JP2010233501A - Reagent composition for assaying glycated protein, reducing influence of fructosyl valine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent composition for assaying a glycated protein, reducing the influence of fructosyl valine. <P>SOLUTION: The reagent composition includes a fructosyl amino acid oxidase having an activity value against the fructosyl valine of not more than 35, when the activity value against fructosyl-valyl histidine is 10. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a reagent composition useful for measurement of glycated protein, particularly HbA1c. More specifically, the present invention relates to a glycated protein measurement reagent composition in which the influence of fructosyl valine is reduced.

For the measurement of blood glucose in diabetic patients, a method of measuring hemoglobin A1c (HbA1c) or glycoalbumin, which is a glycated protein contained in blood protein, as a blood glucose marker is heavily used.
D-glucose present in blood reacts with amino acid residues constituting blood protein to form a glycated protein. The main glycation site in blood proteins is the ε-amino group of lysine residues or the α-amino group of amino acids present at the amino terminus of blood proteins. When measuring HbA1c, the amount of glycated protein produced by binding D-glucose to the α-amino group of valine, which is an amino acid present at the amino terminus of the hemoglobin β chain, is measured.

In recent years, enzymatic measurement methods capable of measuring glycated proteins in blood easily and in a short time have been developed and already commercialized. By using an enzymatic measurement method, it is possible to measure a glycated protein with high throughput, and the enzymatic measurement method is useful in the clinical laboratory field.
In the enzymatic measurement method, first, a glycated protein is hydrolyzed by a protease. Next, glycated amino acids such as fructosyl valine, fructosyl lysine and fructosyl valyl histidine generated by hydrolysis are oxidatively hydrolyzed by fructosyl amino acid oxidase (hereinafter referred to as “FAOD”). Finally, hydrogen peroxide generated by the oxidase reaction is colorimetrically determined by a peroxidase-chromogen reaction system (see, for example, Patent Documents 1 to 11).

  In the measurement of glycated protein by an enzymatic measurement method, the substrate specificity of FAOD, which is the main reaction enzyme, is important. For example, in the measurement of HbA1c, an enzyme (fructosyl valyl histidine oxidase) that acts on fructosyl valyl histidine is desired in order to perform measurement specific to the β chain of glycated hemoglobin. The reason is that both amino acids present at the amino terminus of hemoglobin α-chain and β-chain are valine. Therefore, in order to carry out measurement specific to β-chain, two amino acid residues at the amino terminus (ie, fructose This is because it is necessary to recognize (Silvalylhistidine) (see, for example, Patent Document 12 and Patent Document 13).

JP 2004-129531 A (publication date: April 30, 2004) JP 2004-1113014 A (publication date: April 15, 2004) International Publication No. 2003/064683 Pamphlet (International Publication Date: August 7, 2003) JP 2007-181466 A (publication date: July 19, 2007) JP 2007-289202 A (publication date: November 8, 2007) JP 2005-110657 A (publication date: April 28, 2005) Japanese Patent No. 4045322 (issue date: February 13, 2008) Japanese Patent No. 4014088 (issue date: September 21, 2007) Japanese Patent No. 4039664 (issue date: January 30, 2008) Japanese Patent No. 4010474 (issue date: November 21, 2007) Japanese Patent No. 3971702 (issue date: September 5, 2007) International Publication No. 2005/049857 Pamphlet (International Publication Date: June 2, 2005) International Publication No. 2005/049858 (International Publication Date: June 2, 2005) JP 2003/235585 A (publication date: August 26, 2003) International Publication No. 2003/064683 Pamphlet (International Publication Date: May 26, 2005) Japanese Patent No. 4014088 (issue date: November 28, 2007) JP 2007-289202 A (publication date: November 8, 2007) Pamphlet of International Publication No. 2007/010950 (International Publication Date: January 25, 2007)

Hirokawa K.K. , Gomi K. , Bakke M .; , And Kajiyama N .; , Distribution and properties of novelty engineering for fractosyl peptide in fungi. , Arch. Microbiol. , 2003, 180 (3), 227-231. Hirokawa K.K. , Gomi K. , And Kajiyama N .; , Molecular cloning and expression of novel peptides oxidatives and ther application for the measurement of glycated protein. , Biochem. Biophys. Res. Commun. , 2003, 311 (1), 104-111.

However, the conventional fructosyl valyl histidine oxidase has a problem of low substrate specificity.
So far, fructosyl valyl histidine oxidase has been obtained from several types of filamentous fungi or genetically modified organisms thereof. In addition, genes encoding these fructosyl valyl histidine oxidases have also been isolated (see, for example, Patent Document 14, Non-Patent Document 1, and Non-Patent Document 2). However, they all have the problem of reacting with fructosyl valine.
And when a free saccharified amine is mixed in a sample for some reason, in the measurement using the conventional fructosyl valyl histidine oxidase, a measured value will show an abnormally high value. This problem occurs most often in patients who have been administered high-calorie infusion preparations because they are free in the blood or in the infusion bag when high concentrations of sugar and amino acids are replenished in the body. It is believed that this is because glycated amino acids or glycated peptides are produced. In particular, in the case of HbA1c measurement, if FAOD also reacts with fructosyl valine, an abnormally high amount of glycated protein is exhibited due to the mixing of fructosyl valine.

Thus, a system has been developed in which the glycated amine that has been released separately from the measurement object is erased by FAOD before the measurement object undergoes an oxidation-reduction reaction with FAOD (see, for example, Patent Documents 15 to 18). ).
However, since these methods use FAOD in the elimination reaction, there is a problem that hydrogen peroxide is generated during the elimination reaction. When the hydrogen peroxide is brought into the oxidation-reduction reaction of the measurement object, the measured value increases. Therefore, in this method, it is necessary to remove hydrogen peroxide before the measurement object performs an oxidation-reduction reaction with FAOD.

Therefore, the saccharified amine measuring method including the step of eliminating the free saccharified amine in advance by FAOD includes the following reactions (1) to (4). That means
(1) Reaction for eliminating free amine with FAOD (hereinafter referred to as “elimination reaction”)
(2) Reaction for removing hydrogen peroxide generated in the erasing reaction (hereinafter referred to as “removal reaction”)
(3) Reaction for fragmenting glycated protein into glycated amino acid or glycated peptide by protease (hereinafter referred to as “fragmentation reaction”)
(4) A reaction in which a fragmented glycated amino acid or glycated peptide and a FAOD undergo an oxidation-reduction reaction and thereby develop color (hereinafter referred to as “coloring reaction”).

In the above removal reaction, for example, self-condensation of hydrogen donor and POD (peroxidase) and / or catalase coexist, but hydrogen peroxide cannot be completely removed and the hydrogen peroxide is brought into the color reaction. , May lead to an increase in measured values.
In these reactions, at least the elimination reaction and the removal reaction must be performed before the color development reaction. Further, since the enzyme (FAOD) is decomposed by the protease used in the fragmentation reaction, it is necessary to select an enzyme with high protease resistance, or to perform the elimination reaction and the color development reaction separately from the fragmentation reaction. Therefore, the saccharified amine measuring method has a problem in that the composition of the saccharified amine measuring reagent, the addition time of the saccharified amine measuring reagent, and the reaction order are limited.
Moreover, since glycated albumin is contained in the blood sample, it is necessary to avoid this influence when measuring HbA1c. Since the ε-amino group of lysine residue of albumin is glycated in glycated albumin, the measured value increases when FAOD reacts with fructosyl lysine generated by cleavage with protease. In order to solve this problem, FAOD that reacts with fructosyl valyl histidine but has low reactivity with fructosyl valine and fructosyl lysine is desired, but such FAOD has been reported. Not.

  The present invention has been made in view of the above-mentioned problems, and its object is to newly provide fructosyl valyl histidine oxidase having reduced reactivity to fructosyl valine, which is useful for measurement of glycated proteins, particularly HbA1c. To provide a reagent composition for measuring glycated protein (HbA1c) using the enzyme, and a glycated protein (HbA1c) sensor.

