JP6795214B2 - Method for Purifying Steroid Hormone Membrane Receptor - Google Patents

Description

The present invention relates to a method for purifying a steroid hormone membrane receptor, particularly a method for purifying a steroid hormone membrane receptor expressed in a recombinant cell.

17α, 20β-dihydroxy-4-prognen-3-one (17,20β-DHP), which is a kind of steroid hormone, is known as an egg maturation-inducing hormone in fish. In a study on the mechanism of fish egg maturation induction mediated by membrane receptors, a progestin membrane receptor (mPR), which is a candidate for steroid membrane receptors, was discovered and expressed in various tissues such as the brain and kidneys. (Non-Patent Documents 1 and 2).

mPR is a 7-transmembrane receptor that forms a novel G protein-coupled receptor family consisting of 11 genes that are conserved in spinal animal cells and that are homologous to the adiponQ receptor, and are progestin and adiponQ receptors (progestin and adiponQ receptors). It is named the PAQR) family. There are five types of mPR molecules, α, β, γ, δ, and ε, which correspond to PAQRs 7, 8, 5, 6 and 9, respectively (Non-Patent Documents 3 and 4).

Examining the interaction of chemical substances with steroid membrane receptors has become an important approach in drug discovery research. Currently, such drug discovery research is evaluated by a technique using cultured cells into which a membrane receptor gene has been introduced or a cell membrane fraction thereof. Screening of drug candidates that act on steroid membrane receptors has the advantage of being able to test biological reactions stimulated by hormones because living cells are used. At the same time, there are disadvantages such as low expression level of steroid membrane receptor in cultured cells, use of radioisotope, and need to prepare each cell membrane fraction. Moreover, since the cost of cell culture is high, it is considered difficult to put it into practical use for screening a large number of samples.

Proc. Natl. Acad. Sci. USA, 100, 2221-2236 (2003) Gen. Comp. Endo., 145, 101-108 (2006) Steroids.73, 1160-1173 (2008) Endocrinology, 154, 283-295 (2013)

The present invention provides a simpler method for screening a substance that targets a steroid membrane receptor, and more specifically, a method for purifying a steroid membrane receptor having steroid hormone binding activity. The purpose is to do.

As a result of diligent research to solve the above problems, the present inventors have unexpectedly found that a steroid membrane receptor having steroid hormone binding activity can be purified from a membrane fraction of a cell that forcibly expresses a steroid membrane receptor. The present invention has been completed.

That is, in the present invention, a step of introducing a gene encoding a steroid membrane receptor into a host cell to obtain a recombinant cell and culturing the recombinant cell to express the steroid membrane receptor so that the steroid membrane receptor can be expressed. Provided is a method for purifying a steroid membrane receptor, which comprises a step of expressing the receptor and a step of recovering a membrane fraction containing the steroid membrane receptor and purifying the steroid membrane receptor.

In one embodiment, the steroid membrane receptor can be a progestin receptor (mPR). Also, in other embodiments, the steroid membrane receptor can be the progestin receptor (mPR) α, β, γ, δ, or ε.

In another embodiment, the host cell can be Pichia pastoris.

In a further embodiment, the purification step can be performed by affinity chromatography. The affinity chromatography may utilize a tag selected from His tag, c-Myc tag, GST tag, MBP tag, FLAG tag, HQ tag, HN tag, HAT tag and V5 tag.

In addition, the purification step can be performed with a cellulose gel to which an amino group has been added. The cellulose gel to which the amino group is added can be Cellfine® amino. In addition, the Cellfine® amino can be used for purification without ligand immobilization.

The present invention also provides a steroid membrane receptor purified by the above purification method. The present invention further provides a method of using the steroid membrane receptor in screening for a substance that targets the steroid membrane receptor.

According to the purification method of the present invention, a steroid membrane receptor having steroid-binding activity, particularly mPR, can be easily obtained in a large amount. If this steroid membrane receptor is used, it can be used for screening substances that target the steroid membrane receptor, particularly candidate drugs.

