JP2008099649A - Quinol peroxidase and gene thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new quinol peroxidase using, as a base, quinol physiologically or artificially carrying out electron-transporting reaction in the respiratory chain of an organism; and to provide a gene encoding the new peroxidase. <P>SOLUTION: The new quinol peroxidase (QPO) is isolated and purified by culturing eubacteria of Actinobacillus actinomycetemcomitans ATCC 29522. The QPO gene is obtained by extracting a chromosome DNA from the microbial strain, and carrying out PCR by designing a primer based on the sequence information of the N-terminal amino acid of the purified QPO and the whole chromosome DNA sequence of well-known Actinobacillus actinomycetemcomitans HK5651 strain. The QPO amino acid sequence comprises a sequence having high homology with a cytochrome c peroxidase derived from an existing eubacterium, and a sequence specific to the QPO amino acid sequence. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

Detailed Description of the Invention

The present invention relates to a protein having quinol peroxidase activity derived from the true bacterium Actinobacillus actinomycetemcomitans ATCC 29522, a gene encoding the protein, and the like.

Peroxidase, also called peroxidase, is an enzyme that oxidizes various compounds in the presence of hydrogen peroxide. It can also be said to be an enzyme that reduces hydrogen peroxide with various compounds. Peroxidase is known to be widely distributed in the animal kingdom, plant kingdom, and microbial kingdom, and has already been put into practical use as various analytical reagents such as various enzyme antibody labels. In addition, organochlorine compounds in various waste liquids and wastewater, environmental hormones, etc. It is expected to be used in a wide range of applications such as decomposition / removal of pulp, decolorization of pulp waste liquid, prevention of color transfer of laundry, synthesis of phenol resin, and prevention of food deterioration. Among such peroxidases, peroxidases derived from various microorganisms have been searched in recent years. For example, the basidiomycete Coprinus sinerius (patent document 1), oyster mushroom (non-patent document 1), horotake (patent document 2), white Funerocaete chrysosporium (Non-patent Document 2), Funerocaete Saldida (Non-patent document 3), fungus Alsulomyces ramos (Non-patent document 3), Ascomycete geotricum candyum (Non-patent document 4), Kofukisarokoshikake (Non-Patent Document 5), Kawaratake (Non-Patent Document 6), Seripoliopsis Svemispora (Non-Patent Document 7), Daikomitas Squalens (Non-Patent Document 8), Flavia Radiata (Non-Patent Document 9), And the true bacterium Flavobacterium meningocepticum (non-special It can be mentioned peroxidase derived from literature 10).

JP-A-3-1949 JP-A-8-70862 JP 2000-152786 A Biochim. Biopys. Acta, 1251, 205-209, 1995 Gene, 93, 119-124, 1990 Agric. Biol. Chem, 50, 247, 1986 Journal of Bioscience and Bioengineering, 87, 411, 1999 Biotech. Lett. 23, 103, 2001 Biochem. Biophys. Res. Commun, 179, 428-435, 1991 Gene, 206, 185-193, 1998 Biochim. Biophys. Acta, 1434, 356-364, 1999 Gene, 85, 343-351, 1989 Biochim. Biophys. Acta, 1435, 117-126

Among such peroxidases, cytochrome c peroxidase is a peroxidase that reduces hydrogen peroxide in the presence of reduced cytochrome c, and includes germinating yeast (Non-patent Document 11) and true bacteria Pseudomonas aeruginosa (Non-patent Document). 12) Cytochrome c derived from Pseudomonas nautica (Non-patent document 13), Rhodobacter capslatas (Non-patent document 14), Nitrosomonas europair (Non-patent document 15), Methylococcus capslatas (Non-patent document 16), etc. Peroxidase is known.

J. et al. Biol. Chem. 267, 21802-21807, 1992 Acta Chem. Scand. , 26, 2535-2537, 1972 Biochim. Biophys. Acta, 1434, 248-259, 1999 Eur. J. et al. Biochem. , 258, 29-36, 1998 J. et al. Biol. Chem. , 269, 11878-11886 Arch. Microbiol. , 168, 362-372, 1997

Among such cytochrome c peroxidases, those derived from budding yeast and those derived from true bacteria are greatly different in amino acid sequence and enzymatic properties. The former is a protein having 294 amino acids and one heme b. The amino acid sequence is homologous to many other peroxidases, whereas the latter is a protein that covalently binds 300 to 400 amino acids and two heme c, and is distinct from other peroxidases. It consists of an amino acid sequence.

