JP2002338595A - Method for purifying chromosomal dan duplication initiation protein, dna a, of pathogenic bacterium using affinity between dna a protein and atp, and evaluation of antibacterial activity using affinity between dna a protein and atp - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently purifying a Dna A protein while holding its activity, a method for efficiently evaluating antibacterial activity by using an activity of a Dna A protein purified by the method as an indicator and a method for effectively screening a compound having an antibacterial activity. SOLUTION: A Dna A protein of Staphylococcus aureus is purified to a purity of >=90% from a crude extraction fraction by using an ATP bonding activity as an indicator. The purified Dna A protein exhibits a strong affinity to ATP. The use of the affinity between a purified Dna A protein and ATP enables efficient evaluation of an antibacterial activity and efficient screening of a compound having an antibacterial activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the purification of DnaA protein, the evaluation of antibacterial activity targeting DnaA protein, and the screening of compounds having antibacterial activity.
The present invention relates to a method for utilizing the affinity between a protein and ATP.

[0002]

2. Description of the Related Art In recent years, infectious diseases caused by pathogenic bacteria resistant to antibacterial agents have become a clinical problem. In general, bacterial proteins targeted by antimicrobial agents must have a function that plays an essential role in bacterial growth, and that humans do not have a protein that shares the same structure as the protein. ,is important.

[0003] In Escherichia coli, it has already been found that the DnaA protein is essential for the initiation of chromosomal DNA replication. It is known that mutation of the gene encoding the DnaA protein (dnaA gene) causes abnormal initiation of replication of chromosomal DNA of Escherichia coli. In fact, many temperature-sensitive mutants of the dnaA gene have been reported in E. coli (Hirota, Y., et al. (1968) Cold Spring Harb
or Symp. Quant. Biol. 33: 677). Thus, inhibition of DnaA protein activity inhibits bacterial growth. (Replication initiation protein of bacterial chromosomal DNA, essential for bacterial growth.) On the other hand, DnaA protein is widely found in bacteria including Escherichia coli, but is not present in eukaryotic cells including humans. . These characteristics suggest that the DnaA protein is promising as a target for clinically effective antibacterial agents.

In order to search for an antibacterial agent having a specific target, a method for measuring the activity of the target must be disclosed. Further, in order to avoid the influence of the mixed protein on the activity measurement, the target protein must be purified.

Conventionally, in the purification of bacterial DnaA protein, a method of detecting the presence of the DnaA protein by electrophoresis without measuring the activity of the DnaA protein and purifying the DnaA protein using the same as an index has been generally employed. Was. For example, in Bacillus subtilis, the DnaA protein is detected by electrophoresis and
DnaA protein has been purified (Fukuoka, et al. (1
990) J. Biochem. 107: 732). However, in purifying a protein as a means for searching for an antibacterial agent, it is necessary that the purified protein has activity. This is because, in evaluating the antibacterial activity of the target substance, it is necessary to measure the activity of the purified sample to detect the antibacterial effect. Therefore, purification of DnaA protein using electrophoresis is not suitable as a means for searching for an antibacterial agent.

On the other hand, for the DnaA protein of Escherichia coli, a method of purifying the DnaA protein using the replication activity in a test tube as an index has been reported (Fuller, RS and Kornberg, A. (19).
83) Proc. Natl. Acad. Sci. USA 80: 5817, Sekim
izu, K. et al. (1988) J. Biol. Chem. 263: 7136).
However, this method requires the construction of an in vitro DNA replication system.For this purpose, a plasmid containing the replication initiation site (oriC plasmid) and various replication proteins such as DNA polymerase necessary for the DNA replication reaction are prepared. Must. There are at least ten types of replication proteins required for a DNA replication reaction. In addition, the interaction between replication proteins is important for the function of the replication proteins, and it is impossible to replace one bacterial system such as E. coli with another. Therefore, in order to purify the DnaA protein using the replication activity as an index, it is necessary to prepare a replication protein corresponding to each of the target bacteria. Unfortunately, that is not easy.

[0007] Therefore, in a form suitable for evaluation of antibacterial activity
It has been desired to develop a more efficient method for purifying the DnaA protein. In particular, regarding pathogenic bacteria that are targets for antibacterial drug development, there have been no reports that the DnaA protein has been successfully purified.
Development of the purification method has been strongly desired.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and an object of the present invention is to provide a method for efficiently purifying a DnaA protein while retaining its activity. is there. Still another object of the present invention is to provide a method for efficiently evaluating antibacterial activity and a method for efficiently screening a compound having antibacterial activity, using the activity of the DnaA protein thus purified as an indicator. And In particular, the present invention is directed to Dn
An object of the present invention is to provide a method for purifying aA protein, a method for evaluating antibacterial activity using the protein, and a method for screening a compound having antibacterial activity.

[0009]

Means for Solving the Problems The present inventors have prepared Escherichia coli DnaA.
It has been reported that proteins are activated by binding to ATP (Sekimizu, K. et al. (1987) Cell 50: 25
9) Since there are many proteins having affinity for ATP in the cells, it has been considered difficult for those skilled in the art to purify the DnaA protein using the ATP binding activity as an index. For this reason, the DnaA protein has not been purified using the ATP binding activity of the DnaA protein as an index. However, the present inventors have focused on the advantage that the ATP-binding activity of the DnaA protein can be easily measured quantitatively. We tried to purify the DnaA protein as an indicator. As a result, unexpectedly, Dn of Staphylococcus aureus
We succeeded in purifying aA protein with more than 90% purity.
As a result of quantifying the affinity of the purified DnaA protein for ATP, it was found that the DnaA protein of Staphylococcus aureus had a high affinity for ATP. The successful purification of the DnaA protein was attributed to this high affinity.

