JP2009100718A - Method for producing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a protein catalyzing a reaction forming a corresponding phosphorylated nucleoside by acting on a nucleoside, and to provide a method or the like for measuring a nucleoside by using the protein. <P>SOLUTION: The fact that the protein catalyzing the reaction for forming the phosphorylated nucleoside by acting on the nucleoside can be obtained from Burkholderia thailandensis or the like is confirmed. Therby, provided are the method for producing the protein, the phosphorylation method of the nucleoside by using the protein, the phosphorylation agent for the nucleoside, the method for producing the phosphorylated nucleoside by converting the nucleoside into the phosphorylated nucleoside, and a method for measuring the nucleoside. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to proteins that act on nucleosides.

A nucleoside is a substance involved in many biological reactions that is important as a coenzyme or a component of coenzyme, in addition to being a component of DNA and RNA when bound to phosphoric acid to form a phosphorylated nucleoside. Since the concentration and ratio of nucleoside and phosphorylated nucleoside in cells vary depending on the growth phase or non-growth phase, the state of the cell may be grasped by measuring the amount of intracellular nucleoside. Phosphorylated nucleosides are important as various pharmaceutical raw materials, biochemical research reagents, or nutrient material raw materials.
Conventionally, proteins that act on nucleosides to catalyze reactions that produce phosphorylated nucleosides include 6-phosphofructokinase from Aeropyrum pernix, which is an archaea, and Methanocaldococcus jannachus, which is an archaea. The derived phosphofructokinase-B (Phosphofructokinase-B) is known. (Non-Patent Documents 1 and 2).
On the other hand, the nucleotide sequence of SEQ ID NO: 2 of Burkholderia thailandensis DSM13276 strain was sequence-analyzed by whole genome analysis of the strain (Non-patent Document 3). However, the nature of the protein encoded by the base sequence has not been elucidated so far, and it was only expected to be a ribokinase.
Thomas Hansen et al., Arch. Microbiol. 177, 62-69, 2001 Thomas Hansen et al., Extremeophiles, 11, 105-114, 2007. H. S. Stanley et al., BMC Genomics, 6, 174, 2005

  To provide a method for producing an easily usable protein that catalyzes a reaction that acts on a nucleoside to produce a corresponding phosphorylated nucleoside, a method for measuring a nucleoside using the protein, and the like.

In order to solve the above-mentioned problems, the present inventors have conducted extensive research. As a result, the protein having the amino acid sequence of SEQ ID NO: 1 acts on at least a nucleoside such as adenosine, thymidine, uridine, inosine, or guanosine. It was newly found that it is a protein that catalyzes the reaction to produce the corresponding phosphorylated nucleoside. The amino acid sequence of SEQ ID NO: 1 was a protein encoded by the nucleotide sequence of SEQ ID NO: 2 of Burkholderia thailandensis DSM 13276 strain, but catalyzed the reaction that acts on the above nucleoside to produce the corresponding phosphorylated nucleoside. This has not been known so far, and a very useful method of using a protein is considered. A method for producing the protein has been established, and a method for phosphorylating a nucleoside using the protein, and a nucleoside phosphorylation using the protein. An oxidant, a method for producing a phosphorylated nucleoside using a nucleoside using the protein as a phosphorylated nucleoside, and a method for measuring a nucleoside using the protein have been completed.
That is, the present invention relates to the following.
[1] A method for producing a protein according to any one of (1) and (2) below,
A method for producing the protein, comprising a step of forming the protein based on a base sequence encoding the protein, and a step of obtaining the protein.
(1) a protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a phosphorylation reaction of a nucleoside;
(2) The amino acid sequence of SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence is the amino acid sequence of SEQ ID NO: 5 and the sequence listing SEQ ID NO: A protein comprising 6 amino acid sequences and capable of catalyzing a nucleoside phosphorylation reaction.
[1-1] The production method according to [1], wherein the nucleoside in (1) or (2) is a nucleoside excluding cytidine and xanthosine.
[1-2] The production method according to [1], wherein the nucleoside is at least one of adenosine, thymidine, uridine, inosine, or guanosine.
[1-3] The production method according to any one of [1] to [1-2], wherein the step of forming the protein includes using a cell containing a base sequence encoding the protein.
[1-4] The production method according to any one of [1] to [1-3], wherein the step of forming the protein includes using a transformant into which a base sequence encoding the protein is introduced.
[1-5] The production method according to any one of [1] to [1-4], wherein the step of forming the protein includes using a microorganism having a base sequence encoding the protein.
[2] The step of forming the protein based on the base sequence encoding the protein is a step of using a microorganism that belongs to the genus Burkholderia and produces a protein that can catalyze a phosphorylation reaction of a nucleoside [1] to The production method according to any one of [1-5].
[2-1] The production method according to any one of [1] to [2], wherein the step of forming the protein includes using Burkholderia thailandensis.
[2-2] The production method according to any one of [1] to [2-1], wherein the step of forming the protein includes using Burkholderia thailandensis DSM13276 strain.
[3] The production method according to any one of [1] to [2-2], wherein the protein comprises the amino acid sequence of SEQ ID NO: 1 and can catalyze a nucleoside phosphorylation reaction.
[3-1] The protein comprises an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 are deleted, substituted or added, and is a protein capable of catalyzing a nucleoside phosphorylation reaction. [1] The production method according to any one of [3].
[3-2] The protein comprises an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence is amino acid of SEQ ID NO: 5 A protein comprising the amino acid sequence of the sequence and SEQ ID NO: 6 (wherein Xaa represents any amino acid in SEQ ID NO: 5 and SEQ ID NO: 6), which is a protein capable of catalyzing a phosphorylation reaction of a nucleoside [1] to [ 3-1.] The production method according to any one of the above.
[4] The method according to any one of [1] to [3-2], further comprising a step of purifying the protein when obtaining the protein formed in the step of forming the protein based on the base sequence encoding the protein. The manufacturing method as described.
[4-1] The production method according to any one of [3] to [4], wherein the step of forming the protein includes using a cell containing a base sequence encoding the protein.
[4-2] The production method according to any one of [3] to [4-1], wherein the step of forming the protein includes using a transformant into which a base sequence encoding the protein is introduced.
[5] The production method according to any one of [3] to [4-2], wherein the base sequence encoding the protein is the base sequence represented by SEQ ID NO: 2 in Sequence Listing.
[6] The production method according to any one of [1] to [5], wherein the protein is a protein having the following physicochemical properties <1> to <5>.
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes;
[6-1] The production method according to [6], wherein the phosphorylated nucleoside is a monophosphorylated nucleoside.
[6-2] The production method according to any one of [6] to [6-1], wherein the phosphate donor is ATP.
[6-3] The production method according to any one of [6] to [6-2], wherein the protein has a molecular weight of about 34 kDa as determined by SDS polyacrylamide gel electrophoresis.
[7] A method for phosphorylating a nucleoside using the protein according to any one of (1) to (3) below. However, the nucleoside is a nucleoside excluding cytidine and xanthosine.
(1) A protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a nucleoside phosphorylation reaction.
(2) The amino acid sequence of SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence is the amino acid sequence of SEQ ID NO: 5 and the sequence listing SEQ ID NO: A protein comprising 6 amino acid sequences and capable of catalyzing a nucleoside phosphorylation reaction.
(3) A protein having the following physicochemical properties <1> to <5>.
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes;
[7-1] The method for phosphorylating a nucleoside according to [7], wherein the nucleoside in (1) or (2) is a nucleoside excluding cytidine and xanthosine.
[7-2] The method for phosphorylating a nucleoside according to [7], wherein the nucleoside is at least one of adenosine, thymidine, uridine, inosine, or guanosine.
[7-3] The method for phosphorylating a nucleoside according to any one of [7] to [7-2], wherein the phosphorylated nucleoside is a monophosphorylated nucleoside.
[7-4] The phosphorylation method according to any one of [7] to [7-3], wherein the phosphate donor is ATP.
[7-5] The phosphorylation method according to any one of [7] to [7-4], wherein the protein has a molecular weight of about 34 kDa as determined by SDS polyacrylamide gel electrophoresis.
[8] The method for phosphorylating a nucleoside according to any one of [7] to [7-5], wherein a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing and capable of catalyzing a phosphorylation reaction of a nucleoside is used.
[8-1] The protein comprises an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 are deleted, substituted, or added, and can catalyze a nucleoside phosphorylation reaction. The method for phosphorylating a nucleoside according to any one of [7] to [8].
[8-2] The protein comprises an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence is amino acid of SEQ ID NO: 5 A protein comprising the amino acid sequence of the sequence and sequence listing SEQ ID NO: 6 (wherein Xaa represents any amino acid in SEQ ID NO: 5 and SEQ ID NO: 6), which is a protein capable of catalyzing a phosphorylation reaction of a nucleoside [7] to [ The method for phosphorylating a nucleoside according to any one of [8-1].
[8-3] The method for phosphorylating a nucleoside according to any one of [8] to [8-2], wherein the base sequence encoding the protein is the base sequence represented by SEQ ID NO: 2 in Sequence Listing.
[9] The method for phosphorylating a nucleoside according to any one of [7] to [8-3], wherein a protein having the following physicochemical properties <1> to <5> is used.
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes;
[9-1] The phosphorylation method according to [9], wherein the phosphate donor is ATP.
[9-2] The phosphorylation method according to any one of [9] to [9-1], wherein the protein has a molecular weight of about 34 kDa as determined by SDS polyacrylamide gel electrophoresis.
[10] The method for phosphorylating a nucleoside according to any one of [7] to [9-2], wherein the protein is derived from Burkholderia thylandensis.
[10-1] The method for phosphorylating a nucleoside according to any one of [7] to [10], wherein the protein is derived from Burkholderia thailandensis DSM13276.
[11] Any of [10] to [10-1], wherein the protein uses at least magnesium ion, cobalt ion, nickel ion or manganese ion and catalyzes a reaction of phosphorylating a nucleoside at least at 37 ° C. The method for phosphorylating a nucleoside according to claim 1.
[11-1] The method for phosphorylating a nucleoside according to any one of [10] to [11], wherein the protein uses at least magnesium ion, cobalt ion, nickel ion or manganese ion.
[11-2] The method for phosphorylating a nucleoside according to any one of [10] to [11-1], wherein the protein catalyzes a reaction of phosphorylating a nucleoside at least at 37 ° C.
[12] A nucleoside phosphorylating agent using the protein according to any one of (1) to (3) below. However, the nucleoside is a nucleoside excluding cytidine and xanthosine.
(1) A protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a nucleoside phosphorylation reaction.
(2) The amino acid sequence of SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence is the amino acid sequence of SEQ ID NO: 5 and the sequence listing SEQ ID NO: A protein comprising 6 amino acid sequences and capable of catalyzing a nucleoside phosphorylation reaction.
(3) A protein having the following physicochemical properties <1> to <5>.
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes;
[12-1] The nucleoside phosphorylating agent according to the above [12], which has at least one of the characteristics according to the above [7-1] to [11-2].
[12-2] Any one of the following proteins (1) or (2), metal ion, ADP-dependent hexokinase, glucose, glucose-6-phosphate dehydrogenase, oxidized NAD (P) s and pH A nucleoside phosphorylating agent comprising a buffer.
(1) A protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a nucleoside phosphorylation reaction.
(2) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and include the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6 A protein comprising an amino acid sequence and capable of catalyzing a phosphorylation reaction of a nucleoside.
[12-3] The nucleoside phosphorylating agent according to [12-2], wherein the nucleoside in (1) or (2) is a nucleoside excluding cytidine and xanthosine.
[13] A method for producing a phosphorylated nucleoside using the protein according to any one of (1) to (3) below, wherein the nucleoside is a corresponding phosphorylated nucleoside. However, the nucleoside is a nucleoside excluding cytidine and xanthosine.
(1) A protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a nucleoside phosphorylation reaction.
(2) The amino acid sequence of SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence is the amino acid sequence of SEQ ID NO: 5 and the sequence listing SEQ ID NO: A protein comprising 6 amino acid sequences and capable of catalyzing a nucleoside phosphorylation reaction.
(3) A protein having the following physicochemical properties <1> to <5>.
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes;
[13-1] A method for producing a phosphorylated nucleoside, wherein the nucleoside according to [13] having at least one of the characteristics according to [7-1] to [11-2] is used as the corresponding phosphorylated nucleoside .
[14] A method for measuring a nucleoside comprising the following steps (a) to (c): However, the nucleoside is a nucleoside excluding cytidine and xanthosine.
(A) a step including a first reaction in which any of the following proteins (1) to (3) and a nucleoside that may be contained in a sample are converted to a phosphorylated nucleoside and ADP in the presence of ATP ,
(1) A protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a nucleoside phosphorylation reaction.
(2) The amino acid sequence of SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence is the amino acid sequence of SEQ ID NO: 5 and the sequence listing SEQ ID NO: A protein comprising 6 amino acid sequences and capable of catalyzing a nucleoside phosphorylation reaction.
(3) A protein having the following physicochemical properties <1> to <5>.
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes;
(B) In the presence of a second reaction or a plurality of second reactions different from the first reaction, and a second enzyme or a second plurality of enzymes that catalyzes the reaction, the first reaction A step of causing a change in an object to be measured by a second reaction according to the amount of ADP generated by the reaction;
(C) detecting the amount of change in the measurement target, and measuring the amount of nucleoside that may be contained in the sample;
[14-1] The method for measuring a nucleoside according to [14], wherein the nucleoside in (1) or (2) is a nucleoside excluding cytidine and xanthosine.
[14-2] The method for measuring a nucleoside according to [14], wherein the nucleoside is at least one of adenosine, thymidine, uridine, inosine, or guanosine.
[14-3] The method for measuring a nucleoside according to any one of [14] to [14-2], wherein the phosphorylated nucleoside is a monophosphorylated nucleoside.
[14-4] Any one of [14] to [14-3], wherein the protein in the step (a) comprises a protein having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and capable of catalyzing a nucleoside phosphorylation reaction. A method for measuring a nucleoside according to claim 1.
[14-5] The protein in step (a) is a protein comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 are deleted, substituted or added [14]-[ 14-4] The measuring method of the nucleoside in any one of.
[14-6] The protein in step (a) comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing. A protein that can catalyze a phosphorylation reaction of a nucleoside, including the amino acid sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6 (wherein Xaa represents any amino acid in SEQ ID NO: 5 and SEQ ID NO: 6) [14] The method for measuring a nucleoside according to any one of [14-5].
[14-7] The measurement of the nucleoside according to any one of [14] to [14-6], wherein the protein in step (a) uses a protein having the following physicochemical properties <1> to <5> Method.
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes;
[14-8] The method for measuring a nucleoside according to any one of [14] to [14-7], wherein the protein in step (a) is derived from Burkholderia thailandensis.
[14-9] The method for measuring a nucleoside according to any one of [14] to [14-8], wherein the protein in the step (a) is derived from Burkholderia thalidansis DSM13276 strain.
[14-10] The method for measuring a nucleoside according to any one of [14] to [14-9], wherein the protein in step (a) uses at least magnesium ion, cobalt ion, nickel ion or manganese ion.
[14-11] The method for measuring a nucleoside according to any one of [14] to [14-10], wherein the protein in step (a) catalyzes a reaction of phosphorylating a nucleoside at least at 37 ° C.
[14-12] In the step (b), (b-1) in the presence of ADP-dependent hexokinase, the second reaction is a reaction in which glucose and ADP are converted into glucose-6-phosphate and AMP; 2) In the presence of glucose-6-phosphate dehydrogenase, oxidized NAD (P) and glucose-6-phosphate produced by the above reaction are converted into reduced NAD (P) and glucnolactone-6-phosphorus. The reduced NAD (P) s are increased in accordance with the amount of ADP generated by the first reaction, including the reaction of acidification. In step (c), the increased amount of reduced NAD (P) s The method for measuring a nucleoside according to any one of [14] to [14-11], wherein
[14-13] In the step (b), (b-1) a reaction of changing PEP and ADP to pyruvate and ATP in the presence of pyruvate kinase, and (b-2) in the presence of lactate dehydrogenase, The reduced NAD (P) s and pyruvic acid produced by the above reaction include a reaction in which oxidized NAD (P) s and lactic acid are converted into lactic acid, and reduced NAD (P) depending on the amount of ADP produced by the first reaction. ) Is reduced, and in step (c), the amount of reduced NAD (P) is detected, and the nucleoside measurement according to any one of [14] to [14-12] Method.
[14-14] In the step (b), (b-1) a reaction of changing PEP and ADP to pyruvate and ATP in the presence of pyruvate kinase; and (b-2) oxygen in the presence of pyruvate oxidase. , Including a reaction in which phosphoric acid and pyruvic acid generated by the above reaction are converted into hydrogen peroxide and acetylphosphoric acid, and hydrogen peroxide is increased in accordance with the amount of ADP generated by the first reaction, c) The method for measuring a nucleoside according to any one of [14] to [14-13], wherein in the step, the amount of hydrogen peroxide is detected with a hydrogen peroxide indicator or the like.
[14-15] Detection of the amount of change in step (c) is included in the sample by comparing the amount of change in the measurement target with the known amount of nucleoside after processing in steps (a) and (b). A method for measuring a nucleoside according to any one of [14] to [14-14], which is a step of measuring the amount of nucleoside that may be present.
[14-16] The method for measuring a nucleoside according to any one of [14] to [14-15], wherein the protein has a molecular weight of about 34 kDa by SDS polyacrylamide gel electrophoresis.

