JP2004298188A - Novel thermostable protein with cell division control activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new thermostable protein having cell division control activity. <P>SOLUTION: This protein comprising a specific amino acid sequence has the cell division control activity and ATPase activity. The protein is obtained by cloning a gene estimated to encode the protein having the cell division control activity from a gene sequence of a Sulfolobustokodaii species 7 (JCM10545) which is one species of aerophilic thermoacidophilic crenarchaeon of hyperthermophilic archaebacteria, and then expressing the protein from the cloned product by using Escherichia coil. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel thermostable protein having a cell division controlling activity. This application is an application for a result commissioned by the government.

  In eukaryotes, the function of cell division control proteins is said to be involved in, for example, endoplasmic reticulum-related degradation. It is said to have. Furthermore, it is said that AAA ATPase in the inner membrane of bacteria and mitochondria has a similar function (see Non-Patent Document 1). A protein having an endoplasmic reticulum-associated degradation function (ERAD) is called p97 / VCP in mammals (see Non-Patent Documents 2 and 3). Furthermore, conventionally, as the cell division control proteins, proteins derived from bacteria and yeast are known, and are said to be medically important. Accumulation of unwanted or harmful proteins in cells has been shown to cause disease, such as Alzheimer's disease, Parkinson's disease, and Huntington's chorea. However, a thermostable protein having a cell division controlling activity is not known. The presence of a thermostable protein having cell division controlling activity is expected to expand its industrial use.

On the other hand, there is a study on hyperthermophilic archaea (see Non-Patent Document 4). Sulfolobus tokodaii (JCM10545) (see Non-Patent Document 5), which is a kind of bacteria belonging to the genus Sulfolobus, has a gene whose gene is described. It has already been analyzed (see Non-Patent Document 6). Therefore, if this hyperthermophilic archaea produces a protein having a cell division controlling activity, it is expected that it has excellent heat resistance.
Nature Reviews Molecular Cell Biology, 3,246-255 (2002) Curr. Biol. 12: R182-R184, Traffic, 3 (8): 530-536, Aug 2002 Advances in Protetin Chemistry, Volume 48, Enzymes and Proteins from Hyperthermophilic Microorganisms (M. Adams ed.), Academic Press (1996) Suzuki, T. et al., Extremophiles, 2002 Feb; 6 (1): 39-44 Kawarabayashi, Y. et al., “Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7”, DNA Res. 8 (4), 123-140 (2001)

  The present invention has been made in view of such circumstances, and an object thereof is to provide a novel heat-resistant protein having a cell division controlling activity.

To achieve the above object, the present inventors examined the genome information of Sulfolobus tokodaii (JCM10545), a hyperthermophilic archaeon, and found that the bacterium may produce a protein having a cell division controlling activity. I found something. Based on this finding, the present inventors have further studied and succeeded in expressing a novel heat-resistant protein having a cell division controlling activity from the bacterial gene, and have reached the present invention. Sulfolobus tokodaii (JCM10545) is stored in the Microbial Strain Preservation Facility of the RIKEN Bio-Institute for Biological Research, and can be purchased at the request of a third party. Since the growth temperature of Sulfolobus tokodaii (JCM10545) is 80 ° C and the growth limit temperature is 87 ° C, the protein of the present invention is active even at a high temperature of 80 to 87 ° C.

That is, the protein of the present invention is the following protein (a) or (b).
(A) A thermostable protein consisting of the amino acid sequence of SEQ ID NO: 1.
(B) a heat-resistant protein having an amino acid sequence in which at least one amino acid residue is deleted, substituted, added or inserted in the amino acid sequence of SEQ ID NO: 1 and having a cell division controlling activity and an ATPase activity;

According to the present invention, a novel thermostable protein having a cell division controlling activity can be provided.

As described above, the novel thermostable protein of the present invention is derived from hyperthermophilic archaebacteria, specifically, from Sulfolobus tokodaii (JCM10545). However, the protein of the present invention is not limited to the protein produced by this fungus, and may be produced by other organisms by genetic engineering techniques.