Further, the present inventor has succeeded in purifying the DnaA protein while maintaining the high affinity for ATP in this manner. Therefore, the present inventors evaluated the antibacterial activity using the binding between the DnaA protein and ATP as an index. I thought it could. As mentioned above,
The ATP binding activity of DnaA protein can be easily measured quantitatively. Furthermore, the binding between DnaA protein and ATP is required for DnaA protein replication activity (Sekimizu, K. et al. (1988)
J. Biol. Chem. 263: 7124, Sekimizu, K. et al. (198
7) Cell 50: 259), a substance that inhibits the binding between DnaA protein and ATP is expected to suppress bacterial growth. Therefore, the binding between DnaA protein and ATP is considered to be an effective index for efficient evaluation of antibacterial activity.

[0011] Further, the present inventors have found that DnaA protein and ATP
Effective antibacterial activity based on high affinity with ATP-hydrolyzing activity of ATP-bound DnaA protein as an index or controlling the ATP-binding activity or ATP-hydrolyzing activity of DnaA protein by an activity regulator as an index It was thought that the activity could be evaluated. The use of these antibacterial activity evaluation systems allows efficient screening of candidate compounds for antibacterial agents from many substances.

That is, the present invention provides a method for purifying DnaA protein using affinity between DnaA protein and ATP, a method for evaluating antibacterial activity using affinity between DnaA protein and ATP, and a compound having antibacterial activity. More specifically, (1) a method for purifying a DnaA protein of a pathogenic bacterium, wherein (a) a step of introducing an expression vector having a dnaA gene of a pathogenic bacterium into a host cell; and (b)
Purifying the expressed DnaA protein using ATP binding activity as an indicator, (2) the method according to (1), wherein the pathogenic bacterium is a staphylococcal bacterium, and (3) a staphylococcal genus. The method according to (2), wherein the bacterium is Staphylococcus aureus, (4)
DnaA of a pathogenic bacterium purified by the method according to (1)
Protein, (5) purity after purification is 90% or more (4)
The DnaA protein of the pathogenic bacterium according to (6), wherein the Kd value after purification is 0.3 μM or less;
A protein, (7) the DnaA protein according to any one of (4) to (6), wherein the pathogenic bacterium is a bacterium belonging to the genus Staphylococcus, (8)
A method for evaluating the DnaA protein according to (7), wherein the staphylococcal bacterium is Staphylococcus aureus, (9) a method for evaluating an antibacterial activity of a test sample against pathogenic bacteria, wherein (a) DnaA of pathogenic bacteria is used.
Contacting the test sample with the A protein, (b) detecting the ATP binding activity of the DnaA protein, and (c) comparing the DnaA protein without the test sample with the DnaA protein. Assessing whether the test sample decreases the ATP binding activity of the DnaA protein; (10)
A method for evaluating the antibacterial activity of a test sample against a pathogenic bacterium, comprising: (a) contacting the test sample with a DnaA protein of a pathogenic bacterium to which ATP is bound; (b) ATP of the DnaA protein
Detecting the hydrolysis activity; and (c) determining whether the test sample enhances the ATP hydrolysis activity of the DnaA protein as compared to the case where the test sample is not contacted with the DnaA protein. (11)
A method for evaluating the antibacterial activity of a test sample against a pathogenic bacterium, the method comprising: (a) contacting a test sample with a DnaA protein of a pathogenic bacterium; and (b) a method of controlling the activity of the DnaA protein by a DnaA protein activity regulator. A step of detecting the control of ATP binding activity or ATP hydrolysis activity, and (c) the test sample is a DnaA protein activity controlling factor as compared with a case where the test sample is not contacted with DnaA protein. The DnaA by
Evaluating the control of the ATP binding activity or ATP hydrolyzing activity of the protein.
The method according to any one of (9) to (11), wherein the DnaA protein of the pathogenic bacterium is purified by the method described in (1), (13) the pathogenic bacterium is a staphylococcal bacterium. The method according to any one of (9) to (12), (14)
The method according to claim 1, wherein the staphylococcus is Staphylococcus aureus.
(15) a method for screening a compound having an antibacterial activity against pathogenic bacteria,
(A) a step of evaluating the antimicrobial activity of a plurality of test samples against pathogenic bacteria by the method according to any one of (9) to (14); and (b) the antimicrobial activity of the plurality of test samples from the plurality of test samples. (16) a compound having an antibacterial activity against pathogenic bacteria, which is identified by the method according to any one of (9) to (15), 17) An antibacterial agent comprising the compound according to (16) as an active ingredient.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a method for purifying a DnaA protein of a pathogenic bacterium using the affinity between a DnaA protein and ATP.

[0014] The purification method of the present invention comprises the steps of:
A method comprising the steps of: introducing an expression vector having the A gene into a host cell; and (b) purifying the expressed DnaA protein using ATP binding activity as an index.

In the present invention, the pathogenic bacterium which is a target for purifying the DnaA protein is not particularly limited. In the present invention, the “pathogenic bacterium” means a bacterium capable of infecting a host and causing a disease. When purifying the DnaA protein as a step in the development of an antibacterial agent against infectious diseases of pathogenic bacteria in humans, pathogenic bacteria that target humans are targeted. Pathogenic bacteria include both Gram-negative and Gram-positive bacteria. Examples of gram-negative bacteria include Pseudomonas aeruginosa, cholera, and pathogenic Escherichia coli (O-157), and examples of gram-positive bacteria include staphylococcal bacteria such as Staphylococcus aureus (staphylococcal bacteria). Means 17 types of bacteria already known in the art, including Staphylococcus aureus, Staphylococcus epidermidis and the like), but is not limited thereto.