  According to the present invention, it is possible to provide a protein that catalyzes a reaction that acts on at least a nucleoside such as adenosine, thymidine, uridine, inosine, or guanosine to produce a corresponding phosphorylated nucleoside.

  The nucleoside of the present invention includes a known nucleoside and is not particularly limited, but is a glycoside consisting of a sugar moiety and a base moiety, and examples of the sugar moiety include D-ribose or 2-deoxy D-ribose. Examples thereof include purine and pyrimidine. Preferably, a glycoside whose sugar moiety is D-ribose and whose base moiety is purine or pyrimidine is exemplified. Examples of purines in the present invention include basic heterocyclic nitrogen-containing compounds, such as adenine and guanine, and in some cases, derivatives thereof may be used. In the present invention, examples of the pyrimidine include basic heterocyclic nitrogen-containing compounds such as cytosine, uracil, and thymine. In some cases, derivatives thereof may be used. That is, specific nucleosides in the present invention are not particularly limited as long as they are nucleosides except cytidine and xanthosine, but examples include adenosine, thymidine, uridine, inosine, and guanosine, and these nucleoside derivatives or similar. And in the present invention may be a mixture of these nucleosides. In the present invention, any or all of adenosine, inosine, and guanosine are particularly preferable, and a mixture of these nucleosides is also preferable.

Examples of the protein of the present invention include a protein consisting of the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a nucleoside phosphorylation reaction. In addition, a protein comprising an amino acid sequence substantially equivalent to the amino acid sequence of SEQ ID NO: 1 in the sequence listing, an amino acid sequence in which some amino acids not involved in catalysis are mutated, or various amino acid residues are added. Illustrated. That is, the protein of the present invention comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence is the amino acid of SEQ ID NO: 5 A protein that can catalyze a phosphorylation reaction of a nucleoside, which includes the amino acid sequence of the sequence and SEQ ID NO: 6 (wherein Xaa represents any amino acid in SEQ ID NOs: 5 and 6) can be used. For example, examples include adding a functional protein such as thioredoxin protein or other amino acid sequence to the N-terminal side and / or C-terminal side of the protein of the present invention. Examples include a case where a portion called a tag that can be purified or confirmed by the portion is fused, and in some cases, even if the tag portion is deleted, all or a part of the portion remains. For example, about 20 signal peptides for transporting the protein of the present invention to the outside of the cell or periplasm, or 5 to 10 His for efficient purification may be added, or they may be added in series. It may be added. Further, several protease recognition amino acid sequences can be arranged and added between these amino acid sequences. Similar to the addition examples described above, deletions or substitutions can be made. For example, when there is a domain consisting of several amino acids unrelated to the essential function of the protein of the present invention, or a sequence listing When there is a gap consisting of a plurality of amino acids in the amino acid sequence of SEQ ID NO: 1, these deletions can be combined. It is also possible to combine deletion, substitution or addition as appropriate.
Such amino acid sequences include RREFGGCA (G, A, or T) NI (A, or G) (Y, or F) (A, S, T, or N) L, and DPTG (C, A, V Or I) GDA (F, Y, W, or H) R (G, A, or S) It is more preferable if it is a protein that can catalyze a phosphorylation reaction of a nucleoside. Furthermore, in the amino acid sequence of SEQ ID NO: 1 of the present invention, the N-terminal side and the C-terminal side of the amino acid sequence of SEQ ID NO: 1 contain amino acid residues or polypeptide residues, that is, are added. The additional amino acid residue may be a signal peptide, a TEE sequence, an S tag, or a His tag. When deleting the amino acid of sequence number 1 of a sequence table, the example deleted in order from Met of N terminal side or Lys of C terminal side is mentioned, for example. In the amino acid sequence of SEQ ID NO: 1 in the sequence listing, a protein that has been post-translationally modified such as deletion of Met at the N-terminal or modification of the N-terminal with an acyl group or an alkyl group is also a protein of the present invention. In addition, the protein of the present invention can be chemically modified with succinic anhydride, PEG, or the like by a known method so that the properties of the protein of the present invention such as optimum pH and stability can be easily changed. .

  When the molecular weight of the protein of the present invention changes in the amino acid sequence of SEQ ID NO: 1 when one or more amino acids are deleted, substituted or added, post-translationally modified, or chemically modified For example, when a His tag is added to the N-terminus using pTip QC1 or pTip QC2, which will be described later, the molecular weight may increase by about 1,000.

In addition, the protein of the present invention is preferably a protein having any of the following physicochemical properties and other properties described in the present specification. That is, it preferably has <1> action and <2> substrate specificity of the following physical properties, and the presence or absence of other properties is not particularly limited, but any of the following physicochemical properties and the properties described in the present specification can be used. It is also preferred to have one or more. Particularly preferably, proteins having the following physicochemical properties <1> to <5> are exemplified.
Physicochemical property <1> Action It catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor. Preferably, it is as the following [Formula 1].

