0.7 mM
CaCl ₂ , 0.5 mM MgCl ₂ and 0.6 mM Na ₂ H
Buffer consisting of PO ₄ (hereinafter abbreviated as TBS) 17
To 5 μl, 4 mg of DEAE-dextran (Pharmacia) and 10 μg of the ligated DNA were added. This DEAE-dextran / DNA mixture was added to COS7 cells (1 × 10 ⁸ ) cultured in a D-MEM medium (manufactured by GIPCO) containing 10% fetal bovine serum, and electrocoporation was performed by a conventional method. COS7 by law
Cells were transfected. Then, using the same culture solution, cells were cultured at a concentration of 5% CO ₂ at 37 ° C. This culture was measured delta ^4-3-keto steroids -5β- reductase activity. Transfected COS7
After culturing the cells for 60 hours, the cells are collected and Fureb
isu et al. (Biochim. Biophys. A).
cta, 912, 110-114). That is, after transfection,
The COS7 cells cultured for 60 hours are homogenized in 0.1 M phosphate buffer (pH 7.4), and 10,000 rpm,
After centrifugation for 10 minutes, the supernatant was mixed with 100 mM Tris-hydrochloric acid (pH 7.4), 0.6 μmol NADPH and 0.25 μmolar.
l substrate (androstenedione, cortisone, 7α-
Hydroxy-4-cholestan-3-one) and 37
After reaction at 2 ° C. for 2 hours, the reaction was stopped by adding 1 ml of ethanol, and 5 ml of petroleum ether was added to extract the reaction product. The reaction product was subjected to gas chromatography [column: a methylsilicon capillary of a chemical bond type]. (Model CBP1-M50-0 manufactured by Shimadzu Corporation)
25, 0.25 x 50 m), column temperature: 290 ° C, injection point: 320 ° C]. It was also confirmed immunoreactivity by Western blotting using an anti-serum against delta ^4-3-keto steroids -5β- reductase. As a result, a 5β-reaction product could be detected for each substrate. On the other hand, p
The COS7 cells only transfected SVL vector failed to detect delta ^4-3-keto steroids -5β- reductase at all. Further, as a result of Western blotting, a protein that reacts with the antibody was confirmed (FIG. 4). The purified protein was purified to have a molecular weight of 37,000, androstenedione was used as a substrate to convert 5β-androstenedione and NADP from reduced NADP. the resulting reaction was obtained delta ^4-3-keto steroids -5β- enzyme that catalyzes.

[0047]

According to the present invention, mRN derived from rat liver
From the cDNA library obtained based on A,
Delta ^4-3-keto steroids -5β- reductase screening DNA expressing, the transformed cells obtained by the expression vector constructed is introduced into cells using this efficiently delta ⁴ -3 -Ketosteroid-5β-
Produce reductase. The enzyme is useful, for example, as a material for developing steroid metabolism-related drugs, a steroid hormone inactivating agent, an antigen for producing antibodies such as monoclonal antibodies and polyclonal antibodies as diagnostic agents, and the like. Further, the present invention, delta ^4-3-keto steroids -
Since the entire amino acid sequence of 5β-reductase and the nucleotide sequence of the gene DNA encoding this amino acid sequence could be determined, protein engineering such as conversion of substrate specificity of the enzyme and improvement of heat resistance became possible.

[0048]

[Brief description of the drawings]

FIG. 1 is a diagram showing the amino acid sequence of the delta ^4-3-keto steroids -5β- reductase protein.

[0049]

FIG. 2 shows Δ ⁴ − encoding the amino acid sequence of FIG.
It is a figure which shows the base sequence of 3-ketosteroid-5 (beta) -reductase gene DNA.

[0050]

FIG. 3 shows the construction of plasmid pSV5β-241.

[0051]

FIG. 4 shows SDS-PAGE and Mab-5 of proteins expressed in COS-pSV5β-241.
It is a figure which shows the western-blot analysis result using (beta).

[0052]

Figure 5 shows an amino acid sequence of a peptide fragment obtained by treating lysyl endopeptidase delta ^4-3-keto steroids -5β- reductase preparation from liver rats.

[0053]

Figure 6 is in the embodiment 1, showing the nucleotide sequence of the DNA probe used to screen the cDNA of delta ^4-3-keto steroids -5β- reductase from λgt11 library.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁷ Identification code FI (C12N 9/02 C12R 1:91)

Claims (8)

(57) [Claims] (1) Δ has a amino acid sequence ⁴ -3-keto steroids -5
substantially pure cells producing delta ^4-3-keto steroids -5β- reductase, which is a cell transformed by a recombinant plasmid having a DNA expressing β- reductase. 2. The transformed cell is plasmid pSV5.
2. The cell of claim 1, which has been transformed by β-241.
A cell as described. 3. The cell according to claim 2, wherein the cell transformed with the plasmid pSV5β-241 is COS-pSV5β-241. 4. A substantially pure DN expressing Δ ⁴ -3-ketosteroid-5β-reductase having a nucleotide sequence encoding the amino acid sequence shown in FIG.
A. 5. The DNA according to claim 4, wherein the nucleotide sequence is as shown in FIG. 6. Δ has a amino acid sequence ⁴ -3-keto steroids -5
the recombinant plasmid having a DNA expressing β- reductase cultured medium substantially pure cells producing delta ^4-3-keto steroids -5β- reductase is a cell transformed, then the culture delta ^{4-3-producing} method of ketosteroids -5β- reductase and collecting the delta ^4-3-keto steroids -5β- reductase from the object. 7. The transformed cell is plasmid pSV5.
7. Transformation by β-241.
Delta ^{4-3-producing} method of ketosteroids -5β- reductase according. 8. Plasmid pSV5β-241 method for producing delta ^4-3-keto steroids -5β- reductase according to claim 7, wherein the transformed cells are COS-pSV5β-241 by.