For efficient purification of the DnaA protein, it is preferable that the DnaA protein of the pathogenic bacterium has a high affinity for ATP. A high affinity for ATP generally has a Kd value of 0.3 μM or less, more preferably 0.2 μM or less (eg, 0.15 μM). Staphylococcus bacteria such as Staphylococcus aureus are suitable as targets for purification of DnaA protein because their DnaA protein has high affinity for ATP.

When the nucleotide sequence of the chromosomal DNA of the pathogenic bacterium has been determined, the dnaA gene of these pathogenic bacteria can be easily obtained by a method such as PCR as a gene having a common sequence with the dnaA gene of Escherichia coli. be able to. In addition, pathogenic bacteria, for which identification of the dnaA gene by analysis of the base sequence of chromosomal DNA is difficult, can be easily obtained as a gene homologous to the Escherichia coli dnaA gene by a method such as hybridization using the dnaA gene of Escherichia coli as a probe. It is possible to The present inventors have used this method to isolate the dnaA gene of Staphylococcus aureus for the first time (Katayama, H. et al. (1997) Biol. Pharm. Bull. 2
0: 820). Using these methods, the dnaA gene of the desired pathogenic bacterium can also be cloned.

The dnaA gene can be ligated to an expression vector and introduced into host cells using known genetic engineering techniques. As an expression vector, dnaA
There is no particular limitation as long as expression of the gene can be guaranteed. In this example, pHK011 was used as the expression vector.
Was used. pHK011 is an expression plasmid for the dnaA gene of Staphylococcus aureus having a replication origin derived from pBR322, and has an ampicillin resistance gene. The host cell is not limited as long as it does not stop growing or die due to the expression of the dnaA gene, and includes Escherichia coli, Staphylococcus aureus, Bacillus subtilis, plant cells, animal cells, and the like. In this example, E. coli KA450 strain was used. The KA450 strain is a mutant strain of Escherichia coli lacking the chromosomal DNA replication origin (oriC) and proliferates by a special DNA replication initiation mechanism called stableDNA replication (Kogoma, T. (1986) J. Bacteriol.
166: 361). Expression of the dnaA gene of Staphylococcus aureus suppresses the growth of wild-type Escherichia coli, but does not suppress it (Katayama, H. et al. (1997) Biol. Pharm. Bull.
20: 820). For this reason, E. coli KA450 is
It is suitable as a host cell for expressing the naA gene. Expression of the dnaA gene of other pathogenic bacteria is also expected to suppress the growth of wild-type E. coli, but not this bacteria. The introduction of a vector incorporating the dnaA gene into a host cell can be performed, for example, by the electroporation method (Masamatsu Muramatsu, edited by Lab Manual Genetic Engineering 3rd Edition (1996) Maruzen p1
46-p149) and the rubidium-manganese method (Hanahan, D. (1
983) J. Mol. Biol. 166: 557), but is not limited thereto. Selection of the host cell into which the vector has been introduced can be performed, for example, using a drug resistance marker of the vector. When pHK011 is used as an expression vector, host cells into which the vector has been introduced can be selected on an agar medium containing 50 μg / ml of ampicillin. The E. coli dnaA gene (Fu
ller, RS and Kornberg, A. (1983) Proc. Natl. Aca
d. Sci. USA 80: 5817) and the B. subtilis dnaA gene (Fukuoka, T. et al. (1990) J. Biochem. 107: 732)
Has been reported to be overexpressed in E. coli.

For purification of the DnaA protein expressed in the host cell, generally, a biochemical method for protein purification is used. As a general technique of biochemistry, there is a case where a protein produced in large quantities is identified by electrophoresis, and the protein is purified using the protein as an index. However, when purifying a protein as a target of an antibacterial agent, after purification,
It is necessary to search for substances that show an inhibitory effect on the activity of the protein, and this method is inappropriate. Until now, for DnaA proteins of Escherichia coli and Bacillus subtilis, DnaA
Although it has been reported that it has the property of specifically binding to the box, measuring the activity of this DnaA protein is not only complicated and troublesome, but also has a problem in quantitativeness, and it is an index for searching for inhibitors. Is inappropriate. In the present invention, purification is carried out utilizing the high affinity for ATP possessed by the DnaA protein. D
The naA protein and ATP binding activity are also suitable as purification indicators in that quantitative measurement is easy.

In the detection of ATP binding activity, for example,
ATP radiolabeled at the other part of TP with ³² P or other nuclide
Or other ATP derivatives can be radiolabeled and used. In addition to the radiolabel, a labeling method capable of quantitative measurement such as a fluorescent label can be used. Whether a labeled compound is suitable for quantifying the activity of DnaA protein depends on whether the compound competitively inhibits ATP binding to DnaA protein or whether the compound's binding to DnaA protein is competitively inhibited by ATP. It can be evaluated by detecting. The measurement of the ATP binding activity can be performed, for example, by the filter binding test described in Example 4, but various other methods can be used. For example, gel filtration, Western blotting, and a method using the surface plasmon resonance phenomenon SPR (BIACORE, Setsuko Hashimoto (1997) Bunseki 5: 362) are considered as suitable methods.

The separation and purification of the protein may be performed by using the separation and purification methods used in ordinary protein purification.
It is not limited at all. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation,
Proteins can be separated and purified by appropriately selecting and combining immunoprecipitation, isoelectric focusing, dialysis, recrystallization and the like. Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like. These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC.