[Formula 1]
Nucleoside + ATP-> Phosphorylated nucleoside + ADP

<2> Substrate specificity The protein of the present invention acts on adenosine, inosine, and guanosine. Although the protein of the present invention does not substantially act on cytidine, xanthosine, and ribose, it is usually preferable that the protein does not react under the conditions of Measurement Method 3 in the protein activity measurement method of the present invention. It is also preferable that the reaction does not occur even if the degree of dilution is changed or the concentration of each composition in the reaction reagent mixture is changed.
<3> Optimum pH
The optimum pH at which the protein of the present invention exhibits the maximum activity is in the range of pH 5.5 to 7.5.
<4> pH stability The protein of the present invention retains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours.
<5> Thermal stability The protein of the present invention retains an activity of 80% or more in a 100 mM potassium phosphate buffer pH 7.0 aqueous solution by heat treatment at 50 ° C. for 20 minutes.

  The protein of the present invention can catalyze a nucleoside phosphorylation reaction as described in the above <1> action. In the phosphorylation reaction of the nucleoside, it is preferably at least in the presence of a phosphate donor. The phosphate donor may be CTP, GTP, TTP, polyphosphoric acid or the like, but ATP is particularly desirable. . In the phosphorylation, a phosphorylated nucleoside corresponding to the nucleoside is produced, and the phosphorylated nucleoside is a substance in which a phosphate group is bonded to the nucleoside. The number of phosphate groups may be 1, 2, or 3, but 1, i.e., phosphorylated mononucleoside is preferred. The site that can be phosphorylated is not particularly limited, and examples thereof include 1′-position, 2′-position, and 5′-position, but a ring may be formed. For example, in the case of phosphorylated mononucleoside, 5′-position Is preferred. That is, the phosphorylated nucleoside is preferably a nucleoside 5 'monophosphate. The phosphorylated nucleoside is sometimes called a nucleotide.

  In preparing the protein of the present invention, a step of forming the protein based on the base sequence encoding the protein can be used. The step includes a step of using a cell-free protein synthesis system including a base sequence encoding the protein, preferably a cell including a base sequence encoding the protein, or a microorganism that forms the natural protein and the like. Examples include a process using a microorganism having a base sequence to be used, a process using a transformant into which a base sequence encoding the protein is introduced, and the like.

  A typical method for preparing the protein of the present invention includes an example in which the protein is formed using a natural microorganism that forms the protein. However, a natural microorganism that can prepare the protein of the present invention is preferably used. Burkholderia genus and related Xanthomonas genus, etc., more preferably Burkholderia thaliandensis, and most preferably Burkholderia thaliandensis DSM13276. The Burkholderia thailandensis DSM13276 strain can also be purchased from Deutsche Sammlung von Microorganismen und Zellkulturen GmbH. In addition, the equivalent strains that can be purchased in other strain banks, Epidemiol. Infect. 118, 137-148, using the equivalent strain isolated by the method described in 1995, it is confirmed that the protein having each property of the present invention can be produced and used in the above steps. it can.

  The natural microorganism that forms the protein may be a mutant that has been further treated with a drug such as NTG, ultraviolet light, and / or radiation. The mutant strain can improve the productivity of the protein of the present invention and can form a mutant of the protein of the present invention, and has excellent properties such as stability, productivity, and reactivity. It is also possible to form a body.

  In the above-described production method for preparing the protein, if a base sequence encoding the protein is required, (1) the amino acid sequence of SEQ ID NO: 1 and (2) the amino acid of SEQ ID NO: 1 The sequence comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence includes the amino acid sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6. A base sequence encoding a protein that can catalyze an oxidation reaction or (3) a protein having the physicochemical properties <1> to <5> above may be used.

  The base sequence encoding the amino acid sequence of SEQ ID NO: 1 is not particularly limited. For example, the base sequence of SEQ ID NO: 2 or the base sequence of SEQ ID NO: 2 is used as a codon of a host such as Escherichia coli or actinomycetes. The base sequence changed according to the frequency is illustrated. In the above (2), a base sequence encoding an amino acid sequence substantially equivalent to SEQ ID NO: 1 may be used as long as it can catalyze a nucleoside phosphorylation reaction. SEQ ID NO: 1 amino acid sequence obtained by mutating some amino acids not involved in the catalytic action, for example, a base encoding an equivalent of an amino acid sequence in which one or more amino acids are deleted, substituted or added It may be an array. It is particularly preferable that the nucleotide sequence encoding each amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 6 is included.

  In the preparation of the base sequence encoding the protein, commonly used known gene manipulation means can be used. For example, a site-specific mutagenesis method or a fragment of a specific base of a target gene is replaced with an artificial mutant base. Various methods such as these are exemplified.

  When a cell-free protein synthesis system including a base sequence encoding the protein is employed as the step of forming the protein of the present invention, the base having the above base sequence is selected from wheat germ, Escherichia coli, and rabbit reticulocyte. What is necessary is just to use for well-known systems, such as origin or insect cell origin.

In addition, when adopting a process using a cell containing a base sequence encoding the protein, a transformant is prepared by inserting the above base sequence into a vector and introducing it into a host microorganism. The step of forming the protein by using is exemplified.
The transformant introduced with the base sequence encoding the protein of the present invention includes cells or microorganisms into which a recombinant phage or recombinant plasmid, which is a vector having the base sequence inserted, is introduced into a host. The base sequence encoding the protein of the present invention can be used by synthesizing a part or all of the base sequence. Preferably, the base sequence encoding the protein of the present invention is obtained from a gene donor. The gene donor is not limited as long as it is a cell that forms a protein capable of catalyzing the phosphorylation reaction of a nucleoside, and examples thereof include microorganisms including eubacteria, eukaryotes, and archaea, and preferably the present invention described above. And microorganisms that form the protein.

  As a vector for inserting the base sequence encoding the protein of the present invention, a phage or plasmid constructed for gene recombination among phages or plasmids that can autonomously grow in the host microorganism is suitable. For example, when a microorganism belonging to Escherichia coli is used as a host, λgt · λC, λgt · λB, and the like can be used. In addition, as a plasmid vector, for example, when Escherichia coli is used as a host, Novagen's pET vector, or pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18, pUC19, pUC118, pINI, BluescriptKS +, Bacillus subtilis PW1520, pUB110, pKH300PLK, pIJ680 and pIJ702 when actinomycetes are used as hosts, YRp7, pYC1, YEp13 and the like when yeasts, particularly Saccharomyces cerevisiae are used as hosts. In the present invention, a plasmid vector using E. coli as a host is preferable. The promoter is not particularly limited as long as it can be expressed in the host.

A vector fragment is prepared by cleaving such a vector with a restriction enzyme that produces the same end as the end of the base sequence generated by the restriction enzyme used to cleave the base sequence encoding the protein of the present invention. A fragment of the base sequence encoding the protein of the above and a vector fragment are ligated by DNA ligase according to a conventional method, and the base sequence encoding the protein of the present invention is inserted into the target vector, and a recombinant phage or recombinant plasmid is obtained. Eggplant.
The host into which the recombinant plasmid is introduced may be any cell or microorganism that can stably and autonomously propagate the recombinant plasmid. For example, when the host microorganism is a microorganism belonging to E. coli, E. coli BL21, E. coli DH1, E. coli JM109 Escherichia coli JM101, Escherichia coli W3110, Escherichia coli C600 and the like, Escherichia coli B strain, K strain, C strain and their lysogens can be used. Further, when the host microorganism is a microorganism belonging to the genus Bacillus, Bacillus subtilis, Bacillus megaterium, etc., a microorganism belonging to actinomycetes, Streptomyces lividans TK24, etc., a microorganism belonging to Saccharomyces cerevisiae, Saccharomyces cerevisiae INVSC1 etc. can be used. In the present invention, it is preferable to use Escherichia coli as a host microorganism.

  Another preferred example of the method for forming a protein using a transformant introduced with a base sequence encoding the protein of the present invention is a method for forming a recombinant protein in Rhodococcus bacteria (patent) No. 3,944,577 and Japanese Patent No. 3,793,812). Specifically, a base sequence encoding the protein of the present invention is inserted into a plasmid vector such as pTip QC1 or pTip QC2 suitable for formation of a recombinant protein at low temperature, and the recombinant plasmid is introduced into a lysozyme-sensitive microorganism. A method using the transformant is shown. As the lysozyme-sensitive microorganism, a microorganism belonging to the genus Rhodococcus is preferable (Japanese Patent No. 3876310).

  The protein of the present invention may be formed by culturing a transformant introduced with a base sequence encoding the protein or a microorganism having a base sequence encoding the protein.

  The culture conditions for natural microorganisms that form proteins that can catalyze the phosphorylation reaction of nucleosides and microorganisms for transformants into which a base sequence encoding the protein has been introduced should be determined in consideration of their nutritional physiological properties. It may be selected, and it is usually carried out by liquid culture, but industrially, it is advantageous to carry out deep aeration and agitation culture. As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The culture temperature can be appropriately changed within the range in which the host microorganism grows and the protein of the present invention is formed. In the case of E. coli, the lower limit is 10 ° C. or higher, preferably 20 ° C. or higher, more preferably 25 ° C. or higher, The upper limit is 45 ° C or lower, preferably 42 ° C or lower, more preferably 37 ° C or lower. In the case of actinomycetes, the lower limit is 4 ° C or higher, preferably 10 ° C or higher, more preferably 20 ° C or higher, and the upper limit is 50 ° C or lower, preferably 42 ° C or lower, more preferably 37 ° C or lower. The culture conditions vary slightly depending on the conditions, but the culture may be terminated at an appropriate time in consideration of the time when the protein of the present invention reaches the maximum formation amount. In the case of E. coli, the lower limit is usually 10 hours or more, preferably 12 More than time, more preferably more than 17 hours, upper limit is 60 hours or less, preferably 48 hours or less, more preferably 30 hours or less. In the case of actinomycetes, the lower limit is usually 17 hours or longer, preferably 20 hours or longer, more preferably 24 hours or longer, and the upper limit is 80 hours or shorter, preferably 72 hours or shorter, more preferably 48 hours or shorter. The medium pH can be appropriately changed within the range in which the bacteria grow and form the protein of the present invention. In the case of Escherichia coli and actinomycetes, the lower limit is preferably pH 5.8 or higher, preferably pH 6.2 or higher, and the upper limit is pH 8 0.5 or less, preferably pH 7.5 or less.

  The protein of the present invention can be produced by a method including the step of obtaining the protein of the present invention formed as described above, but it is simply a cell or the like containing cells regardless of whether it is sterilized or non-sterilized. It is also preferable to use a protein that retains impurities from which culture impurities, cell disruptions, etc. have been removed lightly. Furthermore, it is preferable that the crude protein of the protein of the present invention does not substantially contain impurities depending on the purpose and application, but usually, for example, 50% or more, 70% or more, 95% or more of various kinds It is exemplified to make it pure. The purity may be confirmed by a known method such as SDS-PAGE or HPLC.

