JP2005095181A - Method for evaluating safety - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating safety of a compound duplicating a human metabolic system in vitro. <P>SOLUTION: The method for evaluating the safety comprises a step constructing a yeast fungal body expressing (i) the molecular species of a molecular species group consisting of cytochrome P450 1A2, P450 2C9, P450 2E1 and P450 3A4 derived from a human and yeast NADPH-P450 reductase or (ii) the above mentioned molecular species and an artificial fusion enzyme of a yeast NADPH-P450 reductasea in the yeast, a step for reacting the yeast fungal body or a crushed fungal body with a specimen compound, and a step for analyzing the metabolite of the resultant reactional solution, and a step discriminating the presence or absence of the antidotal metabolism of the specimen compound or the presence or absence of the metabolism into a carcinogen. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a safety evaluation method.

In the development of new compounds, it is becoming increasingly necessary to predict and evaluate the metabolism, efficacy and toxicity of compounds in humans at an early stage. In particular, when it is impossible to conduct safety assessment tests by administration to humans, such as agricultural chemicals and general compounds, it is possible to extrapolate the results of safety assessment tests by administration to laboratory animals to humans. We are conducting sex assessment.
However, in recent years, differences among animal species in drug metabolizing enzyme systems such as cytochrome P450 are becoming clear.

  As a result, it has not always been sufficient to use experimental animals for safety assessment tests to predict and evaluate metabolism, drug efficacy and toxicity in humans at an early stage. For this reason, the metabolic system in humans can be obtained without conducting a safety evaluation test in which a new compound is administered to humans, or a safety evaluation test using biological tissues such as human liver that are ethically and technically difficult to obtain. There is a need for an evaluation method that reproduces in vitro systems.

In view of the above situation, the present inventors have intensively studied to find an evaluation method for reproducing a superior human metabolic system in an in vitro system, and as a result, the yeast expression system has an excellent growth of yeast cells. Suitable for mass production of human-derived cytochrome P450 molecular species or an artificial fusion enzyme of said molecular species and yeast NADPH-P450 reductase, and (i) a nucleotide sequence encoding human-derived cytochrome P450 molecular species Yeast expression plasmid comprising a gene having a gene having a nucleotide sequence encoding a gene having a nucleotide sequence encoding a gene having the gene and yeast NADPH-P450 reductase, or (ii) an artificial fusion enzyme of the above molecular species and yeast NADPH-P450 reductase Precise analysis because a very large amount of metabolites can be obtained in a short time and stably by using Further, the present inventors have found that the metabolic system in the human liver can be reproduced in vitro by using an essential combination of certain human cytochrome P450 molecular species.
That is, the present invention relates to (I) all molecular species included in the molecular species group consisting of human-derived cytochrome P450 1A2, human-derived cytochrome P450 2C9, human-derived cytochrome P450 2E1 and human-derived cytochrome P450 3A4.
(1) (a) a promoter for expression in yeast and (b) (i) a gene having a base sequence encoding the above molecular species and a gene having a base sequence encoding yeast NADPH-P450 reductase or ( ii) a step of constructing an expression plasmid in yeast comprising the above-mentioned molecular species and a gene having a base sequence encoding an artificial fusion enzyme of yeast NADPH-P450 reductase (step 1),
(2) By introducing the constructed yeast expression plasmid into yeast, (i) the above molecular species and yeast NADPH-P450 reductase or (ii) the above molecular species and yeast NADPH-P450 reductase in yeast A step of producing a yeast cell for expressing the artificial fusion enzyme (step 2),
(3) a step of reacting the produced yeast cells or the disrupted cells thereof with a compound as a specimen (step 3),
(4) After the reaction, a step of determining the presence or absence of detoxification metabolism of the compound as a sample or the presence or absence of metabolism to a carcinogen by analyzing a metabolite present in the reaction solution (step 4A),
(5) Based on the above determination, (a) in the presence (existence) of the detoxification metabolism of the compound as the specimen or absence (absence) of metabolism to the carcinogen, the compound as the specimen is evaluated as safe, or (B) a step (step 5A) of evaluating the compound as a non-safety in the absence (no) of detoxification metabolism of the compound as a specimen or the presence (present) of metabolism to a carcinogen (step 5A),
An evaluation method characterized by comprising (hereinafter referred to as an evaluation method), and
(II) For all molecular species included in the molecular species group consisting of human-derived cytochrome P450 1A2, human-derived cytochrome P450 2C9, human-derived cytochrome P450 2E1, and human-derived cytochrome P450 3A4,
(1) (a) a promoter for expression in yeast and (b) (i) a gene having a base sequence encoding the above molecular species and a gene having a base sequence encoding yeast NADPH-P450 reductase or ( ii) a step of constructing an expression plasmid in yeast comprising the above-mentioned molecular species and a gene having a base sequence encoding an artificial fusion enzyme of yeast NADPH-P450 reductase (step 1),
(2) By introducing the constructed yeast expression plasmid into yeast, (i) the above molecular species and yeast NADPH-P450 reductase or (ii) the above molecular species and yeast NADPH-P450 reductase in yeast A step of producing a yeast cell for expressing the artificial fusion enzyme (step 2),
(3) a step of reacting the produced yeast cells or the disrupted cells thereof with a compound as a specimen (step 3),
(4) After the reaction, by causing the metabolite present in the reaction solution to act on microorganisms that require nutrient elements, the step of measuring the abundance of microorganisms that are no longer required for the nutrient elements (step 4B) ),
(5) According to the above measurement, (a) microorganisms that have become non-required for nutritional elements are obtained by using the yeast cells into which the above-described yeast expression plasmid has not been introduced (3) and (4) If the sample is present in the same degree as the control (control), the sample compound is evaluated as non-mutagenic, or (b) the microorganism that has become non-required for the nutritional element is contained in the yeast. When a substantial amount is present as compared with the case where the above (3) and (4) are performed using the yeast cells into which the expression plasmid has not been introduced (control), the test compound is evaluated as mutagenic. Step (step 5B),
An evaluation method characterized by comprising (hereinafter referred to as a mutagenicity evaluation method), and
(III) All molecular species included in the molecular species group consisting of human-derived cytochrome P450 1A2, human-derived cytochrome P450 2C9, human-derived cytochrome P450 2E1 and human-derived cytochrome P450 3A4,
(1) (a) a promoter for expression in yeast and (b) (i) a gene having a base sequence encoding the above molecular species and a gene having a base sequence encoding yeast NADPH-P450 reductase or ( ii) a step of constructing an expression plasmid in yeast comprising the above-mentioned molecular species and a gene having a base sequence encoding an artificial fusion enzyme of yeast NADPH-P450 reductase (step 1),
(2) By introducing the constructed yeast expression plasmid into yeast, (i) the above molecular species and yeast NADPH-P450 reductase or (ii) the above molecular species and yeast NADPH-P450 reductase in yeast A step of producing a yeast cell for expressing the artificial fusion enzyme (step 2),
(3) a step of reacting the produced yeast cells or the disrupted cells thereof with a compound as a specimen (step 3),
(4) After the reaction, one kind of (i) human-derived cytochrome P450 molecular species or (ii) human-derived cytochrome P450 molecules included in the molecular species group is analyzed by analyzing metabolites present in the reaction solution. A step of determining the presence or absence of a metabolite specifically present only in the case of an artificial fusion enzyme of seed and yeast NADPH-P450 reductase (step 4C),
(5) According to the above determination, only (a) one type of (i) human-derived cytochrome P450 molecular species or (ii) human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase artificial fusion enzyme If a metabolite that is specifically present is present, the analyte compound is evaluated as effective as a metabolic probe of the cytochrome P450 molecular species, or (b) if a metabolite that is specifically present is absent, A step (step 5C) for evaluating the sample compound as not effective as a metabolic probe of the cytochrome P450 molecular species,
An evaluation method (hereinafter referred to as a metabolic probe evaluation method) is provided.

  The present invention is a safety evaluation method that reproduces a superior human metabolic system in an in vitro system, and has made it possible to predict and evaluate metabolism, drug efficacy, and toxicity at an early stage with high accuracy.

As a safety evaluation method for reproducing a human metabolic system in vitro, a test using a liver homogenate of an experimental animal such as a rat has been conventionally used.
On the other hand, in recent years, a system in which a gene having a base sequence encoding a cytochrome P450 molecular species derived from human is introduced into a human cultured cell has already been constructed. However, the human cultured cell has low proliferation ability and is difficult to culture at high density. For this reason, a large amount of cytochrome P450 molecular species cannot be obtained, which is not suitable for analysis of metabolites. Moreover, compared with microorganisms, such as yeast, culture | cultivation cost is high and an advanced culture | cultivation technique is required.
In order to eliminate the above-described drawbacks, the present invention provides certain (i) human cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) human cytochrome P450 molecular species and yeast NADPH. -A yeast cell that expresses an artificial fusion enzyme of P450 reductase is used. Therefore, a promoter for expression in yeast, a certain (i) gene having a base sequence encoding a cytochrome P450 molecular species derived from human and a gene having a base sequence encoding yeast NADPH-P450 reductase or ( ii) It is necessary to construct a yeast expression plasmid containing a gene having a base sequence encoding a human cytochrome P450 molecular species and yeast NADPH-P450 reductase artificial fusion enzyme.
This will be described below.

In the present invention, it is essential to use in combination all molecular species included in the molecular species group consisting of P450 1A2, P450 2C9, 2E1 and P450 3A4 as human-derived cytochrome P450 molecular species. This is because the types and abundances of various cytochrome P450 molecular species in human liver vary between races and individuals, but in any human case, all cytochrome P450 molecular species included in the above molecular species group are essential. This is because the metabolic system in the human liver can be efficiently reproduced in vitro by using in combination. In the system of the present invention, the above steps 1 to 3 and steps 4A, 4B, and 4C are independent for each cytochrome P450 molecular species (the cytochrome P450 molecular species are connected in parallel in separate systems). Or a state in which a plurality of cytochrome P450 molecular species are mixed (a state in which a plurality of cytochrome P450 molecular species are mixed and simultaneously exist in one system) or an intermediate between the two. In a state (partially independent state, partly present in a mixed state), and then in steps 5A, 5B and 5C, all the above molecular species are examined and evaluated. It doesn't matter. Further, if necessary, in addition to the cytochrome P450 molecular species included in the above molecular species group, for example, other cytochrome P450 molecules such as P450 1A1, P450 2A6, P450 2B6, P450 2C8, P450 2C18, P450 2C19 or P450 2D6 By supplementally combining species, the metabolic system in human liver can be reproduced more accurately in vitro.
The essential combination of human cytochrome P450 molecular species used in the present invention (molecular species group consisting of P450 1A2, P450 2C9, 2E1 and P450 3A4) covers about 85% of the total cytochrome P450 in the human liver. Further, for example, it is estimated that about 90% or more can be covered by supplementary addition of P450 2A6 and / or P450 2C19 and / or P450 2D6 to the molecular species group.
Specifically, for example, P450 3A4 (about 35 ± 10%), P450 P450 2C9 (about 25 ± 10%), P450 1A2 (about 23 ± 10%), P450 2E1 (about 17 ± 10%), etc. A combination of abundance ratios can be given. It goes without saying that it can be increased or decreased as appropriate without taking the above example, and by changing this ratio, it is also possible to more accurately cope with race differences and individual differences in humans.