  Next, the expression vector of the present invention is a vector containing the DNA encoding the protein of the present invention or the DNA of SEQ ID NO: 2.

  Next, the transformant of the present invention is a transformant transformed with the vector of the present invention. The host is not particularly limited, and examples include Escherichia coli.

  Next, the method for producing a protein of the present invention is a production method comprising a step of culturing the transformant of the present invention and a step of collecting the protein expressed in the culturing step.

  Next, a method for controlling cell division by the enzyme of the present invention is a method in which the above-mentioned protein of the present invention is used as the enzyme and the above-mentioned enzyme reaction is carried out at a temperature of 65 to 85 ° C.

  As described above, by using the protein of the present invention, the enzyme reaction can be performed in a high temperature range of 65 to 85 ° C., and cell division can be controlled even under industrial conditions. In this method, the pH of the enzyme reaction is preferably in the range of pH 6 to pH 8.

In the method of the present invention, after performing the enzyme reaction for a certain period of time, the enzyme reaction may be stopped by at least one of a chelating agent and a divalent metal ion. As the chelating agent, ethylenediaminetetraacetic acid (EDTA) is preferable, and as the divalent metal ion, Mn ²⁺ is preferable.

  Hereinafter, the present invention will be described in more detail.

The present inventors have determined the cell division controlling activity from the gene sequence of Sulfolobus tokodaii sp. By cloning a gene (SEQ ID NO: 2) presumed to show the following expression, the novel heat-resistant protein of the present invention was obtained by expressing the gene using E. coli. The method for cloning the gene was carried out as described in Example 1 below. The nucleotide sequence of the cloned gene is as shown in SEQ ID NO: 2, and its deduced amino acid sequence is as shown in SEQ ID NO: 1. In addition, if the heat-resistant protein of the present invention has cell division control activity and ATPase activity, one or more or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 are defective, substituted, or added. Alternatively, it may be inserted. The “deletion, substitution, addition or insertion of an amino acid” in the amino acid sequence can be performed according to a method known to those skilled in the art (for example, a mutagenesis method).

  The protein of the present invention can be produced by the above-described method for producing a protein of the present invention, but is not limited thereto, and may be produced by another production method. For example, as shown in SEQ ID NO: 1, for a protein whose amino acid sequence has been determined, a method known to those skilled in the art based on the sequence, for example, a method of chemically polymerizing individual amino acids to synthesize a protein Can be prepared.

An example of a gene encoding the protein of the present invention is the gene shown in SEQ ID NO: 2. The gene uses, for example, a part of the base sequence represented by SEQ ID NO: 2 from the genome of the hyperthermophilic archaeon Sulfolobus tokodaii (JCM10545) as shown in Example 2 described below as a primer. It can be prepared by a PCR method or a hybridization method using the DNA fragment as a probe. In addition, based on the nucleotide sequence, the gene can be obtained according to a nucleic acid chemical synthesis method known to those skilled in the art, but is not limited thereto.

  The expression vector of the present invention can be obtained by inserting the gene or the DNA of SEQ ID NO: 2 into an appropriate vector. The vector into which the gene of the present invention is inserted is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA, phage DNA, and baculovirus such as AcMNPV. Plasmid DNA can be prepared from Escherichia coli or Agrobacterium by an alkali extraction method or a modification thereof. As a commercially available plasmid, for example, pET-11a (manufactured by Novagen) or a secretory plasmid using a Bacillus host may be used. These plasmids may contain an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and the like.

  Insertion of a gene or the like into a vector includes, for example, a method in which the base sequence of a purified gene is cleaved with an appropriate restriction enzyme, inserted into an appropriate vector DNA at a restriction enzyme site or a multiple cloning site, and ligated to a vector. It can be used, but is not limited thereto. Further, in order to exert the function of the gene of the present invention, the expression vector of the present invention may incorporate a promoter, a terminator, a ribosome binding sequence, and the like in addition to the gene of the present invention. Furthermore, the gene of the present invention may be inserted with a fusion of a sequence encoded by another protein.

