JP5990888B2 - Recombinant producing human methyl sterol oxidase and use thereof - Google Patents

Description

  The present invention relates to a recombinant that produces human-derived methylsterol oxidase and use thereof. In particular, the present invention relates to a recombinant producing a human-derived methyl sterol oxidase, and an analysis method and a production method using the recombinant.

  In drug development, analysis of drug metabolites in the human body is important. Similarly, the metabolism of food components is being studied. So far, analysis of metabolites of various pharmaceuticals or food ingredients has been performed.

  By the way, human methyl sterol oxidase (SC4MOL) is 4,4'-dimethyl-5α-cholest-8-en-3β-ol (4,4'-dimethyl-5α-cholest-8-en) in the cholesterol biosynthesis pathway. -3β-ol) is a physiologically important enzyme that catalyzes the elimination of the 4- and 4'-position methyl groups. It is known that humans deficient in the SC4MOL gene cause various disorders due to accumulation of cholesterol intermediates (Non-patent Document 1). However, it is not known that SC4MOL is involved in the metabolism of pharmaceuticals and food ingredients. However, when SC4MOL is involved in the metabolism of drugs and food ingredients, the drugs and food ingredients that inhibit SC4MOL may have side effects, and it is necessary to investigate both inhibition and metabolism in the early stages of drug development. The present inventors think that there may be.

  The central role in drug metabolism is the Phase I enzyme group centered on cytochrome P450 and the Phase II enzyme group catalyzing the conjugation reaction to give high water solubility. It makes it possible to metabolize and excrete xenobiotics. The present inventors have found that there is a metabolism that cannot be explained only by the metabolism of a drug-metabolizing enzyme that has been known so far in the study of metabolism of a vitamin D derivative, eldecalcitol, which is sold as an osteoporosis therapeutic agent. This situation often occurs in drug metabolism research, but in many cases, it is commercialized without detailed research. Identification of metabolic enzymes is essential for predicting and solving drug-drug interactions and drug-food interactions that cause side-effect problems.

  It was found that the above-mentioned enzyme that metabolizes eldecalcitol requires NADH or NADPH. Therefore, we decided to investigate whether SC4MOL is involved in the metabolism of the vitamin D derivative eldecalcitol. As a means for examining the involvement of SC4MOL, for example, there are compounds called SC4MOL-specific inhibitors. However, this compound was not very specific for SC4MOL. Moreover, although SC4MOL antibody is commercially available, there has been no report of activity inhibition, and decisive experiments could not be easily performed.

  Therefore, first, we decided to search for an expression system for human-derived SC4MOL. An example of SC4MOL that has been expressed so far is a mammal-derived cultured cell (Patent Document 1).

US 2010/0021892 A1

He M, Kratz LE, Michel JJ, Vallejo AN, Ferris L, Kelley RI, Hoover JJ, Jukic D, Gibson KM, Wolfe LA, Ramachandran D, Zwick ME, Vockley J. (2011) J Clin Invest. 121,976-984. Yasuda K, Ikushiro S, Kamakura M, Ohta M and Sakaki T (2010). Drug Metab Dispos. 38 (12): 2117-2123 Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T. (1998). Appl Environ Microbiol. 64,1694-1699

  Since SC4MOL is originally a human-derived enzyme, as described in Patent Document 1, it can be easily expressed in mammalian-derived cultured cells. In addition, since cultured cells derived from mammals have NADH-cyt.b5 reductase, NADPH-P450 reductase and cyt.b5, it can be easily assumed that SC4MOL activity will be expressed. Electrons are transferred from NADH to NADH-cyt.b5 reductase and finally to SC4MOL via cyt.b5, or from NADPH to NADPH-P450 reductase and finally to SC4MOL via cyt.b5. This is because the activity is exhibited when the iron atom of SC4MOL is reduced by the crossing electron transfer system. However, cultured cells derived from mammals are slow to grow and are not suitable for producing a large amount of metabolites in a short time. On the other hand, microorganisms such as yeast and Escherichia coli grow faster than mammalian-derived cultured cells and are suitable for producing a large amount of metabolites in a short time.