Next, the step of constructing an expression plasmid in yeast (step 1) will be described.
The base sequence encoding the above human cytochrome P450 molecular species is disclosed in the base sequences shown in SEQ ID NO: 1 to SEQ ID NO: 7. The gene having the base sequence encoding the cytochrome P450 molecular species derived from human is, for example, (1) (a) preparing an mRNA fraction containing mRNA of the gene, and using reverse transcriptase to prepare cDNA. After the preparation, the target gene from the cDNA library obtained by inserting the cDNA into a phage vector and a plasmid vector, or (b) a commercially available cDNA library derived from human liver, is (i) homologous to the gene. (Ii) a method of cloning according to a conventional method using an antibody that recognizes a protein produced by the gene, or (2) a PCR method from the cDNA library described in (1) above. It can be prepared by a usual method such as a method of cloning using the method.

  On the other hand, for a gene having a nucleotide sequence encoding yeast NADPH-cytochrome P450 reductase, for example, (1) (a) After preparing an mRNA fraction containing the mRNA of the gene and preparing cDNA using reverse transcriptase, A cDNA library obtained by inserting the cDNA into a phage vector and a plasmid vector, or (b) a commercially available yeast-derived cDNA library, and (i) a DNA having homology with the gene. A method of cloning according to a conventional method such as using a fragment, or (ii) an antibody that recognizes a protein produced by the gene, or (2) cloning using a PCR method from the cDNA library described in (1) above It can be prepared by ordinary methods such as Specifically, for example, it can be isolated by the method described in JP-A-62-19085.

  The promoter used in the present invention for expression in yeast is not particularly limited as long as it is a promoter used in a normal yeast expression system. For example, a promoter of a yeast alcohol dehydrogenase gene (hereinafter referred to as ADH) And glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as GAPDH promoter), phosphoglycerate kinase (hereinafter referred to as PGK promoter), and the like. The ADH promoter can be obtained from a yeast expression vector pAAH5 (available from Washington Research Foundation, Ammerer et al., Methods in Enzymology, 101, p. Can be prepared. The yeast ADH1 promoter is included in Washington Research Foundation U.S. Patent No. 299,733 (1981/9/31) and requires license from the rights holder for use in the United States for industrial and commercial purposes. .

  Yeast expression including a promoter for expression in the yeast, a gene having a base sequence encoding the human cytochrome P450 molecular species, and a gene having a base sequence encoding the yeast NADPH-P450 reductase Plasmids can be constructed using conventional genetic recombination methods. For example, a NotI fragment prepared from a yeast expression vector pAAH5N having an ADH promoter and ADH terminator described in JP-A-2-21180 and a base encoding a yeast NADPH-P450 reductase described in JP-A-2-21880 A method of constructing by inserting a gene having a base sequence encoding a cytochrome P450 molecular species derived from human into the Hind III site of the plasmid pAHRR obtained by inserting the gene having a sequence into the NotI site of the plasmid pARRN Can give. In addition, a method of constructing using a vector obtained by exchanging the Hind III site of the pAAH5N vector with another site for restriction enzyme as necessary may be mentioned.

Furthermore, in the present invention, in addition to the gene having the base sequence encoding the human cytochrome P450 molecular species and the gene having the base sequence encoding the yeast NADPH-P450 reductase, the human cytochrome P450 molecular species And yeast NADPH-P450 reductase artificial fusion enzyme (enzyme having the ability to supply monoatomic oxygen of human cytochrome P450 molecular species and the ability to supply reducing power from NADPH of yeast NADPH-P450 reductase in the same molecule) A gene having a base sequence encoding can also be used. Such a gene is obtained by connecting a gene having a base sequence encoding a cytochrome P450 molecular species derived from human and a gene having a base sequence encoding yeast NADPH-P450 reductase by a normal gene manipulation method. Can be constructed. A method for constructing an expression plasmid in yeast can be exemplified by inserting the constructed gene into the Hind III site of the yeast expression vector pAAH5N having the ADH promoter and ADH terminator described in JP-A-2-21180. .
It is also possible to prepare a single yeast expression plasmid that simultaneously expresses multiple types of molecular species.

Next, yeast that expresses (i) a human cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) an artificial fusion enzyme of human cytochrome P450 molecular species and yeast NADPH-P450 reductase in yeast The process for producing the body (process 2) will be described.
A yeast cell that expresses human cytochrome P450 molecular species and yeast NADPH-P450 reductase in yeast or (ii) an artificial fusion enzyme of human cytochrome P450 molecular species and yeast NADPH-P450 reductase is constructed. The yeast expression plasmid can be obtained by introducing it into the yeast by a conventional method such as protoplast method or alkali metal (LiCl) method.
Examples of the yeast strain used in the system of the present invention include Saccharomyces cerevisiae. Saccharomyces cerevisiae AH22 strain (ATCC38626) is preferable.
In addition, a plurality of yeast expression plasmids can be introduced into a single yeast strain so that a plurality of molecular species can be expressed simultaneously.

  By using the yeast cells thus prepared, the electron transfer efficiency in the human cytochrome P450 molecular species is increased, and the human cytochrome P450 or human cytochrome P450 molecular species and yeast NADPH-P450 are used. It becomes possible to significantly improve the monoatomic oxygenation activity of the reductase artificial fusion enzyme.

Next, the step (step 3) of reacting the produced yeast cells or the disrupted cells with a compound as a specimen will be described.
As the yeast cells, live cells are usually used. Examples of the disrupted cells include a cell disruption solution containing microsomes, a microsome fraction, or a fraction containing both microsomes and cytoplasm. These preparation methods may be ordinary methods described in, for example, DNA, Vol. 4, No. 3, p203-p210 (1985). Of the above, it is particularly preferable to react the crushed microbial cell with a compound as a specimen. Among the disrupted bacterial cells, it is generally preferable to react the microsomal fraction with the compound that is the specimen. For example, in biological analysis methods (mutagenicity or toxicity evaluation) as described later, The fraction containing both microsomes and cytoplasm is sometimes preferred.
In the reaction, a compound as a specimen is added to a solution such as a culture solution or a buffer solution containing yeast cells or a disrupted product of the cells, and the mixture is heated at about 10 ° C. to about 40 ° C. for about 0.1 hour to about This can be done by incubating for about 48 hours. In addition, the amount of the abundance and the amount of the compound serving as the sample in the solution of the yeast cells or the disrupted cells vary depending on various conditions such as the reaction temperature, the reaction time, and the type of the compound serving as the sample. The abundance of the cells or the disrupted cells in the solution is the number of P450 molecules of about 10 ¹⁰ to about 10 ¹⁵ per ml of the solution (the number of cells of about 10 ⁵ to about 10 ¹⁰ per ml of the solution of yeast cells). ), Preferably about 10 ¹² to about 10 ¹³ P450 molecules per 1 ml of solution (about 10 ⁷ to about 10 ⁸ cells per solution as yeast cells), A range of about 0.01 μmol to about 1 μmol per 1 ml of the solution is desirable. Needless to say, it can be appropriately increased or decreased without being in the above range.

Next, after the reaction, a step of determining the presence or absence of detoxification metabolism of the compound as a sample or the presence or absence of metabolism to a carcinogen by analyzing a metabolite present in the reaction solution (step 4A) and The process for evaluating safety (process 5A) will be described.
After the reaction, the method of analyzing metabolites present in the reaction solution is supervised by Jiro Shiokawa et al. (2) (Amendment and revised edition), 1st edition, Chemical Dojin (1985), RM SILVERSTEIN et al., Identification method by spectrum of organic compound 4th edition, 3rd edition, published by Tokyo Kagaku Dojin (1984), etc., can be used.
Then, based on the above analysis results, the presence or absence of detoxification metabolism of the compound as a sample or the presence or absence of metabolism to a carcinogen may be determined.

As a method for analyzing metabolites present in the reaction solution, for example, the following biological analysis method can be used (step 4B). In this case, for example, in order to “analyze the metabolite present in the reaction solution”, the “metabolite present in the reaction solution is allowed to act on the nutrient component-requiring microorganism”, so Measure the abundance of the microorganisms that became required, and by the measurement, (a) the microorganisms that became non-required for the nutrient element used yeast cells into which the yeast expression plasmid was not introduced. When (3) and (4) are present to the same extent as in the case of (control), the sample compound is evaluated as mutagenic, or (b) is not required for nutritional elements. If there are substantially more microorganisms than in the case where the above (3) and (4) are performed using the yeast cells into which the yeast expression plasmid is not introduced (control), Some compounds have mutagenic properties It is possible to determine the mutagenicity of the compound is subject by evaluating (step 5B). More specifically, for example, histidine auxotrophy of Salmonella strains [Salmonella typhimurium (his ^-)] or tryptophan auxotrophic E. coli strain [Escherichia coil (trp ^-)] by the action of metabolites present in the reaction solution, the Bacteria that have become non-required for the amino acid of the strain (His ⁺ or Trp ⁺ ), that is, whether or not the revertant bacteria form colonies (whether or not they have reversion mutagenesis) or the number of colonies formed (reversion) Biological analysis methods for investigating mutagenicity). In the case of using the above biological analysis method, (a) a step of reacting the produced yeast cells or the disrupted cells with a compound as a specimen (step 3) and (b) after the reaction, By analyzing metabolites present in the reaction solution, it is possible to simultaneously proceed with the step of determining detoxification metabolism or metabolism to a carcinogen (step 4A).
Since many of the arylamine compounds become mutagenic by undergoing metabolic activation in the human liver, it is advantageous to utilize the present invention using the biological analysis method described above. For example, in the case of a system in which 20 pmol of cytochrome P450 1A2 is present, the mutagenicity of about 0.1 μg of 2-aminoanthracene can be detected.
In addition, a biological analysis method using cells derived from mammals instead of the above microorganisms is also possible. For example, the MCL-5 cell line derived from the L3 cell line, which is a variant of human B lymphoblast cell AHH-1 described in US Pat. No. 4,532,204 (the cytotoxicity of many compounds compared to the parental line) On the other hand, a metabolite present in the reaction solution is allowed to act on a more sensitive cell line), and the increase in the population of the cell line is examined. In the case of the biological analysis method using the above-mentioned microorganisms, the toxicity of the compound as a sample is compared with the increase in the number of cell lines in which the metabolite is acted on and in the cell line in which the metabolite is not acted. Can be evaluated in the same way.