It is a figure which shows the design of the part where the mPRα gene was introduced in the plasmid mPRα-pPICZαC for expressing mPRα protein constructed in Example 1. It is a figure which shows the result of electrophoresis of the PCR product of the Pichia pastoris strain X-33 which introduced the mPRα gene prepared in Example 1. It is a figure which shows the measurement result of the specific binding activity to a steroid performed in Example 3. (A) shows the confirmation result of the expression of mPRα protein by Western blotting, and (B) shows the measurement result of the specific binding of the membrane fraction to the radioligand in the presence of different concentrations of digitonin or colate. (C) shows the measurement results of the membrane fraction of Tr-X-33 and the specific binding to the radioligand by the membrane fraction of X-33 in the presence of jigitonin, and (D) shows the membrane of Tr-X-33. The results of scutchard plot analysis of specific binding to radioligand by fraction are shown. It is a figure which shows SDS-PAGE of the examination result of the solubilization condition of the mPRα-expressing cell membrane fraction in Example 4. It is a figure which shows the result after the cell fine (registered trademark) amino purification in Example 6. (A) shows the absorbance, and (B) shows the results of Western blotting of the sample after purifying the c-Myc tag. It is a figure which shows the result after the c-Myc tag purification in Example 6. (A) shows the results of silver staining of each purification step, and (B) shows the results of Western blotting of the sample after c-Myc tag purification. It is a figure which shows the result of MALDI-TOF-MS of the purified mPRα protein in Example 7. It is a figure which shows the result of the specific binding to 17,20β-DHP by the purified mPRα protein in Example 8.

The method for purifying a steroid membrane receptor of the present invention involves introducing a gene encoding a steroid membrane receptor into a cell so as to express the steroid membrane receptor to obtain a recombinant cell, and the recombinant cell. It includes a step of culturing and expressing a steroid membrane receptor, and a step of collecting a membrane fraction containing the steroid membrane receptor and purifying the steroid membrane receptor.

A conventionally known gene recombination method can be used for the step of introducing a gene encoding a steroid membrane receptor into a host cell to obtain a recombinant cell so that the steroid membrane receptor can be expressed.

Examples of the steroid membrane receptor include progestin receptor (mPR), and for example, progestin receptor (mPR) α, β, γ (including γ-1 and γ-2), δ, or ε is preferable. Can be used. Steroid membrane receptors are conserved in vertebrate cells and include genes for steroid membrane receptors such as goldfish, zebrafish, medaka, Xenopus laevis, chickens, mice, rat rabbits, guinea pigs, pigs, cows, monkeys, etc. Alternatively, human-derived ones can be preferably used, and some sequences of these genes have been identified. Among them, the mPRα gene derived from goldfish has access No. Known as AB12087. Steroid membrane receptors include ligands such as steroid hormones such as 17,20β-DHP, 17-hydroxyprogesterone, progesterone, testosterone, or 17,20β, 21-trihydroxy-4-pregnane-3-one (20β-S). It binds specifically.

The host cell is not particularly limited as long as it can express a steroid membrane receptor on the cell membrane. From the viewpoint of the expression level of the steroid membrane receptor, Pichia pastoris, Sf9 insect cells and the like can be mentioned, and among them, Pichia pastoris is particularly preferably used.

As the vector, in consideration of compatibility between the promoter and the host cell, for example, in the case of Pichia pastoris, pPICZα, pPICZ, pPIC3.5 or the like can be used. In addition, it is preferable that an antibiotic resistance gene such as zeocin is incorporated so that recombinant cells can be easily selected. The steroid membrane receptor gene is inserted so as to produce a fusion protein with a His tag, a c-Myc tag, a FLAG tag, etc. located upstream or downstream thereof in order to facilitate purification by affinity chromatography. Is preferable.

The gene transfer method is not particularly limited, but can be appropriately selected by those skilled in the art depending on the host cell and vector. For example, methods such as electroporation, lipofection, or the calcium phosphate method can be used.

Regarding the step of culturing the recombinant cell and expressing the steroid membrane receptor, it can be carried out by a conventionally known cell culturing method.

The cell culture medium can be appropriately selected by those skilled in the art depending on the host cell, and in the case of Pichia pastoris, BMGY medium or BMMY medium is preferably used. When the recombinant cells have antibiotic resistance, it is preferable to add an appropriate concentration of antibiotics. In the recombinant cell, it is preferable that the promoter, the steroid membrane receptor gene and the tag gene are integrated into the genomic gene of the cell.

Expression of steroid membrane receptors on the membrane of recombinant cells can be confirmed by specific binding activity with 17,20β-DHP. As such a method, for example, a steroid binding experiment using a membrane fraction of a steroid membrane receptor gene-introduced cell can be mentioned. In such an experiment, for example, a specific binding amount of 17,20β-DHP can be measured by a competitive inhibition experiment between a radiation-labeled 17,20β-DHP and an unlabeled 17,20β-DHP.

In the step of recovering the membrane fraction containing the steroid membrane receptor and purifying the steroid membrane receptor, as a method for recovering the membrane fraction, the membrane fraction may be recovered by a conventionally known method. For example, a recombinant cell can be crushed by a cell crusher, and the supernatant obtained by centrifugation at 1000 × g for 7 minutes at 4 ° C. can be centrifuged at 20000 × g for 20 minutes at 4 ° C. and collected as a precipitate. Can be mentioned. The obtained membrane fraction may be solubilized in a buffer such as Lysis buffer (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 10 mM imidazole) containing 0.1% DDM and 1 mM PMSF.

