JP2015109830A - γ-GLUTAMYL CYCLO TRANSFERASE, γ-GLUTAMYL CYCLO TRANSFERASE GENE, METHOD FOR PRODUCING γ-GLUTAMYL CYCLO TRANSFERASE AND USE THEREOF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel γ-glutamyl cyclo transferase (γGCT).SOLUTION: At least one protein selected from the group consisting of the following (P1)-(P3) is used as γGCT: (P1) a protein consisting of a specific amino acid sequence derived from Ralstonia solanacearum; (P2) a protein consisting of an amino acid sequence obtained by deleting, substituting, inserting and/or adding one or some amino acids in the amino acid sequence of the (P1), and having γ-glutamyl cyclo transferase activity; and (P3) a protein consisting of an amino acid sequence which has 80% or more identity to the amino acid sequence of the (P1), and has γ-glutamyl cyclo transferase activity.

Description

  The present invention relates to a method for producing γ-glutamylcyclotransferase, a γ-glutamylcyclotransferase gene, a γ-glutamylcyclotransferase, and uses thereof.

  Glutathione is a tripeptide in which the amino group of cysteine in the cysteine-glycine dipeptide is bound to the carboxyl group at the γ-position of glutamic acid. Glutathione has a strong antioxidant action, and 5-oxoproline and cysteine-glycine dipeptide released from glutathione have been reported to have a skin moisturizing action and a UV oxidative stress suppressing action, respectively.

  A ChaC family protein has been reported as γ-glutamylcyclotransferase that releases 5-oxoproline and cysteine-glycine dipeptide from glutathione (Non-patent Document 1). However, the γ-glutamyl cyclotransferase has low activity against glutathione. For this reason, a new γ-glutamyl cyclotransferase having high activity against glutathione is demanded.

Kumar et. al. , “Mammalian proapoptotic factor ChaC1 and its homologues function as γ-glutamyl cyclotransferases acting specifically on glutathione”, EMBO reports 2012, 13, 1095-1101.

  Accordingly, an object of the present invention is to provide a new γ-glutamyl cyclotransferase.

In order to achieve the above object, the γ-glutamylcyclotransferase of the present invention is at least one protein selected from the group consisting of the following (P1) to (P3).
(P1) a protein comprising the amino acid sequence of SEQ ID NO: 1 (P2) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence of (P1), Protein having glutamyl cyclotransferase activity (P3) A protein having an amino acid sequence having 80% or more identity to the amino acid sequence of (P1) and having γ-glutamyl cyclotransferase activity

  In the present invention, hereinafter, γ-glutamylcyclotransferase is also referred to as “γGCT”, and glutathione is also referred to as “GSH”.

  The γGCT reagent of the present invention includes the γGCT of the present invention and an activator thereof.

The γGCT gene of the present invention is characterized by comprising at least one polynucleotide selected from the group consisting of the following (p1) to (p3).
(P1) a polynucleotide comprising the base sequence of SEQ ID NO: 2 (p2) comprising a base sequence in which one or several bases are deleted, substituted, inserted and / or added in the base sequence of (p1), and γGCT Polynucleotide encoding a protein having activity (p3) A polynucleotide encoding a protein having γGCT activity, comprising a base sequence having 80% or more identity to the base sequence of (p1)

  The method for producing γGCT of the present invention has an expression step of expressing γGCT from a γGCT gene, and the γGCT gene is the γGCT gene of the present invention.

  The γGCT expression vector of the present invention is characterized in that the γGCT gene of the present invention is inserted.

  The method for producing 5-oxoproline of the present invention includes a step of releasing 5-oxoproline from GSH by γGCT, wherein the γGCT is γGCT of the present invention.

  The method for producing a cysteine-glycine dipeptide of the present invention comprises a step of releasing a cysteine-glycine dipeptide from GSH by γGCT, wherein the γGCT is the γGCT of the present invention.

  The screening method for a bacterial wilt control drug candidate substance of the present invention includes a selection step of selecting, from the test substance, an activity inhibitory substance that suppresses γ-GCT activity of γGCT as the control drug candidate substance, and the γGCT Is the γGCT of the present invention.

  The thioredoxin analysis method of the present invention is characterized in that thioredoxin contained in the sample is analyzed by bringing the sample into contact with the γGCT of the present invention and measuring the γGCT activity of the γGCT.

  The γGCT of the present invention has higher activity against glutathione than the known γGCT. Therefore, according to γGCT of the present invention, for example, 5-oxoproline and cysteine-glycine dipeptide can be efficiently produced from glutathione with a smaller amount of protein.

1 is a photograph showing the results of SDS-PAGE of γGCT in Example 1. FIG. FIG. 2 is a graph showing the relative value of glutathione concentration in cells of transformed yeast expressing γGCT in Example 1. FIG. 3 is a graph showing the relative activity of γGCT in Example 1. 4 is a photograph showing colony formation of transformed yeast expressing γGCT and transformed yeast expressing mutant γGCT in Example 2. FIG. FIG. 5 is a photograph showing the results of SDS-PAGE of γGCT in Example 3. 6 is a photograph showing the results of SDS-PAGE of γGCT in Example 4. FIG. FIG. 7 is a graph showing γGCT activity in Example 4. FIG. 8 is a graph showing the cysteine concentration in the reaction solution in the presence of the yeast protein extract in Example 5. FIG. 9 is a graph showing γGCT activity in Example 6 in the presence of various eukaryotic-derived protein extracts. FIG. 10 is a graph showing γGCT activity in the presence of thioredoxin in Example 7. FIG. 11 is a photograph showing the interaction between RSp1022 (wild type) and thioredoxin in Example 8 by the yeast two-hybrid method.

<Γ-Glutamylcyclotransferase and Reagent>
As described above, the γGCT of the present invention is at least one protein selected from the group consisting of the following (P1) to (P3).
(P1) Protein consisting of the amino acid sequence of SEQ ID NO: 1 (P2) In the amino acid sequence of (P1), consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added, and γGCT activity Protein (P3) A protein comprising γGCT activity, comprising an amino acid sequence having 80% or more identity to the amino acid sequence of (P1)

In the present invention, the γGCT may be a protein having a catalytic activity for releasing 5-oxoproline and cysteine-glycine dipeptide from glutathione, and the type and origin thereof are not particularly limited. Examples of the γGCT include those derived from the genus Ralstonia . The Ralstonia genus includes, for example, bacterial wilt ( Ralstonia solanacearum ).

Examples of the γGCT derived from Ralstonia solanacearum include Genbank accession no. A protein having the amino acid sequence (SEQ ID NO: 1) registered by NP — 522583 is exemplified.

The amino acid sequence of SEQ ID NO: 1 in (P1) is as follows. The (P1) can be obtained from, for example, Ralstonia solanacearum . In the following sequence, residues enclosed by two squares are regions involved in catalytic function, one region (YLSL) is a substrate recognition motif of γGCT of the present invention, and the other region (E) is The active center of γGCT of the present invention.

  In the (P2), “1 or several” may be, for example, a range in which the (P2) has the γGCT activity. In the amino acid sequence of (P1), “1 or several” of (P2) is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 9, more preferably 1 to 5. 1 to 3, particularly preferably 1 to 2, most preferably 1 or 2. In the present invention, the numerical range of numbers discloses, for example, all positive integers belonging to the range. That is, for example, the description “1 to 3” means all disclosures of “1, 2, 3” (hereinafter the same).

  In the (P3), the “identity” may be, for example, a range in which the (P3) has the γGCT activity. The “identity” of (P3) is, for example, 80% or more, 85% or more, 90% or more, preferably 95% or more, 96% or more, 97% with respect to the amino acid sequence of (P1). Above, more preferably 98% or more, still more preferably 99% or more. The identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).

  In the proteins (P2) and (P3), it is preferable that residues (YLSL and E) in the two regions involved in the catalytic function are conserved in the amino acid sequence of SEQ ID NO: 1. In the amino acid sequences (P2) and (P3), the two regions can be determined by performing alignment with reference to the amino acid sequence of SEQ ID NO: 1, for example.

  The γGCT of the present invention has the following chemical characteristics, for example.

  The enzyme activity of the γGCT is not particularly limited, and 1 unit (U) is, for example, the amount of enzyme that produces 1 μmol of 5-oxoproline and cysteine-glycine dipeptide from GSH per minute under the conditions of 37 ° C. and pH 8. Can be defined.

  The γGCT only needs to have γGCT activity using GSH as a substrate. For example, the γGCT may further have other catalytic activity.

The γGCT of the present invention may contain, for example, an activator in addition to the protein comprising the amino acid, and both may form a complex. Examples of the activator include thioredoxin (TRX). Examples of the thioredoxin include Trx1, Trx h2, Trx h3, Trx h5, and the like. The origin of the activator is not particularly limited, and may be the same as or different from the protein in the γGCT of the present invention, for example. Specific examples of the activator include yeast-derived and plant-derived. Examples of the yeast include Saccharomyces cerevisiae and Schizosaccharomyces pombe . Examples of the plant include Solanum melongena , Solanum lycopersicum , and Arabidopsis thaliana . Specific examples of the thioredoxin include, for example, Trx1 (SEQ ID NO: 5) derived from Saccharomyces cerevisiae , and Trx h2 (SEQ ID NO: 6), Trx h3 (SEQ ID NO: 7) and Trx h5 (SEQ ID NO: 8) derived from Arabidopsis thaliana Can be given.

The thioredoxin is registered, for example, with an accession number in the following database.
Saccharomyces cerevisiae- derived Trx1: NCBI Accession No._0011181930.1
Arabidopsis thaliana- derived Trx h2: NCBI Accession No._1233588.3
Arabidopsis thaliana- derived Trx h3: NCBI Accession No._123664.3
Arabidopsis thaliana- derived Trx h5: NCBI Accession No. 1033588.4

As said thioredoxin, protein (1) or (2) which consists of the following amino acid sequences can also be used, for example. In the following (2), the identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
(1) a protein comprising any one of the amino acid sequences of SEQ ID NOs: 5 to 8 (2) an amino acid sequence having 70% or more identity to any of the amino acid sequences of SEQ ID NOs: 5 to 8, wherein (1) and Proteins with similar functions

  As described above, the γGCT of the present invention exhibits catalytic activity for releasing 5-oxoproline and cysteine-glycine dipeptide (Cys-Gly) from glutathione. Therefore, the γGCT of the present invention can be used for production of 5-oxoproline and Cys-Gly from glutathione. The γGCT of the present invention can also be used in a method for analyzing glutathione using the catalytic activity.