In addition, metabolic probes can be screened by using the present invention.
The metabolic probe is used to examine the abundance of cytochrome P450 molecular species contained in the molecular species group that plays a central role in drug metabolism in vivo.
That is, by administering to humans a compound that is a specific substrate of a certain human-derived cytochrome P450 molecular species as a metabolic probe, and analyzing a specific metabolite in excreta such as blood and urine, The abundance of the P450 molecular species in the human liver can be estimated. Therefore, one kind of human-derived cytochrome P450 molecular species included in the molecular species group or a metabolite specifically present only in the case of an artificial fusion enzyme of human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase If it can be found, the compound as the sample can be used as a metabolic probe for the molecular species of cytochrome P450 contained in the molecular species group. That is, after the reaction, by analyzing metabolites present in the reaction solution, one type of (i) human-derived cytochrome P450 molecular species or (ii) human-derived cytochrome P450 molecules included in the above molecular species group Only in the case of an artificial fusion enzyme of seed and yeast NADPH-P450 reductase, a step of determining the presence or absence of a metabolite that specifically exists (step 4C), and by the above determination, (a) one type of (i) human origin Cytochrome P450 molecular species or (ii) a human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase artificial fusion enzyme, and there is a metabolite that specifically exists, If it is evaluated as a metabolic probe of a P450 molecular species, or (b) the metabolite specifically present is absent, the analyte compound is Metabolic probes can also be screened by the step of evaluating that they are not effective as metabolic probes for tochrome P450 molecular species (step 5C).

  Hereinafter, examples will be described in more detail. However, the present invention is not limited to these examples.

Example 1 (Acquisition of a gene having a base sequence encoding a human-derived cytochrome P450 molecular species)
A PCR method using a human cytochrome P450 gene cloning primer described in FIGS. 1 to 4 from a commercially available cDNA library derived from human liver (Clontech), a human-derived cytochrome P450 gene cloning linker shown in FIG. Was used to obtain cDNA encoding human cytochrome P450 molecular species. The nucleotide sequence of the obtained cDNA and the test amino acid sequence are shown in the sequence listing.
In addition, the relationship between the sequence number shown below and the cytochrome P450 molecular species derived from human is as follows:
1. Human-derived cytochrome P450 molecular species essential for the present invention (1) SEQ ID NO: 1 1A2
(2) SEQ ID NO: 2 2C9
(3) SEQ ID NO: 3 2E1
(4) SEQ ID NO: 4 3A4
2. Human-derived cytochrome P450 molecular species auxiliary to the present invention (1) SEQ ID NOs: 5, 6, and 7 1A1
(2) SEQ ID NO: 8, 92 A6
(3) SEQ ID NO: 10 2B6
(2) SEQ ID NO: 11, 12, 13 2C8
(4) SEQ ID NO: 14 2C18
(5) SEQ ID NO: 15 2C19
(6) SEQ ID NOs: 16, 17, 18, 19 2D6
It is.

Example 2 (Construction of expression plasmid in yeast: p1A2, p1A2R)
FIG. 6 shows a method for constructing an expression plasmid in yeast of human-derived cytochrome P450 1A2. About 1.5 kb was amplified by PCR using the primers shown in FIG. 1, excluding the N-terminal of about 40 bp of the protein coding region of human-derived cytochrome P450 1A2 gene. The resulting approximately 1.5 kb fragment was cut with SacI and subcloned into the pUC118 vector. The N-terminal of about 40 bp was subcloned into the HindIII and SacI sites of the pUC118 vector using the synthetic linker shown in FIG. A vector having a 1.5 kb fragment was blunted after treatment with HindIII and inserted with an EcoRI linker. This was excised with EcoRI and SacI, ligated with a vector containing N-terminal 40 bp, treated with EcoRI, smoothed, inserted with a Hind III linker, further excised with Hind III, inserted into pAAH5N and pAHRR, and human-derived cytochrome A yeast expression plasmid p1A2 of P450 1A2 and a yeast expression plasmid p1A2R of cytochrome P450 1A2 derived from human and yeast NADPH-P450 reductase were constructed.

Example 3 (Construction of expression plasmid in yeast: p2C9, p2C9R)
FIG. 7 shows a method for constructing a human-derived cytochrome P450 2C9 expression plasmid in yeast. Using the primers shown in FIG. 1, the protein coding region of the human-derived cytochrome P450 2C9 gene was amplified into two fragments of about 0.9 kb and about 0.6 kb by PCR. The obtained about 0.9 kb fragment was cleaved with PstI to change the pUC B vector (the EcoRI site of pUC19 was changed to the HindIII site, and the cloning site formed between the HindIII and HindIII sites was changed to the following cloning site (Formula 1). ):

に交換することにより作製されたサブクローン用のプラスミド）にサブクローン化し、さらに約0.６ｋｂ断片をＸｂａＩ、ＰｓｔＩ部位に組み込んで、２つの断片を連結させた。このプラスミドのＫｐｎＩ部位を平滑化し、ＸｂａＩリンカーを挿入したものからコーディング領域を含むＸｂａＩ断片を切り出し、ｐＵＣベクター中にＡＤＨプロモーター、ターミネーター領域を持つｐＵＣＡＮベクター（市販されるpUC19 ベクターが有するEcoRI 部位とＨindIII部位をそれぞれNotI部位に改変し、形成されたNotI部位とNotI部位の間にpAAH5Nから調製したNotI断片を組み込んで得たベクター）のＨｉｎｄ III部位を平滑化、ＸｂａＩリンカーを導入したｐＵＣＡＮＸに挿入した。これをＮｏｔＩで切り出し、同様にＮｏｔＩ処理したｐＡＡＨ５Ｎ、及びｐＡＨＲＲに挿入し、ヒト由来のチトクロムＰ４５０ ２Ｃ９の酵母内発現プラスミドｐ２Ｃ９及び、ヒト由来のチトクロムＰ４５０ ２Ｃ９と酵母ＮＡＤＰＨ−Ｐ４５０還元酵素の同時酵母内発現プラスミドｐ２Ｃ９Ｒを構築した。

The plasmid was subcloned into a plasmid for subclone prepared by exchanging with (1), and an about 0.6 kb fragment was further incorporated into the XbaI and PstI sites to link the two fragments. The KpnI site of this plasmid was smoothed, an XbaI fragment containing the coding region was excised from the plasmid inserted with the XbaI linker, and a pUCAN vector having an ADH promoter and terminator region in the pUC vector (EcoRI site and HindIII of the commercially available pUC19 vector). Each site was modified to a NotI site, and the HindIII site of a vector obtained by incorporating a NotI fragment prepared from pAAH5N between the NotI site and NotI site was smoothed and inserted into pUCANX into which an XbaI linker was introduced. . This was cut out with NotI, inserted into pAAH5N and pAHRR similarly treated with NotI, and a human-derived cytochrome P450 2C9 expression plasmid p2C9 and a human-derived cytochrome P450 2C9 and yeast NADPH-P450 reductase in the yeast. The expression plasmid p2C9R was constructed.

Example 4 (Construction of expression plasmid in yeast: p2E1, p2E1R)
FIG. 8 shows a method for constructing a human-derived cytochrome P450 2E1 expression plasmid in yeast. Using the primers shown in FIG. 1, the protein coding region of the human-derived cytochrome P450 2E1 gene was amplified into two fragments of about 0.5 kb and about 1.0 kb by PCR. The obtained about 0.5 kb fragment was cleaved with EcoRI and BamHI, subcloned into the pUC118 vector, and the about 1.0 kb fragment was further incorporated into the BamHI and SphI sites to link the two fragments. This was cut with EcoRI and SphI, and once inserted into pUC B, then cut out with Hind III, inserted into pAAH5N and pAHRR vectors, and the human-derived cytochrome P450 2E1 expression plasmid p2E1 in humans and human-derived cytochrome P450 2E1 And yeast NADPH-P450 reductase co-yeast expression plasmid p2E1R was constructed.

Example 5 (Construction of expression plasmid in yeast: p3A4, p3A4R)
FIG. 9 shows a method for constructing a human-derived cytochrome P450 3A4 expression plasmid in yeast. Using the primers shown in FIG. 2, the protein coding region of the human-derived cytochrome P450 3A4 gene was amplified into two fragments of about 0.6 kb and about 0.9 kb by PCR. The obtained approximately 0.6 kb fragment was cut with SacI and subcloned into the pUC118 vector. Thereafter, the 0.9 kb fragment cleaved with XbaI and SacI was incorporated into the product into which XbaI linker had been introduced after being cut and smoothed with EcoRI, and the two fragments were ligated. This plasmid was digested with SphI, blunted, and an XbaI fragment was excised from the plasmid into which the XbaI linker had been introduced, and inserted into the XbaI site of pUCANX. This was cut out with NotI, inserted into pAAH5N and pAHRR similarly treated with NotI, and the yeast-derived plasmid p3A4 of human-derived cytochrome P450 3A4 and the human-derived cytochrome P450 3A4 and yeast NADPH-P450 reductase in the same yeast The expression plasmid p3A4R was constructed.