  By transforming a host organism with the expression vector, the transformant of the present invention can be obtained. The host organism is not particularly limited as long as it can express the gene of the present invention, and includes, for example, prokaryotic cells such as Escherichia coli, but is not limited thereto. As a transformation method, a known calcium chloride method or the like can be used, but is not limited to these methods.

  The method for producing a protein of the present invention includes a step of culturing the transformant and a step of collecting the protein expressed in the culturing step. The culturing method is performed according to a usual method used for culturing host cells. As a medium for culturing a transformant using a microorganism such as Escherichia coli as a host, a medium containing a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the microorganism can be used as long as the transformant can be efficiently cultured. Any of natural media, synthetic media and the like may be used. The recovery of the protein of the present invention is not particularly limited. When the protein is produced in cells or cells, the cells are recovered by crushing the cells or cells. When the protein of the present invention is produced extracellularly or extracellularly, the culture solution may be used as it is, or after the bacterial cells or cells are removed by centrifugation or the like, used for protein isolation and purification. Isolation and purification of the protein of the present invention from the culture by using common biochemical methods, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. can do. When the culture solution is used as it is, heat treatment deactivates proteins other than the protein of the present invention, so that it can be used as a liquid substantially containing only the protein of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Preparation of chromosomal DNA Sulfolobus tokodaii (JCM10545) was cultured overnight in an L medium at 37 ° C. and collected, and 10 mL of an SSC solution (0.15 M NaCl, 0.015 M sodium citrate) was added. , 0.5 M EDTA, 100 mg / ml Chicken egg white lysozyme 0.1 mL and 10% nonionic surfactant Brij-58 were added in an amount of 0.5 mL, allowed to stand at 0 ° C. for 30 minutes, and then treated with Proteinase K (manufactured by Merck) 5 mg. Was dissolved in 0.2 mL of 10% SDS and left at 37 ° C. for a few days. A mixed solution of water-saturated phenol, chloroform and isoamyl alcohol was added to this solution, and the mixture was allowed to stand at 37 ° C. for 1 hour. The aqueous layer was separated, and ethanol was added thereto to precipitate and concentrate DNA. This DNA precipitate was dissolved in 10 mL of a TE solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0)), and 0.25 mL of ribonuclease (final concentration 0.25 mg / mL) was added thereto. , And precipitated with ethanol. Next, after dissolving the DNA in 5 mL of the TE solution, the DNA concentration was determined from the absorbance at 260 nm (Clarke, L. & Carbon, J. (1979) Methods Enzymol. 68, 396-408).

Construction of expression plasmid and gene expression Construction of Expression Plasmid The following DNA primers were synthesized for the purpose of constructing a DNA containing the restriction enzymes BamHI and NotI sites behind the translation region of the thermostable cell division control protein gene, and the thermostable cell division was carried out by PCR using this primer. A restriction enzyme site was introduced after the translation region of the regulatory protein gene. The DNA polymerase used was KOD-plus- (Toyobo).
Forward primer (SEQ ID NO: 3): 5'-TGAGCGCTCAAAGCATGCTAGAAGAGATGGCT-3 '
Reverse primer (SEQ ID NO: 4): 5'-ATATGGATCCGCGGCCGCTTATTAAAGTGCTTTAAACTTTTC-3 '

  After the PCR reaction, the amplified fragment was digested with the restriction enzyme BamHI, and the gene fragment was purified. pET-11a (manufactured by Novagen) was digested with the restriction enzyme NdeI, the ends were blunt-ended with Klenow fragment, and further digested with the restriction enzyme BamHI was purified, and purified with the above structural gene and T4 ligase at 15 ° C., 30 ° C. The reaction was allowed to take place for minutes and ligated. A part of the ligated DNA was introduced into competent cells of Escherichia coli DH5α, spread on an LB agar plate containing ampicillin in an appropriate amount, and cultured at 37 ° C. overnight to obtain a transformant colony. The obtained transformant was cultured for 24 hours in an LB medium (18 mL) containing ampicillin, and the expression plasmid was purified from the culture by a modified alkaline SDS method. The nucleotide sequence of the insert of the purified plasmid was determined using BigDye Terminator kit (registered trademark: manufactured by Applied Biosystems) and ABI PRISM 3700 DNA Analyzer (registered trademark: manufactured by Applied Biosystems). The correct sequence of the fission control protein gene was confirmed.