As a result of intensive studies to achieve the above object, the present inventors have found that the protein has fructosyl valyl histidine oxidase activity useful for the measurement of fructosyl valyl histidine, and is a substrate more than a wild type protein. We succeeded in obtaining mutant proteins with improved specificity.
Furthermore, the present inventors have succeeded in constructing a glycated protein measurement reagent composition in which the influence of fructosyl valine is reduced by using the fructosyl valyl histidine oxidase.

That is, the present invention includes the following inventions.
[Claim 1]
A glycated protein measurement reagent composition comprising a protease and fructosyl amino acid oxidase, wherein the fructosyl amino acid oxidase has the following property (1):
(1) The reactivity with respect to fructosyl valine is 35 or less when the activity value with respect to fructosyl valyl histidine is set to 10.
[Section 2]
A reagent for measuring a glycated protein comprising a protease and fructosyl amino acid oxidase, wherein the fructosyl amino acid oxidase has the following property (2):
(2) The reactivity with fructosyl valine is 1 or less when the activity value with respect to fructosyl valyl histidine is 2.
[Section 3]
Item 2. The reagent composition according to Item 1, wherein the fructosyl amino acid oxidase has any of the following properties (a) to (c).
(A) the amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which isoleucine at position 58 from the amino terminus is substituted with another amino acid, and has fructosyl valyl histidine oxidase activity;
(B) Amino acid sequence (a) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added at a site other than the 58th amino acid from the amino terminus, and has fructosyl valyl histidine oxidase activity ;
(C) an amino acid sequence having 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid corresponding to the 58th isoleucine from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1 is the amino acid A protein substituted with a different amino acid and having fructosyl valyl histidine oxidase activity.
[Claim 4]
The term in which the amino acid substitution is such that the 58th glycine from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1 is substituted with any one selected from the group consisting of methionine, threonine, alanine, asparagine, serine, valine and leucine. 2. The reagent composition according to 1.
[Section 5]
Item 5. The reagent composition according to any one of Items 1 to 4, wherein the glycated protein is HbA1c.
[Claim 6]
Item 5. A glycated protein measurement method using the reagent composition according to any one of Items 1 to 4.
[Claim 7]
Item 5. A glycated protein measurement kit comprising the reagent composition according to any one of Items 1 to 4.
[Section 8]
Item 5. A glycated protein measurement sensor comprising the reagent composition according to any one of Items 1 to 4.

  The reagent composition for measuring a glycated protein according to the present invention uses a protein having fructosyl valyl histidine oxidase activity as described above. Therefore, there is an effect that it is possible to provide a saccharified amine measuring reagent composition that reduces the influence of fructosyl valine.

The result of having quantified fructosyl valyl histidine using the fructosyl valyl histidine oxidase variant (IE353-I58T) is shown. The result of having investigated the influence degree of fructosyl valine using IE353 wild type and the fructosyl valyl histidine oxidase modified body (IE353-I58T) is shown.

  An embodiment of the present invention will be described in detail as follows, but the present invention is not limited to this. In addition, all the nonpatent literatures and patent literatures described in this specification are used as reference in this specification. In addition, “to” in the present specification means “above and below”, for example, if “A to B (in the case where A and B are numerical values or numerical values with units)” is described in the specification. “A or more and B or less” is shown. In the present specification, “and / or” means either one or both.

<1. Reagent composition>
The reagent composition of the present invention is a glycated protein measuring composition comprising a protease and fructosyl amino acid oxidase, wherein the fructosyl amino acid oxidase has the following property (1).
(1) The reactivity with respect to fructosyl valine is 35 or less when the activity value with respect to fructosyl valyl histidine is set to 10.

  The fructosyl amino acid oxidase in the above composition preferably has a reactivity with fructosyl valine of 1 or less when the activity value with respect to fructosyl valyl histidine is 2.

The fructosyl amino acid oxidase in the composition is more preferably the protein described in any of the following (a) to (c). That means
(A) a protein having an amino acid sequence in which the 58th isoleucine from the amino terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with another amino acid and having fructosylvalylhistidine oxidase activity;
(B) the protein according to (a) above, comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added at a site other than the 58th amino acid from the amino terminus; And a protein having fructosyl valyl histidine oxidase activity;
(C) an amino acid sequence having 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid corresponding to the 58th isoleucine from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1 is the amino acid A protein substituted with a different amino acid and having fructosyl valyl histidine oxidase activity.
In addition, in the range of “homology” in this specification, in addition to those in which individual amino acid residues constituting the sequence are the same, those having the same properties of amino acids are included in the range. (For example, isoleucine and leucine are considered the same)
The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of fructosylvalyl histidine oxidase derived from Phaeosphaeria nodorum (for details, see the Examples).
In the protein described in (a) above, the 58th isoleucine from the amino terminus is substituted with another amino acid. The other amino acids are not particularly limited, and can be appropriately substituted with known amino acids. Examples of amino acids to be substituted include, but are not limited to, basic amino acids that are components of natural proteins, modified amino acids that have undergone some chemical modification, and special amino acids derived from basic amino acids.
The 58th isoleucine from the amino terminus is preferably substituted with, for example, methionine, threonine, alanine, asparagine, serine, valine, or leucine. If it is the said structure, the substrate specificity (reactivity with fructosyl valyl histidine) of fructosyl valyl histidine oxidase can further be raised.
From the viewpoint of further increasing the substrate specificity for fructosyl valylhistidine (in other words, greatly reducing the reactivity of fructosyl valine and fructosyl lysine to both substrates) More preferably, the isoleucine is replaced by threonine, serine, valine or alanine.
On the other hand, from the viewpoint of increasing not only the substrate specificity but also the thermal stability, it is more preferable that the 58th isoleucine from the amino terminus is substituted with methionine, serine or alanine.
Therefore, from the viewpoint of further increasing the substrate specificity and thermal stability, it can be said that the 58th isoleucine from the amino terminus is most preferably substituted with serine or alanine.
In the protein described in (b) above, in the protein described in (a) above, one or several amino acids are substituted, deleted, inserted and / or added at a site other than the 58th amino acid from the amino terminus. And a protein having fructosyl valyl histidine oxidase activity.
The place where substitution, deletion, insertion, and / or addition occurs in the protein described in (b) is not particularly limited, and may be any place other than the 58th position from the amino terminus.
In the present specification, the term “one or several amino acids” specifically means the number of amino acids within a range of 10 or less.
The protein described in (c) above is composed of an amino acid sequence having 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and corresponds to the 58th isoleucine from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1. The amino acid is a protein having fructosyl valyl histidine oxidase activity, in which an amino acid different from the amino acid is substituted. The amino acid corresponding to the 58th isoleucine from the amino terminus may be isoleucine or a different amino acid from isoleucine.
Although it does not specifically limit as a specific structure of the said protein, For example, fructosyl valyl histidine oxidase derived from organisms other than Phaeosphaeria nodorum etc. can be mentioned.
The protein described in the above (c) may be any protein as long as it has 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1, more preferably 80% or more. More preferably, it has 90% or more homology, and most preferably 95% or more homology. The protein shown in (c) above may be a protein containing these highly homologous regions as a part of itself.
In the protein described in (c) above, the amino acid corresponding to the 58th isoleucine from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an amino acid different from the amino acid. The amino acid substituted with the amino acid corresponding to the 58th isoleucine from the amino terminus may be any amino acid different from the amino acid, and the specific amino acid is not particularly limited. Examples of amino acids to be substituted include, but are not limited to, basic amino acids that are components of natural proteins, modified amino acids that have undergone some chemical modification, and special amino acids derived from basic amino acids. More specifically, for example, methionine, threonine, alanine, asparagine, serine, valine, or leucine is preferably substituted, but is not limited thereto.
The amino acid corresponding to the 58th isoleucine from the amino terminus can be determined by comparing the amino acid sequence of the protein containing the amino acid with the amino acid sequence shown in SEQ ID NO: 1. For example, a protein having 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1 is searched by a program (for example, GENETYX-WIN (Genetics)). Then, according to the above program (for example, GENETYX-WIN (Genetics)), information on amino acid correspondence when it is determined that the homology is 70% or more (in other words, described in SEQ ID NO: 1). The determination result regarding which amino acid in the protein obtained by the search corresponds to each amino acid in the amino acid sequence can be obtained. Based on this information, the amino acid corresponding to the 58th isoleucine from the amino terminus can be determined.
The database to be searched at this time is not particularly limited, and all known databases (for example, databases of bacteria, yeasts, insects, nematodes, zebrafish, mammals, etc.) can be searched.
In addition, the accuracy of identification of the amino acid corresponding to the 58th isoleucine from the amino terminus should be detected based on the fructosyl valyl histidine oxidase activity measurement method described below for the fructosyl valyl histidine oxidase activity of the searched protein. Can be further increased. For example, it is possible to increase the accuracy of amino acid identification by confirming how the enzyme activity changes when an amino acid thought to correspond to the 58th isoleucine from the amino terminus is substituted.
All the proteins described in the above (a) to (c) have fructosyl valyl histidine oxidase activity. Although the measuring method of fructosyl valyl histidine oxidase activity is not specifically limited, For example, it can measure by the method demonstrated in the section of the "activity measuring method" of the Example mentioned later. In the description of the present invention, “having fructosyl amino acid oxidase activity” preferably means an activity of 0.1 U / mg-protein or more, more preferably an activity of 1.0 U / mg-protein or more. To do.
Further, the protein of the present embodiment may be one in which further substitution has occurred with respect to the protein. The specific location where the substitution occurs and the type of amino acid after substitution are not particularly limited.
The protein of the present embodiment can be appropriately produced using a known method. For example, it can be produced using a gene recombination technique using a polynucleotide and a recombinant vector, which will be described later, or chemically synthesized using an amino acid synthesizer or the like.
A recombinant protein expression system suitably used in the gene recombination technique is not particularly limited, but for example, an E. coli expression system, an insect cell expression system, a mammalian cell expression system, or a cell-free expression system is preferably used.
In addition, the protein according to the present embodiment is, for example, one subjected to intermolecular crosslinking and / or intramolecular crosslinking (for example, disulfide bond), chemical modification (for example, glycosylation, phosphorylation, or other) Functional group added), labeled (eg, His tag, Myc tag, or Flag tag) attached, or fusion protein (eg, streptavidin, cytochrome, GST, GFP, etc.) attached Can be included.
Furthermore, the protein of the present embodiment may also include a chimeric protein constituted by combining several types of protein fragments as long as the fructosyl valyl histidine oxidase activity is substantially maintained.