Example 6 (Construction of expression plasmid in yeast: p1A1, p1A1R)
FIG. 10 shows a method for constructing an expression plasmid in yeast of human-derived cytochrome P450 1A1. The protein coding region of human-derived cytochrome P450 1A1 gene was amplified into two fragments of about 1.0 kb and about 0.5 kb by PCR using the primers shown in FIG. The obtained approximately 1.0 kb fragment was cleaved with XbaI and SacI, and the pUCA vector (pUC19 has an EcoRI site changed to a HindIII site, and the resulting cloning site between the HindIII and HindIII sites was defined as the cloning site below. (Chemical Formula 2):

に交換することにより作製されたサブクローン用のプラスミド）にサブクローンした。増幅した約0.５ｋｂ断片は、一旦ｐＵＣ １９ベクターをＨｉｎｃIIで切断したものにサブクローン化し、ＳａｃＩで切断後、1.０ｋｂ断片を持つベクターと連結した。こうして得られた１Ａ１遺伝子のコーディング領域をＨｉｎｄ IIIで切り出した後、ＡＤＨプロモーターおよびターミネーター領域を有する酵母発現用ベクターｐＡＡＨ５Ｎ、及びその上流に酵母ＮＡＤＰＨ−Ｐ４５０還元酵素遺伝子を有するＰ４５０と酵母ＮＡＤＰＨ−Ｐ４５０還元酵素同時発現用ベクターｐＡＨＲＲのＨｉｎｄ III部位に挿入し、ヒト由来のチトクロムＰ４５０１Ａ１の酵母内発現プラスミドｐ１Ａ１、及びヒト由来のチトクロムＰ４５０１Ａ１と酵母ＮＡＤＰＨ−Ｐ４５０還元酵素の同時酵母内発現プラスミドｐ１Ａ１Ｒを構築した。
さらに、ごく一部の塩基配列のみが異なる２種のヒト由来のチトクロムＰ４５０ １Ａ１遺伝子断片を上記と同様に得て、２種のヒト由来のチトクロムＰ４５０ １Ａ１の酵母内発現プラスミドｐ１Ａ１ Variant１、ｐ１Ａ１ Variant２、及び２種のヒト由来のチトクロムＰ４５０ １Ａ１と酵母ＮＡＤＰＨ−Ｐ４５０還元酵素の同時酵母内発現プラスミドｐ１Ａ１Ｒ Variant１、ｐ１Ａ１Ｒ Variant２を構築した。

Subclone into a subclone plasmid prepared by exchanging The amplified about 0.5 kb fragment was subcloned into a pUC19 vector once cut with HincII, cut with SacI, and ligated with a vector having a 1.0 kb fragment. After the 1A1 gene coding region thus obtained was excised with HindIII, the yeast expression vector pAAH5N having an ADH promoter and terminator region, and P450 having a yeast NADPH-P450 reductase gene upstream thereof and yeast NADPH-P450 reductase It was inserted into the Hind III site of the enzyme co-expression vector pAHRR, and a human-derived cytochrome P4501A1 yeast expression plasmid p1A1 and a human-derived cytochrome P4501A1 and yeast NADPH-P450 reductase co-yeast expression plasmid p1A1R were constructed.
Furthermore, two human-derived cytochrome P450 1A1 gene fragments differing only in a small part of the base sequence were obtained in the same manner as described above, and two human-derived cytochrome P450 1A1 expression plasmids p1A1 Variant1, p1A1 Variant2, And two types of human-derived cytochrome P450 1A1 and yeast NADPH-P450 reductase co-yeast expression plasmids p1A1R Variant1 and p1A1R Variant2 were constructed.

Example 7 (Construction of expression plasmid in yeast: p2A6, p2A6R)
FIG. 11 shows a method for constructing an expression plasmid in yeast of human-derived cytochrome P450 2A6. Using the primers shown in FIG. 3, the protein coding region of the human-derived cytochrome P450 2A6 gene was amplified by dividing it into two fragments of about 0.6 kb and about 0.9 kb. As a result, two types of human-derived cytochrome P450 2A6 gene fragments differing only in a small part of the base sequence were obtained.
The obtained approximately 0.6 kb fragment was cleaved with XbaI and HincII and subcloned into a pUCA vector, and the 0.9 kb fragment was further incorporated into the HincII and KpnI sites to link the two fragments. This was excised with HindIII, inserted into pAAH5N and pAHRR, and two human-derived cytochrome P450 2A6 expression plasmids p2A6, p2A6 Variant1 and two human-derived cytochrome P450 2A6 and yeast NADPH-P450 reductase The plasmids p2A6R and p2A6R Variant1 were simultaneously constructed.

Example 8 (Construction of expression plasmid in yeast: p2B6, p2B6R)
FIG. 12 shows a method for constructing a human-derived cytochrome P450 2B6 expression plasmid in yeast. Using the primers shown in FIG. 3, the human-derived cytochrome P450 2B6 gene whole protein coding region was amplified by PCR. The resulting fragment was cleaved with XbaI and BamHI and subcloned into pUCA. This was partially digested with Hind III, inserted into pAAH5N and pAHRR vector, and the expression cytoplasm P450 2B6 derived from human cytochrome P450 2B6 and the expression of human cytochrome P450 2B6 and yeast NADPH-P450 reductase simultaneously in yeast Plasmid p2B6R was constructed.

Example 9 (Construction of expression plasmid in yeast: p2C8, p2C8R)
FIG. 13 shows a method for constructing a human-derived cytochrome P450 2C8 expression plasmid in yeast. The human-derived cytochrome P450 2C8 gene whole protein coding region was amplified by PCR using the primers shown in FIG. As a result, three human-derived cytochrome P450 2C8 genes differing only in a small part of the base sequence were obtained. The obtained fragment was partially digested with XbaI and once subcloned into pUCA. This was excised with Hind III, inserted into pAAH5N and pAHRR vector, and three human-derived cytochrome P450 2C8 expression plasmids p2C8, p2C8 Variant1, p2C8 Variant2, and three human-derived cytochrome P450 2C8 and yeast. NADPH-P450 reductase co-yeast expression plasmids p2C8R, p2C8R Variant1, and p2C8R Variant2 were constructed.

Example 10 (Construction of expression plasmid in yeast: p2C18, p2C18R)
FIG. 14 shows a method for constructing a human-derived cytochrome P450 2C18 expression plasmid in yeast. Using the primers shown in FIG. 3, the protein coding region of the human-derived cytochrome P450 2C18 gene was amplified by dividing it into two fragments of about 1.4 kb and about 0.9 kb. The obtained about 1.4 kb fragment was cleaved with PstI and subcloned into a pUC A vector, and the about 0.9 kb fragment was further incorporated into the XbaI and PstI sites to link the two fragments. After this plasmid was digested with SmaI, an XbaI fragment was excised from the plasmid into which the XbaI linker had been introduced, and inserted into the XbaI site of pUCANX. This was cut out with NotI, inserted into pAAH5N and pAHRR similarly treated with NotI, and a human-derived cytochrome P450 2C18 expression plasmid p2C18 and a human-derived cytochrome P450 2C18 and yeast NADPH-P450 reductase in the same yeast. The expression plasmid p2C18R was constructed.

Example 11 (Construction of expression plasmid in yeast: p2C19, p2C19R)
FIG. 15 shows a method for constructing a human-derived cytochrome P450 2C19 expression plasmid in yeast. Using primers No.1, No.2, No.3 and No.4, No.5 and No.6, and No.5 and No.7 having the base sequences shown in SEQ ID NOs: 20 to 26, respectively A part of the protein coding region of human-derived cytochrome P450 2C19 gene was amplified by the PCR method (fragments a, b, c). As for other regions, human-derived cytochrome P450 2C9 gene was introduced by introducing a mutation into the human-derived cytochrome P450 2C9 gene by PCR using primers No. 8 to No. 21 having the nucleotide sequences shown in SEQ ID NOs: 27 to 40. A part of the protein coding region of cytochrome P450 2C19 gene is amplified (fragments e and f), and the remaining regions are linkers having the nucleotide sequences shown in SEQ ID NOs: 41 and 42 by directly synthesizing DNA. No. 1 and No. 2 were obtained (fragment d). In this way, a fragment covering the entire protein coding region of human-derived cytochrome P450 2C19 gene was prepared.
In the following, SEQ ID NO: and primer No. Shows the relationship.
Sequence number 20: Primer No. 1 Sequence number 21: Primer No. 2 Sequence number 22: Primer No. 3 Sequence number 23: Primer No. 4 Sequence number 24: Primer No. 5 Sequence number 25: Primer No. 6 Sequence number 26: Primer No. 7 SEQ ID NO: 27: Primer No. 8 SEQ ID NO: 28: Primer No. 9 SEQ ID NO: 29: Primer No. 10 SEQ ID NO: 30: Primer No. 11 SEQ ID NO: 31: Primer No. 12 SEQ ID NO: 32: Primer No. 13 SEQ ID NO: 33: Primer No. 14 SEQ ID NO: 34: Primer No. 15 SEQ ID NO: 35: Primer No. 16 SEQ ID NO: 36: Primer No. 17 SEQ ID NO: 37: Primer No. 18 SEQ ID NO: 38: Primer No .19 SEQ ID NO: 39: Primer No. 20 SEQ ID NO: 40: Primer No. 21 SEQ ID NO: 41: Linker No. 1 SEQ ID NO: 42: Linker No. 2
Next, the fragment a treated with XhoI and BamHI and the fragment b treated with BamHI and PstI are simultaneously inserted into the XhoI and PstI sites of Blue Script (+) to obtain a fragment e treated with XbaI and XhoI. A plasmid containing fragments a, b and e was obtained by inserting into the XbaI and XhoI sites of the resulting plasmid. Further, the fragment c treated with PstI and KpnI and the fragment d, which is a linker, were simultaneously inserted into the PstI and EcoRI sites of Blue Script (+) and then excised again with PstI / EcoRI (fragments c, d And the fragment f treated with EcoRI were simultaneously inserted into the PstI and HincII sites of the aforementioned plasmid (the plasmid containing the fragments a, b and e). In this way, a plasmid containing the entire protein coding region of human cytochrome P450 2C19 gene was constructed. The constructed plasmid was excised with HindIII and similarly inserted into HindIII-treated pAAH5N and pAHRR, and the human-derived cytochrome P450 2C19 expression plasmid p2C19 in human and cytochrome P450 2C19 derived from human and yeast NADPH-P450 reductase A simultaneous yeast expression plasmid p2C19R was constructed.