Purification of the steroid membrane receptor protein is preferably performed by affinity chromatography, especially His tag, c-Myc tag, GST tag, MBP tag, FLAG tag, HQ tag, HN tag, HAT tag, V5 tag and the like. It is preferable to use a tag selected from.

In the purification step, it is particularly preferable to purify using a cellulose gel to which an amino group is added. The cellulose gel to which the amino group is added is preferably Cellfine (registered trademark) amino, and can be used as it is for purification without a ligand being immobilized on the cellfine (registered trademark) amino. preferable.

The steroid membrane receptor of the present invention has been purified by the above purification method. The steroid membrane receptor obtained by the purification method of the present invention is 17,20β-DHP, 17-hydroxyprogesterone, progesterone, testosterone, or 17,20β, 21-trihydroxy-similar to the wild type steroid membrane receptor. It has the activity of specifically binding to ligands such as steroid hormones such as 4-pregnen-3-one (20β-S). Therefore, the steroid membrane receptor can be used in screening for substances that target the steroid membrane receptor, particularly drug candidate substances.

The use of purified steroid membrane receptors in the above screening includes, for example, screening of pharmaceuticals and dietary supplements, safety evaluation of chemicals, preparation of monoclonal antibodies and the like.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.

(reagent)
17,20β-DHP was obtained from Sigma, 3.alpha., Radiolabeled by enzymatic conversion using 20β- hydroxy steroid dehydrogenase (Sigma ^Chemical), to obtain a 3 H-17,20β-DHP. Trypsin, modification (sequence grade) from Promega, α-cyano-4-hydroxysilicate skin acid (CHCA) and peptide calibration standard from Bruker Daltonics, jigitonin from Sigma Aldrich Chemical, DNA polymerase and ligation kit Is from Takara Bio, restriction enzymes are from Wako Pure Chemical Industries, Takara Bio, or New England Biolab, and DNA extraction kits for extracting DNA fragments from agarose gels are from QIAGEN, SDS-polyacrylamide gel electrophoresis. The molecular weight marker for (SDS-PAGE) is from Bio-Rad, the peroxidase (HRP) -labeled anti-rabbit antibody, and the YNB yeast medium without amino acids is from Invitrogen, and the BSA is from Sigma Chemical, an anti-His tag antibody. And anti-c-Myc tag antibodies were obtained from Medical & Biological Laboratories. Other reagents were obtained from Wako Pure Chemical Industries, Ltd. unless otherwise specified. Unless otherwise specified, the reagents to be used were used according to the usage method described in the instruction manual.

(Example 1 Preparation of P. pastoris cells for mPRα protein expression)
The inventors have previously incorporated the cDNA of goldfish mPRα (access No. AB122087) into the pBK-CMV plasmid (Stratagene). This plasmid was cleaved with restriction enzymes EcoRI and XhoI, electrophoresed on a 1.2% agarose gel, and a DNA fragment of about 1100 bp mPRα was recovered and purified by a conventional method.

After treating the purified DNA fragment with the restriction enzymes EcoRI and NotI, the expression vector pPICZαC (Invitrogen) cleaved with the same restriction enzyme was used for DNA Ligation Kit Ver. Ligation was performed by mixing with solutions A and B and incubating at 16 ° C. for 16 hours.

Mix the ligation product with Escherichia coli strain TOP10F'suspended in CaCl ₂ solution (60 mM calcium chloride, 15% glycerol, 10 mM PIPES, pH 7.0) and leave on ice for 20 minutes, 42 ° C., 30 seconds, leave on ice 2 It was transformed into Escherichia coli strain TOP10F'by minute, sown on an LB plate containing 25 μg / ml zeosin, and allowed to stand overnight at 37 ° C. The obtained colonies were inoculated into 400 ml of LB medium containing 25 μg / ml zeocin, cultured with shaking at 37 ° C. overnight, and plasmid DNA was extracted by an alkaline lysis method.

Using the obtained colony as a template, 5'-AOX1 primer (5'-GACTGGCATCCAAATTGACAAGC-3'; SEQ ID NO: 1) and 3'-AOX1 primer (5'-GCAAATTGGCATTCTGACATCC-3'; SEQ ID NO: 2) were used to Ex. After 5 minutes at 94 ° C. using Taq polymerase, 30 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute were repeated for 30 cycles, and then PCR was performed at 72 ° C. for 10 minutes. Colonies into which the mPRα gene expression plasmid had been introduced were selected according to the size of the PCR product (about 1650 bp) and cultured in LB medium containing 25 μg / mL zeocin.