  However, it was extremely questionable whether human-derived SC4MOL can express activity in microorganisms. Because SC4MOL is originally a membrane-bound enzyme that binds to the endoplasmic reticulum membrane, it was expected that expression as a protein would be difficult. In addition, the microorganisms may or may not have the electron transfer systems NADH-cyt.b5 reductase, NADPH-P450 reductase and cyt.b5 necessary for the expression of SC4MOL activity. This is because the structures are different from those of -cyt.b5 reductase, NADPH-P450 reductase and cyt.b5. Even if the SC4MOL protein is expressed in microorganisms, the interaction between the electron transport system NADH-cyt.b5 reductase and cyt.b5 or NADPH-P450 reductase and cyt.b5 does not function properly. It is because it is not possible to obtain.

  Accordingly, an object of the present invention is to provide a recombinant microorganism that expresses human-derived methylsterol oxidase (SC4MOL) and can express SC4MOL activity. Furthermore, the present invention is to provide a method for analyzing and producing a metabolite by SC4MOL using a recombinant microorganism capable of expressing SC4MOL activity.

  The present inventors tried to express a cDNA encoding human-derived SC4MOL in a microorganism. First, SC4MOL protein was expressed using budding yeast having endogenous NADH-cyt.b5 reductase, NADPH-P450 reductase and cyt.b5, and further, expression of SC4MOL activity was attempted. NADH-cyt.b5 reductase, NADPH-P450 reductase and cyt.b5 in budding yeast are homologous to mammalian NADH-cyt.b5 reductase, NADPH-P450 reductase and cyt.b5, respectively. Only 34, 32 and 28%, very low. Therefore, it was very doubtful whether electrons could be successfully donated to the expressed SC4MOL in the budding yeast cells. We also attempted to express SC4MOL and SC4MOL activity in E. coli. There is no NADH-cyt.b5 reductase, NADPH-P450 reductase, or an enzyme corresponding to cyt.b5 in E. coli. Therefore, even if SC4MOL was expressed as a protein, it was expected that SC4MOL activity could not be expressed.

  The present inventors have attempted to clone a cDNA encoding human-derived SC4MOL and to express it in budding yeast and Escherichia coli. As a result, it was unexpectedly found that SC4MOL was expressed and SC4MOL activity was obtained in both recombinant budding yeast and E. coli introduced with cDNA encoding human-derived SC4MOL, and the present invention was completed. .

  Furthermore, the present inventors examined the reactivity to eldecalcitol using a microorganism that expresses SC4MOL activity. As a result, it was found that SC4MOL showed enzyme activity against eldecalcitol, and therefore SC4MOL could be involved in the metabolism of eldecalcitol. Based on this result, it was found that the use of the recombinant budding yeast or Escherichia coli of the present invention capable of expressing human-derived SC4MOL activity makes it possible to produce and analyze various metabolites using SC4MOL, thereby completing the present invention. I let you.

The present invention is as follows.
[1]
A recombinant in which a human-derived methylsterol oxidase (SC4MOL) gene is introduced in a state capable of expressing in a host microbial cell and capable of expressing SC4MOL activity.
[2]
The recombinant according to [1], wherein the host microorganism is budding yeast.
[3]
The recombinant according to [2], which is a budding yeast transformed by introducing a plasmid vector pGYR carrying the SC4MOL gene.
[Four]
The recombinant according to [1], wherein the host microorganism is Escherichia coli.
[Five]
The recombinant according to [4], which is Escherichia coli transformed by introducing a plasmid vector pET17b carrying the SC4MOL gene.
[6]
Incubating the analytical sample with the recombinant according to any one of [1] to [5] to obtain a reaction product of the analytical sample by SC4MOL, and analyzing the obtained reaction product Analysis method of reaction products by SC4MOL of specimens.
[7]
[6] The method according to [6], wherein the sample for analysis is a drug or a drug candidate, or a food ingredient or a food ingredient candidate.
[8]
Incubating a compound that can be a substrate of SC4MOL with the recombinant according to any one of 1 to 5 to obtain a reaction product of SC4MOL of the compound and collecting the obtained reaction product, A method for producing a reaction product by SC4MOL of a compound that can be a substrate.
[9]
[7] The method according to [7], which is a compound that can be a substrate of SC4MOL, a pharmaceutical product or a candidate product for a pharmaceutical product, or a food component or a candidate product for a food component.
[Ten]
[6] to [9], wherein the analyte or the compound that can be a substrate of SC4MOL is dimethyl-5α-cholest-8-en-3β-ol, testosterone, eldecalcitol, sesamin, or episesamin the method of.