The method for producing the protein of the present invention will be described.
The protein of the present invention is obtained by culturing a natural microorganism that forms a protein capable of catalyzing a nucleoside phosphorylation reaction, a transformant microorganism into which a base sequence encoding the protein is introduced, and the like, and obtaining the protein from the culture. Can be manufactured. First, the microorganism or the like is cultured in a nutrient medium to form the protein in the microbial cells or in the culture solution. When formed in the microbial cells, the culture obtained is filtered or centrifuged after the completion of the culture. Collect bacterial cells by means. Next, this bacterial cell is destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, EDTA and / or an appropriate surfactant is added to concentrate or concentrate the protein. The protein of the present invention is precipitated and recovered by applying a fractional precipitation method using an organic solvent such as acetone, methanol, ethanol, or a salting out method using ammonium sulfate, sodium chloride, or the like. The precipitate is subjected to dialysis and isoelectric precipitation, if necessary, and then subjected to gel filtration, adsorption chromatography such as affinity chromatography, ion exchange chromatography or hydrophobic chromatography to obtain the protein of the present invention. Obtainable. Moreover, it can carry out combining these methods suitably. In addition, when the protein of the present invention is formed in the culture solution, the cells are removed by means of filtration or centrifugation of the culture, and the culture solution is the same as that formed in the cells. What is necessary is just to process.

  Proteins obtained by these methods can be used as stabilizers in the form of liquid or solid by adding various salts, saccharides, proteins, lipids, surfactants, etc., with or without ultrafiltration concentration, freeze drying, etc. In addition, sucrose, mannitol, sodium chloride, albumin or the like may be added as a stabilizer in an amount of about 0.5 to 10%.

  The protein of the present invention thus obtained can be distinguished from the following conventional enzymes in terms of properties and sequence. For example, 6-phosphofructokinase derived from Aeropyrum pernix (6-Phosphofructokinase) described in Non-Patent Document 1 and phosphofructokinase-B (Phosphofructokintokine derived from Methanocalkococcus jannaschii described in Non-Patent Document 2). The amino acid homology between each of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 1 of the present invention is as low as 21% and 29%, respectively, which are completely different.

  In addition, Aeropyrum pernix has an optimum growth temperature of 90 ° C. and Methanocaldococcus jannaschii has an optimum growth temperature of 85 ° C. It is uneconomical to obtain many cultures at the same time, and since the high temperature requires safety measures, the production method of the present invention requires less such considerations. It is also preferable as a manufacturing method. Furthermore, since it is known that 6-phosphofructokinase derived from Aeropyrum pernix is difficult to produce using a transformant introduced with a base sequence encoding this enzyme, it is difficult to improve the production method. After all, in addition to being different from the protein of the present invention as a substance, the production method of the protein of the present invention is more preferable as the production method.

  Further, the 6-phosphofructokinase and the phosphofructokinase-B can be distinguished from the protein of the present invention in that they hardly show a catalytic action of a reaction of converting a nucleoside to a phosphorylated nucleoside at 40 ° C. In addition, a method for producing a phosphorylated nucleoside using 6-phosphofructokinase and phosphofructokinase-B as a nucleoside and a nucleoside as a phosphorylated nucleoside is a temperature higher than 40 ° C. Since expensive ATP, which is a coenzyme necessary for catalytic reaction, is unstable at high temperatures, it is uneconomical as a production method.

  On the other hand, the base sequence of SEQ ID NO: 2 was clarified by non-patent document 3 by genome-wide analysis of the strain, but the nature of the protein encoded by the base sequence has not been elucidated so far. Was simply expected as a ribokinase. That is, it has not been known at all that a protein comprising the amino acid sequence of SEQ ID NO: 1 encoded by the base of SEQ ID NO: 2 catalyzes a reaction from a nucleoside to a phosphorylated nucleoside.

  In addition, the method for phosphorylating a nucleoside of the present invention is preferably, for example, a method in which a protein of the present invention is used and a nucleoside is converted to a phosphorylated nucleoside at least in the presence of a phosphate donor. More preferably, at least one of metal ions, magnesium ions, cobalt ions, nickel ions or manganese ions, a nucleoside, a phosphate donor, and the protein of the present invention are contacted. The order of contact is not particularly limited.

  The nucleoside phosphorylation method of the present invention may be carried out in the liquid phase, gas phase, solid phase or the like or on each critical surface, but is preferably carried out in the liquid phase. Examples of the liquid phase include an aqueous phase and an organic solvent phase. Examples of the aqueous phase include water and an appropriate aqueous solution, and an appropriate organic solvent such as methanol and ethanol, or an aqueous medium containing these. However, in these, it is preferable to use an appropriate pH buffer. When a pH buffer is used, the type can be maintained at the desired pH and is not particularly limited as long as the nucleoside is a phosphorylated nucleoside, but Good's buffer, Tris / HCl buffer, potassium phosphate buffer And acetic acid / NaOH buffer solution and citric acid / NaOH buffer solution. The pH at the time of carrying out the nucleoside phosphorylation method of the present invention is not particularly limited as long as the nucleoside is converted to a phosphorylated nucleoside, but the lower limit is exemplified by pH 4 or more, preferably pH 5 or more, more preferably pH 6 or more. The pH is 9 or less, preferably 8 or less, more preferably 7 or less. The concentration of the pH buffer is not particularly limited as long as the target pH can be maintained and the phosphorylation proceeds, but the lower limit is 1 mM or more, preferably 5 mM or more, more preferably 10 mM or more, and the upper limit is 500 mM. Hereinafter, it is preferably 300 mM or less, more preferably 200 mM or less.

  Other preferred liquid phases for carrying out the nucleoside phosphorylation method of the present invention include, for example, sol-gel. In order to obtain a sol-gel, for example, a polysaccharide such as agar can be used. When the sol-gel and the emulsion are distinguished, the emulsion may be implemented as an emulsion. In order to obtain an emulsion, for example, an organic solvent or the like can be used, and if an amphiphilic substance is used, micelles can be used. In any case, when a buffer solution is used, it can be carried out in the same manner as described above.

  The temperature at which the nucleoside phosphorylation method of the present invention is carried out is not particularly limited as long as the nucleoside is converted into a phosphorylated nucleoside, but is preferably within the working temperature range of the protein of the present invention, and the lower limit is preferably 15 ° C. or higher. Is 20 ° C or higher, more preferably 30 ° C or higher, and the upper limit is 70 ° C or lower, preferably 50 ° C or lower, more preferably 40 ° C or lower.

  The amount of the protein of the present invention used in the nucleoside phosphorylation method of the present invention, the amount of phosphate donor, the amount of metal ions, and the reaction time are not particularly limited as long as the nucleoside is converted to a phosphorylated nucleoside, The amount of nucleoside contained in the raw material or sample, the degree of phosphorylation of the target nucleoside, the apparatus used, and / or economic circumstances can be determined so as to obtain a desired result.

  The amount of the protein of the present invention used in the nucleoside phosphorylation method of the present invention is, for example, when the abundance of nucleosides contained in the raw material or sample is 1 mM or less, and when phosphorylating all, the lower limit is 1 mU / ml or more, It is preferably 5 mU / ml or more, more preferably 50 mU / ml or more, and the upper limit is 10 U / ml or less, preferably 3 U / ml or less, more preferably 1 U / ml or less. The amount of the phosphate donor used under the same conditions is, for example, when the phosphate donor is ATP, the lower limit is 1 mM or more, preferably 5 mM or more, more preferably 10 mM or more. Hereinafter, it is more preferably 200 mM or less, particularly preferably 50 mM or less. The amount of the metal ion used under the same conditions varies depending on the concentration of the phosphate donor, and the lower limit relative to the concentration of the phosphate donor is 0.1 equivalents or more, preferably 0.5 equivalents or more. The upper limit is preferably 10 equivalents or less, preferably 5 equivalents or less, and more preferably 3 equivalents or less. The most preferred concentration is 2 equivalents of phosphate donor. The lower limit of the reaction time under the same conditions is 15 seconds or more, preferably 1 minute or more, more preferably 3 minutes or more. Although there is no particular upper limit, it is preferably 30 minutes or less, more preferably 15 minutes or less, and particularly preferably 10 minutes or less.

  In the method for producing a phosphorylated nucleoside using the nucleoside of the present invention as a phosphorylated nucleoside, a phosphorylated nucleoside is produced from the nucleoside by the above-described method for phosphorylating a nucleoside of the present invention. The produced phosphorylated nucleoside may remain mixed with raw materials, other products, etc., but it is also preferable not to substantially contain impurities depending on the purpose or application, etc. For example, various purity of 50% or more, 70% or more, or 95% or more is exemplified. The phosphorylated nucleoside can be subjected to a known purification method, for example, extraction using a solvent, chromatography using silica gel, alumina, and / or celite, and / or recrystallization, etc. It can be illustrated.

The protein of the present invention can be a nucleoside phosphorylating agent.
The nucleoside phosphorylating agent of the present invention can be used in a method for converting a nucleoside into a phosphorylated nucleoside or a method for producing a phosphorylated nucleoside using a nucleoside as a phosphorylated nucleoside. This is shown as a preferred example. Further, it is considered that the nucleoside phosphorylating agent of the present invention is also contained in the composition used in the method for measuring a nucleoside of the present invention described later.
In addition to the protein of the present invention, the phosphorylating agent of the present invention includes a phosphate donor, a metal ion, a pH buffer, other enzymes and reagents, etc., but the protein of the present invention, the phosphate donor, and The amount and type of metal ions added are the same as described above.

The nucleoside phosphorylating agent of the present invention can be provided as a liquid product, a frozen product of a liquid product, a lyophilized product of a liquid product, or a dried product of a liquid product (by heat drying and / or air drying and / or reduced pressure drying). . Liquid products, frozen products of liquid products, and freeze-dried products of liquid products are preferred, freeze-dried products of liquid products and liquid products are more preferred, and liquid products are most preferred. In another embodiment, a frozen liquid product may be preferable. As yet another aspect, lyophilization of a liquid product may be preferable. The nucleoside phosphorylating agent of the present invention may be a single reagent phosphorylating agent, but may be separated into two or more reagents as necessary for the purpose of improving the stability of the reagent and improving the measurement accuracy. good. Further, for the purpose of improving the quality of the reagent, a surfactant or preservative may be mixed. For example, when using POC in capillaries or as an enzyme sensor, the concentration of each component is preferably higher than usual. For example, it is fixed, soaked in paper or film, gel sol It is preferable to use it as a composition.
In the case where the nucleoside phosphorylating agent of the present invention is separated into two or more reagents, for example, for the purpose of stabilizing the reagent, the protein of the present invention, the phosphate donor, the pH buffering agent, etc. are lyophilized to first. In this example, separation is performed by using a metal ion, a pH buffering agent, etc. as a liquid product as a second reagent. For example, when the purpose is to improve the measurement accuracy of the reagent, it is assumed that there is an interfering substance that affects the measured value in the sample, and the first reagent includes a reagent for avoiding the influence of the interfering substance. There is an example of separation which comprises the protein of the present invention, a phosphate donor, a metal ion, a pH buffering agent and the like to form a second reagent.

  The method for measuring a nucleoside of the present invention includes the following steps (a) to (c). (A) a step comprising a first reaction for producing phosphorylated nucleoside and ADP from the above-described protein of the present invention and a nucleoside that may be contained in a sample in the presence of ATP; A second reaction or a plurality of second reactions different from the one reaction, and the ADP produced by the first reaction in the presence of the second enzyme or the second plurality of enzymes catalyzing the reaction. (C) detecting the amount of change in the measurement target and measuring the amount of nucleoside that may be contained in the sample. Process. In addition, although each process of said (a)-(c) may be implemented in a respectively different reaction tank (phase), you may implement in the same reaction tank (phase). The steps (a) to (c) may be performed in the order of steps (a), (b), and (c). Steps (a) and (b) are performed at the same time, and then the steps ( c), or first, step (a) and then steps (b) and (c) at the same time, or steps (a), (b) and (c) at the same time Depending on the device, it can be determined to obtain the desired result. Further, the temperature and time of each step (a) to (c) may be uniform or non-uniform.