The final preparation of a protein consisting of a single polypeptide should show a single band of the expected molecular weight from the gene by SDS polyacrylamide gel electrophoresis. In the present invention, the purity was determined based on whether a single band was shown in SDS polyacrylamide gel electrophoresis. If this condition is satisfied, it can be determined that the purification of the DnaA protein has been completed.

The basic property that the final preparation should exhibit is a high affinity for ATP. Generally, in biochemistry, a protein having a high affinity for a substance means that the Kd value (dissociation constant) for the substance is significantly smaller than that of other proteins. The method of obtaining the Kd value is a basic operation of a biochemical experiment, and various methods can be considered. Dn
As one method for easily determining the Kd value for ATP of aA protein, as shown in Examples, a filter binding test was performed in the presence of various concentrations of radiolabeled ATP,
It is better to perform Scatchard plot and calculate, but it is not limited to this. ATP of Escherichia coli DnaA protein
Is 0.03 μM (Sekimizu, K. et a
l. (1987) Cell 50: 259). Higher affinity for ATP specifically means that the Kd value is less than 0.3 μM. Preferably, it is confirmed whether the yield and the specific activity are increased in each stage of the purification.

The present invention also provides a DnaA protein of a pathogenic bacterium purified by the above purification method. According to the purification method of the present invention, DnaA protein can be purified with high purity while maintaining its activity. Therefore, the DnaA protein purified by the method of the present invention is suitable for evaluation of antibacterial activity and screening of compounds having antibacterial activity. The purity of the purified DnaA protein is preferably 90% or more. The affinity of the purified DnaA protein for ATP is preferably not more than 0.3 μM in Kd value, more preferably not more than 0.2 μM (for example, 0.15 μM). A preferred embodiment of the DnaA protein of the present invention is a DnaA protein derived from a bacterium belonging to the genus Staphylococcus (eg, a DnaA protein derived from Staphylococcus aureus).

Further, the present invention provides a method for evaluating the antibacterial activity of a test sample against a pathogenic bacterium by utilizing the affinity between the DnaA protein of the pathogenic bacterium and ATP.

The first embodiment of the evaluation method of the present invention utilizing the affinity between DnaA protein of pathogenic bacteria and ATP is a method using the ATP binding activity of DnaA protein as an index. The DnaA protein binds to ATP, thereby becoming active. A substance that inhibits the binding between DnaA protein and ATP is considered to inhibit the activation of DnaA protein and thus inhibit the growth of bacteria. Therefore, whether the test sample inhibits the ATP binding activity of the DnaA protein is an important indicator of the antibacterial activity. The first embodiment of the evaluation method of the present invention comprises (a) a step of bringing a test sample into contact with a DnaA protein of a pathogenic bacterium, and (b) an AT of the DnaA protein.
Detecting the P-binding activity, and (c) determining whether the test sample reduces the ATP-binding activity of the DnaA protein, as compared to the case where the test sample is not contacted with the DnaA protein. The step of evaluating

The second embodiment of the evaluation method of the present invention utilizing the affinity between DnaA protein of pathogenic bacteria and ATP is a method using the ATP hydrolysis activity of DnaA protein as an index. The DnaA protein that has become active by binding ATP converts the bound ATP into ADP by its own hydrolytic activity, and becomes inactive. Therefore, it is considered that a substance that enhances the ATP hydrolysis activity of the DnaA protein promotes the inactivation of the DnaA protein, and thus inhibits the growth of bacteria. Therefore, whether the test sample enhances the ATP hydrolysis activity of the DnaA protein,
It is an important indicator of antibacterial activity. In the detection of ATP hydrolysis activity of DnaA protein, high affinity between DnaA protein and ATP is an important basis. This is because the detection of this hydrolysis activity uses a DnaA protein to which ATP is bound. The second aspect of the evaluation method of the present invention, (a)
Contacting the test sample with the DnaA protein of the pathogenic bacterium to which ATP is bound; (b) detecting the ATP hydrolysis activity of the DnaA protein; and (c) Dn not contacting the test sample.
Compared to the case of using the aA protein, the test sample
Evaluating whether to enhance the ATP hydrolysis activity of the DnaA protein.

The third embodiment of the evaluation method of the present invention utilizing the affinity between the DnaA protein of a pathogenic bacterium and ATP is a method using the control of the activity of the DnaA protein by an activity regulator as an index. The activity of DnaA protein in pathogenic bacterial cells is
It is controlled by adenine nucleotides bound to the aA protein. That is, ATP-bound DnaA protein has replication activity, but ADP-bound DnaA protein is inactive (Sekimiz
u, K. et al. (1987) Cell 50: 259)). When DNA replication should not occur, the activity of the replication initiator protein must be suppressed. If this control mechanism is disrupted, pathogenic bacteria will not be able to grow normally (Mizushima T. et al. (1997) EMBO J. 16: 3724, Kata
yama, T. et al. (1998) Cell 94:61). This DnaA protein activity is regulated by the DnaN protein (Katayama, T. et al. (1)
998) Cell 94:61) and other proteins and acidic phospholipids (S
ekimizu, K. et al. (1988) J. Biol. Chem. 263: 713
It is made by interaction with 1). Of the DnaA protein,
Substances that bind to such interaction sites with activity regulators are expected to disrupt the DNA replication control mechanism and consequently suppress the growth of pathogenic bacteria, even if they do not inhibit the activity of DnaA protein Is done. Therefore, whether a test sample inhibits the action of a DnaA protein activity regulator on a DnaA protein is an important indicator of antibacterial activity. In detecting the activity of such an activity regulator, the ATP binding activity of DnaA protein or ATP in the presence of the activity regulator is considered.
The high affinity between DnaA protein and ATP is an important basis for this evaluation method to detect TP hydrolysis activity. The third embodiment of the evaluation method of the present invention, (a) a step of contacting a test sample with a DnaA protein of a pathogenic bacterium, (b) ATP binding activity or ATP hydrolysis of the DnaA protein by a DnaA protein activity regulator Detecting the control of the activity, and (c) the DnaA protein having an ATP-binding activity of the DnaA protein as compared to the case where the DnaA protein is not contacted with the test sample.
Evaluating whether or not the control by the protein activity regulator is suppressed.