The plasmid DNA was extracted from the obtained cells by an alkali lysis method, RNA was degraded by RNase treatment, and then the mPRα gene sequence in the plasmid pBK-CMV was subjected to a DNA sequence using a capillary DNA sequencer CEQ2000XL (Beckman Coulter) and a primer. As a result, it was confirmed using 5'-and 3'-AOX1 primers.

The constructed plasmid for expressing mPRα protein was named mPRα-pPICZαC. As shown in FIG. 1, mPRα-pPICZαC has an α factor, mPRα, c-Myc tag, His tag and AOX1TT gene in order so that they can be expressed downstream of the promoter pAOX1.

Pichia pastoris (P. pastoris) strain X-33 in 400 mL YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%, agar 2%) until OD ₆₀₀ = 1.0-2.0. It was cultured at 37 ° C. overnight. Cells were collected by centrifugation at 1500 xg for 5 minutes at 4 ° C., resuspended in ice-cooled redistilled water, repeated 2-3 times, and finally resuspended in ice-cooled 1M sorbitol. The volume was 4 mL and used for gene recombination.

On the other hand, the above-mentioned expression plasmid mPRα-pPICZαC was treated with the restriction enzyme PmeI, cleaved at one site, and linearized. The linearized plasmid (about 9 μg) was mixed with the obtained X-33 cell suspension for gene recombination and electroporated with Gene Pulser II (Bio-Rad) under the conditions of 1500 V, 25 μF and 200 Ω. Was done. The cells after electroporation were sown on a YPDS (YPD containing 1M sorbitol) plate containing 100 μg / mL zeocin and allowed to stand at 30 ° C. for 2 days. The obtained colonies were replanted on MD plates, allowed to stand at 30 ° C. overnight, and then stored at 4 ° C.

Each colony was inoculated into a test tube containing 1.6 mL MGY medium (Yeast Nitrogen Base 1.34%, glycerol 1%, biotin 4 × ^10-5 %) and cultured in an incubator at 30 ° C. and 175 rpm overnight with shaking. Collect cells and wash twice with sterile ultra-pure water. Resuspend in breaking buffer (50 mM sodium phosphate, pH 7.4, 1 mM EDTA, 5% glycerol) and add equal amounts of phenol / chloroform and glass beads. Crushed with vortex. Spin down by centrifugation for a few seconds and transfer the aqueous layer to a new tube. DNA was precipitated with ethanol, lysed at TE, pH 7.4, and Total DNA was extracted. The extracted Total DNA was used as a template. As a control, PCR products of wild-type cells, mPRα-pPICZαC plasmid and pPICZαC plasmid were used as templates for amplification by PCR using 5'-AOX1 primer and 3'-AOX1 primer. There was.

The PCR product was confirmed by electrophoresis on an agarose gel (Fig. 2). From the left, the line of high molecular weight marker (M), wild type PCR product (X-33), PCR product of recombinant cells (Tr-X-33), mPRα-pPICZαC, pPICZαC, and low molecular weight marker (M). It was confirmed that the gene transfer was successful from the size of the PCR amplification product. The cells of this colony were named mPRαX33.

30 mL of single colony of mPRαX33 in BMGY medium (yeast extract 1%, Bacto peptone 2%, potassium phosphate 100 mM, pH 6.0, amino acid-free yeast nitrogen base 1.34%, biotin 4 × ^10-5 %, glycerol 1 %) Was inoculated into a 200 mL flask and cultured in an incubator at 30 ° C. and 250 rpm with shaking overnight. 5 mL of the culture solution was inoculated into a 500 mL flask containing 100 mL of BMGY medium, and further cultured in an incubator at 30 ° C. and 250 rpm with shaking overnight. Centrifuge at 3000 × g for 5 minutes at room temperature, and settle the precipitate in 2 L of BMMY medium (yeast extract 1%, Bacto peptone 2%, potassium phosphate 100 mM, pH 6.0, amino acid-free yeast nitrogen base 1.34%, biotin. It was suspended in 4 × ^10-5 %, 0.5% methanol), and it was confirmed that the OD was in the range of ₆₀₀ = 1.0 to 5.0. The cells were cultured in an incubator at 20 ° C. and 180 rpm with shaking overnight. The cells were centrifuged at 4000 rpm for 5 minutes at 4 ° C., the cells were collected as a precipitate, frozen in liquid nitrogen, stored at -80 ° C., and used for the expression of the protein of Example 2.