The composition for measurement used for the measurement of the nucleoside of the present invention contains the phosphorylating agent of the nucleoside of the present invention, and the phosphorylating agent of the present invention changes according to the amount of nucleoside, and the amount of change is measured. Any material may be used as long as it contains a substance constituting another reaction system. Examples of such a composition for measurement include a combination of the protein of the present invention, phosphate donor, metal ion, pyruvate kinase, PEP, pyruvate oxidase, phosphate and hydrogen peroxide indicator. Further, a combination of the protein, phosphate donor, metal ion, pyruvate kinase, PEP, lactate dehydrogenase, and reduced NAD (P) of the present invention, or the protein, phosphate donor, metal ion of the present invention , ADP-dependent hexokinase, glucose, glucose-6-phosphate dehydrogenase, and a combination of oxidized NAD (P).
When the measurement composition of the present invention is separated into two or more reagents, the first reagent includes at least the protein of the present invention, a phosphate donor, a metal ion and a pH buffer, and the second reagent includes at least ADP dependency. An example of separation so as to include sex hexokinase, glucose, glucose-6-phosphate dehydrogenase, oxidized NAD (P) s and a pH buffer, and the first reagent contains at least the protein of the present invention, phosphate Donor, metal ion, reduced NAD (P) s and pH buffer, second reagent is separated to contain at least pyruvate kinase, PEP, lactate dehydrogenase and pH buffer, or The first reagent contains at least the protein of the present invention, phosphate donor, metal ion and pH buffer, and the second reagent contains at least pyruvate kinase, PEP, pyruvate oxidase, phosphate, persulfate. Examples can be mentioned to separate to include a hydrogen indicator and pH buffering agents.
The protein of the present invention, phosphate donor, metal ion, ADP-dependent hexokinase, glucose, glucose-6-phosphate dehydrogenase, oxidized NAD (P) used in the measurement composition used for measuring the nucleoside of the present invention The amount and type of reduced NAD (P), pyruvate kinase, PEP, lactate dehydrogenase, rubinate oxidase, phosphate, hydrogen peroxide indicator and pH buffer are as follows: It is the same as the measuring method.

The reaction times of steps (a), (b), and (c) of the present invention are the same as described above.
The origin of the nucleoside of the present invention is not particularly limited, and examples thereof include plasma, serum, urine, research samples and the like, and plasma, serum, urine, research samples and the like that are expected to contain nucleosides are preferable. Examples of other samples include seawater, natural water, fruit juice, beverages, and waste liquids.

In the step (a) in the method for measuring a nucleoside of the present invention, any one or more metal ions of magnesium ion, cobalt ion, nickel ion or manganese ion may be present.
The temperature in step (a) of the present invention is the same as described above.
In the step (a) of the present invention, it is preferable to use water or an appropriate pH buffer, and the conditions are the same as described above.
The amount of the protein of the present invention, the amount of phosphate donor (ATP), the amount of metal ions and the like in the step (a) of the present invention are the same as described above.

  The step (b) in the nucleoside measurement method of the present invention comprises a second reaction or a plurality of second reactions different from the step (a), and a second enzyme or a plurality of enzymes catalyzing the reactions. In the presence of, the amount of change in the measurement target caused by the second reaction is generated according to the amount of ADP produced by the first reaction.

  As an example of step (b) of the present invention, (b-1) in the presence of ADP-dependent hexokinase, the second reaction generates glucose-6-phosphate and AMP from glucose and ADP; -2) In the presence of glucose-6-phosphate dehydrogenase, oxidized NAD (P) s and glucose-6-phosphate produced by the above reaction are converted into reduced NAD (P) s and glucnolactone-6- Reductive NAD (P) s are increased in accordance with the amount of ADP produced by the first reaction, including the reaction to generate phosphoric acid (in this specification, it may be referred to as step (b (a)). And (c) a step of detecting an increased amount of reduced NAD (P) s.

  The origin and origin of the ADP-dependent hexokinase and glucose-6-phosphate dehydrogenase in the step (b (a)) of the present invention are not particularly limited as long as the nucleoside measurement method of the present invention can be carried out. The concentration of ADP-dependent hexokinase and glucose-6-phosphate dehydrogenase in the step (b (a)) is not particularly limited as long as the nucleoside measurement method of the present invention can be carried out, but the lower limit is 0.1 U / ml or more, Preferably it is 1 U / ml or more, more preferably 2 U / ml or more, and there is no particular upper limit, but it is preferably 20 U / ml or less, more preferably 10 U / ml or less. In the step (b (a)), a substrate or metal ion required for the reaction of the ADP-dependent hexokinase may be present. The type and concentration of the substrate or metal ion are not particularly limited as long as the nucleoside measurement method of the present invention can be carried out, but varies depending on the origin and origin of the ADP-dependent hexokinase used. As an example, glucose as a substrate is 0.01 mM or more, preferably 0.1 mM or more, more preferably 1 mM or more, and there is no particular upper limit, but preferably 500 mM or less, more preferably 100 mM or less. Magnesium chloride is used as a metal ion in an amount of 0.1 mM or more, preferably 1 mM or more, more preferably 5 mM or more, and there is no particular upper limit, but it is preferably 100 mM or less, more preferably 10 mM or less. In the step (b (a)), oxidized NAD (P) s which are coenzymes of glucose-6-phosphate dehydrogenase may be present. The kind (oxidized thio-NAD (P), oxidized deamide NAD (P), etc.) and concentration of oxidized NAD (P) are not particularly limited as long as the nucleoside measurement method of the present invention can be carried out. It depends on the origin and origin of 6-phosphate dehydrogenase. As an example, oxidized NAD (P) as a coenzyme is 0.1 mM or more, preferably 1 mM or more, more preferably 3 mM or more, and no upper limit is provided, but preferably 50 mM or less, more preferably 10 mM or less. . Further, DI (reduced NAD (P) oxidase) or formazan dye may be present in the step (b (a)) for the purpose of improving the sensitivity of the reagent. Furthermore, NAD (P) cycling reaction can also be used for the purpose of improving sensitivity.

  As an example of the step (b) of the present invention, (b-1) a reaction of changing PEP to pyruvate in the presence of pyruvate kinase, and (b-2) reduced NAD (in the presence of lactate dehydrogenase) P) and pyruvic acid generated by the above reaction include a reaction that generates oxidized lactic acid (P) and lactic acid, and reduced NAD (P) is generated depending on the amount of ADP generated by the first reaction. (It may be referred to as step (b (b)) in this specification), and in step (c), a step of detecting the reduced amount of reduced NAD (P) s may be mentioned.

  The origin and origin of pyruvate kinase and lactate dehydrogenase in the step (b (i)) of the present invention are not particularly limited as long as the nucleoside measurement method of the present invention can be carried out. The concentration of pyruvate kinase and lactate dehydrogenase in the step (b (i)) is not particularly limited as long as the nucleoside measurement method of the present invention can be carried out, but the lower limit is 1 U / ml or more, preferably 5 U / ml or more, Preferably, it is 10 U / ml or more, and no upper limit is provided. However, pyruvic kinase is required for the reaction in the step (b (b)), preferably 100 U / ml or less, more preferably 50 U / ml or less. Substrate, metal ions may be present. The type and concentration of the substrate or metal ion are not particularly limited as long as the nucleoside measurement method of the present invention can be carried out, but varies depending on the origin and origin of the pyruvate kinase to be used. As an example, as a substrate, PEP is 0.1 mM or more, preferably 0.5 mM or more, more preferably 1 mM or more, and an upper limit is not particularly provided, but preferably 50 mM or less, more preferably 10 mM or less. As the metal ion, magnesium chloride is 0.1 mM or more, preferably 0.5 mM or more, more preferably 1 mM or more, and an upper limit is not particularly provided, but preferably 50 mM or less, more preferably 10 mM or less. In the step (b (b)), reduced NAD (P) s that are coenzymes for lactate dehydrogenase may be present. The type and concentration of reduced NAD (P) s (thio NAD (P) H, deamide NAD (P) H, etc.) and concentration are not particularly limited as long as the nucleoside measurement method of the present invention can be carried out, but lactate dehydrogenase to be used It differs depending on the origin and origin, and also depends on the optical path length of the cuvette used and the performance of the colorimeter. As an example, in the case of a cuvette having an optical path length of 1 cm, 0.01 mM or more, preferably 0.03 mM or more, more preferably 0.07 mM or more, and the upper limit is 0.2 mM or less, preferably 0.17 mM or less, more preferably 0.8. 15 mM or less is exemplified.

  As an example of the step (b) of the present invention, (b-1) a reaction of changing PEP to pyruvate in the presence of pyruvate kinase, and (b-2) oxygen, phosphate and The pyruvic acid generated by the reaction of (b-1) includes a reaction of generating hydrogen peroxide and acetyl phosphoric acid, and the hydrogen peroxide increases according to the amount of ADP generated by the first reaction ( In the present specification, there may be mentioned a step of detecting the amount of hydrogen peroxide with a hydrogen peroxide indicator or the like in the steps (sometimes referred to as b (c)) and (c).

The origin and origin of pyruvate kinase and pyruvate oxidase in the step (b (c)) are not particularly limited as long as the nucleoside measurement method of the present invention can be carried out. The concentrations of pyruvate kinase and pyruvate oxidase in the step (b (c)) are the same as in the case of pyruvate kinase and lactate dehydrogenase described above. The types and concentrations of the substrate and metal ions required for the reaction of pyruvate kinase in the step (b (c)) are the same as described above. The hydrogen peroxide indicator in the step (b (c)) is, together with peroxidase, a coupler such as 4-aminoantipyrine or 3-methyl-2-benzothiazoline hydrazone and a phenol such as chlorophenol or TOOS or N, N-dimethyl. Those skilled in the art can easily conceive such as a condensation reaction with chromogens such as anilines such as aniline and their derivatives, and the use of leuco chromogens. Further, when measuring the consumed oxygen, an oxygen electrode is used.
The temperature in the step (b) of the present invention (including the above steps (b (a)) to (b (c))) is preferably within the working temperature range of the enzyme used in the step (b), and the step (a) The same temperature may be used.

The step (b) of the present invention is not particularly limited as long as the enzyme used in the step (b) can be used, but it is preferable to use water or an appropriate pH buffer, even if the pH is the same as in the step (a). good.
In the method for detecting a change amount of the measurement target in the step (c) in the nucleoside measurement method of the present invention, the change amount of the measurement target is detected by a known method. In general, a method of visually detecting a change in absorption spectrum or light absorption intensity of the measurement object accompanying the change of the measurement object or a method of detecting by an optical method using an apparatus is exemplified. NAD (P ), A method for detecting a change in absorption intensity at a specific wavelength or a change in absorption intensity at a specific wavelength, or a change in absorption spectrum at a specific wavelength or a change in absorption intensity at a specific wavelength may be detected. It is also possible to detect the fluorescence of reduced NAD (P) using an apparatus.