  The γGCT of the present invention can be produced, for example, by the method for producing γGCT of the present invention described later.

  Next, the γGCT reagent of the present invention comprises the γGCT of the present invention and an activator thereof.

  The activator is, for example, a eukaryotic protein extract, and the eukaryote is not particularly limited, and is, for example, at least one selected from the group consisting of animals, plants, fungi, and protists. is there. Examples of the fungi include yeast.

  Examples of the activator include thioredoxin, which is the activator described above, and derivatives thereof, and preferably thioredoxin derived from eukaryotes and derivatives thereof. The thioredoxin is as described above, for example.

  In the γGCT reagent of the present invention, the γGCT and the activator may form, for example, a complex.

  When the γGCT of the present invention is γGCT expressed in a non-eukaryotic transformant, the γGCT is preferably coexisting with the activator, for example.

<Γ-glutamyl cyclotransferase gene>
As described above, the γGCT gene of the present invention is characterized by comprising at least one polynucleotide selected from the group consisting of the following (p1) to (p7).
(P1) a polynucleotide comprising the base sequence of SEQ ID NO: 2 (p2) comprising a base sequence in which one or several bases are deleted, substituted, inserted and / or added in the base sequence of (p1), and γGCT Polynucleotide encoding a protein having activity (p3) Polynucleotide encoding a protein having a γGCT activity consisting of a base sequence having 80% or more identity to the base sequence of (p1) (p4) (P5) a polynucleotide encoding a protein having a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the base sequence (p1) and having a γGCT activity (p5) Polynucleotide encoding a protein comprising the amino acid sequence of (p6) A polynucleotide encoding a protein having a γGCT activity consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of column No. 1 (p7). A polynucleotide comprising an amino acid sequence having 80% identity or more to a sequence and encoding a protein having γGCT activity

  The γGCT gene of the present invention is useful, for example, when the γGCT of the present invention is synthesized by a genetic engineering technique.

The base sequence of SEQ ID NO: 2 is as follows. The base sequence of SEQ ID NO: 2 is the coding sequence of γGCT (P1) of the present invention consisting of the amino acid sequence of SEQ ID NO: 1. The polynucleotide (p1) of SEQ ID NO: 2 can be obtained from, for example, Ralstonia solanacearum . As a coding sequence of γGCT derived from Ralstonia solanacearum , for example, Accession No. A region of 1288854-1290104 in the base sequence registered in AL646053 (including the termination codon) can be mentioned.

The polynucleotide of (p1) (SEQ ID NO: 2)
ATGGAAAGAATCTCGACAAATGCCATCTCCAAGACGGTTGCACGGTCGGACCTGAGCAATGGAAGCTCGGCGCCCCCCAGAAAGAAAAACGGCTCGCCGCCGAGACGCGCCCATGCACCGCTTCTCCCGCTGGCCGACCGCAGCCTGACGGCCATTTCGAAGCCCAACGAAACGCTCGGCAGCCGGCTCGCGCCTGTACCGCAACGCGCCGTGCACGCCAGCGCCGTCCCGAGCGCCGCCGGGAACTGGAACCCGCTGTCCCGCCTGAAACCGGAGGAACTGGCGGGCATTCCTGAAACGGTTGTCGAGAAGTTGGGGAAGGATCTGCACGACCTTTACGAAGATGTGCAAAGCAAGGGACTCAACTACGTACCGGTGTTCGGTTATCTGTCGCTGCGCTACAAGAATTTCCATGAACTCGGCAAGCGCAGCCAGGATGAAGTCCGCGAAGGAAAAGACTGGGTCCCGGCCTCGCTGGACAATCACGCCCTGGATTTCGTGATGAGCACCCAGTATCGGGGCCACCATCAACACCCCGGTGTCGTGGCGGGCCTGAGCCAAAGCGAGGGAAGCGCCATGGACGGGGTCATCCTGAAGCTGCCGGTCGGCAACGCTGAAGAGCTGCTCGCCCGTGTCATCCGCCGGGAACTACTGGACGAGAATGACCTGATCCACCCCGTGCCGGCGACCCCACCAGCGGGAACGACCGGGCTCGCCGGTTCGGCAACTGCCGGTCCGGCAACGCCGCTCTCCCCTAAAAAACCGCGCTCCAACCTGATGTACAGCAGCGCCGTCAGACCGGTCACCCTGCCGAATGGCGCCAAGGTCAGAGCGCTGGTGTTCGTGACCAATCCCGACAGCGCCAAATCGCTGGCGAGCGTGTTCAAGGACCGCGAAGGTGTCTCGCCTCAGCGGTTGGCCTATCTGATGACGAGCACGAGCAAGGGCGATCTGGGAGGACCGGCGATCGACTACTGGAAGCGCTTTGTCGAAACCTGCG AAACAGCCAAGACTGCCGTGCCGCCGATCGTGAGCCAGGCGATCCGGCTCGCCAGCGACTGGCCGGACGCATTCACCGGCGAGCCGGCAGCCCCGGACGACATGCCGCGACAGCACCAGGAAGCGGCATGGTTCCGCGCGATGGCCGGCGCGCGCGCGCGCGCGCGCGCTGGCGCGCGCGCGC

  In the (p2), “1 or several” may be within a range in which the protein encoded by the polynucleotide of (p2) has the γGCT activity. In the base sequence (SEQ ID NO: 2) of (p1), “1 or several” of (p2) is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 9, Preferably 1-5, particularly preferably 1-3, most preferably 1 or 2, may be deleted, substituted, inserted and / or added.

  In the (p3), the “identity” may be, for example, a range in which the protein encoded by the polynucleotide of the (p3) has the γGCT activity. The identity of (p3) is, for example, 80% or more, 85% or more, 90% or more, preferably 95% or more, 96% or more with respect to the base sequence (SEQ ID NO: 2) of (p1). 97% or more, more preferably 98% or more, and still more preferably 99% or more.

In the above (p4), the “hybridizable polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide comprising the base sequence of (p1) (SEQ ID NO: 2). . The hybridization can be detected by, for example, various hybridization assays. The hybridization assay is not particularly limited, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.

In the above (p4), the “stringent conditions” may be, for example, any of low stringent conditions, medium stringent conditions, and high stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. “Medium stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like. "Stringent conditions" are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.

  The polynucleotide (p5) may be any nucleotide sequence in which the protein encoded by the polynucleotide (p5) has the γGCT activity, for example. The base sequence of the polynucleotide (p5) can be designed, for example, by replacing it with the corresponding codon based on the amino acid sequence of SEQ ID NO: 1.

  In the above (p6), “one or several” relating to the amino acid sequence may be within a range in which the protein encoded by the polynucleotide of (p6) has the γGCT, for example. The “1 or several” of (p6) is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 9, more preferably 1 in the amino acid sequence of SEQ ID NO: 1. ~ 5, particularly preferably 1 to 3, most preferably 1 or 2.

  In the above (p7), the “identity” regarding the amino acid sequence may be, for example, within a range in which the protein encoded by the polynucleotide of (p7) has the γGCT activity. The identity of (p7) is, for example, 80% or more, 85% or more, 90% or more, preferably 95% or more, 96% or more, 97% or more with respect to the amino acid sequence of SEQ ID NO: 1. More preferably, it is 98% or more, More preferably, it is 99% or more.

  In the present invention, the various polynucleotides can be produced by, for example, known genetic engineering techniques or synthetic techniques.

<Expression vector of γ-glutamyl cyclotransferase>
As described above, the expression vector of the present invention is characterized in that the γGCT gene of the present invention is inserted. According to the expression vector of the present invention, for example, the γGCT of the present invention can be easily produced by introducing the expression vector into a host described later.

  The expression vector may contain the γGCT gene so that, for example, the γGCT encoded by the γGCT gene of the present invention can be expressed, and other configurations are not limited at all.

  The expression vector can be prepared, for example, by inserting the γGCT gene into a backbone vector (hereinafter also referred to as “basic vector”). The type of the vector is not particularly limited, and can be appropriately determined according to the type of the host. Examples of the vector include viral vectors and non-viral vectors. When the host is transformed by the heat shock method as the introduction method, the vector is preferably a binary vector, for example, a pETDuet-1 vector (Novagen), a pQE-80L vector (QIAGEN), a pUCP26Km vector, pET23d. Examples include vectors (Novagen). When transforming bacteria such as E. coli, examples of the vector include pETDuet-1 vector, pQE-80L vector, such as pET23d vector. When transforming a eukaryote such as yeast, examples of the basic vector include pYE22m, and a commercially available yeast expression vector such as pYES (Invitrogen) or pESC (Stratagene) is used. You can also.

  The expression vector preferably has a regulatory sequence that regulates the expression of the polynucleotide and the γGCT of the present invention encoded by the polynucleotide, for example. Examples of the regulatory sequence include a promoter, terminator, enhancer, polyadenylation signal sequence, origin of replication sequence (ori) and the like. The promoter is not particularly limited, and examples thereof include a galactose inducible promoter. Examples of the galactose-inducible promoter include the GAL1 promoter. In the expression vector, the arrangement of the regulatory sequences is not particularly limited. In the expression vector, the regulatory sequence only needs to be arranged so as to be able to functionally regulate the expression of the polynucleotide and the expression of the γGCT encoded by the polynucleotide, and can be arranged based on a known method. As the regulatory sequence, for example, a sequence provided in advance in the basic vector may be used, the regulatory sequence may be further inserted into the basic vector, and the regulatory sequence provided in the basic vector It may be replaced with the regulatory sequence.