Problems to be solved by the invention

An object of the present invention is to elucidate the base sequence of a gene encoding a novel quinol peroxidase derived from Actinobacillus actinomycetemcomitans ATCC 29522 and to provide a means for more widely utilizing its useful function. It is in.

Means for solving the problem

A novel peroxidase was isolated from Actinobacillus actinomycetemcomitans ATCC 29522 and its enzymatic properties were determined. It was found that it has peroxidase activity using ubiquinol-1 as a substrate, unlike all known peroxidases. I found it. The QPO gene consists of a sequence highly homologous to cytochrome c peroxidase derived from an existing true bacterium and a sequence specific to the QPO gene. The present invention has been completed based on these findings.

That is, the present invention relates to (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or (b) an amino acid wherein one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2. DNA consisting of a sequence and encoding a protein having quinol peroxidase activity (Claim 1), DNA consisting of a base sequence shown in SEQ ID NO: 1 or a complementary sequence thereof, or a sequence containing a part or all of these sequences (Claim 2), DNA that hybridizes with the DNA of Claim 2 under stringent conditions and that encodes a protein having peroxidase activity (Claim 3), and the amino acid sequence shown in SEQ ID NO: 2 In the protein (Claim 4) and the amino acid sequence shown in SEQ ID NO: 2, The few amino acids are deleted, substituted or added in the amino acid sequence, and a protein having a quinol peroxidase activity (claim 5).

Further, the present invention specifically binds to a fusion protein (claim 6) in which the protein according to claim 4 or 5 is bound to a marker protein and / or a peptide tag, or the protein according to claim 4 or 5. An antibody (Claim 7), an antibody according to Claim 7 (Claim 8), wherein the antibody is a monoclonal antibody, or a recombinant vector (Claim) according to any one of Claims 1 to 3 Item 9), a host cell comprising an expression system capable of expressing the protein according to claim 4 or 5 (claim 10), or a host cell comprising the expression system according to claim 10 in a medium. The present invention relates to a method for producing a recombinant protein (Claim 11), which comprises culturing and collecting a recombinant protein having quinol peroxidase activity from the resulting culture.

Examples of the DNA of the present invention include DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, and one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2. DNA encoding an amino acid sequence that encodes a protein having quinol peroxidase activity, DNA consisting of the base sequence shown in SEQ ID NO: 1 or a complementary sequence thereof, or a sequence containing a part or all of these sequences Here, the protein having quinol peroxidase activity may be a peroxidase activity by using quinol as a substrate that can physiologically or artificially bear an electron transfer reaction in the respiratory chain of an organism. Qualitative protein.

As used herein, “quinol capable of physiologically or artificially responsible for an electron transfer reaction in the respiratory chain of a living organism” means benzoquinol represented by ubiquinol-n, rhodoquinol-n, plastquinol-n, and tocokinol- n, menaquinol-n, demethylmenaquinol-n, naphthoquinol represented by phyloquinol-n, and derivatives thereof.

Ubiquinol-n is represented by the following formula:

In the formula, n represents the number of isoprene units in the side chain, and 0 to 10 is preferably used.

Rhodoquinol-n is represented by the following formula:

In the formula, n represents the number of isoprene units in the side chain, and 0 to 10 is preferably used.

Plastokinol-n is represented by the following formula:

In the formula, n represents the number of isoprene units in the side chain, and 0 to 10 is preferably used.

Tocokinol-n is represented by the following formula:

In the formula, n represents the number of isoprene units in the side chain, and 0 to 10 is preferably used.

Menaquinol-n is represented by the following formula:

In the formula, n represents the number of isoprene units in the side chain, and 0 to 10 is preferably used.

Demethylmenaquinol-n is represented by the following formula:

In the formula, n represents the number of isoprene units in the side chain, and 0 to 10 is preferably used.

Philoquinol-n is represented by the following formula:

In the formula, n represents the number of isoprene units in the side chain, and 3 is preferably used.

In addition, hybridization with various DNA libraries under stringent conditions using DNA consisting of the base sequence shown in SEQ ID NO: 1 or a complementary sequence thereof and a sequence containing a part or all of these sequences as a probe. Then, DNA encoding a protein having a novel quinol peroxidase activity can be obtained by isolating DNA that hybridizes to the probe. The DNA thus obtained is also encompassed by the present invention. Examples of hybridization conditions for obtaining the DNA of the present invention include hybridization at 42 ° C. and washing treatment at 42 ° C. with a buffer containing 1 × SSC and 0.1% SDS. More preferably, hybridization at 65 ° C. and washing treatment at 65 ° C. with a buffer containing 0.1 × SSC and 0.1% SDS can be mentioned. In addition, there are various factors other than the above temperature conditions as factors affecting the stringency of hybridization, and those skilled in the art will be able to combine various components as appropriate to achieve the same stringency of hybridization as exemplified above. Stringency can be realized.