<2. Polynucleotide>
The polynucleotide of the present embodiment is the polynucleotide described in (d) or (e) below. That means
(D) a polynucleotide encoding the protein of the present invention;
(E) a polynucleotide that hybridizes under stringent conditions to a complementary polynucleotide of the polynucleotide encoding the protein of the present invention.
As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. The polynucleotide of the present embodiment can be in the form of DNA (eg, cDNA or genomic DNA) or RNA (eg, mRNA). The DNA and RNA may be double-stranded or single-stranded.
The polynucleotide described in (d) above is a polynucleotide encoding the protein of the present invention described above.
The polynucleotide described in (d) above only needs to encode the protein of the present invention, and can be substituted as long as the codons encode the same amino acid. More specifically, when considering introducing the polynucleotide of this embodiment into some host and expressing the protein of the present invention in the host, the codon usage of the host (codon usage) Based on the above, it is also possible to determine a specific base sequence of the polynucleotide of the present embodiment. That is, it is possible to determine a specific base sequence of the polynucleotide of the present embodiment so that the polynucleotide of the present embodiment is formed by codons with high codon usage.
The method for producing the polynucleotide of the present embodiment is not particularly limited, and can be suitably produced by a known method. For example, the polynucleotide of the present embodiment can be prepared by appropriately introducing mutations into the polynucleotide (SEQ ID NO: 2) encoding wild-type fructosyl valyl histidine oxidase. In addition, the polynucleotide of this embodiment can be produced by a chemical synthesis method.
The method for introducing the mutation is not particularly limited, and a mutation may be introduced into the polynucleotide shown in SEQ ID NO: 2, for example, using a commercially available kit as appropriate. Examples of the commercially available kit include KOD-Plus Site-Directed Mutagenesis Kit (manufactured by Toyobo), Transformer Site-Directed Mutagenesis Kit (manufactured by Clonetech), and QuickChange SiteDiet, etc. Although it can, it is not limited to these. A more specific method for introducing a mutation may be performed according to the protocol attached to these kits.
The polynucleotide of the present embodiment is preferably, for example, a polynucleotide encoding an amino acid sequence in which the 58th isoleucine from the amino terminus is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 1. More specifically, in the polynucleotide shown in SEQ ID NO: 2, “att (172nd to 174th)” is preferably a polynucleotide substituted with a codon encoding other than isoleucine.
Although it does not specifically limit as a specific codon which substitutes the said "att", For example, it is preferable that it is a codon which codes methionine, threonine, alanine, asparagine, serine, valine, or leucine. More specifically, the above “att” means “atg (codon encoding methionine)”, “acc (codon encoding threonine)”, “gct (codon encoding alanine)”, “aac (asparagine It is preferably replaced with “coding codon)”, “tcg (codon encoding serine)”, “gtc (codon encoding valine)”, or “ctc (codon encoding leucine)”. It is not limited to. It is of course possible to substitute another codon encoding the amino acid.
Furthermore, in addition to the above substitution, the polynucleotide of the present embodiment is a polynucleotide encoding an amino acid sequence in which the 282nd phenylalanine from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1 is substituted with another amino acid. It is preferable. More specifically, in the polynucleotide shown in SEQ ID NO: 2, “ttc (844th to 846th)” is preferably a polynucleotide substituted with a codon encoding other than phenylalanine.
Although it does not specifically limit as a specific codon which substitutes said "ttc", For example, it is preferable that it is a codon which codes tyrosine. More specifically, the “ttc” is preferably replaced with “tat (codon encoding tyrosine)”, but is not limited thereto. It is of course possible to replace the above “ttc” with another codon encoding tyrosine.
Furthermore, the polynucleotide of the present embodiment is a polynucleotide that encodes an amino acid sequence in which the 110th glycine from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1 is substituted with another amino acid in addition to the above substitution. It is preferable. More specifically, the polynucleotide shown in SEQ ID NO: 2 is preferably a polynucleotide in which “gga (328th to 330th)” is substituted with a codon encoding other than glycine.
The specific codon that replaces the above “gga” is not particularly limited, but is preferably a codon encoding glutamine, for example. More specifically, the “gga” is preferably replaced with “cag (codon encoding glutamine)”, but is not limited thereto. It is of course possible to replace the above “gga” with another codon encoding glutamine.
The polynucleotide described in (e) above is a polynucleotide that hybridizes under stringent conditions to the complementary polynucleotide of the polynucleotide encoding the protein of the present invention described above.
The above “stringent conditions” means that hybridization occurs only when at least 90% identity, preferably at least 95% identity, most preferably 97% identity exists between sequences. Means that.
The hybridization is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained.
The polynucleotide described in (d) or (e) described above may consist only of the polynucleotide encoding the protein of the present invention, but may have other base sequences added thereto.
The base sequence to be added is not limited. For example, a label (for example, His tag, Myc tag, or FLAG tag), a fusion protein (for example, streptavidin, cytochrome, GST, or GFP), a signal sequence (for example, And endoplasmic reticulum translocation signal sequence and secretory sequence), and promoter sequences (eg, yeast-derived promoter sequences, phage-derived promoter sequences, E. coli-derived promoter sequences, etc.) and the like. The site to which these base sequences are added is not particularly limited as long as the desired function of the translated protein is maintained, but is preferably the N-terminus or C-terminus of the translated protein.