As the DnaA protein used in the evaluation method of the present invention, a DnaA protein derived from a desired pathogenic bacterium to be targeted for the development of an antibacterial agent can be used. From the viewpoint of the development of antibacterial agents against infectious diseases of pathogenic bacteria in humans, those that infect humans are targeted as pathogenic bacteria. Pathogenic bacteria include both Gram-negative and Gram-positive bacteria. Gram-negative bacteria, for example, Pseudomonas aeruginosa,
Examples of cholera bacteria and pathogenic Escherichia coli (O-157) are gram-positive bacteria, but include, but are not limited to, bacteria of the genus Staphylococcus such as Staphylococcus aureus. The pathogenic bacterium preferably has a DnaA protein having a high affinity for ATP. High affinity generally means that the Kd value is 0.3 μM or less, more preferably 0.2 μM.
μM or less (for example, 0.15 μM). DnaA protein derived from Staphylococcus spp., Such as Staphylococcus aureus, has a high affinity for ATP, and thus is suitable for evaluation of antibacterial activity in the evaluation method of the present invention. Since the DnaA protein purified by the above-described purification method of the present invention has high purity and high affinity for ATP, it can be suitably used in the evaluation method of the present invention. In addition, the DnaA protein used in the evaluation method of the present invention may be a partial peptide as long as it has an activity that serves as an index for evaluating the antibacterial activity.

The test sample to be brought into contact with the DnaA protein in the evaluation method of the present invention is not particularly limited, and a desired sample to be evaluated for antibacterial activity can be used. The form of the test sample is not particularly limited, and includes, for example, cell extracts, cell culture supernatants, fermentation microorganism products, marine organism extracts, plant extracts, purified or crude proteins, peptides, non-peptide compounds, Synthetic low molecular weight compounds, natural compounds and the like can be exemplified, but not limited thereto.

In the first embodiment of the evaluation method of the present invention,
The ATP-binding activity of the DnaA protein can be detected as described in the above-described purification method of the present invention. In this evaluation method, the ATP binding activity of the DnaA protein detected in the presence of the test sample is significantly (ie, out of the range of the experimental error) compared to the detection in the absence of the test sample. )) If suppressed, the test sample is evaluated as having antimicrobial activity. Whether the inhibition mode of the ATP binding activity of the DnaA protein by a sample evaluated to have antibacterial activity is competitive inhibition or non-competitive inhibition depends on the degree of inhibition when the concentration of ATP is changed. It can be evaluated by checking whether or not to do so. In the former case, it is usually considered to be an analog of ATP; in the latter case, it is usually DnaA
It is considered to be a substance that binds to a site other than the ATP binding site of the protein and inhibits the ATP binding activity of the DnaA protein.

In the second embodiment of the evaluation method of the present invention,
The ATP hydrolysis activity of the DnaA protein can be detected by any of the following methods, but is not limited to these methods. (1) After incubating the complex of [α- ³² P] ATP and DnaA protein, trap the complex on a filter,
After extraction with formic acid, a method of separating into ATP and ADP by thin-layer chromatography and measuring the amount of ADP generated (Sekimizu, K. e.
tal. (1987) Cell 50: 259). (2) After incubating the complex of [α- ³² P] ATP and DnaA protein, the complex was precipitated with an anti-DnaA protein antibody, separated into ATP and ADP by thin layer chromatography, and the amount of ADP generated (Kurokawa, K. et al. (199
8) Biochem. Biophys. Res. Commun. 243: 90). (3) After incubating the complex of [γ- ³² P] ATP and DnaA protein, trap the complex on a filter,
A method for measuring the release of radioactive phosphate (Sekisui et al., Unpublished).

Already, regarding the DnaA protein of Escherichia coli,
Hydrolysis activity of ATP bound to aA protein to ADP (Sekimi
zu, K. et al. (1987) Cell 50: 259). In this evaluation method, when the ATP hydrolysis activity of the DnaA protein detected in the presence of the test sample is significantly enhanced as compared with the case where the ATP hydrolysis activity is detected in the absence of the test sample, the test sample is Is evaluated as having antibacterial activity.

In the third embodiment of the evaluation method of the present invention,
In the detection of the control of the activity of the DnaA protein by the activity regulator, for example, these activities of the DnaA protein are controlled by a system for detecting the ATP binding activity of the DnaA protein or a system for detecting the ATP hydrolysis activity of the DnaA protein. A biological substance may be added, and the effect of the biological substance on the activity of the DnaA protein may be detected. Examples of the biological substance include acidic phospholipids such as DNA polymerase III holoenzyme and cardiolipin, but are not particularly limited as long as they control the activity of DnaA protein. DNA polymerase III holoenzyme promotes the hydrolysis of ATP bound to DnaA protein. DNA
A structure called β-clamp, generated when β, one of the subunits of the polymerase III holoenzyme, is immobilized on DNA, is required for the hydrolysis of ATP bound to DnaA protein. It has also been found that the activity of an unidentified protein is required for this hydrolysis (Katayama, T. et al. (199
8) Cell 94:61). In addition, acidic phospholipids interact with DnaA protein
Inhibits ATP binding (Sekimizu, K. et al. (1988)
J. Biol. Chem. 263: 7131). Dn using acidic phospholipid
Example 5 shows the inhibition of the ATP binding activity of the aA protein. In this evaluation method, the control of DnaA protein activity by an activity control factor, which was detected in the presence of a test sample,
A test sample is evaluated as having antibacterial activity when it is significantly (ie, beyond the range of experimental error) suppressed as compared to the case where detection was performed in the absence of the test sample. .