These DNAs of the present invention are known based on the DNA sequence information disclosed in the present invention, for example, from genomic genes such as Actinobacillus actinomycetemcomitans ATCC 29522 strain and the like such as PCR or hybridization using DNA fragments as probes. It can be prepared by a method. In addition, it can be prepared by chemical synthesis. In order to obtain the DNA of the present invention encoding an artificial mutant protein having quinol peroxidase activity from the DNA of the present invention, that is, the artificial mutant DNA of the present invention at the base sequence level or amino acid sequence level, Mutation means may be used. For example, the mutation can be easily introduced by using a commercially available mutagenesis kit. Further, proteins lacking the first methionine (Met) contained in the amino acid sequence and those lacking the transmembrane region described later are also included in the protein due to the mutation of the amino acid sequence.

The protein of the present invention comprises a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 and an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, The protein of the present invention is not particularly limited as long as it has a quinol peroxidase activity, and the protein of the present invention can be prepared by a known method based on the DNA sequence information and the like, and its origin is not particularly limited. Absent.

The fusion protein of the present invention may be any protein as long as the protein of the present invention is bound to a marker protein and / or a peptide tag. The marker protein is a conventionally known marker protein. There is no particular limitation as long as it is, for example, alkaline phosphatase, antibody Fc region, GFP and the like. Specific examples of peptide tags in the present invention include Myc tag, His tag, FLAG tag, GST. Conventionally known peptide tags such as tags can be specifically exemplified. Such a fusion protein can be prepared by a conventional method, for purification of a protein having quinol peroxidase activity utilizing the affinity between Ni-NTA and His tag, for quantifying an antibody against a protein having quinol peroxidase activity, etc. And also useful as a research reagent in this field.

Specific examples of antibodies that specifically bind to the protein of the present invention include immunospecific antibodies such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and the like. Although all or part of the protein, fusion protein, etc. of the present invention can be prepared by a conventional method, a monoclonal antibody is more preferable in terms of its specificity. Such an antibody such as a monoclonal antibody is useful for elucidating the characteristics of quinol peroxidase and its production mechanism.

The above-described antibodies of the present invention are produced by administering the protein of the present invention or a fragment thereof containing an epitope to an animal (preferably a non-human) using a conventional protocol. Hybridoma method (Nature 256, 495-497, 1975), trioma method, human B-cell hybridoma method (Immunology Today 4,72, 1983) and EBV-hybridoma method (MONOCLONAL method) resulting in antibodies produced by cell line cultures ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) can be used.

Examples of antibodies such as the monoclonal antibody include fluorescent substances such as FITC (fluorescein isocyanate) or tetramethylrhodamine isocyanate, radioisotopes such as ¹²⁵ I, ³² P, ¹⁴ C, ³⁵ S or ³ H, alkaline phosphatase, The functional analysis of the protein of the present invention can be performed by using a fusion protein in which a protein labeled with an enzyme such as β-galactosidase or phycoerythrin or a fluorescent protein such as green fluorescent protein (GFP) is fused. Examples of the immunological measurement method using the antibody of the present invention include RIA method, ELISA method, fluorescent antibody method, plaque method, spot method, hemagglutination method, octalony method and the like.

The recombinant vector of the present invention is not particularly limited as long as it is a vector containing the DNA of the present invention, but preferably includes an expression system capable of expressing the protein of the present invention in a host cell. , Episomal and viral derived expression systems, more specifically bacterial plasmid derived, yeast plasmid derived, papovavirus such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus vector, Examples include vectors derived from bacteriophage, transposon, and combinations thereof, such as those derived from plasmids such as cosmids and phagemids and genetic elements of bacteriophages. This expression system may contain control sequences that not only cause expression but also regulate expression.