2. Expression of recombinant gene Competent cells of Escherichia coli Rosetta-gami (DE3) (Novagen) were thawed and transferred to a Falcon tube at 0.1 mL. Among them, 1. After adding 0.002 mL of the purified expression plasmid solution and leaving it on ice for 20 minutes, heat shock at 42 ° C. for 90 seconds, leaving it on ice for 1 minute, and then LB agar plate containing chloramphenicol and ampicillin , And cultured overnight at 37 ° C to obtain a transformant. The obtained transformant was cultured in an LB medium (5 mL) containing ampicillin for 18 hours to express a thermostable cell division control protein gene. After the culture, the cells were collected by centrifugation (13,000 G, 10 minutes).

  0.2 mL of a disrupted solution (20 mM Tris-HCl, 100 mM KCl, pH 7.5) was added to the collected cells, the cells were disrupted with an ultrasonic generator, and the suspension was added in two 0.1 mL portions. Divided into sample tubes. One sample tube was centrifuged (13,000 G, 10 minutes) to separate into a supernatant and a precipitate, and the precipitate was suspended in 0.1 mL of a crushed liquid. The other sample tube was subjected to heat treatment (70 ° C., 10 minutes) and then centrifuged (13,000 G, 10 minutes) to separate into a supernatant and a precipitate, and the precipitate was suspended in 0.1 mL of a crushed liquid. . Some of these samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) to confirm the expression. The results are shown in the SDS-PAGE photograph of FIG.

  After performing SDS polyacrylamide gel electrophoresis on a sample in which the expression of a thermostable cell division control protein was observed, the sample was transferred to a PVDF membrane by electroblotting and visualized by staining. The protein band was cut out, and the amino terminal sequence was analyzed using a protein sequencer Model492Procise (manufactured by Applied Biosystems). As a result, as shown in SEQ ID NO: 5, the amino terminal sequence of 6 residues was determined. Based on SEQ ID NO: 5, it was confirmed that the expressed protein was subjected to post-translational processing and the first Met was dropped, and that the second and subsequent amino terminal sequences of SEQ ID NO: 5 matched the amino terminal sequences expected from the genomic sequence. As a result, it was confirmed that the expressed protein was a thermostable cell division control protein. This expressed protein was composed of 369 amino acid residues, and its estimated molecular weight was 42.3 kD, which was almost consistent with the result of FIG.