  The expression vector may further include a selection marker coding sequence, for example. Examples of the selection marker include drug resistance markers, fluorescent protein markers, enzyme markers, cell surface receptor markers, and the like.

  The expression vector may further have a coding sequence of a tag sequence, for example. Examples of the tag sequence include a FLAG tag, a 3 × FLAG tag, a Protein A tag, and a His6 tag.

<Method for Producing Gamma-Glutamyl Cyclotransferase>
As described above, the method for producing γGCT of the present invention has an expression step of expressing γGCT from the γGCT gene, and the γGCT gene is the GCT gene of the present invention. The method for producing γGCT of the present invention is characterized in that γGCT is expressed from the γGCT gene of the present invention, and other steps and conditions are not particularly limited.

  In the expression step, for example, the γGCT gene itself may be used as the γGCT gene, or the expression vector of the present invention into which the γGCT gene is inserted may be used.

  The production method of the present invention is characterized in that the γGCT of the present invention is synthesized by γGCT expression from the γGCT gene of the present invention, and other methods and conditions are not limited at all. In the production method of the present invention, a known method can be adopted for the expression of the γGCT of the present invention. For example, a host or a cell-free protein synthesis system may be used.

  In the case of using the former host, the expression step is, for example, a step of expressing the γGCT from the γGCT gene in the host. In the expression step, the host is preferably a transformant introduced with the γGCT gene or the expression vector, for example, and the γGCT is preferably expressed in the host by culturing the transformant. .

  The expression step may include, for example, a step of producing the transformant by introducing the γGCT gene into a host. The transformant that synthesizes γGCT of the present invention can be produced, for example, by introducing the γGCT gene into a host.

The host is not particularly limited, and examples thereof include microorganisms, animal cells, insect cells, or non-human hosts such as cultured cells thereof, isolated human cells or cultured cells thereof. Examples of the microorganism include prokaryotes and eukaryotes. Examples of the prokaryote include Escherichia such as Escherichia coli . Examples of the E. coli include Rosettagami B (DE3) pLysS strain and the like. Examples of the eukaryote include yeast. Examples of the yeast include Saccharomyces cerevisiae and Schizosaccharomyces pombe . Examples of the Saccharomyces cerevisiae include BY4743 strain. Examples of the Schizosaccharomyces pombe include ARC039 strain. Examples of the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21.

  The method for introducing the γGCT gene into the host is not particularly limited, and can be performed by a known method. The introduction method can be appropriately set depending on, for example, the type of the host. Examples of the introduction method include heat shock method, lithium acetate method, introduction method using gene gun such as particle gun, calcium phosphate method, polyethylene glycol method, lipofection method using liposome, electroporation method, ultrasonic nucleic acid introduction method, DEAE -A dextran method, a direct injection method using a micro glass tube, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, a method using Agrobacterium, and the like. Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction aid include atelocollagen, nanoparticles, and polymers.

  The method for culturing the host is not particularly limited, and can be appropriately set depending on the type of the host. The medium used for the culture is not particularly limited and can be appropriately determined according to the type of the host.

  In the case of using the latter cell-free protein synthesis system, the expression step is, for example, a step of expressing the γGCT from the γGCT gene in the cell-free protein synthesis system. The cell-free protein synthesis system can be performed by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which the target polynucleotide has been introduced. Reagent kits can be used.

  The production method of the present invention may further include a purification step of the γGCT, for example. For example, when the γGCT is expressed as an intracellular component, the purification step includes a release step of releasing the intracellular component including the γGCT from the host, and a solid fraction including a cell residue and the γGCT. A solid-liquid separation step for separating the liquid fraction may be included. The method for releasing intracellular components from the host is not particularly limited, and can be carried out by a known method, for example, a method for homogenizing the host. In addition, when the γGCT is expressed as an extracellular component, the purification step includes, for example, a solid fraction containing the host cells and a liquid fraction containing the γGCT in the host culture. A liquid separation step may be included. The γGCT of the present invention uses, for example, the γGCT gene of the present invention in which the above-mentioned polynucleotides (p1) to (p7) are linked to a coding sequence of a signal peptide involved in extracellular secretion. Thus, it can be expressed as an extracellular enzyme.

  In the purification step, the purification method of γGCT is not particularly limited, and examples thereof include salting out and various column chromatography. The solvent used in the various purification steps is not particularly limited, and for example, a buffer solution can be used.

  ΓGCT obtained by the production method of the present invention may be used, for example, as a crude enzyme (non-purified enzyme) as it is, as a partially purified partially purified enzyme, or as a single purified product. It may be used as a purified enzyme.

  In the production method of the present invention, it is preferable that the expressed γGCT coexists, for example, with the above-described activator (specifically, the active factor). In this case, for example, the γGCT and the active factor may be co-expressed in the host by co-expression, or after isolating the γGCT expressed in the host, the active factor and You may coexist. For example, when Escherichia coli is used as the host, the active factor is preferably yeast-derived thioredoxin.

<Method for producing useful decomposition products>
As described above, the method for producing 5-oxoproline of the present invention includes a step of releasing 5-oxoproline from glutathione by γGCT, and the γGCT is the γGCT of the present invention. The production method of the present invention is characterized in that the γGCT of the present invention is used as the γGCT, and other processes and conditions are not limited at all.

  The 5-oxoproline releasing step can be performed, for example, by contacting the GSH and the γGCT. The contact method is not particularly limited, and, for example, a general means using an enzyme can be adopted. As a specific example, a reaction system containing the GSH and the γGCT can be used. In the reaction system, the γGCT may be used, for example, in a free state or in a state immobilized on a carrier. In the case of the latter immobilized enzyme, the type of the carrier is not particularly limited, and a known carrier such as a substrate such as a plate or a container, beads, or a filter can be used. The reaction system is, for example, a reaction solution.

  In the 5-oxoproline releasing step, the amount of γGCT used is not particularly limited, and is preferably 55 to 550 U, more preferably 55 to 400 U, for example, in the case of unit U with respect to 1 g of glutathione. Preferably, it is 100 to 300 U, and in the case of protein amount, it is preferably 155 to 1500 μg, more preferably 155 to 1100 μg, and further preferably 280 to 850 μg.

  In the 5-oxoproline releasing step, the reaction conditions of the γGCT are not particularly limited, and can be appropriately set according to, for example, the chemical characteristics of the γGCT to be used. The reaction temperature is, for example, preferably 25 to 45 ° C, more preferably 30 to 40 ° C, and still more preferably 35 to 37 ° C. For example, the reaction time is preferably 10 to 120 minutes, more preferably 30 to 90 minutes, and further preferably 40 to 60 minutes. The pH of the reaction system is preferably, for example, pH 6-9, more preferably pH 6.5-8.5, and further preferably pH 7-8.

  In addition to glutathione and γGCT, the reaction system may contain, for example, a solvent, a coenzyme for γGCT, an ionic compound, a reducing agent such as dithiothreitol, a chelating agent such as EDTA, and the like. Examples of the solvent include aqueous solvents such as water and buffer. Examples of the buffer include Tris-HCl, potassium phosphate, and sodium phosphate. Depending on the buffer, for example, the reaction can be performed. It is preferable to adjust the pH of the system.

  The method for producing 5-oxoproline of the present invention may further include a step of recovering the 5-oxoproline from the reaction system. The method for recovering 5-oxoproline is not particularly limited, and examples thereof include HPLC and TLC.

  Next, as described above, the method for producing cysteine-glycine dipeptide (Cys-Gly) of the present invention includes a step of releasing Cys-Gly from the GSH by γGCT, and the γGCT is the γGCT of the present invention. It is characterized by being. The Cys-Gly production method of the present invention is characterized in that the γGCT of the present invention is used as the γGCT, and other configurations and processes are not limited at all.

  The Cys-Gly releasing step can be performed in the same manner as the 5-oxoproline releasing step in the method for producing 5-oxoproline of the present invention.

  The method for producing Cys-Gly of the present invention may further include a step of recovering Cys-Gly from the reaction system. The method for recovering the Cys-Gly is not particularly limited, and examples thereof include HPLC and TLC.

  In the production method of the present invention (for example, the production method of 5-oxoproline, the production method of Cys-Gly), the activating agent is preferably allowed to coexist when the γGCT is used. Specifically, for example, in the release step, it is preferable to use the γGCT in the presence of the activator. The activator is, for example, as described above.

<Analytical method of glutathione>
As described above, the method for analyzing glutathione of the present invention includes a step of releasing Cys-Gly from glutathione in a sample by γGCT, a step of releasing cysteine from the released Cys-Gly by dipeptidase, and the cysteine. Including the step of analyzing, wherein the γGCT is the γGCT of the present invention. The present invention is characterized in that the γGCT of the present invention is used as the γGCT, and other processes and conditions are not limited at all. The analysis in the present invention may be, for example, any of qualitative analysis, quantitative analysis, and semi-quantitative analysis.

  The kind in particular of the said sample is not restrict | limited, For example, the sample containing the said GSH, the sample considered to contain, the sample with unknown presence or absence, etc. are object. The sample may be solid or liquid, for example. When the sample is solid, for example, it is preferable to perform pretreatment such as suspension, dispersion or dissolution in a solvent, and to use the sample after pretreatment for the analysis method of the present invention.

  In the Cys-Gly release step, the amount of γGCT used is not particularly limited. For example, in the case of unit U with respect to 1 mL of the sample, 0.2 to 4 U is preferable, and more preferably 0.3 to 3 U. More preferably, it is 0.4-2U, and, in the case of protein amount, 0.6-11.2 μg is preferable, more preferably 0.9-8.4 μg, and still more preferably 1.2. ˜5.6 μg.

  The conditions for the Cys-Gly releasing step are not particularly limited, and the description of the 5-oxoproline releasing step in the method for producing 5-oxoproline of the present invention can be cited.