The present invention also relates to a host cell comprising an expression system capable of expressing the protein of the present invention. Such a DNA encoding the protein of the present invention and a recombinant vector containing the above expression system are introduced into a host cell by Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd END). , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection. (Transection), microinjection, cationic lipid mediated It can be performed by transfection, electroporation, transduction, scrape loading, bullet introduction, infection, and the like. As host cells, bacterial prokaryotic cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, Staphylococcus, fungal cells such as yeast and Aspergillus, insect cells such as Drosophila S2 and Spodoptera Sf9, L cells, Animal cells such as CHO cells, COS cells, HeLa cells, C127 cells, BALB / c3T3 cells (including mutants lacking dihydrofolate reductase and thymidine kinase), BHK21 cells, HEK293 cells, Bowes malignant melanoma cells, A plant cell etc. can be mentioned.

In addition, as a method for producing the recombinant protein of the present invention, any method can be used as long as a host cell comprising the above expression system is cultured in a medium and a recombinant protein having quinol peroxidase activity is collected from the obtained culture. Such a method may be used, and methods for recovering and purifying the protein of the present invention from cell culture include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, and hydrophobic interaction chromatography. , Known methods including affinity chromatography, hydroxyapatite chromatography, and lectin chromatography, preferably a method using high performance liquid chromatography. The column used for the affinity chromatography is, for example, a column to which an antibody against the protein of the present invention is bound, or when a normal peptide tag is added to the protein of the present invention, The protein of the present invention can be obtained by using a column to which a certain substance is bound. The protein purification method of the present invention can also be applied to peptide synthesis.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited by this Example. Example 1 (Preparation of QPO) [1. Cultivation of Actinobacillus actinomycetemcomitans] Actinobacillus actinomycetemcomitans ATCC 29522 was purchased from the American Type Culture Collection (3% Todd Weight) ) A culture medium (THBYE culture medium) consisting of a culture medium (Difco) and 1.2% yeast extract (THBYE culture medium) and stored was used. Pre-culture was performed by culturing the strain overnight in the presence of 5% CO _{2 at} 37 ° C. using 10 ml of THBYE medium. The cells obtained in the preculture were inoculated into a 3 liter flask containing 3 l of YHBYE medium, and main culture was performed. The main culture was carried out by stationary culture at 37 ° C. in the presence of 5% CO ₂ for 18 hours.

[2. Isolation of QPO] To isolate QPO, cells after 18 hours of main culture are separated into medium components and cells by centrifugation, and the cells are suspended in 50 mM phosphate buffer (pH 8.0). Then, it was crushed by ultrasonic waves (processing time: 30 seconds × 12 times). After disrupting the cells, they were separated into a membrane fraction and a supernatant by ultracentrifugation, and the membrane fraction was further added with 50 mM phosphoric acid containing 0.5% of sucrose monolaurate (manufactured by Nacalai) as a surfactant. The membrane-bound peroxidase was solubilized by suspending in a buffer solution (pH 8.0) and further homogenizing with a homogenizer. The solution was further ultracentrifuged to precipitate a solid, and the supernatant was used as a crude enzyme extract for purification. A column (Macroprep Ceramic Hydroxyapatite Type I (manufactured by Bio-Rad)) in which this crude enzyme extract was equilibrated in advance with a 10 mM phosphate buffer (pH 8.0) containing 5% sucrose monolaurate (Bio-Rad) 1.6 cm × 3 cm), and QPO was bound to the column. After the end of pouring, the column was washed with 10 ml of 10 mM phosphate buffer (pH 8.0). QPO was eluted from the column with a linear gradient from 0.1 M phosphate buffer (pH 8.0) to 1.0 M phosphate buffer (pH 8.0). HiTrap was added to the active fraction to a final concentration of 1.7 M and equilibrated with 0.2 M phosphate buffer (pH 8.0) containing 1.7 M ammonium sulfate and 0.25% sucrose monolaurate in advance. Pheny1 HP (Amasham-Pharmacia Biotech) was poured and QPO was bound to the column. After the end of pouring, the column was washed with 10 ml of the buffer used for equilibration of the column. QPO was eluted from the column by a linear gradient into 0.2M phosphate buffer (pH 8.0) containing 0.0M ammonium sulfate and 0.25% sucrose monolaurate. The active fraction is further poured onto an AF-Red-560M column (manufactured by Tosoh Corporation) equilibrated with 0.2 M phosphate buffer (pH 8.0) containing 0.25% sucrose monolaurate, and the passed QPO is recovered. did. This was further equilibrated with 0.2 M phosphate buffer (pH 8.0) containing 0.25% sucrose monolaurate and 0.3 M NaCl. Hiprep 16/60 Sephacryl 1 S-200HR (manufactured by Amasham-Pharmacia Biotech) And then fractionated. When the active fraction finally obtained was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single band was given.