By using the evaluation method of the present invention to evaluate the antibacterial activity of a plurality of test samples and selecting a compound having the antibacterial activity, a compound having the antibacterial activity can be efficiently screened. Becomes The present invention also provides a method for screening for a compound having such antibacterial activity.

The compound having an antibacterial activity identified by the evaluation method or the screening method of the present invention is a strong candidate for an antibacterial agent against pathogenic bacteria.

These compounds are used in humans and other mammals such as mice, rats, guinea pigs, rabbits, chickens,
Cats, dogs, sheep, pigs, cows, monkeys, baboons,
When used as an antibacterial agent for chimpanzees, the compound itself can be formulated and administered by a known pharmaceutical method in addition to directly administering to the patient. For example, tablets, capsules, elixirs, microcapsules, or a sterile solution with water or other pharmaceutically acceptable liquid or suspension as sugar-coated tablets, if necessary. It can be used parenterally in the form of injections. For example, a pharmacologically acceptable carrier or medium,
Specifically, it is generally accepted in appropriate combination with sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder and the like. It can be formulated by mixing in the unit dosage form required for pharmaceutical practice.

Administration to a patient can be performed, for example, by intranasal injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a person skilled in the art. It can be done by a method. The dose varies depending on the patient's symptoms, weight, age, administration method, etc.
It will be possible to select a dose of the drug sufficient to produce the intended antimicrobial effect.

The disease targeted by the antibacterial agent of the present invention is not particularly limited as long as it is a disease caused by pathogenic bacteria. Specifically, opportunistic infections caused by Staphylococcus aureus and Pseudomonas aeruginosa, diarrhea caused by cholera and Shigella, sepsis caused by Pseudomonas aeruginosa, Staphylococcus aureus, skin wound infection caused by S. pyogenes, Streptococcus pyogenes, Mycobacterium tuberculosis , Respiratory tract infections caused by H. influenzae, Legionella bacteria, urinary tract infections caused by gonorrhea, intestinal infections caused by enterohemorrhagic E. coli O-157,
And the like, but are not limited thereto.

[0040]

EXAMPLES The present invention will be described in more detail with reference to the following examples of the DnaA protein of Staphylococcus aureus, but the present invention is not limited to the following examples.

Example 1 Construction of Expression Vector for Purification of Staphylococcus aureus DnaA Protein and Staphylococcus aureus Dn
Induction of aA protein expression in Escherichia coli The dnaA gene of Staphylococcus aureus was transformed into the arabinose operon promoter (Kubota, T., et al. (1997) Biochem. B
232: 130) was constructed (FIG. 1) and the E. coli strain KA450 (Kat) was constructed.
ayama, T. (1994) J. Biol. Chem. 269: 22075). Transformants were ampicillin 100 μg / ml and 5
Isolation was performed on LB agar medium containing 0 μg / ml thymine. This transformant was treated with 4 l of LB liquid medium containing 100 μg / ml of ampicillin and 50 μg / ml of thymine until the _OD660 value reached 0.5, until 30%.
Cultured at a degree. Then, arabinose was added to a final concentration of 1%, and cultured at 30 ° C for 3 hours.
A protein was induced. This was used as a starting material for purification shown below.

[Example 2] Purification of Staphylococcus aureus DnaA protein using ATP binding activity as an index All operations were performed at 4 times unless otherwise noted. The cells were collected by centrifugation (6000 rpm x 10 minutes), and the buffer (50 mM HEPES KOH pH7.6, 1 mM
EDTA, 2 mM dithiothreitol, 0.25 M KCl, 20% (v / v) glycerol) and suspended at -80 ° C. Thaw it slowly on ice and add 0.3mg / ml lysozyme and
20 mM spermidine was added and left on ice for 30 minutes. Samples were frozen by immersion in liquid nitrogen and then slowly thawed on ice. Further, the bacteria were destroyed by sonication, and an extract was obtained by centrifugation (35000 rpm × 30 minutes) (Extract fraction). Ammonium sulfate was added at 0.2 g / ml to the extract and left at 4 degrees for 3 hours. Thereafter, the precipitate was collected by centrifugation (30000 rpm x 20 minutes), and 4 ml of a buffer (50 mM HEPES KOH pH7.6,
Dissolve in 1mM EDTA, 2mM dithiothreitol, 10mM magnesium acetate, 20% (v / v) glycerol (Ammonium sul
fate fraction) and dialyzed against 1 liter of the same buffer for 6 hours. The sample was centrifuged (30000 rpm x 20 minutes) and the supernatant (Dia
lysis sup fraction) was packed in a Mono S column (Amersham Pharmacia Biotech). After washing the column with a buffer (50 mM HEPES KOH pH7.6, 1 mM EDTA, 2 mM dithiothreitol, 10 mM magnesium acetate, 20% (v / v) glycerol), the DnaA protein adsorbed on the column was washed with 0-0.
Eluted with a gradient containing 5M KCl (MonoS colu
mn fraction).