In the cysteine releasing step, the dipeptidase is not particularly limited as long as it has an activity of releasing cysteine from the Cys-Gly, for example. Examples of the dipeptidase include Cys-Gly dipeptidase, and the type and origin thereof are not particularly limited. Examples of the Cys-Gly dipeptidase include those derived from Saccharomyces cerevisiae , and specific examples include Genbank accession no. An example is a protein having an amino acid sequence registered under NP_116702.

  The cysteine releasing step can be performed, for example, by contacting Cys-Gly obtained in the Cys-Gly releasing step with the dipeptidase. In the analysis method of the present invention, the Cys-Gly releasing step and the cysteine releasing step may be performed, for example, after the Cys-Gly releasing step, or the Cys-Gly releasing step and the Cys-Gly releasing step. You may perform a cysteine release process in parallel.

  In the reaction system, the dipeptidase may be used, for example, in a free state or in a state immobilized on a carrier. In the case of the latter immobilized enzyme, the type of the carrier is not particularly limited and is the same as described above.

  In the cysteine releasing step, the amount of the dipeptidase used is not particularly limited. For example, in the case of a protein amount with respect to 1 mL of the sample in the Cys-Gly releasing step, 0.01 to 0.5 μg is preferable, More preferably, it is 0.025-0.2 microgram, More preferably, it is 0.05-0.1 microgram.

  In the cysteine releasing step, the reaction conditions of the dipeptidase are not particularly limited, and can be appropriately set according to the chemical characteristics of the dipeptidase used. The reaction temperature is, for example, preferably 25 to 40 ° C, more preferably 30 to 37 ° C. For example, the reaction time is preferably 10 to 90 minutes, and more preferably 30 to 60 minutes. The reaction pH is, for example, preferably 6-9, more preferably 7-8.

  In addition to the Cys-Gly and the dipeptidase, the reaction system may include, for example, a solvent, a coenzyme for the dipeptidase, an ionic compound, the reducing agent such as dithiothreitol, and the like. Examples of the solvent include aqueous solvents such as water and buffer. Examples of the buffer include Tris-HCl, potassium phosphate, and sodium phosphate. Depending on the buffer, for example, the reaction can be performed. It is preferable to adjust the pH of the system. Examples of the ionic compound include compounds that liberate divalent ions, and examples of the divalent ions include magnesium.

  Next, in the cysteine analysis step, the method for analyzing cysteine is not particularly limited, and can be performed by a known method. Examples of the analysis method include HPLC, a method using an acidic ninhydrin reaction, and the like.

  In the analysis method of the present invention, the cysteine analysis step may be performed, for example, after the cysteine release step, the cysteine release step and the cysteine analysis step may be performed in parallel, or the Cys − The Gly release step, the cysteine release step, and the cysteine analysis step may be performed in parallel.

  In the analysis method of the present invention, the activating agent is preferably allowed to coexist when the γGCT is used. Specifically, for example, in the Cys-Gly releasing step, it is preferable to use the γGCT in the presence of the activator. The activator is, for example, as described above.

<Analytical reagent for glutathione>
As described above, the analysis reagent of the present invention is a GSH analysis reagent, and includes γGCT and dipeptidase, and the γGCT is the γGCT of the present invention.

  According to the analysis reagent of the present invention, the GSH analysis method of the present invention can be performed more simply. The analysis reagent of the present invention is characterized in that the γGCT of the present invention is used as the γGCT, and other configurations are not particularly limited. The γGCT and the peptidase of the present invention are as described above.

  The analysis reagent of the present invention may further include, for example, components that can be used in the analysis method of the present invention. The component is the same as described above, for example.

  For example, the analysis reagent of the present invention preferably further contains the activator. The activator is, for example, as described above. In the analysis reagent of the present invention, for example, the activating agent is preferably allowed to coexist when γGCT is used.

  In the analysis reagent of the present invention, for example, each component may be stored in one container, or each component may be stored separately in a plurality of containers. In the latter case, the analysis reagent of the present invention is also referred to as an analysis kit, for example. The analysis kit of the present invention may further include instructions for use, for example.

<Screening method>
As described above, the screening method of the present invention is a screening method for a bacterial wilt control drug candidate substance, wherein an activity inhibitory substance that suppresses γGCT activity of γGCT is used as the control drug candidate substance from the test substance. Including a selection step of selecting, wherein the γGCT is the γGCT of the present invention. The screening method of the present invention is characterized in that the target of the candidate substance for control agent is the γGCT of the present invention, and other steps and conditions are not particularly limited.

  Examples of the activity inhibitor include low molecular weight compounds, peptides, proteins, nucleic acids and the like.

  In the method for screening for an activity inhibitory substance of the present invention, for example, in a reaction system containing the γGCT, the GSH and the test substance, an activity detection step for detecting the γGCT activity of the γGCT, and a decrease in the γGCT activity of the γGCT. A candidate substance selecting step of selecting the test substance thus made as the control drug candidate substance.

  In the screening method of the present invention, in the selection step, the γGCT activity is preferably an activity in the presence of the activator of γGCT. The activator is, for example, as described above.

<Method for analyzing thioredoxin>
As described above, the method for analyzing thioredoxin of the present invention comprises analyzing a thioredoxin contained in the sample by bringing the sample into contact with the γGCT of the present invention and measuring the γGCT activity of the γGCT. And In the present invention, the analysis may be, for example, qualitative analysis or quantitative analysis.

  As described above, it has been found that the γGCT of the present invention exhibits γGCT activity by forming a complex with the activator, for example, as described above. Therefore, when the sample is brought into contact with the γGCT of the present invention and the activity of the γGCT is measured, the presence or absence of the activator in the sample can be analyzed by the presence or absence of the γGCT activity (qualitative analysis). The amount of the activator in the sample can be analyzed according to the degree of the γGCT activity (quantitative analysis).

  In the analysis method of the present invention, the γGCT to be brought into contact with the sample is, for example, γGCT in an unbound state with thioredoxin. Such γGCT can be obtained, for example, by expressing the γGCT gene of the present invention in the thioredoxin non-expressing host. Examples of the thioredoxin non-expressing host include the prokaryotes as described above, and a specific example is E. coli.

  In the analysis method of the present invention, for example, thioredoxin can be quantified from the measured γGCT activity using a correlation prepared in advance. The correlation is, for example, the relationship between the thioredoxin concentration and the γGCT activity in the presence of the thioredoxin at the concentration. As a specific example, a correlation equation, a calibration curve, or the like indicating the correlation can be used.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

[Example 1]
In this example, a transformed yeast introduced with a γGCT (RSp1022 (wild type)) gene derived from Ralstonia solanacearum was prepared, and the expression and activity of γGCT were confirmed.

(1) Construction of expression vector
RSp1022 cDNA (wild type, SEQ ID NO: 2) encoding γGCT (SEQ ID NO: 1) was amplified using the genomic DNA of Ralstonia solanacearum as a template. The obtained RSp1022 cDNA (wild type) was ligated to the cloning site of the self-prepared Gateway binary vector pMT1294 to construct an RSp1022 (wild type) expression vector. In the pMT1294, a galactose-inducible promoter (GAL1 promoter) is arranged on the 5 ′ side of the cloning site, and a base sequence encoding a 3 × FLAG tag and a purification tag (Protein A tag) is functionally located on the 3 ′ side. They are linked and arranged so as to express.

  A mutant RSp1022 (E216Q) expression vector was prepared from the RSp1022 (wild type) expression vector by site-directed mutagenesis. In the mutant RSp1022 (E216Q) expression vector, RSp1022 (E216Q) cDNA encoding a mutant γGCT (RSp1022 (E216Q)) in which the 216th glutamic acid in γGCT (SEQ ID NO: 1) is substituted with glutamine is inserted. As will be described later, the 216th glutamic acid of γGCT (SEQ ID NO: 1) is the active center of γGCT, and the mutant RSp1022 (E216Q) is an inactive γGCT having low γGCT activity due to substitution with glutamine. It is.

On the other hand, an expression vector for expressing a ChaC protein derived from yeast was constructed as γGCT of the comparative example. Specifically, YER163c cDNA (SEQ ID NO: 4) encoding ChaC protein (YER163c) (SEQ ID NO: 3) was amplified using Saccharomyces cerevisiae genomic DNA as a template. The obtained YER163c cDNA was similarly ligated to the cloning site of the vector to construct a YER163c expression vector.

YER163c (SEQ ID NO: 3)
MTNDNSGIWVLGYGSLIYKPPSHYTHRIPAIIHGFARRFWQSSTDHRGTPANPGRVATLIPYEDIIRQTAFLKNVNLYSESAPIQDPDDLVTIGVVYYIPPEHAQEVREYLNVREQNGYTLHEVEVHLETNREHEAVERGITALQLPRHNKDEGY
YER163c cDNA (SEQ ID NO: 4)
ATGACTAATGACAACAGTGGTATCTGGGTCCTAGGCTACGGATCTCTGATTTACAAGCCTCCGTCTCATTACACGCATAGAATACCGGCAATAATTCACGGTTTTGCTCGTCGGTTCTGGCAAAGTTCCACTGATCATAGGGGTACACCTGCAAACCCAGGAAGAGTGGCCACACTAATCCCGTATGAGGATATTATAAGGCAAACTGCCTTCTTGAAGAATGTGAATTTGTATAGTGAAAGTGCACCTATCCAGGATCCCGACGACTTAGTCACCATAGGGGTAGTGTATTATATACCACCAGAACATGCTCAAGAGGTTAGAGAGTATTTGAACGTAAGAGAACAAAACGGGTACACTCTACATGAGGTTGAGGTGCATCTAGAAACTAATCGGGAGCACGAGGCAGAATTGGGTGAAGCATTAGAACAGTTGCCTCGGCACAACAAATCGGGTAAACGGGTACTACTTACTTCGGTGTACATTGGTACAATAGATAATGAGGCCTTCGTCGGACCAGAGACCGTGGATGAAACTGCGAAAGTAATTGCCGTGTCGCATGGACCTAGTGGATCTAACTATGAATACCTTGCAAAACTGGAGCAAGCGCTAGCGCAAATGCCCATCATGAAAGAGCGAGGGCGTATTACCGATCATTATCTGACTGCTCTATTGGAGACTGTAAATAAATACAGGTGA

(2) Production of transformed yeast
The RSp1022 (wild type) expression vector, the RSp1022 (E216Q) expression vector, and the YER163c expression vector were independently introduced into Saccharomyces cerevisiae BY4743 strain by the lithium acetate method. Each obtained transformant was cultured at 26 ° C. for 72 hours using a uracil-free synthetic (SD) medium, and transformed yeast was recovered.