<3. Recombinant vector>
The recombinant vector of the present embodiment contains the polynucleotide of the present invention.
The recombinant vector of the present embodiment only needs to contain the polynucleotide of the present invention, and its specific configuration is not particularly limited. As a base vector constituting the recombinant vector of the present embodiment, a plasmid, phage, cosmid, adenovirus, retrovirus or the like can be used, but is not limited thereto.
The recombinant vector of the present embodiment can contain an expression control region (for example, a promoter, a terminator, and / or an origin of replication) depending on the host to be introduced. Examples of the promoter include, but are not limited to, viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.).
The recombinant vector of this embodiment preferably contains at least one selectable marker. Examples of such markers include dihydrofolate reductase and neomycin resistance gene. By using the above selection marker, it can be confirmed whether or not the polynucleotide of the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell.
The recombinant vector of the present embodiment can be produced according to a conventional method using a restriction enzyme and / or ligase.
When using a plasmid as the vector, for example, pBluescript (registered trademark) or pUC18 can be used. In this case, examples of the host microorganism or host cell into which the vector is introduced include yeast, E. coli (Escherichia coli W3110 (Escherichia coli), Escherichia coli C600, Escherichia coli JM109, Escherichia coli DH5α), insect cells and Mammalian cells can be used.
The method for introducing the recombinant vector of the present embodiment into the above-described host microorganism or host cell is not particularly limited, but conventionally known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, etc. For example, when a recombinant vector is introduced into a microorganism belonging to the genus Escherichia as a host microorganism, it is preferable to use an electroporation method, but is not limited thereto. In addition, it is also possible to introduce a recombinant vector into a host using a commercially available competent cell (for example, competent high JM109, competent high DH5α; manufactured by Toyobo).

<4. Transformant>
The transformant of this embodiment is one into which the recombinant vector of the present invention has been introduced.
The host to be transformed is not particularly limited, and various conventionally known cells can be suitably used.
Specifically, for example, bacteria such as Escherichia coli (for example, Escherichia coli), yeast (for example, budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe, etc.), insect cells, nematodes (for example, Caenorhabditis elegans, Africa, etc.) Examples include, but are not limited to, oocytes (eg, Xenopus laevis), animal cells (eg, CHO cells, COS cells, and Bowes melanoma cells), and various human cultured cells. Further, as used herein, the term “transformant” includes not only cells, tissues or organs but also individual organisms.
A method for introducing the above-described recombinant vector into a host, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method may be preferably used. it can.

<5. Method for producing fructosyl valyl histidine oxidase>
The method for producing fructosyl valyl histidine oxidase according to the present embodiment includes the step of culturing the transformant of the present invention described above (referred to as “culturing step”).
The fructosyl valyl histidine oxidase production method of the present embodiment may include other steps that can be included in protein production using the transformant in addition to the above-described culturing step. Examples of the other steps include, but are not limited to, a recovery step for recovering the protein produced by the transformant after the culture step and / or a purification step for purifying the protein. Below, each process is demonstrated.

(5-1) Culture process In the culture process, the transformant of the present invention is cultured in a nutrient medium or the like, whereby the protein of the present invention is stably produced in a large amount.
The culture form of the transformant (eg, culture method, culture conditions, etc.) can be selected depending on the host. For example, it is preferable to culture the transformant by a liquid culture method. Industrially, it is advantageous to perform aeration and agitation culture.
As a nutrient source of the nutrient medium used in the culturing step, those commonly used for culturing can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, lactose, molasses, or pyruvic acid may be used. The nitrogen source may be any assimitable nitrogen compound, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, or soybean meal alkaline extract may be used. In addition to these, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron and manganese zinc, specific amino acids, specific vitamins and the like can be added to the medium as necessary.
The culture temperature of the transformant can be appropriately changed as long as the transformant is within the range in which the protein of the present invention can be produced. For example, when Escherichia coli is used as a host, It is about 20-42 degreeC. In addition, in the said temperature range, it can be said that 20-30 degreeC is still more preferable. Within the temperature range, a large amount of active protein can be produced.
Moreover, although culture | cultivation time is not specifically limited, What is necessary is just to complete culture | cultivation when the protein of this invention reaches the maximum yield, and is about 6 to 48 hours normally.
In addition, the pH of the medium is not particularly limited, and can be appropriately changed within a range in which the transformant is suitably grown and the protein of the present invention can be produced. The pH is preferably about 6.0 to 9.0.

(5-2) Recovery step In the recovery step, the protein of the present invention produced by the transformant during the culture step is recovered.
When the transformant secretes the protein outside the cell, the culture solution contains the protein of the present invention. Therefore, in this case, the culture solution may be recovered in the recovery step. The culture solution can be used as it is as the protein of the present invention. At this time, the culture solution and the transformant may be further separated by, for example, filtration or centrifugation.
In addition, when the protein of the present invention is present in the transformant without being secreted outside the cell, it is transformed from the culture solution obtained by culturing the transformant using means such as filtration or centrifugation. The body is separated. After the separated transformant is crushed by a mechanical method or an enzymatic method such as lysozyme, the target protein may be recovered from the crushed solution. If necessary, a chelating agent (for example, EDTA) and a surfactant (for example, Triton-X100) are added to the crushed liquid to solubilize the protein of the present invention, and the target protein is recovered as an aqueous solution. It is also possible to do.

(5-3) Purification step The purification step is a step of purifying the protein obtained by the recovery step.
Although the specific method of the said refinement | purification process is not specifically limited, For example, the solution containing the protein of this invention concentrate | evaporates under reduced pressure, membrane concentration, salting out (for example, using ammonium sulfate and sodium sulfate etc.), Alternatively, it may be subjected to a fractional precipitation method using a hydrophilic organic solvent (for example, methanol, ethanol and acetone). By these operations, the protein of the present invention can be precipitated and purified.
In the purification step, purification may be performed using heat treatment, isoelectric point treatment, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, reverse phase chromatography, or a combination thereof.
The purified product (purified enzyme) containing the protein of the present invention obtained by using these techniques is preferably purified to such an extent that it shows a single band in electrophoresis (SDS-PAGE).
The purified product can be pulverized by freeze drying, vacuum drying, spray drying, or the like. If it is the said structure, it will become easy to distribute | circulate a refined material.
Moreover, when using a refined | purified substance, it can be used in the state melt | dissolved in the buffer suitably according to the use. The buffer can be suitably selected depending on the nature of the protein and / or the experimental conditions or environment. As the buffer solution, for example, a borate buffer solution, a phosphate buffer solution, a Tris-HCl buffer solution, a GOOD buffer solution, or the like is preferably used. Furthermore, by adding amino acids (for example, glutamic acid, glutamine, lysine and the like), serum albumin and the like to the purified product, the protein contained in the purified product can be stabilized.