Example 12 (Construction of expression plasmid in yeast: p2D6, p2D6R)
FIG. 16 shows a method for constructing a human-derived cytochrome P450 2D6 expression plasmid in yeast. Using the primers shown in FIG. 4, except for the N-terminal of about 200 bp of the protein coding region of human-derived cytochrome P450 2D6 gene, 1.3 kb is divided into two fragments of about 0.4 kb and about 0.9 kb. Amplified separately. The resulting approximately 0.9 kb fragment was cut with KpnI and subcloned into pUCA. The N-terminal 200 bp uses the three synthetic linkers shown in FIG. 5. First, the two linkers on the N-terminal side are incorporated into the XbaI and PstI sites of the Blue Script (+) vector, and then another linker is added. Incorporated into SmaI and PstI sites. Further, an approximately 0.4 kb fragment obtained by PCR was inserted into the PstI and HincII sites of this plasmid, then cleaved with NspV and XbaI, inserted into a plasmid containing a 0.9 kb fragment, and the coding region was ligated. This was excised with HindIII, inserted into pAAH5N and pAHRR vectors, and a human-derived cytochrome P450 2D6 expression plasmid p2D6, and a human-derived cytochrome P450 2D6 and yeast NADPH-P450 reductase co-yeast expression plasmid p2D6R Built.
Furthermore, three human-derived cytochrome P450 2D6 gene fragments differing only in a small part of the base sequence were obtained in the same manner as described above, and three human-derived cytochrome P450 2D6 expression plasmids p2D6 Variant1, p2D6 Variant2, The plasmids p2D6R Variant1, p2D6R Variant1, and p2D6R Variant3 were constructed in p2D6 Variant3 and two human-derived cytochrome P450 2D6 and yeast NADPH-P450 reductase simultaneously in yeast.

Example 13 (Construction of an expression plasmid in yeast containing an artificial fusion enzyme gene)
A plasmid was constructed according to FIG. An XbaI-XhoI fragment was obtained using plasmid p3A4 as a template and the primers shown in FIG. In addition, the approximately 2.1 kb XhoI-HindIII fragment obtained from plasmid pBFCR1 (Japanese Patent Application No. 4-209226) was inserted into the XhoI and HindIII sites of the commercially available vector Blue Script (+) and then digested simultaneously with restriction enzymes XhoI and XbaI. And a fragment was obtained. Both of these fragments were simultaneously inserted into the vector pUCAN at the XbaI site, and the resulting plasmid was digested with the restriction enzyme NotI to obtain an approximately 5.6 kb fragment. By ligating this fragment with the approximately 10.5 kb NotI fragment obtained from the vector pAAH5N, the yeast expression plasmid pF3A4 containing the target artificial fusion enzyme gene was obtained. The artificial fusion enzyme consists of 1156 amino acid residues, and its structure is the entire amino acid sequence (503 residues) encoding human liver cytochrome P450 3A4 from the N-terminus, a sequence derived from a linker (Ala-Arg-Ala), yeast NADPH -Continued from the 42nd to the C-terminal of the N-terminal cytochrome P450 reductase.

Example 14 (Production of transformed yeast cells)
Saccharomyces cerevisiae AH22 strain is inoculated into 1.0 ml of YPD medium (1% yeast extract, 2% polypeptone, 2% glucose), shaken at 30 ° C. for 18 hours, and then collected by centrifugation (5000 × g, 10 minutes). did. The obtained cells were suspended in 1.0 ml of 0.2M LiC1 solution and then centrifuged again (5000 × g, 10 minutes). The obtained pellet was added to 20 μl of 1M LiC1 solution, 30 μl of 70% polyethylene glycol 4000. About 1.0 μg of a solution, various (i) human-derived cytochrome P450 and yeast NADPH-P450 reductase co-expression plasmids in yeast or (ii) human-derived cytochrome P450 and yeast 10 μl of a solution containing each NADPH-P450 reductase artificial fusion enzyme expression plasmid in yeast was added. After thoroughly mixing this, it was incubated at 30 ° C. for 1 hour, and further 140 μl of sterilized water was added and stirred. This solution was plated on an SD synthetic medium plate (2.0% glucose, 0.67% nitrogen source amino acid-free (Nitrogen base w / o amino acids, manufactured by Difco), 20 μg / ml histidine 2.0% agar) at 30 ° C. Incubated for 3 days to select transformed yeast cells having the above-mentioned yeast expression plasmid. Thus, in yeast, (i) human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) various human fusion enzymes that express human-derived cytochrome P450 and yeast NADPH-P450 reductase are expressed. Yeast cells were prepared.

Example 15 (Quantification of human-derived cytochrome P450 expressed in yeast)
Various yeasts that express the human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) the artificial fusion enzyme of human-derived cytochrome P450 and yeast NADPH-P450 reductase prepared according to Example 14 200 ml of each cell culture medium (SD synthesis medium, cell concentration of about 1.5 × 10 ⁷ cells / ml) was collected, and the cells were collected in 10 ml of 100 mM potassium phosphate buffer (pH 7.0). Was suspended and then centrifuged (5000 × g, 10 minutes). The obtained pellet was newly suspended in 2.0 ml of 100 mM potassium phosphate buffer (pH 7.0), and 1.0 ml was dispensed into two cuvettes. After carbon monoxide was blown into the cuvette on the sample side, 5-10 mg of dithionite was added to both cuvettes, and after stirring, the difference spectrum of 400-500 nm was measured, and (i) human-derived cytochrome present in yeast The artificial fusion enzyme concentration of P450 or (ii) human-derived cytochrome P450 and yeast NADPH-P450 reductase was calculated. Expression levels of various types of (i) human-derived cytochrome P450 or (ii) human-derived cytochrome P450 and yeast NADPH-P450 reductase in various transformed yeast cells are all about 10 ^{5 to} about 10 ⁶ molecules / cell. It was the level of.

Example 16 (Preparation of yeast S-9 Mix fraction, cytoplasmic fraction and microsomal fraction)
Various yeasts that express the human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) the artificial fusion enzyme of human-derived cytochrome P450 and yeast NADPH-P450 reductase prepared according to Example 14 3.8 l of each cell culture medium (SD synthesis medium, cell concentration of about 1.0 × 10 ⁸ cells / ml) was collected, and the resulting cells were collected with 400 ml of buffer A (10 mM Tris− It was suspended in HCl (pH 7.5), 2M sorbitol, 0.1 mM DTT, 0.2 mM EDTA, 160 mg zymolyase 100,000 (zymolyase 100T) was added, and the mixture was incubated at 30 ° C. for 60 minutes. Spheroplasts obtained by centrifugation (5000 × g, 10 minutes) were suspended in 100 ml of buffer A and then centrifuged (5000 × g, 10 minutes). After repeating the same centrifugation operation once again to wash the spheroplasts, the spheroplasts were suspended in 200 ml of a buffer (10 mM Tris-HCl (pH 7.5), 0.65 M sorbitol, 0.1 mM DTT) Sonication (50 W, 5 minutes) was performed. The crushed material was centrifuged (9000 × g, 20 minutes), and the supernatant was collected to obtain a yeast S-9 Mix fraction. Further, this was further centrifuged (125000 × g, 70 minutes), and the precipitate was collected and resuspended in 10 ml of 0.1 M potassium phosphate buffer (pH 7.4) to obtain a microsomal fraction. On the other hand, the cytoplasmic fraction was obtained by collecting the supernatant.

Example 17 (Construction of expression plasmid in yeast using GAPDH promoter and expression in yeast)
FIG. 18 shows a method for constructing an expression plasmid in yeast using the GAPDH promoter. After cutting pUC19 with EcoRI and smoothing, the HindIII fragment (about 3.0 Kb) obtained from pARRN was inserted into the HindIII site of the plasmid pUN obtained by inserting NotI linker to obtain pUR. On the other hand, the XhoI site of the plasmid pAAH5 was blunted and the XbaI linker was inserted, then cleaved with restriction enzymes XbaI and SalI, and the resulting fragment (about 2.2 Kb) was inserted into the XbaI and SaII sites of pUC19. A fragment (about 2.2 Kb) obtained by cutting the obtained plasmid with XbaI and PstI, an XbaI-PstI fragment (about 1.3 Kb) excised from 2 μm DNA prepared from Saccharomyces cerevisiae AH22 strain, and pUR with PstI A pURL was obtained by ligating three fragments obtained by cutting. Furthermore, the HindIII site was eliminated by ligating pURL with HindIII, smoothing and then ligating. Next, a NotI fragment (about 1.6 Kb) containing the GAPDH promoter and terminator [Agric. Biol. Chem., 51, 1641-1647 (1987), J. Biol. Chem., 267, 16497-16502 (1992)] PURLG was obtained by inserting into the NotI site of pURL. By inserting the human-derived cytochrome P450 2D6 cDNA obtained in accordance with Example 9 into the HindIII site of pURLG, a simultaneous yeast expression plasmid pG2D6R of human-derived cytochrome P450 2D6 and yeast NADPH-P450 reductase is obtained. It was. When this plasmid was introduced into Saccharomyces cerevisiae AH22 according to Example 14, production of human-derived cytochrome P450 2D6 was observed.

  Next, in order to efficiently reproduce the metabolic system in human liver in vitro, all the molecular species included in the molecular species group consisting of P450 1A2, P450 2C9, P450 2E1 and P450 3A4 as human-derived cytochrome P450 molecular species. It is essential that P450 2A6 and / or P450 2C19 and / or P450 2D6 be supplementarily added to the above molecular species group, and P450 1A1, P450 2A6, P450 2B6, A number of test examples are described below to show that accuracy is improved by supplemental combination of other cytochrome P450 molecular species such as P450 2C8, P450 2C18, P450 2C19 or P450 2D6. First, a test was conducted in which the yeast cells produced in Example 14 or 16 or the disrupted cells were reacted with various compounds.