(Example 2 Crushing and solubilizing mPRα protein-expressing cells)
20 mL of Lysis buffer (0.1% digitonin, 1 mM PMSF) was added to the mPRαX33 cells obtained in Example 1. 5 mL suspension and 1.5 mL zirconia beads were placed in 5 crushing tubes. After crushing with a cell crusher MS-100R at 4000 rpm for 300 seconds, the operation of leaving the cells on ice for 5 minutes was repeated 6 times. Centrifugal at 1000 × g for 7 minutes at 4 ° C. The supernatant was collected in a centrifuge tube (Sup1).

Next, 4 mL of Lysis buffer was added to each crushing tube containing the precipitate, and the cells were similarly crushed with a cell crusher MS-100R at 4000 rpm for 300 seconds, and then left on ice for 5 minutes, and the operation was repeated 6 times. Centrifuge at 1000 xg for 7 minutes at 4 ° C. and the supernatant was collected in a centrifuge tube (Sup2).

Subsequently, 4 mL of Lysis buffer was added to each crushing tube containing the precipitate, and the cells were similarly crushed with a cell crusher MS-100R at 4000 rpm for 300 seconds, and then left on ice for 5 minutes, which was repeated 6 times. Centrifuge at 1000 xg for 7 minutes at 4 ° C. and the supernatant was collected in a centrifuge tube (Sup3).

Finally, 4 mL of Lysis buffer was added to each crushing tube containing the precipitate, and the cells were similarly crushed with a cell crusher MS-100R at 4000 rpm for 300 seconds, and the obtained suspension was recovered as a sample for electrophoresis. (Debris).

The collected supernatants (Supp1 to 3) were centrifuged at 20000 × g for 20 minutes at 4 ° C., and the precipitate was suspended in Lysis buffer to obtain a 200 mL suspension (cell membrane fraction). The protein concentration was measured with a protein assay stain (Bio-Rad).

(Example 3 Expression of mPRα protein and verification of specific binding activity to 17,20β-DHP)
SDS-PAGE was performed on the cell membrane fraction (20,000 × g, 20 minutes, centrifugation fraction at 4 ° C.) prepared from the X33 cells and mPRαX33 cells obtained in Example 2, and the mPRα protein was obtained by Western blotting. Expression was confirmed (A in FIG. 3). Western blotting with an anti-His tag antibody detected a non-specific protein band in the original X33 cell fraction, whereas the mPRαX33 cell fraction detected two other protein bands. The two bands indicated by the arrows are mPRα, the upper band is mPRα to which the α factor signal sequence is bound, and the lower band is mPRα to which the α factor signal sequence is cleaved, which is later MALDI-TOF. / Confirmed by MS.

Next, the experimental conditions under which the membrane fraction of cells expressing the mPRα protein and the specific binding of the radioligand can be detected were examined. For membrane fraction samples suspended in HEAD buffer (HEPES 25 mM, NaCl 10 mM, dithioerythritol 1 mM, EDTA 1 mM), add final concentrations of 0.001%, 0.01%, 0.1% digitonin or cholic acid salt. The following operations were performed for the case where nothing was added and the case where nothing was added. The GF / B filter was immersed in a HEAW buffer (HEPES 25 mM, NaCl 10 mM, EDTA 1 mM, pH 7.4) containing 2.5% TWEEN 80 for 30 minutes. 4 μL of 1 μM DHP or EtOH was placed in a test tube. ^[3 H] ^-17,20β-DHP of 4nM there (40 Ci / mmol) were placed one by 200 [mu] L (this one series 3), then put in a protein sample by 200 [mu] L, and allowed to stand for 30 minutes at 4 ° C. with gentle shaking. GF / B was set in the suction device, and 400 μL of the sample was dropped in the center of the GF / B. It was washed 3 times at 4 ° C. using a HEAW buffer. The GF / B was removed into a vial. Liquid scintillation was placed in a vial, shaken vigorously for 15 minutes and measured with a scintillation counter. The measurement results are shown in B of FIG. As a result, the highest binding activity was detected when the final concentration of 0.1% digitonin was added, so the specific binding of the mPRα protein expressed under these conditions to 17,20β-DHP was measured. The result is shown in C of FIG.