ADVANTAGE OF THE INVENTION According to this invention, recombinant microorganisms, such as colon_bacillus | E._coli and yeast, which expressed the activity of human methyl sterol oxidase (SC4MOL) can be provided. Furthermore, various metabolites can be produced by adding various pharmaceuticals or food ingredients to a culture solution containing the cells of these recombinant microorganisms or a buffer solution containing glucose and incubating them. Therefore, there are the following advantages.
(1) The human-derived SC4MOL-expressing microorganism of the present invention can be used for predicting how a drug, food ingredient, environmental substance and the like undergo metabolism in the human body.
(2) It is possible to manufacture a large amount of metabolites and investigate their safety and physiological functions.
(3) Some of these metabolites may show useful properties that are not found in the original compound, and are considered to be a tool for new drug development.

  Our finding that the enzyme SC4MOL, which has a physiologically important function, metabolizes various pharmaceuticals, food ingredients, etc. is an unexpected result. In the future, this enzyme is one of the important drug-metabolizing enzymes. Is likely to be positioned as one. Currently, identification of metabolic enzymes is essential in the development of pharmaceuticals, and development may be stopped if metabolic enzymes are not known. Therefore, the technology we have established will be indispensable for future drug development.

FIG. 3 is a schematic diagram showing SC4MOL gene insertion into a yeast expression vector pGYR-HindIII. FIG. 4 shows the HPLC results of metabolites of eldecalcitol (a), episesamin (b), paclitaxel (c), and testosterone (d) after 24 hours of culture of SC4MOL-expressing yeast. It is the figure which showed the mass spectrometry result of the eldecalcitol metabolite by SC4MOL expression yeast culture, and the estimated metabolism.

<Recombinant>
The recombinant of the present invention is a recombinant in which a human-derived methylsterol oxidase (SC4MOL) gene is introduced in a state capable of expressing in a host microbial cell and capable of expressing SC4MOL activity. The base sequence and amino acid sequence of the human-derived SC4MOL gene can be obtained from the following site, and are described as SEQ ID NOS: 1 and 2 in the sequence listing.
http://www.ncbi.nlm.nih.gov/protein/5803157
NCBI Reference Sequence: NP_006736.1

  SC4MOL has an EC number of 1.14.13.72, and as described above, the 4 and 4 ′ positions of the 4,4′-dimethyl-5α-cholest-8-en-3β-ol in the cholesterol biosynthesis pathway are removed. It is an enzyme that catalyzes a release reaction to produce 4β-hydroxymethyl-4α-methyl-5α-cholest-7-en-3β-ol.

  In the recombinant of the present invention, microorganisms used as host microorganism cells are limited to microorganisms that can express SC4MOL activity when introduced in a state where the SC4MOL gene can be expressed. Examples of such microorganisms include budding yeast and E. coli. In addition to the expression of SC4MOL as a protein, the expression of SC4MOL activity requires that the electron transport system NADH-cyt.b5 reductase and cyt.b5 be present in or near the cell and be able to donate electrons to SC4MOL. is there. Microorganisms having such a function were budding yeast and Escherichia coli as confirmed by the present inventors. The strain of budding yeast is not particularly limited, and examples thereof include AH22 strain, SHY3 strain, NA87-11A strain and the like. The E. coli strain is not particularly limited, and examples thereof include JM109, DH5α, TOP10 derived from E. coli K12 strain, BL21 strain derived from E. coli B strain, and the like.