Examples of the method for measuring the amount of nucleoside in the sample in the step (c) of the present invention include a measurement method for measuring or quantifying a nucleoside that may be contained in the sample by qualitative analysis. A measuring method is preferred.
The measurement method for quantification is as follows. A sample that may contain a nucleoside and a sample that contains a nucleoside at a known concentration are individually processed in steps (a) to (b), and the amount of each change is detected in step (c). . The concentration of the nucleoside in the sample that may contain nucleoside is the amount of change detected in step (c) for the sample that may contain nucleoside and the sample that contains nucleoside at a known concentration. Quantification is performed by comparing the amount of change detected in step (c).

EXAMPLES Hereinafter, although this invention is demonstrated based on an Example etc., the scope of the present invention is limited to the following Examples etc. and is not interpreted. The techniques described in accordance with the conventional method include, for example, the method of Maniatis et al. (Maniatis, T., et al. Molecular Cloning. Cold Spring Harbor Laboratory 1982, 1989) and basic experimental methods for proteins and enzymes (revised) 2nd edition, Takeo Horio, 1994 Nankodo), or can be carried out according to the procedures attached to various commercially available enzymes and kits. In addition, the measurement values shown below can vary depending on the measurement conditions and the accuracy of the equipment used.
The reagents used in the present invention are those manufactured by Wako Pure Chemical Industries, Ltd., Sigma-Aldrich, Takara Bio, etc. unless otherwise specified, and those that are readily available in the market may be used. it can. It goes without saying that the manufacturer and purity of the reagent are not particularly limited.
When measuring the activity amount of the protein of the present invention, the following measurement method 1, measurement method 2, or measurement method 3 is used according to the purpose.
Since measurement method 1 has a small number of reaction steps, an accurate activity value can be easily measured. However, since measurement method 1 measures absorbance at 340 nm, it is easily subject to limitations of the measurement apparatus. Since measurement method 2 is a method of measuring absorbance at 550 nm, it is difficult to be restricted by the measurement apparatus. However, since the dye is sparingly water-soluble, care must be taken when measuring an accurate activity value. Whereas measurement method 1 and measurement method 2 are measurements using inosine as a substrate, measurement method 3 can be measured using any substance as a substrate, but it is a method for measuring absorbance at 340 nm, so the limitations of the measurement apparatus Because it is easy to receive and there are many reagents used, care must be taken when measuring an accurate activity value.
In measurement method 1 and measurement method 2, IMPDH refers to inosine 5 ′ monophosphate dehydrogenase. If IMPDH is not commercially available, S.I. I. Kim et al., Biosci. Biotechnol. Biochem. 64, 1210-1216, 2000, and the like.
In addition, in this specification, when it is necessary to specify the amount of protein activity, all are expressed as the amount of activity in Measurement Method 3 unless otherwise specified. In the conversion, the same specimen can be measured by each measuring method and converted from the ratio of the change amount of the value. The ratio of the amount of change may vary depending on the measurement conditions and the accuracy of the equipment used, but measurement method 1: measurement method 2: measurement method 3 = 3.0: 5.5: 0.7 is exemplified. The

[Measurement method 1]
In a 1.0 cm cuvette made of quartz, 1.0 ml of the reaction reagent mixture 1 is weighed and preheated at 37 ° C. for 2 minutes. 0.05 ml of the protein solution of the present invention diluted to an appropriate concentration with 30 mM potassium phosphate buffer (pH 7.0) is added and mixed, and the reaction is started at 37 ° C. Five minutes later, 1.0 ml of the reaction reagent mixture 2 is mixed, and the absorbance at 340 nm is measured to determine the change in absorbance. The enzyme activity (U / ml) is calculated by the following [Equation 2], where As / min is the determined change in absorbance and Ab / min is a blind test using purified water instead of the protein solution of the present invention.

[Formula 2]
Enzyme activity (U / ml) = {(As / min−Ab / min) /6.22} × 2.05 / 0.05 × dilution factor

[Reaction reagent mixture 1]
30 mM potassium phosphate buffer, pH 7.0
10 mM ATP (pH 7)
20 mM magnesium chloride 5 mM inosine 50 mM potassium chloride [reaction reagent mixture 2]
100 mM Tris / HCl buffer pH 9.0
13U / ml IMPDH
20 mM NAD
50 mM potassium chloride 1 mM DTT
200 mM EDTA (pH 9)

[Measurement method 2]
Weigh 1.0 ml of the reaction reagent mixture in a quartz 1.0 cm cuvette and preheat at 37 ° C. for 2 minutes. 0.01 ml of the protein solution of the present invention diluted to an appropriate concentration with 30 mM potassium phosphate buffer (pH 7.0) is added and mixed, and the reaction is started at 37 ° C. Five minutes after the start of the reaction, 2 ml of 0.1N hydrochloric acid is mixed to stop the reaction, and the absorbance at 550 nm is measured to determine the change in absorbance. The enzyme activity (U / ml) is calculated by the following [Equation 3], where As / min is the determined change in absorbance and Ab / min is a blind test using purified water instead of the protein solution of the present invention.

[Formula 3]
Enzyme activity (U / ml) = {(As / min−Ab / min) / 19} × 3.01 / 0.01 × dilution factor

[Reaction reagent mixture]
30 mM Tris / HCl buffer pH 9.0
5 mM ATP (pH 7)
10 mM magnesium chloride 2.5 mM inosine 5 U / ml IMPDH
10 mM NAD
50 mM potassium chloride 5 U / ml DI (Asahi Kasei Pharma Corporation)
0.025% NTB
0.5% TX-100

[Measurement method 3]
Weigh 1.3 ml of the reaction reagent mixture in a quartz 1.0 cm cuvette and preheat at 37 ° C. for 2 minutes. 0.03 ml of the protein solution of the present invention diluted to an appropriate concentration with 20 mM Tris / HCl buffer (pH 7.5) is added and mixed, and the reaction is started at 37 ° C. After starting the reaction, the absorbance at 340 nm is measured to determine the change in absorbance per minute during which the reaction is linear. The enzyme activity (U / ml) is calculated by the following [Equation 4], with the obtained change in absorbance as As / min, blinded using purified water instead of the protein solution of the present invention as Ab / min.

[Formula 4]
Enzyme activity (U / ml) = {(As / min−Ab / min) /6.22} × 1.33 / 0.03 × dilution factor

[Reaction reagent mixture]
20 mM Tris / HCl buffer pH 7.5
20 mM glucose 1 mM ATP (pH 7)
1mM magnesium chloride 1mM NADP
50 mM potassium chloride 5 U / ml glucose 6-phosphate dehydrogenase (Asahi Kasei Pharma Corporation)
5U / ml ADP-dependent HK (manufactured by Asahi Kasei Pharma Corporation)
1 mM inosine

[Example 1]
<Substrate specificity of the protein of the present invention>
The protein of the present invention prepared according to Example 17 described later was used. In the activity measurement method 3 described above, the relative activity of the protein of the present invention was measured using each substrate in Table 1 instead of inosine. Table 1 shows the substrate specificity comparing the relative activities.

[Example 2]
<Optimum pH of the protein of the present invention>
A buffer solution of the protein of the present invention prepared in Example 17, which will be described later, in the reaction reagent mixture 1 of the measuring method 1, pH 4.5 to pH 6.0 is a citric acid / NaOH buffer solution (circle mark in FIG. 1). ), PH 6.0 to pH 7.5 in the range of potassium phosphate buffer (circles in FIG. 1), pH 7.0 to pH 9.0 in the range of Tris / HCl buffer (Δ in FIG. 1), pH 9. 5 was changed to a glycine / NaOH buffer solution (marked with □ in FIG. 1), activity was measured, and the optimum pH was measured. FIG. 1 shows the relative activity (%) with the maximum activity as 100%. The optimum pH at which the protein of the present invention exhibits the maximum activity is in the range of pH 5.5 to 7.5.

[Example 3]
<PH stability of the protein of the present invention>
100 mM of the protein of the present invention prepared in Example 17 described later at a pH of 0.475 mg / ml, pH 4.5 to pH 6.0 in the range of citric acid / NaOH buffer (circle mark in FIG. 1), pH 6 The range of 0.0 to pH 7.5 is potassium phosphate buffer (marked with ● in FIG. 2), the range of pH 7.0 to pH 9.0 is Tris / HCl buffer (marked with Δ in FIG. 2), pH 9.5 to 11 0.0 was diluted in glycine / NaOH buffer (marked in FIG. 2) and heated at 37 ° C. for 3 hours, and then the residual activity of the protein of the present invention was measured by Method 1. As shown in FIG. 2, the protein of the present invention retained at least 70% activity in the range of pH 6 to 10 at 37 ° C. for 3 hours.

[Example 4]
<Thermal stability of the protein of the present invention>
The protein of the present invention prepared in Example 17, which will be described later, was diluted in 100 mM potassium phosphate buffer pH 7.0 to 0.475 mg / ml and heat-treated at each temperature for 20 minutes. Residual activity was measured by Measurement Method 1. As shown in FIG. 3, the protein of the present invention retained an activity of 80% or more in a 100 mM potassium phosphate buffer pH 7.0 aqueous solution by heat treatment at 50 ° C. for 20 minutes.

[Example 5]
<Molecular weight of the protein of the present invention>
As shown in FIG. 4, the molecular weight of the protein of the present invention prepared in Example 17, which will be described later, by SDS polyacrylamide gel electrophoresis was about 34 kDa. The calculated value from the amino acid sequence of SEQ ID NO: 1 was 33,994.

[Example 6]
<Working temperature range of the protein of the present invention>
The reaction rate of phosphorylating inosine of the protein of the present invention was measured by measuring the activity of the protein of the present invention prepared in Example 17, which will be described later, by changing the reaction temperature from 15 to 70 ° C. FIG. 5). The results are shown as Arrhenius plots in FIG. As shown in FIG. 5, the working temperature range was at least 15-70 ° C. Moreover, from FIG. 6, the activation energy between 20-45 degreeC was about 64 kJ / mol. In FIG. 6, T represents an absolute temperature. In FIG. 5, the relative activity does not seem to increase at 50 ° C. or higher, but this is considered to be the result of the stability of the protein of the present invention in the reaction solution or the decomposition of ATP.

[Example 7]
<Utility of metal of protein of the present invention>
In the following [mixture] using the protein of the present invention prepared in Example 17 to be described later, the rate at which AMP was produced by the protein of the present invention when the metals shown in Table 2 were used was measured, and the results were obtained. The relative ratio is shown in Table 2.
[Mixture] Adenosine was added to 1 ml to a final concentration of 1 mM, and adenosine phosphorylation was performed at 37 ° C. for 30 minutes. The [mixture] after the adenosine phosphorylation method was diluted 10-fold with purified water, and 25 μl was separated by HPLC. Asahipak GS-320 manufactured by Showa Denko KK was used for HPLC, the mobile phase was 200 mM sodium phosphate buffer pH 3.0, the flow rate was 0.5 ml / min, and the reaction was carried out at room temperature. The amount of AMP produced was absorbed at 260 nm. Measured with
[blend]
50 mM Tris / HCl buffer pH 7.5
2.5 mM ATP
5 mM Metal compound shown in Table 2 5 mU / ml Protein of the present invention prepared according to Example 17

  The action of the protein of the present invention acting on ATP and nucleoside to catalyze the reaction to produce ADP and phosphorylated nucleoside uses at least one of magnesium ion, cobalt ion, nickel ion and manganese ion. I understood it.