[3. Affinity of QPO for ubiquinol-n] To 1 ng of purified QPO, 1 ml of a reaction solution [100 mM Tris-HCl buffer (pH 7.5), 80 μM hydrogen peroxide, 20-400 μM ubiquinol-1] was added, and a spectrophotometer DU640 ( The change in quinol peroxidase activity relative to the base mass was measured by following the production of ubiquinone-1 by increasing the absorbance at 278 nm using Beckman). By plotting on the basis of the results to Hans-Woolf plots were calculated _{K m} values for ubiquinol -1 QPO. The results are shown in FIG. As can be seen from FIG. 1, it was revealed that purified QPO has a K _m value of about 107 μM for ubiquinol-1. The K _m for ubiquinol-1 of cytochrome bo and bd, which are quinol oxidases of E. coli, is known to be 48 and 110 μM, respectively, and is similar to the above K _m value of QPO for ubiquinol-1. Therefore, it was considered that the enzyme group using quinol as a substrate shared the quinol of the respiratory chain by having similar affinity for quinol.

Since it is difficult to measure the affinity for “quinol capable of physiologically or artificially responsible for an electron transfer reaction in the respiratory chain of an organism” other than ubiquinol-n, the reaction solution [100 mM Tris-HCl buffer (pH 7) is used. 5), 25 μg / ml membrane fraction, 80 μM hydrogen peroxide], NADH or succinic acid was added to a final concentration of 0.1 mM, and the apparent peroxidase activity (reduction in hydrogen peroxide concentration) was measured. . A PeroxoQuant kit (Pierce) was used for measuring the hydrogen peroxide concentration. In this experiment, NADH and succinic acid are oxidized by NADH: quinone oxidoreductase and succinic acid: quinone oxidoreductase present in the membrane fraction, respectively, and the quinone present in the membrane fraction is reduced to quinol, It becomes a substrate for QPO. As a result of the experiment, peroxidase activity of 341,811 nmol / mg / min could be measured using NADH and succinic acid as substrates. Since the only quinol capable of carrying out an electron transfer reaction in the respiratory chain of Actinobacillus actinomycetemcomitans is demethylmenaquinol-n, the peroxidase activity shown in FIG. It could be said that. From the above experiment, it was shown that the QPO of the present invention can use not only benzoquinol such as ubiquinol-n but also naphthoquinol such as demethylmenaquinol-n as a substrate.

Example 2 (Determination and preparation of QPO gene) N-terminal amino acid sequence analysis of QPO was entrusted to Apro Science and analyzed by a conventional method using Procise 494 cLC (manufactured by Applied Biosystems). As a result, the amino acid sequence of SEQ ID NO: 3 was determined. Based on this N-terminal amino acid sequence, a BLAST search was performed on the entire chromosomal DNA sequence of known Actinobacillus actinomycetemcomitans. As a result, an ORF (open) starting from a sequence that completely matches the amino acid sequence of SEQ ID NO: 3 reading frame), which was designated as the QPO gene.

For obtaining the QPO gene, a PCR (polymerase chain reaction) method was used. Based on the chromosomal DNA sequence, two types of primers were prepared: primer 1: 5′-TGAGCGCTCTAAATATAGATTTAATTACT-3 ′ (SEQ ID NO: 4) and primer 2: 5′-ACAGCGTTTTAAAAACCGCTTG-3 ′ (SEQ ID NO: 5). In 1 μl of chromosomal DNA solution, 0.3 μM primer 1, 0.3 μM primer 2, 0.2 mM dNTP (deoxyribonucleoside triphosphate), 1 mM MgSO ₄ , 1 U KOD (manufactured by TOYOBO), 5 μl 10 × KOD Buffer was added to bring the final volume to 50 μl. PCR was performed using MJ Research PTC-200 (manufactured by MJ Research) under the conditions of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 3 minutes, and 35 cycles.