(3) Confirmation of Recombinant Protein Expression The transformed yeast was cultured at 26 ° C. for 18 hours using SD medium containing 20 mg / mL glucose until OD _600nm = 0.2. The transformed yeast after the culture was centrifuged at 2000 rpm for 5 minutes to collect the pellet. The pellet was centrifugally washed with sterilized water three times. Next, the washed transformed yeast was inoculated on a synthetic (SGal) medium containing galactose and cultured at 26 ° C. for 18 hours to express the recombinant protein. Then, the cultured transformed yeast was recovered and suspended in a buffer solution (1 mol / L sorbitol, 0.1 mol / L EDTA, pH 8.0) containing 1 mmol / L DTT.

1 μL of zymolyase (trade name: 20T, Nacalai Tesque) per OD _{600 nm} = 10 of the transformed yeast was added to the suspension, and the mixture was reacted at 30 ° C. for 1 hour to lyse cell walls and make spheroplasts. Next, the spheroplast was recovered by centrifugation and washed with the buffer. Furthermore, 1 mL of lysis buffer (50 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.1% NP-40, pH 7.5) was added per spheroplast OD _{600 nm} = 10, and a Dounce homogenizer was used. The spheroplast was crushed by moving the stroke up and down. And the said crushing liquid was centrifuged for 30 minutes at 15000 rpm, and the supernatant (cell-free extract) containing a recombinant protein was collect | recovered.

  The cell-free extract and IgG-binding Sepharose beads (GE Healthcare) were mixed to bind protein A in the recombinant protein and IgG of the beads. Then, after collecting the beads by centrifugation, they were washed by centrifugation three times with the lysis buffer to prepare a purified sample that was bound to the beads.

  Next, the purified sample was subjected to SDS-PAGE to confirm the expression of the recombinant protein. That is, the purified sample bound to the beads was boiled in an SDS sample buffer, the recombinant protein was released from the beads, and SDS-PAGE was performed using a 10% gel. The SDS buffer is a self-prepared Laemmli sample buffer (2 × stock: 0.125 mol / L, Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, 2.5% saturated bromophenol blue-containing ethanol. )It was used. After the electrophoresis, the recombinant protein was detected using an anti-G6PDH antibody (Sigma) and an HRP-labeled anti-rabbit IgG antibody (Cell Signaling Technology). As a control, a purified sample was prepared and the recombinant protein was detected by SDS-PAGE in the same manner except that a vector into which the various cDNAs were not inserted (control expression vector) was used.

  These results are shown in FIG. FIG. 1 is a photograph showing the results of SDS-PAGE of the purified sample. In FIG. 1, the left side of the photograph shows the molecular weight, and in each lane, from the left, the traits introduced with the control expression vector, the YER163c expression vector, the RSp1022 (wild type) expression vector, and the mutant RSp1022 (E216Q) expression vector The result of the purified sample derived from converted yeast is shown.

  As shown in FIG. 1, recombinant protein expression was not observed in the control expression vector. In the YER163c expression vector, bands were observed in the vicinity of about 48 KDa, about 45 KDa, and about 43 KDa. These molecular weights approximated the total theoretical value of 48.3 KDa of the molecular weights of YER163c and protein A. In the RSp1022 (wild type) expression vector, a band was observed in the vicinity of about 65 KDa, and in the mutant RSp1022 (E216Q) expression vector, a band was observed in the vicinity of about 65 KDa. The total theoretical value of the molecular weight of RSp1022 (wild type) and protein A is 66.6 KDa, the total theoretical value of the molecular weight of RSp1022 (E216Q) and protein A is 66.6 KDa, and each detected band is The molecular weight was almost the same as the estimated molecular weight (total theoretical value). From these facts, it was confirmed that the YER163c, the Rs1022 and the Rs1022 (E216Q) were expressed by the transformed yeast, and each was purified to be single.

(4) Confirmation of GSH expression level in transformed yeast Glutathione quantification using DTNB (5-5'-dithiobis [2-nitrobenzoic acid]) for the cell-free extract collected in (3) above (See Ralman et al., Nat Protoc: 3159-3165. 2006). Then, the GSH concentration of the control cell-free extract was set to 1, and the relative value of the GSH concentration of each cell-free extract was calculated.

  These results are shown in FIG. FIG. 2 is a graph showing the relative value of the GSH concentration of the cell-free extract. In FIG. 2, the horizontal axis indicates the type of protein expressed in the cell-free extract, and the vertical axis indicates the relative value of the GSH concentration in the cell-free extract (relative GSH level). , Shows the relative value of the GSH concentration. As shown in FIG. 2, the cell-free extract expressing the YER163c, the RSp1022 (wild type), and the mutant RSp1022 (E216Q) with respect to the control has a decreased relative value of the GSH concentration. In the cell-free extract expressing RSp1022 (wild type), the relative value of the GSH concentration was significantly reduced. From these results, it was found that the RSp1022 (wild type) showed extremely high γGCT activity as compared with the YER163c. Moreover, since the relative value of the GSH concentration of the mutation RSp1022 (E216Q) is higher than that of the RSp1022 (wild type), the 216th glutamic acid in SEQ ID NO: 1 is RSp1022 (wild type). It was found to be important for γGCT activity.

(5) Confirmation of γGCT activity 50 μL of the purified sample of (3) and 50 μL of 5 mmol / L GSH solution were mixed and reacted at 37 ° C. for 30 minutes to release 5-oxoproline and Cys-Gly from GSH. . Next, 10 μL of 0.5 μg / mL Cys-Gly dipeptidase (Dug1, derived from Sacchromyces cerevisiae, self-prepared) was added to the mixed solution and reacted at 37 ° C. for 60 minutes to liberate cysteine from Cys-Gly. And about the said liquid mixture after reaction, the density | concentration of the said cysteine was measured by acidic ninhydrin reaction. Then, the relative value of the γGCT activity of each purified sample was calculated with the cysteine concentration of the purified sample of YER163c as 100%.

  These results are shown in FIG. FIG. 3 is a graph showing the γGCT activity of the purified sample. In FIG. 3, the horizontal axis indicates the protein type of the purified sample, and the vertical axis indicates the relative value of γGCT activity. As shown in FIG. 3, the RSp1022 (wild type) showed γGCT activity 4.5 times that of the YER163c. In this example, the protein amount of the purified sample of YER163c was about 5 to 10 times the protein amount of the purified sample of Rsp1022 (wild type). That is, it can be said that Rsp1022 (wild type) is an enzyme having a very excellent specific activity, which is 5 to 1/10 of the protein of YER163c and exhibits γGCT activity 4.5 times as much. In addition, since the inactive mutant RSp1022 (E216Q) showed almost no γGCT activity, it was found that the 216th glutamic acid in SEQ ID NO: 1 is important for the γGCT activity of RSp1022 (wild type). .

[Example 2]
In this example, amino acids necessary for γGCT activity were confirmed for γGCT (RSp1022 (wild type)) derived from Ralstonia solanacearum .

(1) Preparation of expression vector In the same manner as in Example 1 (1), the following expression vector expressing the mutant RSp1022 was prepared from the RSp1022 (wild type) expression vector by site-directed mutagenesis.
Mutant RSp1022 (Y129A) expression vector: Vector mutant RSp1022 (L130A) expression vector expressing the mutant γGCT (RSp1022 (Y129A)) in which the 129th tyrosine of SEQ ID NO: 1 is replaced by alanine: 130th leucine of SEQ ID NO: 1 Vector mutant RSp1022 (L130G) expressing a mutant γGCT (RSp1022 (L130A)) in which is substituted with alanine: expressing a mutant γGCT (RSp1022 (L130G)) in which the 130th leucine of SEQ ID NO: 1 is replaced with glycine Vector mutation RSp1022 (S131A) expression vector: Vector mutation RSp1022 that expresses mutant γGCT (RSp1022 (S131A)) in which the 131st serine of SEQ ID NO: 1 is substituted with alanine 132A) expression vector: vector 132 th leucine of SEQ ID NO: 1 expresses mutations γGCT substituted with alanine (RSp1022 (L132A))

(2) Production of transformed yeast In the same manner as in Example 1 (2), each of the expression vectors was introduced alone to produce a transformed yeast.

(3) Confirmation of the active site of γGCT
In Saccharomyces cerevisiae , the proliferation ability decreases when the intracellular GSH concentration decreases. Therefore, whether or not the transformed yeast can form colonies was confirmed, and using this as an index, the presence or absence of γGCT activity and the amino acid sequence important for γGCT activity were confirmed.

Each transformed yeast, in sterile water, a predetermined ratio (10 ^0-fold, 10 x ^1, 10 ² times, 10 ³ fold) was diluted. Then, each of the transformed yeasts after dilution was individually sown on an SD medium containing 200 mg / mL galactose and cultured at 26 ° C. for 72 hours. And the presence or absence of colony formation in the culture medium after the said culture | cultivation was confirmed visually. For the control, the presence or absence of colony formation was confirmed in the same manner except that a transformed yeast introduced with a control expression vector was used instead of the transformed yeast. Moreover, the presence or absence of the colony formation of the said transformed yeast was confirmed similarly except having used the culture medium containing glucose instead of the said galactose as a control which canceled galactose induction.