<6. Method for measuring glycated protein>
The reagent composition for measuring glycated protein according to the present invention can be used in a method for measuring fructosyl amino acids. In addition, the method for measuring fructosyl amino acids using the reagent composition according to the present invention can be applied to the measurement of glycated proteins such as HbA1c. The reagent composition according to the present invention can be suitably used for a method for measuring fructosyl valyl histidine, and can be suitably applied to measurement specific to the β chain of glycated hemoglobin. Hereinafter, a method for measuring a glycated protein using the protein according to the present invention (hereinafter referred to as “the measuring method of the present invention”) will be described.
The measurement method of the present invention is characterized in that the reagent composition according to the present invention is allowed to act on at least a glycated protein. One embodiment of the measurement method of the present invention is shown below, but the present invention is not limited to this.
One embodiment of the measurement method according to the present invention is:
(1) A step of reacting a sample with a protease to decompose a glycated protein in the sample and preparing a glycated amine derived from the glycated protein in the sample (referred to as “first step” for convenience),
(2) a step of causing the protein according to the present invention to act on a glycated amine derived from a glycated protein in the sample obtained by the step (1) (referred to as “second step” for convenience), and
(3) A method for measuring a glycated protein comprising a step of measuring the amount of hydrogen peroxide generated or consumed oxygen in the step (2) (referred to as “third step” for convenience). .
One specific example in this embodiment is an enzymatic method. In the enzyme method, a glycated protein in a sample is fragmented to the amino acid or peptide level using an enzyme (for example, protease) (first step). Next, FAOD is added to the resulting glycated amino acid and / or glycated peptide, and hydrogen peroxide is generated by an oxidation-reduction reaction (second step). Peroxidase (POD) and a reducing agent that develops color by oxidation are added to this sample, and an oxidation-reduction reaction is caused between hydrogen peroxide and the reducing agent using POD as a catalyst (third step). The amount of hydrogen peroxide can be measured by coloring the reducing agent by oxidation-reduction reaction and measuring the color intensity (third step).

(6-1) First Step The first step is a step of degrading the glycated protein in the sample to prepare a glycated amine derived from the glycated protein in the sample.
As used herein, “glycated protein” means a protein in which a sugar is bound (glycated) to a part or all of the amino acid residues constituting the protein. Although it does not specifically limit as glycated protein, For example, the thing (for example, HbA1c) etc. by which the alpha amino group of the amino terminal of protein was glycated are mentioned. The HbA1c exemplified above is used as an index for clinical diagnosis such as diabetes diagnosis.
The protease used in the first step is not particularly limited as long as it can decompose the glycated protein contained in the sample into glycated amino acid or glycated peptide. For example, proteases used in clinical tests can be preferably used.
The “sample” used in the present measurement method is not particularly limited as long as it is a target for which the presence or concentration of glycated protein is to be detected. For example, in addition to whole blood, plasma, serum and blood cells, etc. And biological samples such as urine and cerebrospinal fluid (that is, samples collected from the living body), and drinking water such as juice, and foods such as soy sauce and sauce. Since the method of the present invention can be applied to the diagnosis of diabetes, it is particularly useful when measuring a whole blood sample and a blood cell sample among the above. Although not particularly limited, when measuring glycated hemoglobin in erythrocytes, whole blood is lysed as it is, or erythrocytes separated from whole blood are lysed, and this hemolyzed sample is used as a sample for measurement. And it is sufficient.
The specific conditions for reacting the sample with the protease are not particularly limited as long as the desired saccharified amine can be prepared, and depending on the concentration and type of the sample and the concentration and type of the protease. What is necessary is just to employ | adopt after considering suitable conditions suitably.
The concentration of the protease that can be suitably used in the measurement method of the present invention is, for example, 0.1 U to 1 MU / ml, more preferably 1 U to 500 KU / ml, and most preferably 5 U to 100 KU / ml. The concentration of the protease is not limited, and can be suitably determined for the experimenter or the user depending on the reaction conditions, the type and state of the sample, the procedure of the experimenter, the type of reagent used, and the like.
Examples of the “glycated amine” on which the reagent composition according to the present invention acts include glycated amino acids and glycated peptides derived from glycated proteins contained in a sample. The length of the glycated peptide is not particularly limited, and examples include a length that the protein of the present invention can act on, for example, a range of 2 to 6 amino acid residues.

(6-2) Second Step The second step is a step of allowing the protein according to the present invention to act on the glycated amine derived from the glycated protein in the sample obtained in the first step.
The FAOD activity that the protein according to the present invention can have is not particularly limited, but the higher the FAOD activity, the more sensitive the glycated amine can be detected and the less the amount of FAOD protein used. Is preferred. The concentration of FAOD protein that can be suitably used in the measurement method of the present invention is, for example, 0.1 to 500 U / ml, preferably 0.5 to 200 U / ml, and most preferably 1.0 to 100 U / ml. It is. However, the concentration of the FAOD is not particularly limited, and can be appropriately determined according to the reaction conditions, the type and state of the sample, the procedure of the experimenter, the type of reagent to be used, and the like.
When the reagent composition according to the present invention acts on a saccharified amine, oxygen is consumed by oxidative hydrolysis reaction and hydrogen peroxide is generated.

(6-3) Third Step The third step is a step of measuring the amount of hydrogen peroxide generated in the second step or the amount of consumed oxygen. As long as the third step is a method capable of measuring the amount of hydrogen peroxide or the amount of oxygen, the specific method is not particularly limited. Therefore, known methods can be applied as appropriate.
When using the enzymatic method, the amount of hydrogen peroxide is measured by adding POD and a reducing agent that develops color by oxidation to the sample obtained in the second step, and measuring the color intensity of the reducing agent. .
POD suitable in this case is derived from horseradish, microorganisms and the like. Moreover, the suitable use density | concentration of POD is 0.01-100 units / mL.
As a suitable method for measuring hydrogen peroxide in the third step, a coupler (such as 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone (MBTH)) in the presence of POD is used. On the other hand, a Trinder reagent that produces a dye by oxidative condensation reaction of a phenolic, aniline, or toluidine chromogen, and a leuco dye that directly oxidizes and colors in the presence of POD can be used. These methods are known to those skilled in the art and can be readily used in general.
Although it does not limit as a Trinder reagent, For example, phenol and its derivative (s) can be used.
Examples of couplers that can be suitably used in the third step include 4-aminoantipyrine, aminoantipyrine derivatives, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH), and sulfonated methylbenzthiazolinone hydrazone (SMBTH). Can be mentioned.
Although it does not specifically limit as a leuco pigment | dye which can be used conveniently in a 3rd process, For example, a triphenylmethane derivative, a phenothiazine derivative, a diphenylamine derivative, etc. can be used.
In the measurement of the amount of hydrogen peroxide in the third step, in addition to the color development method using POD or the like, measurement methods using various sensor systems are generally known to those skilled in the art. 2001-204494). Specifically, examples of electrodes used in various sensor systems include oxygen electrodes, carbon electrodes, gold electrodes, and platinum electrodes. In the present invention, a buffer solution under conditions suitable for the present invention is used together with a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / Cl electrode) using these electrodes on which an enzyme is immobilized as a working electrode. While being inserted and kept at a constant temperature, a constant voltage can be applied to the working electrode, and a sample can be added to measure the increase in current due to hydrogen peroxide resulting from the enzyme reaction.
Further, as a method for measuring by an amperometric system using a carbon electrode, a gold electrode, a platinum electrode, or the like, there is a system using an immobilized electron mediator. For example, an enzyme and an electron mediator such as potassium ferricyanide, ferrocene, osmium derivatives, and phenazine methosulfate are adsorbed or immobilized on a polymer matrix by a covalent bond method as a working electrode, and a counter electrode (for example, a platinum electrode) Etc.) and a reference electrode (for example, an Ag / AgCl electrode, etc.) are inserted into a buffer solution under conditions suitable for the present invention and kept at a constant temperature. Subsequently, a constant voltage can be applied to the working electrode, and a sample can be added to measure the increase in current due to hydrogen peroxide resulting from the enzyme reaction.
The amount of saccharified amine can also be measured by measuring the amount of oxygen consumed in the third step (for example, but not limited to, see JP-A-2001-204494). Specifically, an oxygen electrode is used, oxygen is immobilized on the electrode surface, inserted into a buffer solution under conditions suitable for the present invention, and kept at a constant temperature. A sample is added here, and the decrease value of the current is measured.
When amperometric measurement is performed using a carbon electrode, a gold electrode, a platinum electrode, etc., these electrodes on which an enzyme is immobilized are used as the working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, the like) , Ag / AgCl electrode, etc.), and the increase in current including the mediator is measured. As the mediator, potassium ferricyanide, ferrocene, osmium derivatives, phenazine methosulfate, and the like can be used.
In the measurement method of the present invention, an inactive protein may be added to the system for performing each step in order to increase the stability of the protein of the present invention. Inactive proteins include serum albumins, globulins and fibrous proteins. A preferred protein is bovine serum albumin, with a preferred concentration in wt / vol being 0.05-1%. Preferred inactive proteins are those that do not contain protease impurities that cause enzymatic degradation.
The measurement of fructosyl amino acid concentration is performed using a specific volume of the sample and a specific volume of the reagent. Absorbance measurement is performed as soon as possible after mixing and before significant absorbance changes due to fructosyl amino acids occur to measure the sample blank. A first absorbance measurement after 0.5-5 seconds is appropriate. The second absorbance measurement is typically 3-5 minutes at 37 ° C. at a fructosyl amino acid concentration of 1 mg / dL after the absorbance has become steady. Typically, the reagent is standardized with an aqueous or serum solution having a known fructosyl amino acid concentration.