The GF / B filter was then immersed in a HEAW buffer containing 2.5% TWEEN80 for 30 minutes for specific binding of the expressed mPRα protein to 17,20β-DHP. A 100-fold molecular excess of DHP or EtOH was placed in a test tube in an amount of 4 μL each. Furthermore, ^[3 H] -17,20β-DHP of 2.5~10nM the (40 Ci / mmol) were placed in each 200 [mu] L (this one series 3). 200 μL of a membrane fraction sample containing 0.2% digitonin was added, and the mixture was gently shaken and allowed to stand at 4 ° C. for 30 minutes. GF / B was set in the suction device, and 400 μL of the sample was dropped in the center of the GF / B filter. It was washed 3 times at 4 ° C. using a HEAW buffer. The GF / B filter was removed into a vial. Liquid scintillation was placed in a vial, shaken vigorously for 15 minutes and measured with a scintillation counter. The measurement results are shown in D of FIG. The inset in the upper right is data showing the amount of bound saturation at each radioligand concentration, and this data is shown as a Scatchard plot. From this result, it was determined that the maximum binding amount of 17,20β-DHP was 67 pmol / g and the binding affinity was 9.4 nM. This result is almost in agreement with the data of the receptor on the goldfish egg membrane and the goldfish mPRα protein expressed in cultured cells reported by Tokumoto et al. (Endocrinology, 148, 3459-3467 (2007)), which is normal. It was confirmed that the mPRα protein having a three-dimensional structure could be expressed.

(Example 4 Examination of solubilization conditions for mPRα-expressing cell membrane fraction)
The protein concentration of the cell membrane fraction prepared from the mPRαX33 cells obtained in Example 2 was diluted to 1 mg / mL or less (usually 0.8 mg / mL), and the solubilization conditions of the mPRα protein were examined. .. Various surfactants (MEGA-10, FOS, OTG, Tween-20, Tween-80, DDM, DDA or OG) were added to the cell membrane fraction so that the final concentration was 0.1% or 0.01%. It was. After reacting at 4 ° C. for 30 minutes, the mixture was centrifuged at 20000 × g for 20 minutes at 4 ° C., and 120 μL of the supernatant was transferred to another tube to prepare a sample for electrophoresis. 120 μL of Lysis buffer was added to the remaining precipitate to prepare a sample for electrophoresis in the same manner. SDS-PAGE was performed on these samples, and the solubilization status of mPRα protein was examined by Western blotting (Fig. 4).

(Example 5 Solubilization of mPRα protein)
The protein concentration of 200 mL of the cell membrane fraction prepared from the mPRαX33 cells obtained in Example 2 was diluted to 1 mg / mL or less (usually 0.8 mg / mL), and the final amounts of DDM, PMSF and glycerol were reduced. It was added to 0.1% DDM, 1 mM PMSF, and 10% glycerol. It was transferred to a beaker and stirred with a stirrer at room temperature for 20 minutes or longer. Centrifugation was carried out at 20000 × g for 30 minutes at 4 ° C., and the supernatant was collected as a solubilized sample and used for purification of mPRα protein.

(Example 6 Purification of mPRα protein)
<Ni-NTA purification>
25 mL of Ni-NTA resin was replaced with Lysis buffer (0.01% DDM, 1 mM PMSF) and loaded into a column. Lysis buffer was run through the column with a perista pump (flow rate 1 mL / min or less) until the absorbance stabilized and equilibrated. A 10 mL Lysis buffer (0.01% DDM, 1 mM PMSF) was flowed, and the absorbance after equilibration was set to 0.

The entire amount of the solubilized sample for purification of the mPRα protein obtained in Example 5 was applied to the column at a flow rate of 0.5 mL / min or less, and the thru was collected in a beaker. Lysis buffer (0.01% DDM, 1 mM PMSF) was run through the column until the absorbance stabilized and the thru was collected in a beaker. Start gradient elution with 250 mL of Lysis buffer containing 10-250 mM imidazole and 250 mL of Elution buffer (0.01% DDM, 1 mM PMSF, 250 mM imidazole) at a flow rate of 1 mL / min, after which all eluates have a fraction collector. 20 mL each was collected using. Finally, 60 mL of Elution buffer was flowed. For each of the obtained fractions, the absorbance was measured using BIO-MINI UV MONITOR AC-5200L.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on each fraction, and the elution pattern was confirmed by CBB staining and Western blotting. SDS-PAGE was performed according to the Laemmli method under denaturing conditions using a 12% polyacrylamide gel. For CBB staining, soak in a staining solution (50% trichloroacetic acid solution containing 0.25% Coomassie Brilliant Blue R-250) for 3 to 5 minutes, wash with water for about 10 minutes, and then destaining solution (5% methanol, 7.5). The destaining solution with (% acetic acid solution) was exchanged several times and performed overnight. Western blotting was performed after transcribing the SDS-PAGE protein pattern onto an Immunobillon membrane (Millipore). Membranes were blocked at room temperature for 1-2 hours with 20 mM Tris buffer containing 5% skim milk, pH 7.6, TBS containing 0.1% Tween. It was then reacted with the anti-His tag antibody of the primary antibody diluted 500-fold with TBS buffer, washed and reacted with the HRP-labeled anti-rabbit antibody of the secondary antibody diluted 2000-fold with TBS buffer. An enhanced chemiluminescence (ECL) detection kit (PerkinElmer) was used to visualize the mPRα protein of interest. The signal was digitized using a CCD camera system (Luminescent Image Analyzer LAS-4000 mini, Fujifilm).