The ATP binding activity of each sample at each stage of purification was measured by the method described below. Buffer (5
2 μl of a sample diluted with 0 mM HEPES KOH pH7.6, 1 mM EDTA, 2 mM dithiothreitol, 10 mM magnesium acetate, 20% (v / v) glycerol) was added to a binding buffer containing 1 μmol [α- ³² P] ATP (40 mM HEPES KOH pH7.6, 0.5mM EDTA,
1 mM dithiothreitol, 10% (w / v) sucrose, 100 mM potassium glutamate, 0.05 mg / ml bovine serum albumin) 50
μl and left on ice for 15 minutes. The sample was filtered through a filter (Millipore, HA, 0.45 μm, diameter 25 mm),
The trapped radioactivity was quantified using a liquid scintillation counter (Sekimizu, K. et al. (1987) Cell
50: 259). Compared to the first fraction (Extract fraction), the final sample
The specific activity (activity per unit protein mass) of ATP binding activity of the (MonoS column fraction) increased about 20-fold (Table 1). AT
The yield of P-binding activity was 30%. 2 mg of the final preparation was obtained from the cells obtained by the 4 l culture shown above.

[0044]

[Table 1]

Quantification of protein in each purified fraction in the table was determined by Micr
o Performed using the Lowry method. The amount of protein, ATP binding activity, specific activity of ATP binding and yield at each purification step are shown.
The ATP binding assay was performed on 2 μl of a 100-fold dilution of each purified fraction, and the ATP binding activity of each purified fraction as a whole was determined.

Example 3 Analysis of Purity of Staphylococcus aureus DnaA Protein in Each Purification Step by SDS Polyacrylamide Gel Electrophoresis 5 μg of the protein in the fraction in each purification step was 1% SDS, 2% Heat treated at 90 ° C for 4 minutes in the presence of 2-mercaptoethanol,
Electrophoresis in 0% polyacrylamide gel (Laemml
i, UK (1970) Nature 227: 680). The proteins in the gel were stained with Coomassie Brilliant Blue R250 (FIG. 2). The final sample (MonoS column fraction) has a molecular weight of 52 kDa
And its purity was estimated to be greater than 90%.

Example 4 Purified Staphylococcus aureus DnaA
Quantification of protein affinity for ATP DnaA protein 2.4 pmo of final preparation (MonoS column fraction) for purification
In the presence of various concentrations (0.1 μM) of ATP, the amount of ATP bound was quantified by the filter binding assay described above. The Kd value was calculated from the Scatchard plot (FIG. 3) and calculated to be 0.15 μM. Although the affinity of the DnaA protein for ATP differs depending on the origin of the DnaA protein, it was found from this example that the DnaA protein of S. aureus has a high affinity for ATP.

Example 5 Purified Staphylococcus aureus DnaA
Inhibitory effect of acidic phospholipids on ATP binding activity of protein ATP binding activity of DnaA protein is required for DNA replication initiation activity of this protein (Mizushima, T. et al. (1996) J. B
iol. Chem. 271: 25178). Therefore, the pathogenic bacteria D
Substances that inhibit the ATP binding activity of the naA protein are thought to inhibit the initiation of DNA replication in pathogenic bacteria, and are candidates for compounds having antibacterial activity. The present invention provides an effective method for screening such an antibacterial agent.
It has been shown that cardiolipin inhibits ATP-binding activity in the DnaA protein of Escherichia coli (Sekimiz
u, K. et al. (1988) J. Biol. Chem. 263: 7131). Cardiolipin is an acidic phospholipid present in the cell membrane of pathogenic bacterial cells.

Cardiolipin derived from bovine heart was purchased from Sigma. After evaporating the solvent, it was suspended in water by sonication (Branson ultrasonic machine 450). Various amounts of cardiolipin and 5 picomoles of purified DnaA protein (MonoS
The column fractions), buffer (40 mM HEPES KOH pH 7.6 containing [α- ^{3 2} P] ATP in the 1 [mu] M, 40 mM KCl, magnesium 40μM acetate, 0.5 mM EDTA, 2 mM dithiothreitol, 10% (w /
v) Sucrose, 100 mM potassium glutamate, 0.05 mg / ml
Bovine serum albumin) (50 μl) and left on ice for 2 hours. Filter the sample (Millipore, HA, 0.45μ)
m, diameter 25 mm) and the trapped radioactivity was quantified by a liquid scintillation counter (Sekimizu,
K. et al. (1987) Cell 50: 259). The ATP binding activity of the DnaA protein was almost completely inhibited by 10 μg of cardiolipin (FIG. 4).

[0050]

The DNA replication is essential for the growth of pathogenic bacteria. Furthermore, the DnaA protein is the initiation protein for DNA replication. Therefore, substances that inhibit the activity of DnaA protein may inhibit the growth of pathogenic bacteria. Also, Dn
The activity of the aA protein is controlled by the binding of ATP to this protein. If the control mechanism is abnormal, pathogenic bacterial cells cannot grow (Katayama, T. et al.
(1998) Cell 94:61). Inhibitors of the activity and control mechanism of DnaA protein are expected to inhibit the growth of pathogenic bacterial cells by inhibiting DNA replication or causing abnormal replication of pathogenic bacteria. According to the present invention, it has become possible to efficiently search for candidate compounds for such inhibitors. Further, according to the present invention, it has become possible to efficiently purify a high-purity DnaA protein having activity, which is useful for searching for a candidate compound for such an inhibitor.