[Example 8]
<Protein concentration measurement>
The protein concentration of the protein of the present invention was measured according to the method described in the instruction manual using a protein assay kit manufactured by Bio-Rad, and calculated using BSA as a standard. The specific activity of the protein of the present invention can vary depending on the purity of the protein, the measurement conditions, the precision of the equipment used, etc., but in the case of the protein of the present invention prepared in Example 17 described later, it is about 0. It was 5 U / mg.

[Example 9]
<Vmax and Km, part 1>
The substrate concentration of the protein of the present invention prepared in Example 17, which will be described later, was adjusted to 5 or more different concentrations between 1/10 and 10 times the Km value by the activity measurement method of Measurement Method 3. Each apparent Km value was calculated by a line Weber-Burk reciprocal plot. The results are shown in Table 3. Vmax and Km in Table 3 are apparent Vmax and Km under the conditions of Measurement Method 3.

[Example 10]
<Vmax and Km, part 2>
The inosine concentration and NAD concentration of the protein of the present invention prepared in Example 17, which will be described later, in the activity measurement method of Measurement Method 1 so that the concentration is 5 or more different between 1/10 times and 10 times the Km value. Was prepared, and the Km value for inosine was calculated by a line Weber-Burk reciprocal plot. As a result, Vmax = 1.59 μmol / min / mg and Km = 0.18 μmol / min / mg.

[Example 11]
<Extraction of DNA>
A 1 mg / ml lysozyme solution containing 50 mM Tris / HCl buffer (pH 8.0), 50 mM EDTA, 15% sucrose, and cells of Burkholderia thalidansis DSM13276 collected by culturing using LB medium at 30 ° C. for 2 days After treatment at 37 ° C. for 10 minutes, SDS was added to a final concentration of 0.25% to dissolve the cells. Further, an equal amount of a 1: 1 mixture of phenol / chloroform was added and stirred for 30 minutes, and then centrifuged (12,000 rpm, 15 minutes) to recover the aqueous layer. A 1/10 volume of 3M sodium acetate (pH 5.5) was mixed with the recovered aqueous layer, and then twice the amount of ethanol was gently overlaid, and the genomic DNA was wrapped around a glass rod and separated. The separated genomic DNA is dissolved in 10 mM Tris / HCl buffer (pH 8.0), 1 mM EDTA aqueous solution (hereinafter sometimes referred to as TE), an appropriate amount of RNase A is added, and the mixture is incubated at 37 ° C. for 1 hour and mixed. RNA was degraded. Next, an equal amount of a 1: 1 mixture of phenol / chloroform was added and treated in the same manner as above to separate the aqueous layer. One-tenth volume of 3M sodium acetate (pH 5.5) and twice the volume of ethanol were added to the collected aqueous layer, and genomic DNA was separated once again by the method described above. This chromosome was dissolved in TE, a 1: 1 mixture of TE-saturated phenol and chloroform was added to suspend the whole, and the same centrifugation was repeated, and the upper layer was again transferred to another container. To the separated upper layer, 3M sodium acetate buffer (pH 5.5) and ethanol were added, stirred, cooled at -70 ° C for 5 minutes, centrifuged (2,000 G, 4 ° C, 15 minutes), and precipitated. The chromosomes were washed with 75% ethanol and dried under reduced pressure. The DNA preparation of Burkholderia thailandensis DSM13276 strain was obtained by the above operation.

[Example 12]
<Amplification of gene shown in SEQ ID NO: 2 by PCR method>
Sequence Listing SEQ ID NO: 3 and Sequence Listing Sequence so that the gene of Sequence Listing SEQ ID NO: 2 is inserted into the multiple cloning site NdeI and BamHI sites of pTip QC1 or pTip QC2 (Patent No. 3944777 and Patent No. 3793812) The primer number 4 was designed. For PCR, KOD DNA polymerase (manufactured by Toyobo) was used. PCR conditions followed the method described in the instruction manual. The obtained PCR product of about 1.0 kbp was purified according to a conventional method such as using QIAGEN QIAquick PCR Purification Kit.

[Example 13]
<Ligation with expression vector>
The PCR product purified in Example 12 was digested with NdeI and BamHI according to a conventional method. This insert was purified according to a conventional method. The insert was ligated with pTip QC1 or pTip QC2 purified by restriction enzyme treatment with NdeI and BamHI according to a conventional method to prepare pTip QC1 / BthNK and pTip QC2 / BthNK. pTip QC1 / BthNK and pTip QC2 / BthNK were transformed into E. coli DH5α according to a conventional method, and the recombinant plasmid purified from a positive clone by colony direct PCR was confirmed to have the correct insert sequence by DNA sequencing.

[Example 14]
<Transformation of pTip QC1 / BthNK and pTip QC2 / BthNK to Rhodococcus erythropolis>
Transformation of pTip QC1 / BthNK and pTip QC2 / BthNK into Rhodococcus erythropolis was performed by electroporation (electroporation) according to the method described in JP-A-2004-073116. The electroporation conditions were as follows. The competent cell was melted and mixed with pTip QC1 / BthNK or pTip QC2 / BthNK, placed in a 2 mm dedicated chamber and pulsed with 7.5 mS, 2500 V, 25 μF, 400Ω. Thereby, three strains of pTip QC1 / BthNK and pTip QC2 / BthNK were obtained, respectively.

[Example 15]
<Expression check>
Three strains of each of the transformants obtained in Example 14 were inoculated into an LB medium containing 34 μg / ml chloramphenicol. The seed was cultured at 30 ° C. for 2 days. 1/10 amount of the above seed was inoculated in LB medium containing 34 μg / ml chloramphenicol and 1 μg / ml thiostrepton, and cultured at 30 ° C. for 1 day. The culture broth is collected by centrifugation, suspended in 20 mM Tris / HCl buffer (pH 7.5), 1/5 volume of the culture broth, sonicated, centrifuged, and the resulting supernatant Was used as a crude protein solution. SDS-PAGE of the crude protein solution is shown in FIG. This confirmed the mass expression of the protein of the present invention. The arrow in FIG. 7 (A) is the protein of the present invention expressed in the pTip QC1 / BthNK and the arrow in FIG. 7 (B) is the pTip QC2 / BthNK transformant. Similar results were obtained for all three strains of the transformant obtained in Example 14.

[Example 16]
<Purification of the protein of the present invention from pTip QC1 / BthNK transformant>
The crude protein solution obtained by culturing the pTip QC1 / BthNK transformant in Example 15 was added with 50 mM phosphate buffer (pH 8.0) containing 0.1 M nickel chloride and 0.3 M sodium chloride. It was made to adsorb | suck to Equilibrated Charging Sepharose FF (made by GE Healthcare Bioscience). After thoroughly washing with 50 mM phosphate buffer (pH 8.0) containing 0.3 M sodium chloride, 50 mM phosphate buffer (pH 6.0) containing 10% glycerol and 0.3 M sodium chloride. Washed thoroughly. Elution was performed with 50 mM phosphate buffer (pH 6.0) containing 0.4 M imidazole, 10% glycerol and 0.3 M sodium chloride. The active fraction was dialyzed against 30 mM phosphate buffer (pH 7.0) containing 0.03 M sodium chloride, 0.05 M potassium chloride and 1 mM DTT to obtain the purified protein of the present invention. About 80 mg of purified protein was obtained in 1 L of culture.

[Example 17]
<Purification of the protein of the present invention from pTip QC2 / BthNK transformant>
The crude protein solution obtained by culturing the pTip QC2 / BthNK transformant in Example 15 was equilibrated with 10 mM Tris / HCl buffer (pH 8.0). It was adsorbed on BB (manufactured by GE Healthcare Bioscience). After thorough washing with 10 mM Tris / HCl buffer (pH 8.0), elution was performed with a linear gradient using 10 mM Tris / HCl buffer (pH 8.0) containing 0 and 0.5 M potassium chloride. did. Phenyl sep. Equilibrated with 10 mM potassium phosphate buffer pH 7.5 containing 15% ammonium sulfate was added to the active fraction to a final concentration of 15%. It was adsorbed on FF (manufactured by GE Healthcare Bioscience) and eluted with a linear gradient using 10 mM potassium phosphate buffer (pH 7.5) containing 15 and 0% ammonium sulfate. The active fraction was desalted with G-25 (manufactured by GE Healthcare Bioscience) equilibrated with 10 mM potassium phosphate buffer (pH 7.5), and then equilibrated with 10 mM potassium phosphate buffer (pH 7.5). DEAE sep. It was made to adsorb | suck to FF (made by GE Healthcare Bioscience). After thoroughly washing with 10 mM potassium phosphate buffer (pH 7.5), elution was performed with a linear gradient using 10 mM potassium phosphate buffer (pH 7.5) containing 0 and 0.5 M potassium chloride. . The active fraction was desalted with G-25 equilibrated with 10 mM potassium phosphate buffer (pH 7.0) to obtain the purified protein of the present invention. About 50 mg of purified protein was obtained in 1 L of culture.

[Example 18]
<Expression of protein of the present invention using pET system (manufactured by Novagen)>
The insert obtained in Example 13 was ligated with pET21a (+) purified by restriction enzyme treatment with NdeI and BamHI according to a conventional method to prepare pET21a (+) / BthNK. pET21a (+) / BthNK was transformed into Escherichia coli BL21 (DE3) according to a conventional method, and the recombinant plasmid purified from the positive clone by colony direct PCR was DNA sequenced to confirm that the insert sequence was correct.
The obtained three transformant strains were inoculated into an LB medium containing 50 μg / ml ampicillin. The seed was cultured at 30 ° C. for 1 day. 1/100 amount of the above seed was inoculated in LB medium containing 50 μg / ml ampicillin and cultured at 30 ° C. for one day. IPTG was added so that the density | concentration in a culture solution might be 1 mM, and also it culture | cultivated at 30 degreeC for 3 hours. The culture broth is collected by centrifugation, suspended in 20 mM Tris / HCl buffer (pH 8.5), 1/5 times the volume of the culture, sonicated, centrifuged, and the resulting supernatant Was used as a crude protein solution. The SDS-PAGE of the crude protein solution is shown in FIG. From this, large-scale expression of the protein of the present invention was confirmed. In addition, the arrow in a figure is the protein of this invention.