Mass cultivation of recombinant Escherichia coli Using the plasmid DNA (pET-11a vector) prepared in the same manner as in Example 2, Escherichia coli DH5α strain was transformed according to a conventional method. Plasmid DNA was extracted from the transformed Escherichia coli DH5α using the alkaline SDS method. This plasmid DNA was used to transform Escherichia coli Rosetta-Gami (DE3) strain. Three loops of the colony growing on the plate were weighed and 5 mL of LBL medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% lactose, 50 μg / mL ampicillin, 40 μg / mL) (ml chloramphenicol) and precultured at 37 ° C. for about 6 hours until immediately before the start of culture. The total volume of the preculture was added to 3 L of 4 × LBL medium (4% peptone, 2% yeast extract, 2% NaCl, 50 μg / mL ampicillin), and the mixture was placed at 37 ° C. The cells were cultured under the control of a computer program at a pH of 7.2 and a pressure of 0.02 Pa. The pH was adjusted with autoclaved 2M HCl (manufactured by Wako Pure Chemical Industries, Ltd.) and 2M NaOH (manufactured by Wako Pure Chemical Industries, Ltd.). About 20 hours before collection, an autoclaved 300 mL expression induction solution (10% lactose, 20% glycerol) was added. When the growth of Escherichia coli reached the stationary phase (41 hours after the start of culture), the cells were collected using a large centrifuge (AvantiHP-30I manufactured by Beckman). The collected cells were stored at −30 ° C. At this time, a small amount of the cells is separately taken, dissolved and suspended in 150 mM NaCl, 20 mM Tris-HCl (pH 8), 5 mM β-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.). 201). This solution was divided into two equal parts, one was centrifuged at 9100 G at 4 ° C. for 10 minutes, separated into a supernatant and a precipitate, and the other was placed in a thermostat (TAITEC DryThermounit DTU-1C) set at 75 ° C. for 10 minutes. After heating, the mixture was centrifuged at 9100 G and 4 ° C. for 10 minutes to separate a supernatant and a precipitate. A denaturant (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 2.4%) was added to these four kinds of supernatants and precipitates (the precipitates were resuspended in a cell lysate). β-mercaptoethanol, 0.005% bromophenol blue (manufactured by Wako Pure Chemical Industries, Ltd.) were added, and the mixture was denatured by heating at 95 ° C. for 5 minutes. These denatured protein solutions were added to 12.5% or 15% (depending on the molecular weight of the protein to be expressed) polyacrylamide gel, and subjected to electrophoresis by SDS-PAGE. Using a staining solution (Quick-CBB, manufactured by Wako Pure Chemical Industries), the gel after electrophoresis was stained and decolorized to confirm the expression of the target protein.

Purification of Recombinant Protein In Example 3, the cells stored at −30 ° C. were dissolved and suspended in 150 mM NaCl, 20 mM Tris-HCl (pH 8), and 5 mM β-mercaptoethanol. The mixture was crushed with UD-201 (TOMY), heated in a thermostat (TAITEC, DryThermoUnit DTU-1C) set at 75 ° C. for 10 minutes, and then quickly cooled. Next, the disrupted cell fluid was centrifuged at 100,000 G for 1 hour using a large centrifuge (Avanti HP-30I manufactured by Beckman), and the supernatant was collected.

Next, this supernatant was replaced with a buffer solution of 20 mM Tris-HCl (pH 8.0) and 5 mM β-mercaptoethanol using a protein purification device (AKTA explorer, manufactured by Amersham Biosciences), and then an anion exchange column was used. (RESOURCE ^™ Q 6 ml, manufactured by Amersham Biosciences). Elution was performed with sodium chloride, and each eluted fraction was confirmed by SDS-PAGE, and a fraction of the target protein was collected.

  Next, after replacing the collected fraction with a buffer solution of 20 mM MES (2-morpholinoethanesulfonic acid) pH 6.0 and 5 mM β-mercaptoethanol, a cation exchange column (TSK-GEL Bioassist S manufactured by Tosoh Corporation) was used. Passed. Elution was performed with sodium chloride, and each eluted fraction was confirmed by SDS-PAGE electrophoresis, and a fraction of the target protein was collected.

  Next, the collected fraction was replaced with a 10 mM phosphate buffer solution, and then passed through a hydroxyapatite column (Bioscale CHT-10I manufactured by BIO-RAD). Elution was performed with a high-concentration phosphate buffer solution, and the eluted fractions were confirmed by SDS-PAGE electrophoresis, and the target protein fraction was collected.

  Next, the collected fraction was concentrated by centrifugation using a centrifugal concentration tube (VIVASPIN10000, manufactured by Millipore), and 150 mM NaCl, 10 mM phosphate buffer, pH 7.0, using a protein purification device (manufactured by GILSON). The gel was passed through a gel filtration column (manufactured by Tosoh Corporation, TSK-gel G2000SW) equilibrated in step 1. The eluted fraction was confirmed by SDS-PAGE electrophoresis, and the target protein fraction was collected.

  Next, the collected fraction was replaced with 10 mM phosphate buffer solution (PH 7.0) and 0.5 mM β-mercaptoethanol, and then concentrated by centrifugation using a centrifugal concentration tube (VIVASPIN10000 manufactured by Millipore). It was used for activity measurement.