The resulting reaction solution was separated by agarose electrophoresis, and a DNA fragment of about 1.5 kbp was recovered from the gel using Perfectprep Gel Cleanup (manufactured by Eppendorf). The DNA fragment was ligated to a pZErO-2 vector (manufactured by Invitrogen) using an EcoRV site according to the instruction manual, and Escherichia coli Top10 strain was transformed using this vector, and the transformed Escherichia coli was added to 50 mg / l of kanamycin. Screening was by drug resistance. From the transformant, the plasmid was purified using FastPlasmid Mini (manufactured by Eppendorf) and then digested using PstI and NotI (both manufactured by Takara) to obtain a plasmid that yielded DNA fragments of about 1.5 kbp and 3.3 kbp. The clone was judged to contain the desired DNA fragment, and the sequence of the DNA fragment inserted into the plasmid was determined. The DNA sequence was determined by using a primer having complementary DNA sequences to the Sp6 promoter / priming site in the pZErO-2 vector and the M13 (-20) Forward priming site, and was requested to Shimadzu's single-pass sequencing service. . The sequence extending from the 121st adenine to the 1503rd adenine of the DNA sequence thus obtained (SEQ ID NO: 1) was considered to be a gene encoding QPO. The QPO amino acid sequence deduced from the sequence (SEQ ID NO: 2) was predicted to encode 460 amino acids. The 10 amino acid sequences on the N-terminal side of the amino acid sequence (SEQ ID NO: 2) completely matched the sequence (SEQ ID NO: 3) obtained from the result of the protein N-terminal amino acid sequence analysis obtained above.

The amino acid sequence (SEQ ID NO: 2) predicted from the QPO gene sequence (SEQ ID NO: 1) was subjected to protein secondary structure prediction using the SOSUI system. As a result, the 26th from phenylalanine (Phe) corresponding to the fourth position was detected. It was predicted that sequences spanning tyrosine (Tyr) are likely to be localized in the cytoplasmic membrane. From this, it was predicted that QPO is a single-transmembrane membrane protein. It was considered that the transmembrane region does not participate in quinol peroxidase activity.

As a result of motif analysis of the QPO amino acid sequence (SEQ ID NO: 2), the 56th cysteine (Cys) to the 60th histidine (His), the 203rd cysteine (Cys) to the 207th histidine (His) ), And the 345 th cysteine (Cys) to the 349 th histidine (His) were found to have a heme c binding motif.

When a homology search was performed on the QPO amino acid sequence (SEQ ID NO: 2), there was a gene showing significant homology. Genes having homology with QPO are roughly divided into two groups: bacterial cytochrome c peroxidase genes and genes with unknown functions having three heme c-binding regions.

In the QPO amino acid sequence, high homology was confirmed between the region from the 170th to the 460th region and the full-length cytochrome c peroxidase gene. Specifically, Pseudomonas aeruginosa (1EB7, 42%), Paracoccus pantotrophas (2C1V, 40%), Pseudomonas nautica (1MNL, 40%), Rhodobacter capsulatus (1ZZH, 41%), Nitrosomonas Examples include, but are not limited to, cytochrome c peroxidase derived from europair (1IQC, 41%). Alphanumeric characters in parentheses indicate the PDB accession number and homology (Identities%) of each sequence.

For genes with unknown function having three heme c-binding regions that had high homology with the full length of the QPO amino acid sequence, E. coli (AAC76543.1, 46%), Pest bacteria (AAM84433.1, 54%) ), Salmonella enterica (AAO711193.1, 46%), Shigella flexneri (AAN45044.2, 46%), Bacteroides fragilis (AAL09840.1, 46%), Hemophilus duclayi (AAF14629.1, 63%), etc. As an example. Alphanumeric characters in parentheses indicate the Genbank accession number and homology (Identities%) of each sequence. Although the functions of these genes are not yet clear, it is highly likely that the gene products of these genes also have quinol peroxidase activity because they have high homology with the QPO amino acid sequence of the present invention.

It is a figure which shows the reaction rate of QPO of this invention in the various density | concentrations of ubiquinol-1. It is a figure showing the apparent peroxidase activity by the respiratory chain substrate which the membrane fraction of Actinobacillus actinomycetemcomitans shows.

Claims (11)

DNA encoding the following protein (a) or (b): (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) an amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and quinol peroxidase Protein with activity. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or a complementary sequence thereof, or a sequence containing a part or all of these sequences. A DNA that hybridizes with the DNA of claim 2 under stringent conditions and encodes a protein having peroxidase activity. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2. A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having quinol peroxidase activity. A fusion protein in which the protein according to claim 4 or 5 is bound to a marker protein and / or a peptide tag. An antibody that specifically binds to the protein according to claim 4 or 5. The antibody according to claim 7, wherein the antibody is a monoclonal antibody. A recombinant vector comprising the DNA according to any one of claims 1 to 3. A host cell comprising an expression system capable of expressing the protein according to claim 4 or 5. A method for producing a recombinant protein, comprising culturing a host cell comprising the expression system according to claim 10 in a medium, and collecting a recombinant protein having quinol peroxidase activity from the obtained culture.

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