  The result is shown in FIG. FIG. 4 is a photograph showing the presence or absence of colony formation of each transformed yeast. In FIG. 4, the left side of the photograph shows the type of protein expressed by the transformed yeast, the upper side of the photograph shows the presence or absence of galactose in the medium (ON is galactose added, OFF is glucose added), and the lower side of the photograph Indicates the dilution factor of the transformed yeast. As shown in FIG. 4, colony formation could be confirmed in any transformed yeast in the medium containing glucose (OFF). On the other hand, in the culture medium (ON) containing galactose, the transformed yeast expressing RSp1022 (wild type) cannot form colonies, whereas the mutant RSp1022 (E216Q), the mutant RSp1022 (Y129A), and the mutant RSp1022 (L130A), the mutant RSp1022 (L130G), the mutant RSp1022 (S131A) and the transformed yeast expressing the mutant RSp1022 (L132A) were all able to form colonies. Since the transformed yeast in which the colony was formed showed that the GSH concentration was maintained, these results indicate that the amino acid sequence at positions 129 to 132 (YLSL) and the amino acid residue at position 216 (E ) Was found to be important for γGCT activity.

[Example 3]
In this example, transformed Escherichia coli into which a γGCT (RSp1022 (wild type)) gene derived from Ralstonia solanacearum was introduced was prepared, and the expression of γGCT was confirmed.

The RSp1022 cDNA (wild type, SEQ ID NO: 2) of Example 1 (1) was ligated at the cloning site of the pET23d vector (Novagen, the same applies hereinafter) to construct an RSp1022 (wild type) expression vector for E. coli. Next, E. coli Escherichia coli Rosetta gamiB (DE3) pLysS strain (Novagen) was used as the host instead of yeast, and the transformation was carried out by the heat shock method in the same manner as in Example 1 (2). A transformant was prepared. Then, the transformant was cultured, and a cell-free extract was prepared in the same manner as in Example 1 (3). The transformant was cultured in an LB medium containing 100 μg / mL ampicillin at 37 ° C. for 18 hours. Furthermore, in order to induce protein expression, IPTG was added so as to be 72 μg / mL, followed by culturing at 18 ° C. for 6 hours to express the protein. Then, using a nickel affinity column, RSp1022 (wild type) was purified from the cell-free extract, and the purified RSp1022 (wild type) was subjected to SDS-PAGE. YER163c was purified in the same manner except that the YER163c expression vector was used instead of the RSp1022 (wild type) expression vector, and subjected to SDS-PAGE.

  The result is shown in FIG. FIG. 5 is a photograph showing the results of SDS-PAGE of the purified protein. In FIG. 5, the left side of the photograph shows the molecular weight, and each lane is a purified sample derived from transformed Escherichia coli introduced with the molecular weight marker (M), the YER163c expression vector, and the RSp1022 (wild type) expression vector from the left. Results are shown. As shown in FIG. 5, in the same manner as in Example 1, in the YER163c expression vector, a single band can be confirmed at the total theoretical value of 27.6 KDa of the molecular weight of YER163c and His6 tag, and the RSp1022 (wild type) In the expression vector, a single band was confirmed at the total theoretical value of 46 KDa of the molecular weight of RSp1022 (wild type) and His6 tag.

[Example 4]
In this example, a transformed yeast and a transformed Escherichia coli into which a γGCT (RSp1022 (wild type)) gene derived from Ralstonia solanacearum was introduced were prepared, and the expression and activity of γGCT were confirmed.

(1) Expression vector The RSp1022 (wild type) expression vector, the mutant RSp1022 (E216Q) expression vector, and the YER163c expression vector constructed in Example 1 (1) were used as expression vectors for yeast introduction. In addition, an expression vector for introducing E. coli was constructed in the same manner as the expression vector for introducing yeast in Example 1. Specifically, a cDNA in which a Protein A tag is fused to the C-terminus of the RSp1022 (wild type), the RSp1022 (E216Q), or the YER163c, was used in the same manner as in Example 3 to obtain a pET23d vector (Novagen, hereinafter Similarly, the RSp1022 (wild type) expression vector for E. coli, the RSp1022 (E216Q) expression vector for E. coli, and the YER163c expression vector for E. coli were constructed.

(2) Production of transformed yeast and transformed Escherichia coli In the same manner as in Example 1 (2) above, the Saccharomyces cerevisiae BY4743 strain was transformed into the RSp1022 (wild type) expression vector, the mutant RSp1022 as the expression vector for yeast introduction. The (E216Q) expression vector and the YER163c expression vector were each introduced alone, and the transformed yeast was recovered. Further, in the same manner as in Example 3, the RSp1022 (wild type) expression vector and the RSp1022 (E216Q) expression vector were used as the expression vector for Escherichia coli in Escherichia coli Rosetta gamiB (DE3) pLysS strain (Novagen). The YER163c expression vector was introduced alone, and transformed E. coli was recovered.

(3) Confirmation of recombinant protein expression The transformed yeast was cultured in the same manner as in Example 1 (3), and the transformed Escherichia coli was cultured in the same manner as in Example 3. . Next, in the same manner as in Example 1 (3), the respective recombinant proteins were purified from the transformed yeast and the transformed Escherichia coli, separated by SDS-PAGE, and then rabbit IgG was used. The recombinant protein to which the Protein A tag was added was detected by Western blotting.

  These results are shown in FIG. FIG. 6 is a photograph showing the results of Western blotting of the purified sample. In FIG. 6, the right side of the photograph shows the molecular weight, and in each lane, the control expression vector, the YER163c expression vector, the RSp1022 (wild type) expression vector, and the mutant RSp1022 (E216Q) expression vector are introduced from the left. The result of the purified sample derived from the transformed yeast and the purified sample derived from the transformed E. coli mother is shown.

  As shown in FIG. 6, the following was confirmed in both the transformed yeast and the transformed E. coli. That is, recombinant protein expression was not observed in the control expression vector. In the YER163c expression vector, a band was observed in the vicinity of about 48 KDa, and this molecular weight was close to the total theoretical value of 48.3 KDa of the molecular weight of YER163c and protein A. In addition, in the RSp1022 (wild type) expression vector and the mutant RSp1022 (E216Q) expression vector, a band is observed in the vicinity of about 65 kDa, and the molecular weight of these is the total theory of the molecular weights of RSp1022 (wild type) and protein A. The value was 66.6 KDa, and approximated the total theoretical value of 66.6 KDa for the molecular weight of RSp1022 (E216Q) and protein A. From these facts, it was confirmed that the RSp1022 (wild type) and the RSp1022 (E216Q) were expressed by the transformed yeast and the transformed Escherichia coli, and each was purified to be single.

(4) Confirmation of γGCT activity Except that the purified sample derived from the transformed yeast and the purified sample derived from the transformed E. coli were used, the cysteine concentration was measured in the same manner as in Example 1 (5), The relative value of γGCT activity of each purified sample derived from the transformed yeast was calculated with the cysteine concentration of the purified sample derived from YER163c derived from the transformed yeast as 100%, and the cysteine concentration of the purified sample of YER163c derived from the transformed E. coli The relative value of the γGCT activity of each purified sample derived from the transformed E. coli was calculated.

  These results are shown in FIG. FIG. 7 is a graph showing the γGCT activity of the purified sample. In FIG. 7, the horizontal axis represents the protein type of the purified sample, and the vertical axis represents the relative value of γGCT activity. As shown in FIG. 7, in the purified sample derived from the transformed yeast, only the RSp1022 (wild type) showed an extremely high γGCT activity as compared with the YER163c, as in Example 1 (5). It was. On the other hand, as shown in FIG. 7, in the purified sample derived from the transformed Escherichia coli, the YER163c showed the same γGCT activity as the transformed yeast, but the RSp1022 (wild type) was the transformed yeast. The γGCT activity like that could not be confirmed. Thus, even with the same amino acid sequence, RSp1022 (wild type) derived from the transformed E. coli does not exhibit γGCT activity, and RSp1022 (wild type) derived from the transformed yeast exhibits γGCT activity. It was suggested that the protein extract fraction of yeast is an activated fraction that activates the RSp1022 (wild type).

[Example 5]
In order to confirm that the yeast protein extract fraction is an activated fraction that activates the RSp1022 (wild type), in this example, a purified sample derived from transformed E. coli and a yeast protein extract were used. The purified sample was checked for γGCT activity.

(1) Confirmation of Recombinant Protein Expression 15 mL of a transformed Escherichia coli culture solution into which the RSp1022 (wild type) expression vector for E. coli and the mutant RSp1022 (E216Q) expression vector were introduced was collected in the same manner as in Example 4. The suspension was suspended in 1 mL of lysis buffer, and protein was extracted from E. coli by sonication. The composition of the lysis buffer was 10 mmol / L Tris-HCl (PH 8.0), 150 mmol / L NaCl, 0.1% surfactant NP-40 (manufactured by SIGMA). A solution containing 25 μL of IgG-binding beads (GE Healthcare) was added to 0.5 mL of the extracted crude protein solution, and mixed while rotating at 4 ° C. for 3 hours, so that the recombinant protein bound to the IgG-binding beads was mixed. A simple purified sample was prepared. Then, in the same manner as in Example 1 (3), SDS-PAGE and Western blotting were performed on the purified sample, and the recombinant protein was detected.

  As a result, similar to the result of Example 4, a single band was observed at the same density in the RSp1022 (wild type) and the mutant RSp1022 (E216Q). From this, it was confirmed that the RSp1022 (wild type) and the RSp1022 (E216Q) both have the same level of expression.

(2) Preparation of yeast protein extract A yeast protein extract was prepared by the following method. That is, Saccharomyces cerevisiae BY4743 strain was cultured at 26 ° C. using SD medium containing 20 mg / mL glucose until OD _{600 nm} = 1, and the yeast was collected. 20 mL of the yeast culture solution was collected, suspended in the lysis buffer of (1) above containing protease inhibitors Pepstatin A (2 μmol / L) and PMSF (1 mmol / L), and 0.5 mL of glass bead solution was added. The mixture was shaken vigorously using a vortex mix for 1 minute. After shaking, the mixture was immediately placed on ice, cooled for 1 minute, and again vigorously shaken for 1 minute with the vortex mixture. After repeating this operation 10 times, the crushed yeast and the glass beads were separated and removed from the mixed solution by centrifugation to obtain a supernatant as a protein extract. The protein concentration of the supernatant was quantified using a micro BCA assay kit manufactured by Thermo Fisher Scientific, and the supernatant (yeast protein extract) was stored at -80 degrees.