Test Example 1 (7-ethoxycoumarin O-deethylation reaction and metabolite analysis using living transformed yeast cells)
Various yeasts that express the human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) the artificial fusion enzyme of human-derived cytochrome P450 and yeast NADPH-P450 reductase prepared according to Example 14 Add 7-ethoxycoumarin to a final concentration of 0.5 mM in 6 ml of each cell culture medium (SD synthesis medium, cell concentration approximately 2.0 × 10 ⁷ cells / ml), and add 2 at 30 ° C. Alternatively, after incubation for 5 hours, the supernatant was obtained by centrifugation (5000 × g, 10 minutes). 62.5 μl of 15% TCA and 2 ml of chloroform were added thereto, and the mixture was stirred well and then centrifuged (5000 × g, 10 minutes). The chloroform layer was recovered from the separated layers, and 4 ml of 0.01N NaOH containing 0.1 M NaCl was added thereto, and after stirring well, the mixture was centrifuged again (5000 × g, 10 minutes). After collecting the supernatant, fluorescence (ex. 366 nm, em. 452 nm) was measured for the supernatant fraction, and the produced 7-hydroxycoumarin was quantified. As a result, 7-ethoxycoumarin O-deethylation activity was observed in all of the various yeast cells expressing 11 types of human cytochrome P450 molecular species. The degree of activity in each molecular species was strong in P450 1A1 and P450 2B6, and sufficient in P450 1A2, P450 2E1, P450 2A6, and P450 2D6. P450 2C8, P450 2C9, P450 3A4, P450 2C18, and P450 2C19 were weak.

Test Example 2 (Tolbutamide hydroxylation reaction and metabolite analysis using living transformed yeast cells)
As in Test Example 2, (i) human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) various yeasts that express human-derived cytochrome P450 and yeast NADPH-P450 reductase artificial fusion enzyme Tolbutamide was added to each culture solution to a final concentration of 1.0 mM and incubated at 30 ° C. for 15 hours, followed by centrifugation (5000 × g, 10 minutes) to obtain a supernatant. 2 ml of dichloromethane was added to this, and after stirring well, it was centrifuged again (5000 × g, 10 minutes). The dichloromethane layer was collected from the separated layers, and the solvent was removed under reduced pressure. The obtained residue was dissolved in 100 μl of acetonitrile, and the solution was analyzed by HPLC under the following conditions. As a result, tolbutamide hydroxide was detected in human-derived cytochrome P450 2C8, P450 2C9 P450, 2C18 and P450 2C19 expressing yeast cells. The degree of activity in each molecular species was strong in P450 2C9 and sufficient in P450 2C19. P450 2C8 and P450 2C18 were weak. On the other hand, it was not detected in yeast cells expressing other molecular species.
<HPLC conditions>
Column: μ Bondapak C18 (Waters)
Carrier: 10-70% acetonitrile-dist. Water
(Linear concentration gradient 20 minutes)
Temperature: 50 ° C
Detection: UV 230nm
Sample volume: 50 μl

Test Example 3 (Testosterone hydroxylation reaction using living transformed yeast cells and analysis of metabolites)
As in Test Example 1, (i) human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) various yeasts expressing human-derived cytochrome P450 and yeast NADPH-P450 reductase artificial fusion enzyme Testosterone was added to each culture solution to a final concentration of 0.05 mM and incubated at 30 ° C. for 15 hours, followed by centrifugation (5000 × g, 10 minutes) to obtain a supernatant. 2 ml of dichloromethane was added to this, and after stirring well, it was centrifuged again (5000 × g, 10 minutes). The dichloromethane layer was collected from the separated layers, and the solvent was removed under reduced pressure. The obtained residue was dissolved in 100 μl of acetonitrile, and the solution was analyzed by HPLC under the following conditions. As a result, testosterone hydroxide was detected in human-derived cytochrome P450 1A1, P450 2C8 and P450 3A4-expressing yeast cells. The degree of activity in each molecular species was strong in P450 3A4 and weak in P450 1A1 and P450 2C8. On the other hand, it was not detected in yeast cells expressing other molecular species.
<HPLC conditions>
Column: μ Bondapak C18 (Waters)
Carrier: 20-70% acetonitrile-dist. Water (linear concentration gradient 25 minutes)
Temperature: 50 ° C
Detection: UV 254nm
Sample volume: 50 μl

Test Example 4 (Analysis of chlorozoxazone hydroxylation and metabolites using living transformed yeast cells and microsomal fraction)
When living transformed yeast cells are used, as in Test Example 1, (i) human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) human-derived cytochrome P450 and yeast NADPH-P450 Chloroxoxazone was added to each culture solution of various yeast cells expressing a reductase artificial fusion enzyme to a final concentration of 0.5 mM, incubated at 30 ° C. for 15 hours, and then centrifuged (5000 × g, For 10 minutes) to obtain a supernatant. 2 ml of dichloromethane was added to this, and after stirring well, it was centrifuged again (5000 × g, 10 minutes). The dichloromethane layer was collected from the separated layers, and the solvent was removed under reduced pressure. The obtained residue was dissolved in 100 μl of acetonitrile, and the solution was analyzed by HPLC under the following conditions.
On the other hand, when the microsomal fraction is used, (i) human cytochrome P450 molecular species and yeast NADPH-P450 reductase obtained in Example 16 or (ii) human cytochrome P450 and yeast NADPH-P450 reduction NADPH and chlorozoxazone were added to various yeast microsomal fractions expressing the enzyme artificial fusion enzyme to final concentrations of 0.5 mM and 250 μM, respectively, and incubated at 37 ° C. for 10 minutes. Thereafter, trichloroacetic acid was added to a final concentration of about 10% (v / v). 2 ml of dichloromethane was added to this, and after stirring well, it was centrifuged again (15000 × g, 5 minutes). The dichloromethane layer was collected from the separated layers, and the solvent was removed under reduced pressure. The obtained residue was dissolved in 100 μl of acetonitrile, and the solution was analyzed by HPLC under the following conditions.
As a result, chlorozoxazone hydroxide was detected in all of the various yeast cells expressing 11 human cytochrome P450 molecular species. The degree of activity in each molecular species was strong in P450 2E1 and sufficient in P450 1A1, P450 1A2, P450 2A6, and P450 2D6. P450 2C8, P450 2C9, P450 2B6, P450 2C18, P450 2C19, and P450 3A4 were weak.
<HPLC conditions>
Column: μ Bondapak C18 (Waters)
Carrier: 20-70% acetonitrile-dist. Water (linear concentration gradient 25 minutes)
Temperature: 50 ° C
Detection: UV 254nm
Sample volume: 50 μl

Test Example 5 (2-aminoanthracene hydroxylation reaction and metabolite analysis using yeast S-9 Mix fraction and microsome fraction: Ames test)
The Ames test method followed the usual method described in Mutat. Res. (1975) 31,347 etc. In addition, 2-aminoanthracene which is an arylamine type compound was used as a compound which is a specimen. However, as metabolic activation sources, (1) a yeast S-9 Mix fraction obtained from various human-derived cytochrome P450-expressing yeast cells, and (2) a microsomal fraction prepared from the yeast S-9 Mix fraction. Or (3) Rat S-9 Mix containing about 1200 pmol of human-derived cytochrome P450 molecular species per sample [Comparative Example: obtained by homogenizing the liver and then centrifuging (9000 × g, 10 minutes) The clear fraction (manufactured by Kikkoman)] was used for the Ames test. As a result, human-derived cytochrome P450 2A6-expressing yeast cells (Saccharomyces cerevisiae AH22 / p2A6R), human-derived cytochrome P450 2C8-expressing yeast cells (Saccharomyces cerevisiae AH22 / p2C8R) and human-derived cytochrome P450 3A4 expressing yeast cells When using the yeast S-9 Mix fraction obtained from the body (Saccharomyces cerevisiae AH22 / p3A4R) and the microsomal fraction prepared from the yeast S-9 Mix fraction, no backmutant colonies were detected. However, human-derived cytochrome P450 1A2-expressing yeast cells (Saccharomyces cerevisiae AH22 / p1A2R) and human-derived cytochrome P450 2E1-expressing yeast cells (Saccharomyces cerevisiae AH22 / p2E1R) ) And a microsome fraction prepared from the yeast S-9 Mix fraction, 1,000 or more in a test amount of 1 μg / plate (diameter 90 mm) Of back mutation colonies were detected. Judging from the number of backmutant colonies detected, the P4501A2 molecular species had strong metabolic activity, while the P450 2E1 molecular species had only weak metabolic activity.
On the other hand, in comparison with rat S-9 Mix, a yeast S-9 Mix fraction obtained from a human-derived cytochrome P450 1A2 expressing yeast (Saccharomyces cerevisiae AH22 / p1A2R) and the yeast S-9 Mix fraction were obtained. When the prepared microsomal fraction was used, the amount of P450 tested was smaller (1/500 and 1/30 or less) than when rat S-9 Mix was used. More than double back mutation colonies were detected.

Test Example 6 (Acetanilide hydroxylation reaction and metabolite analysis using living transformed yeast cells)
As in Test Example 1, (i) human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) various yeasts expressing human-derived cytochrome P450 and yeast NADPH-P450 reductase artificial fusion enzyme Acetanilide was added to each culture broth to a final concentration of 5 mM, incubated at 30 ° C. for 15 hours, and then centrifuged (5000 × g, 10 minutes) to obtain a supernatant. The obtained supernatant fraction was analyzed by HPLC under the following conditions. As a result, acetanilide hydroxide was detected in all of the various yeast cells expressing 11 types of human cytochrome P450 molecular species. The degree of activity in each molecular species was strong in P450 1A2 and P450 2D6, and was sufficient in P450 1A1, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C18, P450 2C19, and P450 2E1. . P450 3A4 was weak.
<HPLC conditions>
Column: μ Bondapak C18 (Waters)
Carrier: methanol: water: acetic acid = 15: 84: 1
Temperature: 30 ° C
Detection: UV 245nm
Sample volume: 50 μl

Test Example 7 (Coumarine 7-position hydroxylation using transformed yeast cells and analysis of metabolites)
Various yeasts that express the human-derived cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) the artificial fusion enzyme of human-derived cytochrome P450 and yeast NADPH-P450 reductase prepared according to Example 14 Coumarin was added to 6 ml of each cell culture solution (SD synthesis medium, cell concentration of about 2.0 × 10 ⁷ cells / ml) to a final concentration of 0.5 mM, and at 30 ° C. for 2 or 5 hours. After incubation, the supernatant was obtained by centrifugation (5000 × g, 10 minutes). 62.5 μl of 15% TCA and 2 ml of chloroform were added thereto, and the mixture was stirred well and then centrifuged (5000 × g, 10 minutes). The chloroform layer was recovered from the separated layers, and 4 ml of 0.01N NaOH containing 0.1 M NaCl was added to the chloroform layer. After stirring well, the mixture was centrifuged again (5000 × g, 10 minutes). After collecting the supernatant, fluorescence (ex. 366 nm, em. 452 nm) was measured for the supernatant fraction, and the produced 7-hydroxycoumarin was quantified. As a result, the activity was specifically detected only in the human P450 2A6-expressing yeast cells and not detected in the other human P450-expressing yeast cells.