<7. Glycated protein measurement kit and glycated protein measurement sensor>
(7-1) Glycated protein measurement kit The glycated protein measurement kit according to the present invention (hereinafter referred to as “kit of the present invention”) is characterized by comprising at least the reagent composition according to the present invention. Although the form of the reagent composition according to the present invention provided in the kit of the present invention is not particularly limited, for example, a form such as an aqueous solution, a suspension or a lyophilized powder can be adopted. The lyophilized powder can be made according to conventional methods.
The method for blending the above-mentioned additives is not particularly limited. For example, a method of blending an additive in a buffer solution containing a protein according to the present invention, a method of blending a protein according to the present invention into a buffer solution containing an additive, or a protein and a stabilizer according to the present invention in a buffer solution Although the method of mix | blending simultaneously is mentioned, It is not limited to these.
The kit of this invention may be comprised by the reagent composition provided with the protein which concerns on this invention, peroxidase, and a chromogen at least.
In the measurement method of the present invention, a peroxidase reaction is used to detect hydrogen peroxide derived from oxidation of fructosyl amino acids. Therefore, peroxidase and chromogen (hydrogen peroxide coloring reagent) are preferably used for the reagent composition. The peroxidase and chromogen (hydrogen peroxide coloring reagent) are not limited at all.
The chromogen (hydrogen peroxide coloring reagent) used in the present invention is preferably stable in solution and low in bilirubin interference. Examples of the chromogen (hydrogen peroxide coloring reagent) that can be preferably used in the present invention include 4-aminoantipyrine or 3-methyl-2-benzothiazoline hydrazone (MBTH) and phenol or a derivative thereof or aniline or a derivative thereof. The reagent which consists of these combinations is mentioned. The chromogen (hydrogen peroxide coloring reagent) used in the present invention may be benzidines, leuco dyes, 4-aminoantipyrine, phenols, naphthols and aniline derivatives.
The peroxidase preferably used in the present invention is preferably a horseradish peroxidase. This peroxidase is commercially available in high purity and low cost. The enzyme concentration must be high enough for a rapid and complete reaction, preferably 1,000 to 50,000 U / L.
In addition to the above configuration, the other reagent composition may contain a buffer (for example, borate buffer, phosphate buffer, Tris-HCl buffer, GOOD buffer, etc.). Furthermore, the reagent composition includes chelating reagents (such as EDTA and O-dianisidine) that capture ions that interfere with the enzyme reaction, ascorbic acid oxidase that eliminates ascorbic acid, which is a substance that interferes with the determination of hydrogen peroxide, and various interfaces. Active agents (such as Triton X-100 and NP-40) as well as various antibacterial and preservatives (such as streptomycin and sodium azide) and the like may be included. These reagents may be a single reagent or a combination of two or more reagents.
Although it does not specifically limit as a buffering agent, Arbitrary buffering agents which have sufficient buffering ability in the pH range of 6-8.5 can be used. Buffers in this pH range include phosphate, tris, bis-trispropane, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 2-morpholinoethanesulfonic acid monohydrate (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (piperazine-1,4-bis (2-ethanesulfonic acid)) (PIPES), 2- [4- (2-hydroxyethyl) -1 -Piperazinyl] ethanesulfonic acid (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) (HEPES), and 3- [N-tris (hydroxymethyl) methylamino] -2-hydroxypropanesulfonic acid (TAPSO) Particularly preferred buffering agents are MES and PIPES. In particular, the preferred concentration range is 20 to 200 mM, and the preferred pH range is pH 6 to 7.
The kit of the present invention includes, for example, FAOD, buffer solution, protease, POD, coloring reagent, chelating reagent that captures ions that interfere with the enzyme reaction, ascorbic acid that eliminates ascorbic acid that interferes with the determination of hydrogen peroxide. Includes oxidases, surfactants, stabilizers, excipients, antibacterial agents, preservatives, well plates, fluorescent scanners, automatic analyzers, and instruction manuals describing the measurement method according to the present invention on paper media It may be.
The kit of the present invention can be used for the method for measuring fructosyl amino acids and the measurement of glycated protein according to the present invention, and can be particularly preferably used for measuring glycated hemoglobin.

(7-2) Glycated protein measurement sensor A glycated protein measurement sensor according to the present invention (hereinafter referred to as “sensor of the present invention”) is a sensor used for detecting a glycated protein, and includes at least the protein according to the present invention. Yes. The sensor of the present invention is used for carrying out the measuring method of the present invention. In particular, it can be suitably used for measurement of glycated hemoglobin. Therefore, the sensor of this invention may be comprised by the article | item used for implementation of the measuring method of this invention. For the description of the article, the description of the buffering agent and the like in the section of the measurement method of the present invention and the kit of the present invention can be used.
One embodiment of the sensor of the present invention can be used with the protein according to the present invention immobilized on a support. The support is not particularly limited as long as it can immobilize the protein according to the present invention, and a shape and material suitable for the properties of the protein may be used. The shape of the support is not particularly limited as long as it has a sufficient area for immobilizing proteins, and examples thereof include substrates, beads, and membranes. Examples of the support material include inorganic materials, natural polymers, and synthetic polymers.
Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference.

<1. Activity measuring reagent>
Table 1 shows the compositions of the activity measuring reagents used in the following examples. The reagents used in the examples were those purchased from Nacalai Tesque unless otherwise specified.

<2. Activity measurement method>
The method for measuring FAOD activity is shown below.
The enzyme activity of FAOD for each substrate was measured by increasing the absorbance of the dye produced by the peroxidase reaction using hydrogen peroxide generated by the enzyme reaction.
First, 3 ml of the activity measurement reagent was heated at 37 ° C. for 5 minutes, and then the activity measurement reagent was preliminarily added to the enzyme diluent (50 mM potassium phosphate buffer (pH 6.5) containing 0.1% Triton X100). The reaction was started by adding 0.1 ml of the diluted enzyme solution.
After reacting at 37 ° C. for 5 minutes, the change in absorbance at 500 nm per unit time was measured (ΔOD _test / min). As a blind test, 0.1 ml of enzyme dilution solution was used in place of the enzyme solution, and the change in absorbance was measured by performing the same operation (ΔOD _blank / min).
Based on the following calculation formula, the enzyme activity was calculated from the change in absorbance obtained. The amount of enzyme that oxidizes 1 micromole of substrate per minute under the above conditions was defined as 1 unit (U).
(a formula)
Activity value (U / ml) = {((ΔOD _test / min) − (ΔOD _blank /min))×3.1 ml × dilution factor} / (13 × 1.0 cm × 0.1 ml)
In the above formula, “3.1 ml” represents the total liquid volume, “13” represents the millimolar extinction coefficient, “1.0 cm” represents the optical path length of the cell, and “0.1 ml” represents the enzyme sample. Indicates the liquid volume.