  The SC4MOL gene is introduced into a host microbial cell in a state where it can be expressed. Specifically, a plasmid into which the SC4MOL gene has been introduced can be introduced into the host microorganism cell downstream of the promoter sequence of the gene expression plasmid. Examples of gene expression plasmid vectors include pGYR, YEp352, Yep51, and pSH19 in budding yeast, and pkk223-3, pKSNdl, pCW, and pET17b in Escherichia coli. The method for introducing the plasmid into the host microorganism cell is not particularly limited, and a conventional method can be used depending on the type of the host microorganism. In addition, NADPH-P450 reductase gene is contained in pGYR used when transforming budding yeast in Examples. However, there is the same gene on the genome of the budding yeast as a host. It is considered that there are about 20 copies of the vector per yeast in the yeast cells, and the yeast AH22 strain is a haploid yeast, so the expression level of NADPH-P450 reductase in the cells into which pGYR was introduced Is estimated to be about 20 times that of the wild type. On the other hand, pET17b used as an expression vector when transforming Escherichia coli in Examples does not contain a reductase gene or a coenzyme gene.

  The host microbial cells into which the SC4MOL gene has been introduced can be appropriately cultured and grown, and then collected to obtain the recombinant of the present invention. The collected recombinant of the present invention can be used in any state of wet cells and dry cells.

<Analysis method>
The present invention includes a method for analyzing a reaction product by SC4MOL of a sample for analysis. This analysis method is a method comprising incubating an analysis sample with the recombinant of the present invention to obtain a reaction product of the analysis sample by SC4MOL, and analyzing the obtained reaction product.

  The sample for analysis can be, for example, a drug or a drug candidate, or a food ingredient or a food ingredient candidate. However, these are not intended to be limited. The analyte for analysis can in particular be a methyl sterol compound that is expected to undergo metabolism by SC4MOL.

Incubation of the sample for analysis and the recombinant of the present invention can be appropriately carried out according to the type of host microorganism used for the recombinant, and further according to the type of sample for analysis. The incubation conditions are not particularly limited, but are as follows, for example.
When adding a specimen for analysis to the growing cell culture, in recombinant yeast (AH22 strain), SD liquid medium (2% glucose, 0.67% N-base w / o amino acid, 20 μg / ml L-histidine) ) Incubate at 30 ° C for 24 hours. In recombinant E. coli, TB medium containing ampicillin 50 μg / ml and kanamycin 25 μg / ml (tryptone 12 g, yeast extract 24 g, glycerol 5 g was dissolved in distilled water 900 mL, and then 1M phosphate buffer (pH 7.0) was added. Add 100mL to make 1L medium) Incubate at 37 ° C for 24 hours. Recombinant yeast or recombinant E. coli cells collected by methods such as centrifugation are suspended in 0.1 M potassium phosphate buffer (pH 7.4) containing 8% glucose, and an analytical sample is added for several hours. The reaction product can be obtained by incubating for ~ 24 hours.

  The reaction product by SC4MOL produced by incubation with the recombinant of the invention is subjected to a predetermined analysis as it is or after being appropriately purified. The predetermined analysis is not particularly limited, and for example, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrometer, NMR, etc. can be used.

  According to the analysis method of the present invention, it is possible to identify and quantify a reaction product by SC4MOL of a sample for analysis.

<Manufacturing method>
The present invention includes a method for producing a reaction product obtained by converting a compound that can be a substrate of SC4MOL by SC4MOL. This production method includes incubating a compound that can be a substrate of SC4MOL with the recombinant of the present invention to obtain a reaction product of the compound that can be a substrate of SC4MOL by SC4MOL, and collecting the obtained reaction product. .

  The compound that can be a substrate of SC4MOL is not particularly limited. For example, 4,4′-dimethyl-5α-cholest-8-en-3β-ol, which is the original substrate, and its analogs (for example, 4β-hydroxymethyl- 4α-methyl-5α-cholest-7-en-3β-ol, 3β-hydroxy-4β-methyl-5α-cholest-7-en-4α-carbaldehyde), testosterone and its analogs, eldecalcitol (ED71) And lignans such as sesamin and episesamin.

  Incubation of the compound that can be a substrate of SC4MOL and the recombinant of the present invention can be appropriately carried out according to the type of host microorganism used for the recombinant, and further according to the type of compound that can be a substrate of SC4MOL. For the incubation conditions, the description in the above analysis method can be referred to.

  The reaction product of SC4MOL, which can be a substrate of SC4MOL obtained by incubation, is collected by a conventional method. Examples of conventional methods include high performance liquid chromatography (HPLC) and thin layer chromatography (TLC).