Activity measurement Assay Method The protein purified in Example 4 has a cell division controlling activity based on sequence homology. The activity of this protein was evaluated by measuring the reaction of decomposing ATP into ADP in the presence of metal ions. In this measurement, the phosphorylation reaction of the purified protein of Example 4 itself or of another factor was not directly detected. This measuring method is as follows. That is, a 50 mM buffer solution containing 100 mM KCl, 20 mM MgCl ₂ , 1 mM or 2 mM ATP (manufactured by Oriental Yeast), and 2 μM of the purified protein of Example 4 (as described below, Was different for 10 minutes, and then 0.1 mL of 0.1 M H ₃ PO ₄ was added to stop the reaction. The reaction was started by adding the purified protein of Example 4 to the buffer solution, and the buffer solution was held at each reaction temperature described below for 1 minute before the protein was added. After completion of the reaction, the solution was temporarily transferred onto ice, and after removing proteins by filtration, separation and quantification of the substrate (ATP) and the product (ADP) were performed by HPLC. As the column, an ion pair column (TSK-GEL DEAE-2SW, manufactured by Tosoh Corporation) was used, and a gradient of phosphate ions (50 to 500 mM phosphate buffer solution (pH 3.0), 20% acetonitrile) was performed. . The peak values of the detected ATP (substrate) and ADP (product) were summed, and the amount of the product was determined by multiplying the ratio occupied by ADP by the substrate concentration. Each measured value is a value excluding the amount of spontaneous degradation of ATP, based on the result of a control experiment in which the above measurement was performed without adding the purified protein of Example 4.

2. At each temperature of the optimum temperature 25,45,65,85 ° C., subjected to the measurement, by comparing the respective _{k app,} it was determined specific activity at each temperature. The measurement was performed in two ways, at an ATP concentration of 1 mM and 2 mM. Tris-HCl whose pH was adjusted at 25 ° C. so that the pH became 7.5 at each temperature was used as the buffer solution. The specific activity at each temperature is based on the value measured at 65 ° C. where the _kapp is the maximum at the ATP concentration of 1 mM, and the measured value at 85 ° C. where the _kapp is the maximum at the ATP concentration of 2 mM. Was calculated as a reference (100%). The measurement results are shown in the graph of FIG. As shown in the figure, the specific activities at the ATP concentrations of 1 mM and 2 mM were almost the same. The activity reached its maximum (k _app = 0.15) around 65 ° C., and was kept almost the same at 85 ° C. Incidentally, who _{k app} in ATP concentration 2mM is slightly larger than the _{k app} in ATP concentration 1 mM, _{k app} in ATP concentration 2mM, 85 ° C. was 0.04. Since it was considered that the reaction was not saturated at an ATP concentration of 1 mM, all subsequent measurements were performed at an ATP concentration of 2 mM.

3. Optimum pH
In each condition of PH5.90,6.42,6.86,7.27,7.90,8.50, it performs the measurement, by comparing the respective _{k app,} a specific activity at each pH I asked. As a buffer solution, Tris-HCl was used at a pH of 7.27 or less, and Glycine-NaOH was used at a pH of 7.90 or more. The reaction temperature was 85 ° C. Each of the buffer solutions was adjusted at 25 ° C. so as to have a desired pH at 85 ° C. The specific activity at each pH _{is, k app} was calculated measurements at pH6.42 became maximum reference (100%). The measurement results are shown in the graph of FIG. The activity was maximized around pH 6.5, and decreased when it shifted to the acidic side or the alkaline side, indicating that the optimum pH was between 6 and 8.

4. Thermal Stability Prior to the measurement, 150 μL of the protein solution purified in Example 4 (20 mM Tris-HCl, pH 7.5 at 25 ° C.) was pretreated at 85 ° C. At 30, 60, 90, 120, 150 and 180 minutes after the start of the pretreatment, a part of each solution was withdrawn and allowed to stand on ice. After sufficiently lowering the temperature, the measurement was performed using the solution, and the activity was measured. The reaction temperature was 85 ° C. In addition, the pretreatment time of 0 minute is a time when the pretreatment was not performed, and the specific activity at each pretreatment time was calculated using this as a reference (100%). The measurement results are shown in the graph of FIG. As shown in the figure, no decrease in activity was observed, indicating that the protein purified in Example 4 was stable at 85 ° C.