<3. Substrate specificity evaluation method>
The method for evaluating the substrate specificity of FAOD is described below.
An activity measurement reagent was prepared so that the concentration of each substrate (FK: fructosyl lysine, FV: fructosyl valine, F-VH: fructosyl valyl histidine) was 2 mM, and the activity measurement method described above The activity value ratio (substrate specificity) was calculated according to the following formula.
(F−V / F−VH) = (activity value when FV is used as a substrate) ÷ (activity value when F−VH is used as a substrate)
(F-K / F-VH) = (activity value when F-K is used as substrate) / (activity value when F-VH is used as substrate)
In addition, it is determined that the smaller the activity value ratio defined by the formula is, the better the specificity for F-VH is.

<5. Cloning of Fructosylvalylhistidine Oxidase Gene>
In order to perform cDNA cloning of fructosylvalyl histidine oxidase from Phaeosphaeria nodorum, the genome database of Phaeosphaeria nodorum (http: //www.ncbi.nlm.nih.govil/stable_genctable=genctomgtable=genomgtable=genctomg32=genctomgtable=genctomgtable=genctomgeb32) Searched for.
A gene predicted to encode FAD-dependent oxidoreductase was extracted from the genome database. As a result of further narrowing down from the viewpoint of having the possibility of encoding an enzyme that substantially acts on fructosyl valyl histidine among various genes obtained from the search results, GenBank no. AAGI01000177239512 bp Phaeosphaeria nodorum SN15 cont1.177, whole genome shotgun Sequence (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?tool=portal&db=nuccore&term=&query%5Fkey=11&dopt=gb&dispmax=20&page= 1 & qty = 1 & WebEnv = 0x3fThZQ1ubv2E4QutcTFzS2yHOV3ljOuR6kUwGyNOVEiqCGdFac8yYAlfzZG% 2DvpBxXqNrPNESW3sk% 402645540Fv The gene corresponding to the sequence of 76,625 bp from 75,143 bp of 1) was cloned candidate genes.
Next, with respect to the cDNA of the above cloning candidate gene, use of a splicing site (exon-intron) prediction tool (http://www.fruitfly.org/seq_tools/splice.html) By comparing with the tosyl amino acid oxidase gene, the entire cDNA sequence corresponding to the protein coding portion was deduced. Based on this information, the total cDNA sequence was synthesized by total synthesis by PCR of gene fragments, which is a conventional method, that is, by performing reverse transcription through preparation of total RNA and extraction of mRNA. The cDNA was blunted using Blunting high (Toyobo Co., Ltd.) and subcloned into the SmaI site of pUC118. As a result of sequence analysis of the cDNA portion, the base sequence shown in SEQ ID NO: 2 was obtained. The coding region of cDNA in the base sequence shown in SEQ ID NO: 2 is from the first base to the 1314th base, the start codon is from the first base to the third base, and the stop codon TAG is from the 1312th base to the 1314th base. It corresponds to the base of The amino acid sequence clarified from the base sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1.

<6. Large-scale expression and purification of fructosyl valyl histidine oxidase gene in Escherichia coli, enzyme assay>
A large-scale expression in Escherichia coli of a plasmid containing the fructosyl valyl histidine oxidase gene having the base sequence shown in SEQ ID NO: 1 was attempted. The full-length cDNA region excluding the stop codon of the fructosyl valyl histidine oxidase gene (from the first base to the 1311th base of the base sequence shown in SEQ ID NO: 2) was amplified by PCR. At that time, an NdeI cleavage site was inserted using the primer P1 (5′-GGAATTCCATATGGCGCCCTCCAGAGCAAACACCAGGTTCATT-3 ′) (“CATATG” in the above base sequence is an NdeI site) shown in SEQ ID NO: 3 on the N-terminal side of the amino acid sequence, An XhoI cleavage site was introduced using primer P2 (5′-CCGCTCCGAGCAAGTTCGCCCTCGGCTTATCATGATTCCAACC-3 ′) (“CTCGAG” in the above base sequence is an XhoI site) shown in SEQ ID NO: 4 on the side. This DNA fragment was subcloned into the NdeI-XhoI site of the pET-23b vector (Novagen) so as to be in the forward direction with the T7 promoter. The prepared plasmid was named pIE353 as a fructosyl valyl histidine oxidase expression plasmid.
Escherichia coli BL21 CodonPlus (DE3) -RIL (Stratagene) was transformed with the plasmid pIE353. Subsequently, transformant BL21 CodonPlus (DE3) -RIL (pIE353) showing ampicillin resistance was selected.
As a medium for culturing the transformant, 200 ml of TB medium was dispensed into a 2 L Sakaguchi flask and autoclaved at 121 ° C. for 20 minutes. After allowing the medium to cool, ampicillin, which was separately sterile filtered, was added to the medium to a final concentration of 100 μg / ml. This medium was inoculated with 2 ml of a culture solution of BL21 CodonPlus (DE3) -RIL (pIE353) previously cultured for 16 hours at 30 ° C. in an LB medium containing 100 μg / ml ampicillin.
Subsequently, aeration and agitation culture was performed at 30 ° C. for 24 hours. After completion of the culture, the cells were collected by centrifugation, suspended in 50 mM potassium phosphate buffer (pH 7.5), and the cells were sonicated.
Next, the suspension was centrifuged to obtain a supernatant as a crude enzyme solution. It measured using the enzyme activity measuring reagent which contains fructosyl valyl histidine as a substrate, and confirmed having fructosyl valyl histidine oxidase activity. The obtained crude enzyme solution was purified using MagExtractor-His-tag- (manufactured by Toyobo) according to a prescribed protocol to obtain a purified enzyme preparation (IE353). The IE353 standard obtained by this method was confirmed to be single by SDS polyacrylamide gel electrophoresis.
The enzyme activity of the IE353 preparation was measured using an enzyme activity measuring reagent containing fructosyl valyl histidine as a substrate, and confirmed to have fructosyl valyl histidine oxidase activity.

<7. Construction, purification, and enzyme assay of a protein having fructosyl valyl histidine oxidase activity by functional modification of the fructosyl valyl histidine oxidase gene>
Using the expression plasmid pIE353 and a synthetic oligonucleotide (SEQ ID NO: 5 and 6) designed so that the 58th isoleucine of the amino acid sequence shown in SEQ ID NO: 1 is randomly substituted with another amino acid, KOD-Plus Site- Using Directed Mutagenesis Kit (manufactured by Toyobo Co., Ltd.), a mutation treatment operation was performed according to a prescribed protocol, and the nucleotide sequence was confirmed. Although the base sequences of 100 recombinant plasmids were confirmed, since the 58th isoleucine of the amino acid sequence shown in SEQ ID NO: 1 is randomly substituted, the type of encoded amino acid is the same isoleucine as the wild type A plasmid substituted with 19 amino acids except (I) was obtained. At the same time, BL21 CodonPlus (DE3) -RIL was transformed with these 100 plasmids, and then transformants showing ampicillin resistance were selected, and only those having fructosylvalylhistidine oxidase activity were selected.
For example, a base sequence (att) encoding the 58th amino acid sequence shown in SEQ ID NO: 1 was substituted with a base sequence (acc) encoding threonine (T) (pIE353-I58T). The purified enzyme preparation obtained from the plasmid is referred to as IE353-I58T. (The same is applied to other cases shown below.)
Similarly, a plasmid (pIE353-I58A) substituted with a base sequence (gct) encoding alanine (A), a plasmid (pIE353-I58V) substituted with a base sequence (gtc) encoding valine (V), leucine ( L) a plasmid (pIE353-I58L) substituted with a nucleotide sequence (ctc) encoding, a plasmid (pIE353-I58M) substituted with a nucleotide sequence (atg) encoding methionine (M), and a phenylalanine (F) A plasmid (pIE353-I58F) substituted with a base sequence (ttc), a plasmid (pIE353-I58S) substituted with a base sequence (tcg) encoding serine (S), and a base sequence encoding glutamine (Q) ( caa) substituted plasmid (pIE353-I58) ), A plasmid (pIE353-I58C) substituted with a base sequence (tgc) encoding cysteine (C), a plasmid (pIE353-I58Y) substituted with a base sequence (tac) encoding tyrosine (Y), asparagine ( N) A plasmid (pIE353-I58N) substituted with a base sequence (aac) encoding, a plasmid (pIE353-I58K) substituted with a base sequence (aag) encoding lysine (K), and a histidine (H) A plasmid (pIE353-I58H) substituted with the base sequence (cac), a plasmid (pIE353-I58R) substituted with the base sequence (cgc) encoding arginine (R), and a base sequence encoding aspartic acid (D) (Gac) substituted plasmid (pIE353- 58D), a plasmid (pIE353-I58E) substituted with a base sequence (gaa) encoding glutamic acid (E), a plasmid (pIE353-I58W) substituted with a base sequence (tgg) encoding tryptophan (W), proline A plasmid (pIE353-I58P) substituted with the base sequence (ccc) encoding (P) was obtained.
BL21 CodonPlus (DE3) -RIL was transformed with each of the obtained plasmids, and then transformants showing ampicillin resistance were selected.
In the same manner as in Reference Example 2, each transformant was cultured, and the resulting crude enzyme solution was purified to obtain each purified enzyme preparation (IE353-I58T, IE353-I58A, IE353-I58V, IE353-I58L). IE353-I58I, IE353-I58M, IE353-I58F, IE353-I58S, IE353-I58Q, IE353-I58C, IE353-I58Y, IE353-I58N, IE353-I58K, IE353-I58H, IE353-I58R, IE353E35D -I58E, IE353-I58W, IE353-I58P). Each sample obtained by this method was confirmed to be single by SDS polyacrylamide gel electrophoresis.