  Furthermore, the collected compounds can be purified by conventional methods.

  The analytical sample used in the analysis method and the production method of the present invention and the compound that can be a substrate of SC4MOL can be, for example, a pharmaceutical product or a drug candidate product, or a food component or a food component candidate product. Examples of the analyte or compound for analysis may include eldecalcitol, a pharmaceutical product, and sesamin, a food ingredient. However, these are merely examples.

  Hereinafter, the present invention will be described in more detail with reference to examples.

1． Cloning of human SC4MOL gene
The gene encoding SC4MOL was amplified by PCR using a human liver cDNA library as a template and primers 1 and 2 (SEQ ID NOs: 3 and 4). PCR was performed in reaction 1 (94 ° C 2 minutes, 94 ° C 15 seconds, 52 ° C 30 seconds, 68 ° C 1 minute, 30 cycles; template DNA 10 ng, reaction solution, dNTP 0.2 mM, KOD-plus-DNA polymerase (TOYOBO ) 1 U, 10 × PCR Buffer for KOD-Plus-5 μl, each primer 0.2 μM, final volume 50 μl). As a result of electrophoresis of the PCR product on a 1% agarose gel, amplification specific to the target size (about 0.9 kb) was observed (hereinafter, electrophoresis on a 1% agarose gel is simply referred to as electrophoresis). The PCR product was ligated to the pTA2 vector according to the instruction manual of TArget CloneTM-Plus- (TOYOBO). Escherichia coli JM109 was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and LB agar medium containing 50 μg / ml ampicillin (polypeptone 10 g, yeast extract 5 g, NaCl 5 g and agar) 15 g was dissolved in 1 L of distilled water). Several colonies of the obtained E. coli colonies were selected, and colony PCR was performed using them as templates. PCR was performed according to the instruction manual of EmeraldAmp (registered trademark) PCR Master Mix (Takara). Reaction 2 (98 ° C 10 seconds, 55 ° C 30 seconds, 72 ° C 1 minute, 30 cycles; reaction solution, EmeraldAmp PCR Master Mix (2 × Premix) (Takara) 5 μl, M13-M3 primer and M13-Rv primer each 0.2 μM, final solution volume 10 μl). The obtained PCR product was electrophoresed to obtain several clones in which specific amplification was observed at the expected size (about 1.0 kb). Sequencing was performed in order to confirm that the insert sequence of the obtained clone did not contain any mutation due to PCR. First, the obtained Escherichia coli colony was inoculated into 5 ml of LB medium (containing 50 μg / ml of ampicillin), followed by shaking culture at 37 ° C. and 200 rpm for 16 hours. Thereafter, the plasmid was extracted using Wizard (registered trademark) Plus SV Minipreps DNA Purification System (Promega), and the plasmid concentration was measured using absorbance at 260 nm to obtain template DNA. The cycle sequencing reaction was performed using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). As a result of sequencing, it was confirmed that the sequence was unique to the gene encoding SC4MOL. This plasmid was named pTA2-SC4MOL.

Sequence number 3: 5'-ATATAAGCTTAAAAAAATGGCAACAAATGAAAGTGT-3 '
Sequence number 4: 5'-ATATAAGCTTTTATTCAGTCTTTTTCTC-3 '