Test Example 8 (Debrixone hydroxylation reaction using yeast microsome fraction and analysis of metabolites)
Various yeasts expressing (i) a human cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) an artificial fusion enzyme of human cytochrome P450 and yeast NADPH-P450 reductase prepared according to Example 16 microsomal fraction NADPH and ^{1 4} C-labeled Deburikison each final concentration 6 mM, was added to a 100 [mu] M, and incubated at 37 ° C. 30 min. Thereafter, perchloric acid was added to a final concentration of 10% (v / v), the mixture was stirred well, and centrifuged (15000 × g, 5 minutes) to obtain a supernatant. The obtained supernatant fraction was analyzed by HPLC under the following conditions. As a result, debrixone hydroxide was detected in the human-derived cytochrome P4501A1, P450 2D6-expressing yeast microsome fraction. The degree of activity in each molecular species was strong in P450 2D6 and sufficient in P450 1A1. On the other hand, other human-derived cytochrome P450-expressing yeast cells were not detected.
<HPLC conditions>
Column: COSMOSIL 5C18 (manufactured by Nacalai Tesque)
Carrier: Liquid A: Acetonitrile, Liquid B: 20 mM sodium perchlorate (pH 2.5)
0-15 minutes A / B = 9/91
15-30 minutes A / B = 9/91 to 25/75, linear gradient 30-32 minutes A = 100%
32-42 minutes A / B = 9/91
Column temperature: room temperature Detector: RI ^{(1 4} C)
Sample volume: 100 μl

Test Example 9 (S-mephetonin hydroxylation reaction using yeast microsomal fraction and analysis of metabolites)
Various yeasts expressing (i) a human cytochrome P450 molecular species and yeast NADPH-P450 reductase or (ii) an artificial fusion enzyme of human cytochrome P450 and yeast NADPH-P450 reductase prepared according to Example 16 microsomal fraction NADPH and ^{1 4} C-labeled S- Mefeitonin each final concentration 3 mM, was added to a 25 [mu] M, and incubated at 37 ° C. 30 min. Thereafter, the same amount of methanol as the reaction solution was added and stirred well, followed by centrifugation (15000 × g, 5 minutes) to obtain a supernatant. The obtained supernatant fraction was analyzed by HPLC under the following conditions. As a result, the activity was specifically detected only in human-derived cytochrome P450 2C19-expressing yeast cells, and was not detected in other human-derived cytochrome P450-expressing yeast cells.
<HPLC conditions>
Column: COSMOSIL 5C18 (manufactured by Nacalai Tesque)
Carrier: A solution: methanol / 10 mM potassium phosphate buffer (pH 7.0) = 40/60 mixed solution, B solution: methanol 0-18 minutes A = 100%
18-20 minutes B = 100%
20-35 minutes A = 100%
Column temperature: room temperature Detector: RI ^{(1 4} C)
Sample volume: 100 μl

The results of Test Example 1 to Test Example 9 are summarized in the following table.
In order to reproduce the metabolic system in human liver in vitro, as many cytochrome P450 molecular species as possible should be selected. In order to reproduce efficiently, the contribution of each molecular species to the metabolic system in human liver In simple terms, one having a high metabolic activity and / or one that metabolizes as many compounds as possible may be appropriately selected.
Therefore, selection of cytochrome P450 molecular species having high metabolic activity against tolbutamide was first examined. Cytochrome P450 molecular species having metabolic activity were P4502C9, P450 2C8, P450 2C18, and P450 2C19. Of these, the only cytochrome P450 molecular species with particularly high metabolic activity was P450 2C9, so this molecular species was selected. Next, selection of cytochrome P450 molecular species having high metabolic activity against testosterone was examined. Cytochrome P450 molecular species having metabolic activity were P450 2C8, P450 3A4, and P450 1A1. Among these, since the only cytochrome P450 molecular species having high metabolic activity was P450 3A4, this molecular species was selected. Furthermore, selection of cytochrome P450 molecular species having high metabolic activity against 2-aminoanthracene and chlorozoxazone was examined. Cytochrome P450 molecular species having metabolic activity for 2-aminoanthracene were P450 1A2 and P450 2E1. Among these, the cytochrome P450 molecular species having the highest metabolic activity was P450 1A2, and this molecular species was selected. For chlorozoxazone, all P450 molecular species had metabolic activity. Among these, the cytochrome P450 molecular species having the highest metabolic activity was P450 2E1, and this molecular species was selected. Next, selection of cytochrome P450 molecular species having high metabolic activity against acetanilide and 7-ethoxycoumarin was examined. As a result, it was found that the four kinds of molecular species selected above can be used, so it was decided not to select other molecular kinds. Thus, metabolic reaction was made possible with respect to 6 representative test compounds out of 9 test compounds with the 4 molecular species thus selected. Therefore, selection of cytochrome P450 molecular species having high metabolic activity for the remaining three test compounds (coumarin, debrisoquin, S-mefetonin) was examined. In any of the test compounds, specific molecular species are involved in metabolism, and it is not always essential to efficiently reproduce the metabolic system in human liver in vitro. It was found that the metabolic system in the human liver can be reproduced more accurately in vitro by auxiliary combination with the four selected molecular species. From the above, it can be seen that, in the present invention, it is essential to use in combination all molecular species included in the molecular species group consisting of P450 1A2, P450 2C9, P450 2E1 and P450 3A4 as human-derived cytochrome P450 molecular species.

Example 18 (Measurement of metabolic activity of chlorozoxazone using mixed microsomal fraction)
The microsomal fractions of various human-derived cytochrome P450-expressing yeast prepared in Example 16 were mixed and used to measure the hydroxylation activity of chlorozoxazone. The mixing ratio of various human cytochrome P450 molecular species is
(1) P450 3A4 (35%), P450 2C9 (25%), P450 1A2 (23%), P450 2E1 (17%) (hereinafter referred to as mixed system A)
(2) P450 3A4 (33%), P450 2C9 (5.8%), P450 2C8 (5.8%), P450 2C18 (5.8%), P450 2C19 (5.8%), P450 1A2 (19 %), P450 2E1 (15%), P450 1A1 (2.4%), P450 2A6 (3.0%), P450 2B6 (2.4%), P450 2D6 (2.4%) (hereinafter, mixed system) (B)
Was used. Yeast microsomal fraction mixed with each mixture system was added as a substrate in ^{1 4} C labeled Kurorozokisazon coenzyme NADPH to respective concentrations 382MyuM, and 3 mM. After incubation at 37 ° C. for 30 minutes, 1 mL of dichloromethane was added to stop the reaction. After stirring, the dichloromethane layer was recovered by centrifugation (10,000 × g, 5 minutes), and the solvent was removed with a nitrogen stream. The resulting residue was dissolved by adding 54 μL of acetonitrile and 146 μL of water, and the resulting solution was analyzed by HPLC under the following conditions. As a result, chlorozoxazone similar to that reported by Peter et al. In the metabolism of chlorozoxazone using human liver microsomal fractions (Chem. Res. Toxicol. Vol. 3, p566-573, 1990) for both mixed systems A and B Metabolite patterns were obtained.
<HPLC conditions>
Column: COSMOSIL 5C18 (manufactured by Nacalai Tesque)
Carrier: A liquid: acetonitrile / water = 27/73 mixed liquid, B liquid: acetonitrile 0-15 minutes A = 100%
15-17 minutes B = 100%
17-25 minutes A = 100%
Column temperature: room temperature Detector: RI ^{(1 4} C)
Sample volume: 100 μl
Furthermore, the turnover rate of human cytochrome P450 in the yeast microsome fraction and human liver microsome fraction (reported by Peter et al.) Mixed in each mixed system was calculated. In the case of the mixed system A, it was 1.8 [product nmol / nmol P450 / min], and in the case of the mixed system B, it was 1.6 [product nmol / nmol P450 / min]. On the other hand, in the case of the human liver microsome fraction, the results of calculation of the turnover rate V by the following method using Vmax and Km and the substrate concentration [S] described in the literature (shown in Table 2), the fluctuation range due to individual differences However, the lower limit was 1.0 [product nmol / nmol P450 / min] and the upper limit was 5.9 [product nmol / nmol P450 / min]. Within this range, the turnover speed V in the mixed system B as well as the turnover speed V in the mixed system A according to the present invention are sufficiently included (they are completely in order). there were.)
Therefore, it was found that the metabolic system in the human liver can be sufficiently reproduced in vitro even with only the four selected molecular species.
<Calculation method>
The Vmax and Km described in Peter et al. And the substrate concentration [S] used in this example were determined by the Michaelis-Menten equation (Equation 1).
V = (Vmax * [S]) / (Km + [S])
By substituting into, the turnover rate V of human-derived cytochrome P450 at an arbitrary substrate concentration [S] is calculated.

Example 19 (Measurement of metabolic activity of debrisoquin using mixed microsomal fraction)
Microsomal fractions of various human-derived cytochrome P450-expressing yeast prepared in Example 16 were mixed, and the hydroxylation activity of debrisoquine was measured using this. The mixing ratio of various human-derived cytochrome P450 molecular species is P450 3A4 (33%), P450 2C9 (5.8%), P450 2C8 (5.8%), P450 2C18 (5.8%), P450 2C19. (5.8%), P450 1A2 (19%), P450 2E1 (15%), P450 2B6 (2.4%), P450 2D6 (2.4%) were used. The mixed yeast microsomal fraction was added as a substrate in ^{1 4} C labeled debrisoquine coenzyme NADPH to as respectively 100 [mu] M, a 6 mM. After incubating at 37 ° C. for 30 minutes, 50 μL of 60% perchloric acid (12.5% (v / v) as final concentration) was added to stop the reaction. After stirring, the supernatant was collected by centrifugation (15,000 × g, 5 minutes), and the reaction product was analyzed by HPLC (analysis conditions are the same as those described in Test Example 8). As a result, a pattern of debrisoquin metabolites similar to that reported by Kronbach et al. In the metabolism of debrisoquin using human liver microsomes (Methods in Enzymology, Vol. 206, p509-517 (1991)) was obtained.