(3) Confirmation of γGCT activity The purified sample bound to the IgG-bound beads of (1) was mixed with the yeast protein extract of (2) to prepare a mixed sample. The mixed sample was added with 480 μL of the diluted protein of yeast protein (2) (protein amount 0, 50, 100, 150, 200 μg) to 20 μL of the purified sample, so that the total amount was 500 μL. The mixed sample was then shaken at 4 ° C. for 2 hours. The purified sample reacted with the yeast protein extract of various protein concentrations is washed three times with the lysis buffer of (1), and the beads bound to the purified sample are used as the purified sample after the extract treatment. It was collected. Using the purified sample after the treatment, a reaction was carried out in the same manner as in Example 1 (5), and the cysteine concentration in the reaction solution was measured.

  These results are shown in FIG. FIG. 8 is a graph showing the cysteine concentration in the reaction solution in the presence of the yeast protein extract. In FIG. 8, the horizontal axis represents the protein concentration of the yeast protein extract in the mixed sample. In FIG. 8, the vertical axis indicates the cysteine concentration in the reaction solution, which corresponds to the γGCT activity of the purified sample. As shown in FIG. 8, in the RSp1022 (wild type), cysteine was detected by adding the yeast protein extract, and the cysteine concentration increased depending on the addition concentration of the yeast protein extract. That is, γGCT activity was confirmed by the addition of the yeast protein extract, and the activity increased depending on the concentration of the yeast protein extract. On the other hand, cysteine was not detected in RSp1022 (E216Q) even when the yeast protein extract was added, and γGCT activity could not be confirmed. From these results, it was found that the yeast protein extract activated the transformed Escherichia coli-derived RSp1022 (wild type).

Example 6
In order to examine whether or not the RSp1022 (wild type) γGCT is activated by a protein extract derived from a eukaryote other than yeast, in this example, a purified sample derived from transformed Escherichia coli, yeast, bacteria, animal Alternatively, the purified protein was reacted with a plant protein extract to confirm the γGCT activity of the purified sample.

(1) Preparation of various protein extracts Yeast protein extracts were prepared in the same manner as in Example 5 (2).

Bacterial protein extract was prepared from Ralstonia solanacearum GMI1000 by the following method. That is, the cells were cultured overnight at 26 ° C. using 5 mL of BG liquid medium (1% peptone, 0.1% yeast extract, 0.1% casamino acid, 0.5% glucose). The obtained culture solution was transferred to 90 mL of BG liquid medium, and further cultured at 26 ° C. overnight. After culturing, the cells are collected by centrifugation, and the cells after collection are washed once with 10 mL of sterilized water, suspended in 5 mL of the lysis buffer of Example 5 (1), and then ultrasonically disrupted. Cells were disrupted. The obtained disrupted bacterial solution was centrifuged to separate the supernatant and unbroken cells. The protein concentration of the supernatant was quantified with the micro BCA assay kit, and the supernatant was stored at −80 ° C. as a bacterial protein extract.

  Animal protein extracts were prepared from HeLa cells by the following method. That is, HeLa cells were cultured until they became confluent in a culture dish having a diameter of 10 cm, and then scraped with a cell scraper and suspended in 10 mL PBS. The suspension was centrifuged to recover the cells, 1 mL of the lysis buffer was added to the cells, 400 μL of glass bead solution was further added, and the mixture was vigorously shaken for 1 minute using a vortex mixture. After shaking, the mixture was quickly cooled on ice for 1 minute. After repeating this operation 10 times, the crushed yeast and the glass beads were separated and removed from the mixed solution by centrifugation to obtain a supernatant as a protein extract. The protein concentration of the supernatant was quantified with the micro BCA assay kit, and the supernatant was used as an animal protein extract and stored at −80 ° C.

The plant protein extract was prepared from Arabidopsis thaliana by the following method. That is, 0.5 g of Arabidopsis thaliana leaves were placed in a screw-cap type 2 mL plastic tube, one stainless steel ball having a diameter of 5 mm was added, and it was quickly frozen with liquid nitrogen. The sample after freezing was crushed at 1,200 rpm for 30 seconds using a bead beater (Central Scientific Trade) and quickly cooled with liquid nitrogen. This operation was repeated three times, and 0.5 mL of the lysis buffer was added to the Arabidopsis thaliana sample that was powdered by crushing, and further separated into a supernatant and uncrushed cells by centrifugation. The protein concentration of the supernatant was quantified using the micro BCA assay kit, and the supernatant was used as a plant protein extract and stored at −80 ° C.

(2) Confirmation of γGCT activity The various protein extracts of (1) were mixed with the purified sample bound to the IgG-bound beads of Example 5 (1) to prepare a mixed sample. The mixed sample is obtained by adding the yeast protein extract, the bacterial protein extract, the animal protein extract, and the plant protein extract of (1) to 20 μL of the purified sample of Example 5 (1). 480 μL (protein amount 100 μg) was added to make the total amount 500 μL. The mixed sample was shaken at 4 ° C. for 2 hours. The purified sample reacted with protein extracts from various organisms was washed three times with the lysis buffer, and the beads to which the purified sample was bound were collected as purified samples after the various extract treatments. Using the purified sample after the treatment, a reaction was carried out in the same manner as in Example 1 (5), and the cysteine concentration in the reaction solution was measured.

  These results are shown in FIG. FIG. 9 is a graph showing the cysteine concentration in the reaction solution in the presence of the various protein extracts. In FIG. 9, the horizontal axis shows the types of the various protein extracts. In FIG. 9, the vertical axis indicates the cysteine concentration in the reaction solution, which corresponds to the γGCT activity of the purified sample. As shown in FIG. 9, the addition of eukaryotic animal and plant protein extracts confirmed the γGCT activity of the RSp1022 (wild type) as in the case of the yeast protein extract. On the other hand, γGCT activity of the RSp1022 (wild type) could not be confirmed by addition of a bacterial protein extract not applicable to eukaryotes.

Example 7
The inventors identified thioredoxin as an activator of the RSp1022 (wild type) in eukaryotic protein extract fractions. In this example, in order to confirm that the activator is thioredoxin, a purified sample derived from transformed E. coli was reacted with yeast thioredoxin, and the γGCT activity of the purified sample was confirmed.

(1) Confirmation of γGCT activity The purified sample of Example 3 was mixed with yeast thioredoxin to prepare a mixed sample. The yeast thioredoxin was self-prepared as follows. Specifically, the TRX1 gene encoding the yeast thioredoxin (ScTrx1, SEQ ID NO: 5) was incorporated into a pET23d vector, an expression vector was constructed, and Escherichia coli Rosetta gami B (DE3) pLysS strain was transformed with the expression vector. After the obtained transformed Escherichia coli was cultured in LB medium, the expression of the yeast thioredoxin protein was induced by IPTG (isopropyl-β-thiogalactopyranoside). The induced yeast thioredoxin protein was extracted from the transformed Escherichia coli by sonication, and affinity-purified with a nickel affinity resin using a His6 tag fused to the C-terminus of the yeast thioredoxin protein.

ScTrx1 (SEQ ID NO: 5)
MVTQFKTASEFDSAIAQDKLVVVDFYATWCGPCKMIAPMIEKFSEQYPQADFYKLDVDELGDVAQKNEVSAMPTLLLFKNGKEVAKVVGANPAAIKQAIAANA

  In the mixed sample, the protein concentration of the purified sample of (1) is 0.2 μmol / L, and the concentration of thioredoxin is 0.01, 0.02, 0.04, 0.1, 0.2 μmol / L. It was. And the cysteine density | concentration in a reaction liquid was measured like the said Example 1 (5) except having used 50 microliters of said mixed samples.

  These results are shown in FIG. FIG. 10 is a graph showing the cysteine concentration in the reaction solution in the presence of thioredoxin. In FIG. 10, the horizontal axis represents the concentration of thioredoxin in the mixed sample. In FIG. 10, the vertical axis indicates the cysteine concentration in the reaction solution, which corresponds to the γGCT activity of the purified sample. As shown in FIG. 10, in the RSp1022 (wild type), cysteine was detected by addition of thioredoxin, and the cysteine concentration increased depending on the addition concentration of thioredoxin. That is, γGCT activity was confirmed by the addition of thioredoxin, and the activity increased in a thioredoxin concentration-dependent manner. From this result, it was found that thioredoxin activates the transformed Escherichia coli-derived RSp1022 (wild type).

[Example 8]
In this example, the interaction between RSp1022 (wild type) and thioredoxin was examined by the enzyme two-hybrid method.

  The fusion proteins shown in Table 1 below were set to Bait and Prey, and the interaction between RSp1022 (wild type) and thioredoxin was confirmed as follows.