2． Production of yeast expressing SC4MOL gene
About 1 μg of the pTA2-SC4MOL plasmid prepared in 1 was treated with HindIII at 37 ° C. for 1 hour and electrophoresed. An about 0.9 kb DNA fragment was separated by electrophoresis and recovered from the gel using the QIAquick Gel Extraction Kit (QIAGEN) to obtain an insert fragment. As a yeast expression vector, pGYR, which has a proven record in the expression of cytochrome P450, an endoplasmic reticulum membrane enzyme, was used (Non-patent Document 2 *). The pGYR vector was treated with HindIII at 37 ° C. for 1 hour, and an approximately 11 kb DNA fragment was separated by electrophoresis and recovered from the gel. The HindIII-treated pGYR vector was dephosphorylated with Calf Intestinal Alkaline Phosphatase (Takara), and a vector fragment was prepared using QIAquick PCR Purification Kit (Takara). After confirming the concentration of the insert fragment and pGYR vector fragment by electrophoresis, mix them so that their molar ratio is about 3: 1 to 10: 1, and then add an equal volume of DNA Ligation Kit Ver. 2 (TaKaRa) solution I And reacted at 16 ° C. for 30 minutes. Thereafter, Escherichia coli JM109 strain was transformed by the calcium chloride method, and applied to an LB agar medium containing 50 μg / ml of ampicillin. Several colonies of the obtained E. coli colonies were selected, and colony PCR was performed using them as templates. PCR was performed under the conditions of Reaction 2 according to the instruction manual of EmeraldAmp (registered trademark) PCR Master Mix (Takara). Primers 1 and 3 (SEQ ID NO: 3 and SEQ ID NO: 5) were used. The obtained PCR product was electrophoresed to obtain several clones showing specific amplification at the expected size (about 1.8 kb). The obtained Escherichia coli colony was inoculated into 5 ml of LB medium (containing 50 μg / ml of ampicillin), and cultured with shaking at 37 ° C. and 200 rpm for 16 hours. Thereafter, a plasmid was extracted using Wizard (registered trademark) Plus SV Minipreps DNA Purification System (Promega), a portion thereof was treated with HindIII at 37 ° C. for 1 hour, and electrophoresis was confirmed to introduce the insert. This plasmid was named pGYR-SC4MOL. Saccharomyces cerevisiae AH22 strain was transformed by the lithium chloride method and applied to SD agar medium (2% glucose, 0.67% N-base w / o amino acid, 1.5% agar, 20 μg / ml L-histidine). The obtained colony was used as the SC4MOL gene-expressing yeast in the following production test.
SEQ ID NO: 5: 5'-GACGTTGTAAAACGACGGCAGTG-3 '

3． SC4MOL gene-expressing E. coli
Using the pTA2-SC4MOL plasmid prepared in 1 as a template, PCR was performed using primers 2 and 4 (SEQ ID NOs: 4 and 6) under the conditions of reaction 1 by PCR. As a result of electrophoresis of the obtained PCR product, amplification specific to the target size (about 0.9 kb) was observed. The PCR product was ligated to the pTA2 vector according to the instruction manual of TArget CloneTM-Plus- (TOYOBO). Escherichia coli JM109 was transformed by the calcium chloride method and applied to an LB agar medium containing 50 μg / ml of ampicillin. Several colonies of the obtained E. coli colonies were selected, and colony PCR was performed using them as templates. PCR was performed under the conditions of Reaction 2 according to the instruction manual of EmeraldAmp (registered trademark) PCR Master Mix (Takara). The obtained PCR product was electrophoresed to obtain several clones in which specific amplification was observed at the expected size (about 1.0 kb). Sequencing was performed in order to confirm that the insert sequence of the obtained clone did not contain any mutation due to PCR. First, the obtained Escherichia coli colony was inoculated into 5 ml of LB medium (containing 50 μg / ml of ampicillin), followed by shaking culture at 37 ° C. and 200 rpm for 16 hours. Thereafter, the plasmid was extracted using Wizard (registered trademark) Plus SV Minipreps DNA Purification System (Promega), and the plasmid concentration was measured using absorbance at 260 nm to obtain template DNA. The cycle sequencing reaction was performed using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). As a result of sequencing, it was confirmed that the sequence was unique to the gene encoding SC4MOL. About 1 μg of the obtained plasmid was treated with NdeI and HindIII at 37 ° C. for 1 hour and electrophoresed. An about 0.9 kb DNA fragment was separated by electrophoresis and recovered from the gel using the QIAquick Gel Extraction Kit (QIAGEN) to obtain an insert fragment. The pET17b vector was treated with NdeI and HindIII at 37 ° C. for 1 hour, and a DNA fragment of about 3 kb was separated by electrophoresis, then recovered from the gel, and a vector fragment was prepared using QIAquick PCR Purification Kit (Takara). After confirming the concentration of the insert fragment and pET17b vector fragment by electrophoresis, mix them so that their molar ratio is about 3: 1 to 10: 1, and then add an equal volume of DNA Ligation Kit Ver. 2 (TaKaRa) solution I And reacted at 16 ° C. for 30 minutes. Thereafter, Escherichia coli JM109 was transformed by the calcium chloride method and applied to an LB agar medium containing 50 μg / ml of ampicillin. Several colonies of the obtained E. coli colonies were selected, and colony PCR was performed using them as templates. PCR was performed under the conditions of Reaction 2 in which only the primer sequence was changed according to the instruction manual of EmeraldAmp (registered trademark) PCR Master Mix (Takara). The primer used was a T7-promoter primer and a T7-terminator primer. The obtained PCR product was electrophoresed to obtain several clones in which specific amplification was observed at the expected size (about 1.1 kb). The obtained Escherichia coli colony was inoculated into 5 ml of LB medium (containing 50 μg / ml of ampicillin), and cultured with shaking at 37 ° C. and 200 rpm for 16 hours. Thereafter, the plasmid was extracted using Wizard (registered trademark) Plus SV Minipreps DNA Purification System (Promega), a portion thereof was treated with NdeI and HindIII at 37 ° C. for 1 hour, and electrophoresis was confirmed to introduce the insert. . This plasmid was named pET17b-SC4MOL. Escherichia coli BL21 strain into which pGro12 (Non-patent Document 3), a GroEL / ES expression vector, was introduced was transformed by the calcium chloride method, and the pET17b-SC4MOL plasmid was introduced. The resulting colonies were applied to an LB agar medium containing 50 μg / ml of ampicillin and 25 μg / ml of kanamycin, and the obtained colonies were used as SC4MOL gene-expressing E. coli in the following production test.
Sequence number 6: 5'-ATATCATATGGCAACAAATGAAAGTGTCAGCATCTTTA-3 '