<8. Characterization of each fructosyl valyl histidine oxidase protein>
Wild type fructosyl valyl histidine oxidase (IE353), and fructosyl valyl histidine oxidase variants (IE353-I58T, IE353-I58A, IE353-I58V, IE353-I58L, IE353-I58I, IE353-I58M, IE353-I58F, (IE353-I58S, IE353-I58Q, IE353-I58C, IE353-I58Y, IE353-I58N, IE353-I58K, IE353-I58H, IE353-I58R, IE353-I58D, IE353-I58E, IE353-I58W, IE353-I58P) As a result of measuring the enzyme activity of the enzyme preparation using an activity measuring reagent, the one having fructosyl valyl histidine oxidase activity is IE353, E353-I58T, IE353-I58M, IE353-I58S, IE353-I58V, IE353-I58A, a IE353-I58N, IE353-I58L, was performed substrate specificity evaluation. The results are shown in Table 2.

  As is clear from the results in Table 2, the mutant obtained by substituting the 58th isoleucine of the amino acid sequence shown in SEQ ID NO: 1 with another amino acid was compared to the FV / V / V-type fructosyl varishistidine oxidase protein. F-VH and F-K / F-VH are reduced.

<9. Measurement of fructosylvalylhistidine with a modified fructosylvalylhistidine oxidase>
Next, it was confirmed whether fructosyl valyl histidine can be measured with a modified fructosyl valyl histidine oxidase (IE353-I58T).
<Reaction solution composition>
50 mM MES (Doujin Chemical) pH 6.5
0.1% Triton X100 (manufactured by Nacalai Tesque)
・ 02% 4-aminoantipyrine (manufactured by Nacalai Tesque)
・ 01% TOOS (manufactured by Nacalai Tesque)
20 U / mL horseradish peroxidase (trade name PEO-301, manufactured by Toyobo)
1U / mL IE353-I58T
MES: 2-morpholinoethanesulfonic acid monohydrate TOOS: N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine <Reaction procedure>
After adding 10 μL of a sample (each concentration fructosyl valyl histidine aqueous solution) to 300 μL of the reaction solution which has been preliminarily set to 37 ° C., the reaction is performed after exactly 1 minute. The absorbance at 546 nm was measured before the sample addition and 1 minute after the sample addition, and the absorbance change (ΔAbs / min) for 1 minute was calculated.
The results are shown in FIG.
As is clear from FIG. 1, there is a correlation between the fructosyl valyl histidine to be measured and the change in absorbance, and the fructosyl valyl histidine can be quantified using a modified fructosyl valyl histidine oxidase (IE353-I58T). it can.

<10 (comparative example). Evaluation of the effect of fructosyl valine on the measurement of fructosyl valyl histidine using wild-type fructosyl valyl histidine oxidase>
8. Using wild type fructosyl valyl histidine oxidase (IE353) A reagent for measuring fructosyl valyl histidine was prepared with the same composition as the above. Using this reagent, a sample: 0.8 mM fructosyl valyl histidine further added with each concentration of fructosyl valine was measured, and the degree of influence of each fructosyl valine was compared. The result is 11. The results are shown in FIG.
<Evaluation method of impact>
Influence (%) = ((measured value with added FV) − (measured value without added FV)) / (measured value without added FV) × 100

<11. Evaluation of the influence of fructosyl valine in the measurement of fructosyl valyl histidine using a modified fructosyl valyl histidine oxidase>
9. Using a modified fructosyl valyl histidine oxidase (IE353-I58T) A reagent for measuring fructosyl valyl histidine was prepared with the same composition as the above. Using this reagent, a sample: 0.8 mM fructosyl valyl histidine further added with each concentration of fructosyl valine was measured, and the degree of influence of each fructosyl valine was compared. The result is 10. The results are shown in FIG.
<Evaluation method of impact>
Influence (%) = ((measured value with added FV) − (measured value without added FV)) / (measured value without added FV) × 100

  As is clear from FIG. 2, IE353-I58T has a reduced influence of fructosyl valine compared to the wild type. Therefore, if a FAOD having a low F-V / F-VH is used, the effect of fructosyl valine can be reduced.

  According to the present invention, a fructosyl valyl histidine oxidase can be provided by using a fructosyl valyl histidine oxidase having a low effect on fructosyl valine. Can be used to provide a method for measuring fructosyl valyl histidine. Therefore, the present invention can be widely used in industries in the life science field including the clinical examination field based on preventive medicine, the diagnostic medical field, the pharmaceutical field, and the health medical field.

Claims (8)

A glycated protein measurement reagent composition comprising a protease and fructosyl amino acid oxidase, wherein the fructosyl amino acid oxidase has the following property (1):
(1) The reactivity with respect to fructosyl valine is 35 or less when the activity value with respect to fructosyl valyl histidine is set to 10. A reagent for measuring a glycated protein comprising a protease and fructosyl amino acid oxidase, wherein the fructosyl amino acid oxidase has the following property (2):
(2) The reactivity with fructosyl valine is 1 or less when the activity value with respect to fructosyl valyl histidine is 2. The reagent composition according to claim 1, wherein the fructosyl amino acid oxidase has any of the following properties (a) to (c).
(A) the amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which isoleucine at position 58 from the amino terminus is substituted with another amino acid, and has fructosyl valyl histidine oxidase activity;
(B) Amino acid sequence (a) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added at a site other than the 58th amino acid from the amino terminus, and has fructosyl valyl histidine oxidase activity ;
(C) an amino acid sequence having 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid corresponding to the 58th isoleucine from the amino terminus in the amino acid sequence shown in SEQ ID NO: 1 is the amino acid A protein substituted with a different amino acid and having fructosyl valyl histidine oxidase activity.   In the amino acid substitution shown in SEQ ID NO: 1, the 58th glycine from the amino terminus is substituted with any one selected from the group consisting of methionine, threonine, alanine, asparagine, serine, valine and leucine. Item 4. The reagent composition according to Item 1.   The reagent composition according to any one of claims 1 to 4, wherein the glycated protein is HbA1c.   A glycated protein measurement method using the reagent composition according to claim 1.   A glycated protein measurement kit comprising the reagent composition according to claim 1.   A glycated protein measurement sensor comprising the reagent composition according to claim 1.