[Example 19]
<Nucleoside phosphorylation method and phosphorylated nucleoside production>
Using the protein of the present invention prepared in Example 17, the following [mixture] was prepared as a nucleoside phosphorylating agent, and adenosine phosphorylation was carried out to produce phosphorylated adenosine (AMP) from adenosine. . [Mixture] Adenosine was added to 1 ml to a final concentration of 1 mM, and adenosine phosphorylation was performed at 37 ° C. for 30 minutes. The [mixture] after the adenosine phosphorylation method was diluted 10-fold with purified water, and 25 μl was separated by HPLC. Asahpak GS-320 manufactured by Showa Denko KK was used for HPLC, the mobile phase was 200 mM sodium phosphate buffer pH 3.0, the flow rate was 0.5 ml / min at room temperature, and the absorbance at 260 nm was monitored. A chromatogram after the method for phosphorylating adenosine is shown in FIG. 9 (C), and a chromatogram before implementation is shown in FIG. 9 (B). (A) of FIG. 9 is a chromatogram obtained by separating a standard ATP, ADP, AMP, and adenosine mixture by HPLC under the same HPLC conditions. Using the nucleoside phosphorylating agent using the protein of the present invention, phosphorylation adenosine (AMP) was able to be produced from adenosine by carrying out a method for phosphorylating adenosine. A blind test was performed using purified water instead of the protein of the present invention obtained in Example 17, and it was confirmed that the chromatogram before and after the execution was unchanged from FIG. 9B.
[blend]:
50 mM Tris / HCl buffer pH 7.5
2.5 mM ATP
5 mM magnesium chloride 5 mU / ml Protein of the present invention prepared according to Example 17

[Example 20]
<Measurement method 1 of nucleoside>
Using the protein of the present invention prepared in Example 17, the following [mixture 1] and [mixture 2] were prepared as nucleoside phosphorylating agents, and a method for measuring a mixture of adenosine, inosine and guanosine was carried out. A Hitachi 7080 automatic analyzer was used for the measurement. Parameters are 5 μl of sample, 100 μl of [mixture 1] as R1, 100 μl of [mixture 2] as R2, measurement main wavelength is 340 nm, measurement subwavelength is 405 nm, 1 point end, 10 minutes reaction, 0-31, purified water Minus the blank. FIG. 10 shows the measurement results obtained by adjusting a 1: 1: 1 mixture of adenosine, inosine, and guanosine as a sample in the range of the horizontal axis of FIG. A mixture of adenosine, inosine, and guanosine could be measured with a nucleoside phosphorylating agent using the protein of the present invention.
[Mixture 1]:
20 mM potassium phosphate buffer, pH 7.0
10 mM ATP (pH 7)
20 mM magnesium chloride 0.25 U / ml Protein of the invention prepared according to Example 17 50 mM potassium chloride [mixture 2]:
20 mM potassium phosphate buffer, pH 7.0
20 mM glucose 20 mM magnesium chloride 50 mM potassium chloride 5 mM NADP
5 U / ml glucose 6-phosphate dehydrogenase 5 U / ml ADP-dependent HK

[Example 21]
<Method 2 for measuring nucleoside>
Using the protein of the present invention prepared in Example 17, the following [mixture] was prepared as a nucleoside phosphorylating agent, and a method for measuring adenosine, inosine, and guanosine was carried out. A Hitachi 7080 automatic analyzer was used for the measurement. The parameters were 3 μl of sample, 130 μl of [mixture] as R1, measurement main wavelength was 340 nm, measurement subwavelength was 405 nm, rate A, 5 minutes reaction, 7-9, and a blank with purified water was subtracted. The results obtained by adjusting adenosine, inosine, and guanosine as samples in the range of the horizontal axis in FIGS. 11 to 13 are shown in FIGS. A nucleoside phosphorylating agent using the protein of the present invention was prepared, and adenosine, inosine, and guanosine could be measured.
[blend]:
50 mM potassium phosphate buffer, pH 7.0
50 mM potassium chloride 1 mM ATP (pH 7)
0.5mM magnesium chloride 0.5mM PEP
0.1 mM NADH
50 U / ml pyruvate kinase 15 U / ml lactate dehydrogenase 2 mM DTT
25 mU / ml protein of the present invention prepared according to Example 17

[Example 22]
<Method 3 for measuring nucleoside>
Using the protein of the present invention prepared in Example 17, the following [mixture] was prepared as a nucleoside phosphorylating agent, and a method for measuring adenosine, inosine, and guanosine was carried out. A Hitachi 7080 automatic analyzer was used for the measurement. The parameters were 3 μl of sample, 130 μl of [mixture] as R1, measurement main wavelength was 340 nm, measurement subwavelength was 405 nm, rate A, 5 minutes reaction, 7-9, and a blank with purified water was subtracted. The results obtained by adjusting adenosine, inosine, and guanosine as samples in the range of the horizontal axis of FIGS. 14 to 16 are shown in FIGS. A nucleoside phosphorylating agent using the protein of the present invention was prepared, and adenosine, inosine, and guanosine could be measured.
[blend]:
20 mM potassium phosphate buffer, pH 7.0
20 mM glucose 1 mM ATP (pH 7)
1mM magnesium chloride 1mM NADP
50 mM potassium chloride 5 U / ml glucose 6-phosphate dehydrogenase 5 U / ml ADP-dependent HK
6 mU / ml protein of the present invention prepared according to Example 17

[Example 23]
The purified protein of the present invention prepared in Example 18 and the purified protein of the present invention prepared in Example 18 and the crude protein solution of the present invention prepared in Example 18 were purified in the same manner as in Example 17. The above physicochemical properties <1> to <5> were compared. As a result, it was confirmed that the two kinds of purified proteins of the present invention were proteins having the above-mentioned physicochemical properties <1> to <5>.

  The present invention provides a method for producing a protein that catalyzes a reaction that acts on a nucleoside to produce a phosphorylated nucleoside. The protein of the present invention can be used for the production of phosphorylated nucleosides, which are important as various pharmaceutical raw materials, biochemical research reagents, nutrient raw materials, and the like, and also for the measurement of nucleosides in biological samples.

It is a figure which shows optimal pH of the protein of this invention. It is a figure which shows the pH stability of the protein of this invention. It is a figure which shows the thermal stability of the protein of this invention. It is a figure which shows the result of SDS-PAGE of the protein of this invention. It is a figure which shows the relative activity which phosphorylates inosine in each temperature of the protein of this invention. It is a figure which shows the reaction rate which phosphorylates inosine in each temperature of the protein of this invention. (A) It is a figure which shows the result of SDS-PAGE of the crude protein liquid obtained by culture | cultivating pTipQC1 / BthNK transformation Rhodococcus erythropolis. (B) SDS-PAGE of a crude protein solution obtained by culturing pTip QC2 / BthNK-transformed Rhodococcus erythropolis. It is a figure which shows the result of SDS-PAGE of the crude protein liquid which culture | cultivated pET21a (+) / BthNK transformed colon_bacillus | E._coli BL21 (DE3). (A) A chromatogram obtained by separating a standard ATP, ADP, AMP, and adenosine mixture by HPLC under the conditions described in Example 19. (B) A chromatogram before carrying out the method for phosphorylating adenosine described in Example 19. (C) A chromatogram after carrying out the method for phosphorylating adenosine described in Example 19. It is a figure which shows the result of having measured the adenosine, inosine, and guanosine mixture using the nucleoside phosphorylating agent of Example 20. FIG. (Example 20) It is a figure which shows the result of having measured adenosine using the phosphorylating agent of the nucleoside of Example 21. (Example 21) It is a figure which shows the result of having measured inosine with the phosphorylating agent of the nucleoside of Example 21. (Example 21) It is a figure which shows the result of having measured guanosine with the phosphorylating agent of the nucleoside of Example 21. (Example 21) It is a figure which shows the result of having measured adenosine with the nucleoside phosphorylating agent of Example 22. FIG. (Example 22) It is a figure which shows the result of having measured inosine with the phosphorylating agent of the nucleoside of Example 22. (Example 22) It is a figure which shows the result of having measured guanosine with the phosphorylating agent of the nucleoside of Example 22. FIG. (Example 22)

Claims (8)

A method for producing a protein according to any one of (1) to (3) below:
A method for producing the protein, comprising a step of forming the protein based on a base sequence encoding the protein, and a step of obtaining the protein.
(1) a protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a phosphorylation reaction of a nucleoside;
(2) The amino acid sequence of SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence is the amino acid sequence of SEQ ID NO: 5 and the sequence listing SEQ ID NO: A protein comprising 6 amino acid sequences and capable of catalyzing a phosphorylation reaction of a nucleoside;
(3) a protein having the following physicochemical properties <1> to <5>;
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes; 2. The production according to claim 1, wherein the step of forming the protein based on the base sequence encoding the protein is a step of using a microorganism that produces a protein belonging to the genus Burkholderia and capable of catalyzing a phosphorylation reaction of a nucleoside. Method. The production method according to claim 1, further comprising a step of purifying the protein when the protein formed in the step of forming the protein based on a base sequence encoding the protein is obtained. The production method according to claim 3, wherein the base sequence encoding the protein is the base sequence represented by SEQ ID NO: 2 in Sequence Listing. A method for phosphorylating a nucleoside using the protein according to any one of (1) to (3) below. However, the nucleoside is a nucleoside excluding cytidine and xanthosine.
(1) A protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a phosphorylation reaction of a nucleoside (2) In the amino acid sequence of SEQ ID NO: 1, one or more amino acids are deleted, substituted or added A protein capable of catalyzing a phosphorylation reaction of a nucleoside comprising the amino acid sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6;
(3) a protein having the following physicochemical properties <1> to <5>;
<1> Action Catalyzes a reaction in which a nucleoside is converted to a phosphorylated nucleoside in the presence of at least a phosphate donor;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes; The method for phosphorylating a nucleoside according to claim 5, wherein the protein is derived from Burkholderia thailandensis. The protein is a protein having a molecular weight of about 34 kDa as determined by SDS polyacrylamide gel electrophoresis, or a protein utilizing at least magnesium ion, cobalt ion, nickel ion or manganese ion, or a nucleoside at least at 37 ° C. The method for phosphorylating a nucleoside according to claim 6, wherein the protein catalyzes a reaction that phosphorylates nucleoside. The measuring method of the nucleoside including each process of following (a)-(c). However, the nucleoside is a nucleoside excluding cytidine and xanthosine.
(A) a first reaction in which a protein of any one of (1) to (3) below and a nucleoside that may be contained in a sample in the presence of ATP generates a phosphorylated nucleoside and ADP Process,
(1) a protein comprising the amino acid sequence of SEQ ID NO: 1 and capable of catalyzing a phosphorylation reaction of a nucleoside;
(2) The amino acid sequence of SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence is the amino acid sequence of SEQ ID NO: 5 and the sequence listing SEQ ID NO: A protein comprising 6 amino acid sequences and capable of catalyzing a phosphorylation reaction of a nucleoside;
(3) a protein having the following physicochemical properties <1> to <5>;
<1> Action Acts on ATP and nucleoside to catalyze the reaction producing ADP and phosphorylated nucleoside;
<2> Substrate specificity It acts on adenosine, inosine, and guanosine, but does not substantially act on cytidine and xanthosine. Also has virtually no effect on ribose;
<3> Optimum pH
pH 5.5-7.5;
<4> pH stability Maintains an activity of 70% or more in the range of pH 6 to 10 at 37 ° C. for 3 hours;
<5> Thermal stability 100% potassium phosphate buffer solution in an aqueous solution of pH 7.0, retaining an activity of 80% or more by heat treatment at 50 ° C. for 20 minutes;
(B) In the presence of a second reaction or a plurality of second reactions different from the first reaction, and a second enzyme or a second plurality of enzymes that catalyzes the reaction, the first reaction A step of causing a change in an object to be measured by a second reaction according to the amount of ADP generated by the reaction;
(C) detecting the amount of change in the measurement target, and measuring the amount of nucleoside that may be contained in the sample;