Four. Metabolism of various substrates using SC4MOL gene-expressing yeast
The SC4MOL gene-expressing yeast was inoculated into 5 mL of SD liquid medium (2% glucose, 0.67% N-base w / o amino acid, 20 μg / ml L-histidine), and cultured with shaking at 30 ° C. and 200 rpm. When the OD ₆₁₀ value reached 0.25-0.3, the substrate was added at a final concentration of 10 μM. The substrates used this time are eldecalcitol (Chugai Pharmaceutical Co., Ltd.), testosterone, paclitaxel, sesamin, and episesamin, but are not limited thereto. After addition of the substrate, the culture was further continued, and 2 mL of chloroform: methanol = 3: 1 was added to 500 μL of the culture solution 24 hours later, and after vigorous stirring, the chloroform layer was collected and dried under reduced pressure. This was dissolved in acetonitrile, centrifuged at 14500 rpm for 15 minutes, and the supernatant was analyzed by HPLC. (Condition: Column: YMC-Pack ODS-AM manufactured by YMC Co., Ltd. (inner diameter: 4.6 mm × length: 300 mm); flow rate: 1.0 ml / min; column temperature: 40 ° C., detection wavelength and elution conditions differ depending on the substrate as follows. )

HPLC condition 1: Eldecalcitol water / acetonitrile system; 25-95% acetonitrile linear concentration gradient (25 minutes) Detection wavelength: 265 nm
HPLC condition 2: Testosterone water / acetonitrile system; 20-100% acetonitrile linear concentration gradient (25 minutes) Detection wavelength: 290 nm
HPLC condition 3: Paclitaxel water / methanol system; 10-95% methanol linear concentration gradient containing 0.01% trifluoroacetic acid (16 minutes) followed by 95% methanol containing 0.01% trifluoroacetic acid (8 minutes) Detection wavelength: 227 nm
HPLC condition 4: Sesamin, episesamin water / methanol system; 10-90% methanol linear concentration gradient (30 minutes) Detection wavelength: 280 nm

  Fig. 2 shows an example of an HPLC chart of metabolites. The metabolites of sesamin and episesamin have been confirmed to match the metabolites of their respective P450s (Non-patent Document 2) and HPLC detection times, and are presumed to be monocatechol bodies in which the methylenedioxyphenyl group is cleaved. it can. In addition, the metabolite of testosterone matches that of CYP3A4 and the detection time of HPLC, and the metabolite is considered to be 6β-hydroxide.