Concentration and buffer exchange Fractions in which elution of mPRα was confirmed were put together in a beaker and mixed. It was filtered using an ultrafiltration filter CentriprepYM3, centrifuged at 2500 rpm for 20 minutes at 4 ° C., and concentrated to a liquid volume of 14 mL. 50 mM Tris was added, centrifugation and concentration were repeated 3 times, and the mixture was replaced with Tris-HCl buffer and pH 8.0 so that the NaCl concentration became 1 mM or less, and the final liquid volume was 47.5 mL.

<Celfine (registered trademark) amino purification>
5 mL of Cellfine Amino resin (JNC) was replaced with Starting buffer (0.01% DDM, 1 mM PMSF) and loaded into a column. The Starting buffer was run through the column at a flow rate of 1 mL / min or less using a perista pump until the absorbance stabilized and equilibrated. A 10 mL Starting buffer was flowed, and the absorbance after equilibration was set to 0.

The entire volume of the concentrated and buffered fraction mixture was applied to a Cellfine® amino column at a flow rate of 1 mL / min or less and the thru was collected in a beaker. The Starting buffer was run through the column until the absorbance stabilized and the thru was collected in a beaker.

Start gradient elution at a flow rate of 1 mL / min with a Starting buffer containing 50 mL 0-0.5 M NaCl and a 50 m Elution buffer (0.01% DDM, 1 mM PMS, 0.5 M NaCl), and all eluates thereafter. Collected 5 mL each using a fraction collector. Finally, 15 mL of Elution buffer was flowed.

For each fraction, the absorbance was measured in the same manner as above. The result is shown in FIG. 5 (A). SDS-PAGE was performed and the elution pattern was confirmed by Western blotting. The results are shown in FIG. 5 (B). The two bands indicated by the arrows are mPRα, the upper band is mPRα to which the α factor signal sequence is bound, and the lower band is mPRα to which the α factor signal sequence is cleaved, which is later MALDI-TOF. / Confirmed by MS.

Concentration and buffer exchange Fractions in which elution of mPRα was confirmed were put together in a beaker and mixed. It was filtered using an ultrafiltration filter Centriprep YM3, centrifuged at 2500 rpm for 20 minutes at 4 ° C., and concentrated to a liquid volume of 3 mL. PBS was added and centrifugation / concentration was repeated 3 times to replace with PBS buffer, and the final volume was 1.6 mL. It was frozen in liquid nitrogen and stored at −80 ° C.

<C-Myc tag purification>
The concentrated and buffer-exchanged samples were thawed and centrifuged at 20000 xg for 20 minutes at 4 ° C. to collect the supernatant. A mini-column for nucleic acid was packed with 10 μL of anti-c-Myc tag beads (Protein Mild Purification Kit, MBL). The obtained supernatant was applied to a 100 μL column, allowed to stand for 10 minutes, and then flush-centrifuged. 100 μL of PBS was flushed once and flush centrifuged. 100 μL of 1 × Wash buffer was run twice. A 50 μL c-Myc tag peptide solution was run once and eluted. C-Myc tag purified protein, along with Ni-NTA purified protein and Celfine® amino purified protein, CBB stained or 2D-silver stained reagent II "first" (first chemistry) as described above. Silver staining was performed according to the manual of Yakuhin Co., Ltd.), and the protein after c-Myc tag purification was confirmed by Western blotting in the same manner as above. For the His tag, a rabbit anti-His tag was used as the primary antibody, an HRP-labeled anti-rabbit antibody was used as the secondary antibody, and for the c-Myc tag, a mouse anti-c-Myc tag was used as the primary antibody and an HRP-labeled anti-mouse antibody was used as the secondary antibody. Using.

The results are shown in FIG. From (A), it was confirmed that the mPRα protein that had undergone Ni-NTA purification, Cellfine (registered trademark) amino purification, and c-Myc tag purification could be specifically purified and concentrated as compared with other proteins. .. The two bands indicated by the arrows in (B) are mPRα, the upper band is mPRα to which the α factor signal sequence is bound, and the lower band is mPRα to which the α factor signal sequence is cleaved. Confirmed by MALDI-TOF / MS.