[Brief description of the drawings]

FIG. 1 is a diagram (left) and a photograph (right) showing the expression of S. aureus DnaA protein by a S. aureus DnaA protein mass production system. On the left, the mass-producing plasmid pHK011
The structure of is shown. S. aureus DnaA protein is induced by arabinose. The right side of the figure shows the bacterial cells before and after induction.
4 shows the analysis results of SDS polyacrylamide gel electrophoresis. The protein in the gel is Coomassie Brilliant Blue R250
Stained. It can be seen that the Staphylococcus aureus DnaA protein having a molecular weight of 52 kDa is induced.

FIG. 2. Staphylococcus aureus DnaA as the purification step progresses
4 is a photograph showing an increase in protein content. 5 μg of protein from each purification step fraction was analyzed by SDS polyacrylamide gel electrophoresis. The proteins in the gel were stained with Coomassie Brilliant Blue R250.

FIG. 3 shows the dissociation constant (Kd) of S. aureus DnaA protein with respect to ATP. 2.4 pmol of DnaA protein (MonoScol) purified in the presence of various concentrations of radiolabeled ATP
The ATP binding activity of the umn fraction) was measured. on the right,
3 shows a Scatchard plot. The Kd value was calculated to be 0.15 μM.

FIG. 4 shows the inhibition of ATP binding of DnaA protein by acidic phospholipids. Various amounts of cardiolipin and 5 pmol of purified DnaA protein (MonoS column fraction) were added to 1 μmol
Add 50 μl of buffer containing [α- ³² P] ATP and add on ice
Left for hours. Filter the sample (Millipore, H
A, 0.45 μm, diameter 25 mm) and the trapped radioactivity was quantified using a liquid scintillation counter.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12N 15/09 C12Q 1/68 Z 4H045 C12P 21/02 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33 / 15 33/53 D 33/50 C12N 15/00 A 33/53 A61K 37/22 F term (reference) 4B024 AA11 BA80 CA02 DA06 EA04 GA11 HA03 4B063 QA01 QA18 QQ98 QR42 QR48 QS24 QS32 QX07 4B064 AG01 CA02 CA19 CC24 CE02 CE04 CE09 DA13 4C084 AA02 AA17 BA47 ZB352 4H011 AA02 BB17 4H045 AA10 AA20 AA30 CA11 EA50 FA74 GA06 GA20 (54) [Name of the present invention] Purification method, method for evaluating antibacterial activity utilizing affinity between DnaA protein and ATP, and method for screening compound having antibacterial activity

Claims (17)

[Claims] 1. A method for purifying a DnaA protein of a pathogenic bacterium, comprising: (a) introducing an expression vector having a dnaA gene of a pathogenic bacterium into a host cell; and (b) converting the expressed DnaA protein into a host cell. A step of purifying using ATP binding activity as an index,
A method that includes 2. The method according to claim 1, wherein the pathogenic bacterium is a bacterium belonging to the genus Staphylococcus. 3. The method according to claim 2, wherein the staphylococcus is Staphylococcus aureus. 4. A DnaA protein of a pathogenic bacterium purified by the method according to claim 1. 5. The DnaA protein of a pathogenic bacterium according to claim 4, wherein the purity after purification is 90% or more. 6. The DnaA protein of a pathogenic bacterium according to claim 4, wherein the Kd value after purification is 0.3 μM or less. 7. The DnaA protein according to claim 4, wherein the pathogenic bacterium is a staphylococcal bacterium. 8. The DnaA protein according to claim 7, wherein the staphylococcus is Staphylococcus aureus. 9. A method for evaluating the antibacterial activity of a test sample against a pathogenic bacterium, comprising: (a) contacting the test sample with a DnaA protein of the pathogenic bacterium;
Detecting the TP binding activity; and (c) determining whether the test sample reduces the ATP binding activity of the DnaA protein as compared to the case where the test sample is not contacted with the DnaA protein. Assessing. 10. A method for evaluating the antibacterial activity of a test sample against a pathogenic bacterium, comprising: (a) contacting the test sample with DnaA protein of a pathogenic bacterium to which ATP is bound; (b)
Detecting the ATP hydrolysis activity of the DnaA protein; and (c) comparing the DnaA protein with the DnaA protein in which the DnaA protein is not contacted with the test sample.
Evaluating whether or not the P hydrolysis activity is enhanced. 11. A method for evaluating the antibacterial activity of a test sample against a pathogenic bacterium, comprising: (a) contacting the test sample with a DnaA protein of the pathogenic bacterium; and (b) using a DnaA protein activity controlling factor. ATP binding activity of the DnaA protein or A
Detecting the control of TP hydrolysis activity; and (c) comparing the DnaA protein with a DnaA protein activity control factor, Evaluating whether the control of ATP binding activity or ATP hydrolysis activity is suppressed. 12. The method according to claim 1, wherein the DnaA protein of the pathogenic bacterium is purified by the method according to claim 1.
A method according to any one of the preceding claims. 13. The method according to claim 9, wherein the pathogenic bacterium is a bacterium belonging to the genus Staphylococcus. 14. The method according to claim 13, wherein the staphylococcus is Staphylococcus aureus. 15. A method for screening a compound having an antibacterial activity against a pathogenic bacterium, wherein (a) the method according to any one of claims 9 to 14, wherein the method comprises the steps of: And (b) selecting a compound evaluated to have antimicrobial activity from a plurality of test samples. 16. A compound having antibacterial activity against a pathogenic bacterium, which is identified by the method according to claim 9. 17. An antibacterial agent comprising the compound according to claim 16 as an active ingredient.