Example 20 (Measurement of metabolic activity of S-mefetonin using mixed microsomal fraction)
The microsomal fractions of various human-derived cytochrome P450-expressing yeast prepared in Example 16 were mixed and used to measure the hydroxylation activity of S-mefetonin. As the mixing ratio of various human-derived cytochrome P450 molecular species, those shown in the mixed system B described in Example 18 were used. The mixed yeast microsomal fraction, as a substrate in ^{1 4} C labeled S- Mefeitonin coenzyme NADPH, respectively 28 .mu.M, was added to a 6 mM. After incubating at 37 ° C. for 30 minutes, 250 μL of methanol was added to stop the reaction. After stirring, the supernatant was collected by centrifugation (15,000 × g, 5 minutes), and the reaction product was analyzed by HPLC (analysis conditions are the same as those described in Test Example 9). As a result, a pattern of S-mephetonin metabolites similar to that reported by Goldstein et al. In the metabolism of S-mefetonin using human liver microsomes (Biochemistry Vol.33, p1743-1752, (1994)) was obtained. It was.

  The present invention is a safety evaluation method that reproduces a superior human metabolic system in an in vitro system, and has made it possible to predict and evaluate metabolism, drug efficacy, and toxicity at an early stage with high accuracy.

SEQ ID NO: 20
Oligonucleotide primer designed for PCR (Primer No. 1)
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Oligonucleotide primer designed for PCR (Primer No. 2)
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Oligonucleotide primer designed for PCR (Primer No. 3)
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Oligonucleotide primer designed for PCR (Primer No. 4)
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Oligonucleotide primer designed for PCR (Primer No. 5)
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Oligonucleotide primer designed for PCR (Primer No. 6)
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Oligonucleotide primer designed for PCR (Primer No. 7)
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Oligonucleotide primer designed for PCR (Primer No. 8)
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Oligonucleotide primer designed for PCR (Primer No. 9)
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Oligonucleotide primer designed for PCR (Primer No. 10)
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Oligonucleotide primer designed for PCR (Primer No. 11)
SEQ ID NO: 31
Oligonucleotide primer designed for PCR (Primer No. 12)
SEQ ID NO: 32
Oligonucleotide primer designed for PCR (Primer No. 13)
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Oligonucleotide primer designed for PCR (Primer No. 14)
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Oligonucleotide primer designed for PCR (Primer No. 15)
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Oligonucleotide primer designed for PCR (Primer No. 16)
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Oligonucleotide primer designed for PCR (Primer No. 17)
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Oligonucleotide primer designed for PCR (Primer No. 18)
SEQ ID NO: 38
Oligonucleotide primer designed for PCR (Primer No. 19)
SEQ ID NO: 39
Oligonucleotide primer designed for PCR (Primer No. 20)
SEQ ID NO: 40
Oligonucleotide primer designed for PCR (Primer No. 21)
SEQ ID NO: 41
Oligonucleotide designed for linker (Linker No.1)
SEQ ID NO: 42
Oligonucleotide designed for linker (Linker No. 2)

It is a figure which shows the primer for human-derived cytochrome P450 1A2, P450 2C9 and P4502E1 gene cloning. It is a figure which shows the primer for human-derived cytochrome P450 3A4, P450 1A1, and P4502A6 gene cloning. It is a figure which shows the primer for cytochrome P450 2B6 of human origin, P450 2C8, and P4502C18 gene cloning. It is a figure which shows the primer for human-derived cytochrome P450 2C19, P450 2D6 and P450 3A4 (XbaI-XhoI fragment) gene cloning. It is a figure which shows the synthetic linker used for cytochrome P450 1A2 of human origin, and P450 2D6 gene cloning. It is a figure which shows the construction method of the expression plasmid (p1A2, p1A2R) in yeast containing the cytochrome P450 1A2 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p2C9, p2C9R) in yeast containing the cytochrome P450 2C9 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p2E1, p2E1R) in yeast containing the cytochrome P450 2E1 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p3A4, p3A4R) in yeast containing the cytochrome P450 3A4 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p1A1, p1A1R) in yeast containing the cytochrome P450 1A1 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p2A6, p2A6R) in yeast containing the cytochrome P450 2A6 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p2B6, p2B6R) in yeast containing the cytochrome P450 2B6 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p2C8, p2C8R) in yeast containing the cytochrome P450 2C8 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p2C18, p2C18R) in yeast containing the cytochrome P450 2C18 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p2C19, p2C19R) in yeast containing the cytochrome P450 2C19 gene derived from a human. It is a figure which shows the construction method of the expression plasmid (p2D6, p2D6R) in yeast containing the cytochrome P450 2D6 gene derived from a human. It is a figure which shows the construction method of the expression plasmid in yeast (pF3A4) containing the artificial fusion enzyme gene of human-derived cytochrome P450 3A4 and yeast NADPH-P450 reductase. It is a figure which shows the construction method of the expression plasmid in yeast using the GAPDH promoter.

Claims (6)

Analyzes the metabolites present in the reaction solution after contact between the sample compound and the following reagents to determine whether the sample compound is detoxified or metabolized to carcinogens. According to (a) the presence or absence of detoxification metabolism of a compound as a specimen or the absence (absence) of metabolism to a carcinogen, the compound as a specimen is evaluated as safe, or (b) is a specimen. Yeast cells produced by the following steps as a reagent for evaluating the sample compound as unsafe in the absence (no) of detoxification metabolism of the compound or in the presence of metabolism to a carcinogen (yes) Or use of this microbial cell crushed material.
<Production process>
For all molecular species included in the molecular species group consisting of human-derived cytochrome P450 1A2, human-derived cytochrome P450 2C9, human-derived cytochrome P450 2E1, and human-derived cytochrome P450 3A4,
(1) (a) a promoter for expression in yeast and (b) (i) a gene having a base sequence encoding the above molecular species and a gene having a base sequence encoding yeast NADPH-P450 reductase or ( ii) a step of constructing an expression plasmid in yeast comprising the above-mentioned molecular species and a gene having a base sequence encoding an artificial fusion enzyme of yeast NADPH-P450 reductase, and (2) the constructed yeast expression plasmid in yeast Introducing a yeast cell that expresses (i) the above molecular species and yeast NADPH-P450 reductase or (ii) an artificial fusion enzyme of the above molecular species and yeast NADPH-P450 reductase in yeast Process The abundance of microorganisms that have become non-required for the nutritional element by causing the metabolite present in the reaction solution after contacting the compound as the specimen to the following reagent to act on the microorganism that requires the nutritional element. , And by the measurement, (a) a microorganism that has become non-required for the nutritional element is subjected to the same measurement as the above measurement using yeast cells into which the yeast expression plasmid has not been introduced. When present in the same degree as in the case of control (control), the test compound is evaluated as mutagenic, or (b) microorganisms that are not required for nutritional elements are expressed in the yeast as described above. In order to evaluate the compound as a mutagenic agent when there is a substantial amount compared to the case where the same measurement as described above (control) is performed using yeast cells into which a plasmid has not been introduced. As a reagent of The use of yeast cells produced by serial processes or microbial cells disruption product.
<Production process>
For all molecular species included in the molecular species group consisting of human-derived cytochrome P450 1A2, human-derived cytochrome P450 2C9, human-derived cytochrome P450 2E1, and human-derived cytochrome P450 3A4,
(1) (a) a promoter for expression in yeast and (b) (i) a gene having a base sequence encoding the above molecular species and a gene having a base sequence encoding yeast NADPH-P450 reductase or ( ii) a step of constructing an expression plasmid in yeast comprising the above-mentioned molecular species and a gene having a base sequence encoding an artificial fusion enzyme of yeast NADPH-P450 reductase, and (2) the constructed yeast expression plasmid in yeast Introducing a yeast cell that expresses (i) the above molecular species and yeast NADPH-P450 reductase or (ii) an artificial fusion enzyme of the above molecular species and yeast NADPH-P450 reductase in yeast Process By analyzing a metabolite present in the reaction solution after contact between the compound as a specimen and the following reagent, one type of (i) human-derived cytochrome P450 molecular species or (ii ) Only in the case of an artificial fusion enzyme of cytochrome P450 molecular species derived from human and yeast NADPH-P450 reductase, the presence or absence of a metabolite that specifically exists is determined, and according to the determination, (a) one type of (i) human When there is a metabolite that specifically exists only in the case of an artificial fusion enzyme of cytochrome P450 molecular species derived from human or cytochrome P450 molecular species derived from human and yeast NADPH-P450 reductase, the sample compound is designated as cytochrome When it is evaluated that it is effective as a metabolic probe of P450 molecular species, and (b) when a metabolite specifically present is absent, Using beam P450 as a reagent for assessing the ineffective as molecular species metabolic probe, yeast cells produced by the following process or said cell-product.
<Production process>
For all molecular species included in the molecular species group consisting of human-derived cytochrome P450 1A2, human-derived cytochrome P450 2C9, human-derived cytochrome P450 2E1, and human-derived cytochrome P450 3A4,
(1) (a) a promoter for expression in yeast and (b) (i) a gene having a base sequence encoding the above molecular species and a gene having a base sequence encoding yeast NADPH-P450 reductase or ( ii) a step of constructing an expression plasmid in yeast comprising the above-mentioned molecular species and a gene having a base sequence encoding an artificial fusion enzyme of yeast NADPH-P450 reductase, and (2) the constructed yeast expression plasmid in yeast Introducing a yeast cell that expresses (i) the above molecular species and yeast NADPH-P450 reductase or (ii) an artificial fusion enzyme of the above molecular species and yeast NADPH-P450 reductase in yeast Process The produced yeast cells or the disrupted cells thereof are yeast cells or yeast cell fragments obtained by mixing yeast cells in all the molecular species. Use according to any claim. The produced yeast cell or the disrupted cell thereof has a turnover rate that is in order coincident with the turnover rate of cytochrome P450 derived from human possessed by the microsomal fraction prepared from human liver, The use according to any one of claims 1 to 4, which is a crushed bacterial cell. The turnover rate of human-derived cytochrome P450 contained in the microsomal fraction prepared from human liver is within the range of 1.0 to 5.9 [product nmol / nmol P450 / min]. Use according to claim 5, characterized in that

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