5. Influence of chelating agent, protein and metal ion on the activity 1) Based on the activity (100%) when the above measurement was carried out using 20 mM MgCl ₂ as usual, 2) further adding 0.01% BSA 3) When the above measurement was conducted by further adding 0.2 M EDTA 4) When the above measurement was conducted by further adding 0.1 M MnCl ₂ 5) Excluding 20 mM MgCl ₂ The effects of chelating agents, proteins and metal ions on the activity were examined by comparing the activities of the two methods. The reaction temperature was 85 ° C. The measurement results are shown in the graph of FIG. As shown, the activity was reduced by adding 0.01% BSA. In addition, when 0.2 M EDTA was added and when 0.1 M MnCl ₂ was added, no activity was observed, indicating that the chelating agent and Mn ²⁺ ion were effective in inhibiting the reaction. Was. When the reaction was performed without 20 mM MgCl ₂ , almost no activity was observed, indicating that Mg ²⁺ was essential for the reaction.

  According to the present invention, a novel thermostable protein having a cell division controlling activity can be provided. The protein of the present invention can be used at a high temperature, and can be used for many purposes such as increasing the substrate concentration, improving the reaction efficiency, removing contaminating microorganisms, extending the storage period and the service life, as well as expanding industrial applications. Benefits are brought.

FIG. 1 is an SDS-PAGE photograph of the recombinant protein in one example of the present invention. FIG. 2 is a graph showing a temperature-specific activity relationship in one example of the present invention. FIG. 3 is a graph showing a relationship between pH and specific activity in one example of the present invention. FIG. 4 is a graph showing the relationship between pretreatment time at 85 ° C. and specific activity in one example of the present invention. FIG. 5 is a graph showing the effect of a chelating agent, a protein, and a metal ion on the activity of a purified protein in one example of the present invention.

SEQ ID NO: 1: Amino acid sequence of thermostable cell division control protein SEQ ID NO: 2: Nucleotide sequence of thermostable cell division control protein SEQ ID NO: 3: Introduction of restriction enzyme sites BamHI and NotI at ends of structural gene of heat-resistant cell division control protein 1 shows a forward primer for performing the following.
SEQ ID NO: 4 shows a reverse primer for introducing restriction enzyme sites BamHI and NotI into the end of the structural gene of the thermostable cell division control protein.
SEQ ID NO: 5: N-terminal amino acid sequence

Claims (10)

The following heat-resistant protein of (a) or (b).
(A) A thermostable protein consisting of the amino acid sequence of SEQ ID NO: 1.
(B) a heat-resistant protein comprising an amino acid sequence in which one or more amino acid residues have been deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 1 and having a cell division controlling activity and an ATPase activity; The protein according to claim 1, which is derived from a hyperthermophilic archaeon. The protein according to claim 2, wherein the hyperthermophilic archaeon is Sulfolobus tokodaii (JCM10545). A vector comprising the DNA encoding the protein according to any one of claims 1 to 3 or the DNA according to SEQ ID NO: 2. A transformant transformed by the vector according to claim 4. A method for producing the protein according to any one of claims 1 to 3, comprising a step of culturing the transformant according to claim 5, and a step of collecting the protein expressed in the culturing step. . A method for controlling cell division with an enzyme, wherein the enzyme reaction is performed at a temperature of 65 to 85 ° C using the protein according to any one of claims 1 to 3 as the enzyme. The method according to claim 7, wherein the pH of the enzymatic reaction is in a range of pH 6 to 8. The method according to claim 7 or 8, wherein after performing the enzyme reaction for a certain period of time, the enzyme reaction is stopped by at least one of a chelating agent and a divalent metal ion. The method according to claim 9, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA), and the divalent metal ion is Mn2 ⁺ .

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