  Eldecalcitol was analyzed by LC-MS to estimate the metabolite structure. LC-MS was performed using Finnigan LCQ ADVANTAGE MIX (ThermoFisher SCIENTIFIC, Waltham, MA, USA) in APCI method and positive mode. The LC conditions are as follows. Column; ODS (2 mm × 150 mm Develosil ODS-HG-3, Nomura Chemical Co. Ltd., Aichi, Japan); mobile phase, acetonitrile: methanol: water = 3: 4: 3; flow rate, 0.2 mL · min; UV detection Wavelength, 265nm. The LC / MS results are shown in FIG. The molecular ion peak (M + H) was found to be m / z = 433, and the metabolite molecular weight was 432. Compared with the molecular weight 490 of eldecalcitol, it was found to be 58 mass smaller, suggesting that the metabolite has a structure in which the hydroxypropoxy group at the 2-position is dehydroxypropylated (FIG. 3).

Five. Metabolism of eldecalcitol using Escherichia coli expressing SC4MOL gene.
SC4MOL gene-expressing E. coli is dissolved in 900 mL of distilled water after 5 mL of TB medium (trypton 12 g, yeast extract 24 g, glycerol 5 g containing ampicillin 50 μg / ml and kanamycin 25 μg / ml, then 1M phosphate buffer (pH 7.0) Was inoculated into a 1 L medium) and cultured with shaking at 37 ° C. and 200 rpm. When OD ₆₁₀ reached 0.5 to 1.0, IPTG (final concentration 1 mM), arabinose was final concentration (4 mg / mL), and eldecalcitol (final concentration 10 μM) was added as a substrate. The culture was further continued, and 2 mL of chloroform: methanol = 3: 1 was added to 500 μL of the culture solution after 24 hours, and after vigorous stirring, the chloroform layer was collected and dried under reduced pressure. This was dissolved in acetonitrile, centrifuged at 14500 rpm for 15 minutes, and the supernatant was analyzed by HPLC.

  As a result, a metabolite peak was detected at the same position as in the case of the SC4MOL gene-expressing yeast, and it was confirmed that the same metabolism as in the SC4MOL gene-expressing yeast was observed even when the SC4MOL gene-expressing Escherichia coli was used.

Reference example 1
None of the activities was observed in the control strain into which the vector pGYR containing no SC4MOL cDNA was introduced. From these results, the metabolite of eldecalcitol in the above examples is considered to be an activity derived from SC4MOL expressed in yeast.

Reference example 2
No activity was observed in the control strain into which the vector pET17b containing no SC4MOL cDNA was introduced. From these results, the metabolite of eldecalcitol in the above examples is considered to be an activity derived from SC4MOL expressed in E. coli.

  Since the present invention provides an expression system for the enzyme SC4MOL having physiologically important functions, it is useful in the field of pharmaceuticals and foods.

Claims (5)

At least one compound serving as a substrate for human methyl sterol oxidase (SC4MOL) selected from the group consisting of dimethyl-5α-cholest-8-en-3β-ol, testosterone, eldecalcitol, sesamin, and episesamin ; Incubating with a recombinant yeast, which is a yeast transformed with a plasmid vector carrying the human-derived SC4MOL gene in an expressible state, to obtain a reaction product of SC4MOL of the compound and collect the obtained reaction product A process for producing a reaction product using SC4MOL. The production method according to claim 1, wherein the plasmid vector carrying the SC4MOL gene in an expressible state is a plasmid vector carrying the yeast-derived NADPH-P450 reductase gene in an expressible state. The production method according to claim 2, wherein the plasmid vector carrying the SC4MOL gene and the yeast-derived NADPH-P450 reductase gene in an expressible state is a plasmid vector pGYR carrying the SC4MOL gene. Express human-derived SC4MOL gene with at least one compound that is a substrate of human-derived SC4MOL selected from the group consisting of dimethyl-5α-cholest-8-en-3β-ol, testosterone, eldecalcitol, sesamin, and episesamin SC4MOL reaction comprising incubating with recombinant E. coli, which is E. coli transformed with a plasmid vector loaded as possible, to obtain a reaction product of SC4MOL of said compound and collecting the resulting reaction product Product manufacturing method. The production method according to claim 4, wherein the plasmid vector carrying the SC4MOL gene in an expressible state is the plasmid vector pET17b carrying the SC4MOL gene.

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