When a pGBKT7 vector (Clontech) is used as a Bait expression vector and the RSp1022 (wild type) gene is incorporated into this vector, growth inhibition occurs. Therefore, a bait expression vector was constructed by incorporating the RSp1022 (wild type) active center mutant (E216Q) gene having no γGCT activity into the pGBKT7 vector. On the other hand, a pGADT7 vector (Clontech) was used as a Prey expression vector, and the Saccharomyces cerevisiae Trx1 gene (ScTrx1), an Arabidopsis thaliana- derived h-type thioredoxin, AtTrx h2 cDNA, Trx h Thus, a Prey expression vector was constructed. The yeast AH109 strain (Clontech) was transformed with the Bait expression vector and the Prey expression vector so that the combinations described in the following table were obtained. The obtained transformed yeast was seeded on a yeast synthesis medium SD (-Trp, -Leu) not containing tryptophan or leucine, thereby obtaining a transformed colony. The obtained transformed colony was suspended in 100 μL of sterilized water, and 10 μL of the suspension was spotted on the following plate media (1), (2), and (3), and the growth on each plate was observed. Interaction was evaluated.
(1) Yeast synthesis medium SD (-Trp, -Leu) not containing tryptophan and leucine
(2) Yeast synthesis medium SD not containing tryptophan, leucine and histidine (-Trp, -Leu, -His)
(3) Yeast synthesis medium SD (-Trp, -Leu, -His, + 3-AT) which does not contain tryptophan, leucine and histidine but contains 3-AT which is an analog of histidine

The medium (1) is a control medium in which yeast transformed with all combinations of Bait expression vector and Prey expression vector can grow. The media (2) and (3) are media that allow the yeast to grow only when Bait and Prey interact. Specifically, in the medium (2), the yeast can grow only when the reporter histidine synthase His3 is expressed by the interaction of Bait and Prey and the histidine auxotrophy is restored. In addition, the medium of (3) has stricter conditions than the medium of (2). Specifically, the combination of Bait and Prey has a stronger interaction by adding 3AT, which is a histidine analog. Is a medium that allows the yeast to grow. In Table 1 below, GAL4DBD is a DNA binding domain of GAL4 (DBD), AD is an activator domain (AD), ScTrx1 is yeast-derived thioredoxin (SEQ ID NO: 5), and AtTrx h2 (SEQ ID NO: 6). ), AtTrx h3 (SEQ ID NO: 7), and AtTrx h5 (SEQ ID NO: 8) are h-type thioredoxins derived from Arabidopsis thaliana , respectively.

AtTrx h2 (SEQ ID NO: 6)
MGGALSTVFGSGEDATAAGTESEPSRVLKFSSSARWQLHFNEIKESNKLLVVDFSASWCGPCRMIEPAIHAMADKFNDVDFVKLDVDELPDVAKEFNVTAMPTFVLVKRGKEIERIIGAKKDELEKKVSKLRA
AtTrx h3 (SEQ ID NO: 7)
MAAEGEVIACHTVEDWTEKLKAANESKKLIVIDFTATWCPPCRFIAPVFADLAKKHLDVVFFKVDVDELNTVAEEFKVQAMPTFIFMKEGEIKETVVGAAKEEIIANLEKHKTVVAAA
AtTrx h5 (SEQ ID NO: 8)
MAGEGEVIACHTLEVWNEKVKDANESKKLIVIDFTASWCPPCRFIAPVFAEMAKKFTNVVFFKIDVDELQAVAQEFKVEAMPTFVFMKEGNIIDRVVGAAKDEINEKLMKHGGLVASA

  These results are shown in FIG. FIG. 11 is a photograph showing colony formation in each combination of the Bait and the Prey. In FIG. 11, lane 1 “-Trp, -Leu” is the result of the medium of (1), and lane 2 “-Trp, -Leu, -His” is the result of the medium of (2). Lane 3 “-Trp, -Leu, -His, + 3AT” is the result of the medium of (3) above.

  As shown in FIG. 11, colony formation was confirmed in lane 2 in the combination of RSp1022 (wild type) and thioredoxin (ScTrx1, AtTrx h2, AtTrx h3 and AtTrx h5). In particular, among thioredoxins, in the case of AtTrx h5, colony formation in lane 3, which is a severe condition, was confirmed, and a strong interaction was confirmed.

  While the present invention has been described with reference to the embodiments and examples, the present invention is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  This application claims the priority on the basis of Japanese application Japanese Patent Application No. 2013-225795 for which it applied on October 30, 2013, and takes in those the indications of all here.

  As described above, the γ-glutamyl cyclotransferase of the present invention has higher activity against glutathione than the known γ-glutamyl cyclotransferase. Therefore, according to the γ-glutamyl cyclotransferase of the present invention, for example, 5-oxoproline and cysteine-glycine dipeptide can be efficiently produced from glutathione.

Claims (34)

A γ-glutamyl cyclotransferase, which is at least one protein selected from the group consisting of the following (P1) to (P3).
(P1) a protein comprising the amino acid sequence of SEQ ID NO: 1 (P2) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence of (P1), Protein having glutamyl cyclotransferase activity (P3) A protein having an amino acid sequence having 80% or more identity to the amino acid sequence of (P1) and having γ-glutamyl cyclotransferase activity The γ-glutamyl cyclotransferase according to claim 1, which is derived from bacterial wilt. A γ-glutamyl cyclotransferase reagent comprising the γ-glutamyl cyclotransferase according to claim 1 or 2 and an activator thereof. The reagent according to claim 3, wherein the activator is a eukaryotic protein extract. The reagent according to claim 3 or 4, wherein the activator is a eukaryotic thioredoxin. The reagent according to claim 4 or 5, wherein the eukaryote is at least one selected from the group consisting of animals, plants, fungi and protists. A γ-glutamyl cyclotransferase gene comprising at least one polynucleotide selected from the group consisting of the following (p1) to (p3).
(P1) a polynucleotide comprising the base sequence of SEQ ID NO: 2 (p2) comprising a base sequence in which one or several bases have been deleted, substituted, inserted and / or added in the base sequence of (p1), and γ -Polynucleotide encoding a protein having glutamyl cyclotransferase activity (p3) A protein comprising a base sequence having 80% or more identity to the base sequence of (p1), and having a γ-glutamyl cyclotransferase activity Encoding polynucleotide An expression vector for γ-glutamyl cyclotransferase, wherein the γ-glutamyl cyclotransferase gene according to claim 7 is inserted. an expression step of expressing γ-glutamylcyclotransferase from a γ-glutamylcyclotransferase gene,
The method for producing γ-glutamyl cyclotransferase, wherein the γ-glutamyl cyclotransferase gene is the γ-glutamyl cyclotransferase gene according to claim 7. The method for producing γ-glutamyl cyclotransferase according to claim 9, wherein the expression step causes γ-glutamyl cyclotransferase to be expressed from the γ-glutamyl cyclotransferase gene in a host. The method for producing γ-glutamylcyclotransferase according to claim 10, wherein the host is yeast or Escherichia coli. The method for producing γ-glutamyl cyclotransferase according to claim 10 or 11, wherein, in the expression step, the host is a transformant into which the γ-glutamyl cyclotransferase gene has been introduced. The γ according to any one of claims 10 to 12, wherein the host is a transformant into which the γ-glutamyl cyclotransferase gene has been introduced by an expression vector into which the γ-glutamyl cyclotransferase gene has been inserted. -Method for producing glutamyl cyclotransferase. Furthermore, the manufacturing method of (gamma) -glutamyl cyclotransferase as described in any one of Claims 10-13 including the release process which releases an intracellular component from the said host after the said expression process. liberating 5-oxoproline from glutathione by γ-glutamylcyclotransferase,
The method for producing 5-oxoproline, wherein the γ-glutamylcyclotransferase is the γ-glutamylcyclotransferase according to claim 1 or 2. The method for producing 5-oxoproline according to claim 15, wherein in the release step, the γ-glutamylcyclotransferase is used in the presence of an activator of the γ-glutamylcyclotransferase. The method for producing 5-oxoproline according to claim 16, wherein the activator is a eukaryotic protein extract. The method for producing 5-oxoproline according to claim 16 or 17, wherein the activator is a eukaryotic thioredoxin. The method for producing 5-oxoproline according to claim 17 or 18, wherein the eukaryote is at least one selected from the group consisting of animals, plants, fungi, and protists. releasing cysteine-glycine dipeptide from glutathione by γ-glutamylcyclotransferase,
A method for producing a cysteine-glycine dipeptide, wherein the γ-glutamylcyclotransferase is the γ-glutamylcyclotransferase according to claim 1 or 2. 21. The method for producing a cysteine-glycine dipeptide according to claim 20, wherein in the release step, the γ-glutamylcyclotransferase is used in the presence of an activator of the γ-glutamylcyclotransferase. The method for producing a cysteine-glycine dipeptide according to claim 21, wherein the activator is a eukaryotic protein extract. The method for producing a cysteine-glycine dipeptide according to claim 21 or 22, wherein the activator is a eukaryotic thioredoxin. The method for producing a cysteine-glycine dipeptide according to claim 22 or 23, wherein the eukaryote is at least one selected from the group consisting of animals, plants, fungi, and protists. A selection step of selecting an activity inhibitory substance that suppresses γ-glutamylcyclotransferase activity of γ-glutamylcyclotransferase from a test substance as a candidate for a control agent,
A screening method for a candidate drug for controlling bacterial wilt disease, wherein the γ-glutamylcyclotransferase is the γ-glutamylcyclotransferase according to claim 1 or 2. 26. The screening method for a candidate agent for controlling bacterial wilt disease according to claim 25, wherein, in the selection step, the γ-glutamylcyclotransferase activity is an activity in the presence of an activator of the γ-glutamylcyclotransferase. 27. The screening method for a bacterial wilt control drug candidate substance according to claim 26, wherein the activator is a eukaryotic protein extract. 28. The screening method for a bacterial wilt control drug candidate substance according to claim 26 or 27, wherein the activator is a eukaryotic thioredoxin. 29. The screening method for a bacterial wilt control drug candidate substance according to claim 27 or 28, wherein the eukaryote is at least one selected from the group consisting of animals, plants, fungi, and protists.   Analyzing the thioredoxin contained in the sample by bringing the sample into contact with the γ-glutamyl cyclotransferase according to claim 1 or 2 and measuring the γ-glutamyl cyclotransferase activity of the γ-glutamyl cyclotransferase. A method for analyzing thioredoxin. The thioredoxin analysis method according to claim 30, wherein the analysis is a qualitative analysis or a quantitative analysis. 32. The method for analyzing thioredoxin according to claim 30 or 31, wherein the γ-glutamylcyclotransferase to be contacted with the sample is γ-glutamylcyclotransferase in an unbound state with thioredoxin. The thioredoxin analysis method according to any one of claims 30 to 32, wherein the γ-glutamylcyclotransferase to be contacted with the sample is γ-glutamylcyclotransferase expressed in a thioredoxin non-expressing host. The thioredoxin analysis method according to any one of claims 30 to 33, wherein the γ-glutamylcyclotransferase is γ-glutamylcyclotransferase expressed in a prokaryotic host.