(Example 7 Mass fingerprinting (PMF) analysis of peptide of purified mPRα protein)
The purified mPRα protein was treated with trypsin and PMF analysis by MALDI-TOF-MS was performed using autoflex (Bruker d'Altonics). As a database for analysis, Mascot (Matrics Science) was used.

The detection peak in MALDI-TOF-MS is shown in FIG. As a result of PMF analysis, the two protein bands detected by Western blotting in the Mascot database are goldfish progestin membrane receptor alpha (accession number 40714507, theoretical mass 41647, theoretical isoelectric point 6.95, number of matched peptides). 6. The sequence coverage area was 18%), and it was confirmed that the purified protein was mPRα protein.

(Example 8 Specific binding to 17,20β-DHP by mPRα protein-expressing cell membrane)
The mPRαX33 cells obtained in Example 1 were cultured as described above to express the mPRα protein. As a control, X33 cells not transformed into mPR were used. 200 μL of the cell culture solution recovered after expression was suspended in HEAD buffer (HEPES 25 mM, NaCl 10 mM, dithioerythritol 1 mM, EDTA 1 mM) containing 1.5 mL of 0.1% digitonin, and then transferred to a 2 mL tube and 200 μL zirconia. Beads were added. After crushing with a cell crusher MS-100R at 4000 rpm for 300 seconds, the operation of leaving the cells on ice for 5 minutes was repeated 6 times. Centrifugation was carried out at 1000 × g for 7 minutes at 4 ° C., and the supernatant was transferred to a 1.5 mL tube and centrifuged at 20000 × g for 20 minutes at 4 ° C. The precipitate is resuspended in 1 mL of HEAD buffer to a liquid volume of 4 mL and suspended until the grains disappear. Protein was quantified in the same manner as above, and diluted with HEAD buffer containing 0.2% digitonin to give a final concentration of 1 mg protein / mL.

The mPRα protein was purified in the same manner as in Example 6, and the concentrated sample after purification was protein quantified and then replaced with a HEAD buffer.

The GF / B filter was immersed in a HEAW buffer (HEPES 25 mM, NaCl 10 mM, EDTA 1 mM, pH 7.4) for 30 minutes. 4 μL of 1 μM DHP or EtOH was placed in a test tube. Furthermore, ³ H-17,20β-DHP of 4nM the (40 Ci / mmol) were placed in each 200 [mu] L (this one series 3). 200 μL of each protein sample to which 20 mg / mL BSA was added was added, and the mixture was gently shaken and allowed to stand at 4 ° C. for 30 minutes. GF / B was set in the suction device, and 400 μL of the sample was dropped in the center of the GF / B. It was washed 3 times at 4 ° C. using a HEAW buffer. The GF / B was removed into a vial. Liquid scintillation was placed in a vial, shaken vigorously for 15 minutes and measured with a scintillation counter. Corresponds to the maximum binding of standard ligand ^{([3 H] -17,20β-DHP} ) of samples containing EtOH is absence nonradioactive ligand (17,20β-DHP), a sample was added to 1 [mu] M DHP It corresponds to the binding amount of the standard ligand in the presence of a 100-fold molecular excess of the non-radiative ligand, and the difference between these two values corresponds to the specific binding amount.

The measurement results are shown in FIG. From FIG. 8, it was confirmed that the purified mPRα protein has a binding activity to a ligand as a progestin receptor.

According to the purification method of the present invention, a steroid membrane receptor having steroid-binding activity, particularly mPR, can be easily obtained in a large amount. If this steroid membrane receptor is used, it can be used for screening substances that target the steroid membrane receptor, particularly candidate drugs.

Claims (2)

A process of introducing a gene encoding a steroid membrane receptor into a host cell to obtain a recombinant cell so that the steroid membrane receptor can be expressed, and
The process of culturing transgenic cells to express steroid membrane receptors,
The process of collecting the membrane fraction containing the steroid membrane receptor and purifying the steroid membrane receptor,
A steroid membrane receptor purified by a purification method containing
The steroid membrane receptor is a fusion protein of a progestin membrane receptor (mPR) and a tag , and has an activity of binding to a ligand as a progestin receptor.
The tag is selected from His tag, c-Myc tag, GST tag, MBP tag, FLAG tag, HQ tag, HN tag, HAT tag and V5 tag.
The purification step is performed by affinity chromatography using a tag and a cellulose gel to which an amino group is added, and when the cellulose gel to which the amino group is added is used, the amino group is added to a sample containing mPR. A steroid membrane receptor that is purified by being adsorbed on a cellulose gel and then eluted with an elution buffer and recovered. The method of using the steroid membrane receptor according to claim 1 in screening for a substance that targets the steroid membrane receptor.