JP2005278537A - New polypeptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a target polypeptide for searching new antimicrobial agents that can develop excellent effects against Staphylococcus aureus that is cited as a typical refractory infectious disease because of its resistance to multiple drugs and deterioration of medical sensitivity to diseases. <P>SOLUTION: This peptide originating from Staphylococcus aureus is essential for the growth or proliferation of bacteria, Staphylococcus aureus or the like, further has the ATP-hydrolytic activity. This peptide is represented by a specific sequence. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

In recent years, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and the like that have become resistant or reduced in sensitivity to multiple drugs have increased and are regarded as a cause of intractable infections. As a drug that exerts excellent efficacy against such existing drug resistant / low-sensitive bacteria, a drug with a new skeleton (new chemical structure) having a new mechanism of action different from existing drugs is most promising.

Recent approaches to drug discovery are changing from conventional methodologies where the structural modification of compounds is the starting point. Proteins that can be targeted by drugs are identified by genetic analysis, etc., and substances that affect their functions or activities are produced. Methodologies to search by chemical methods are important. In addition, the drug discovery research paradigm is changing in a more comprehensive and efficient direction by the advancement of many technologies such as high-speed screening technology using robots, virtual screening, and structure-based drug design technology. However, even in such a situation, it is an important issue to identify and verify new drug discovery target candidates and to provide promising drug discovery targets (proteins).

At present, the genomic DNA base sequences of many bacteria have been decoded, but it goes without saying that such genomic information is useful in drug discovery research. For example, genome information of Staphylococcus aureus was revealed in 2001 in two strains (N315 strain and Mu50 strain) (Non-patent document 1: Kuroda, M. et al., Lancet, 357: 1225-1240). , 2001). In 2002, another strain (MW2 strain) was reported (Non-patent Document 2: Baba, T. et al., Lancet, 359: 1819-1827, 2002). In addition, at present, the database published on the Internet contains the genomic DNA base sequence information of at least 4 types (252 strain, 476 strain, COL strain, and NCTC8325 strain) of Staphylococcus aureus strains (for example, non-patent literature). 3: A homology search is possible at http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?).
Lancet, 357: 1225-1240, 2001 Lancet, 359: 1819-1827, 2002 http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi? J. Biol. Chem., 277: 28380-28383, 2002 Proc. Natl. Acad. Sci. USA, 100: 4678-4683, 2003 Science, 293: 2266-2269, 2001 Mol. Micobiol., 43: 1387-1400, 2002 EMBO J., 22: 427-437, 2003

The subject of this invention is providing the protein (polypeptide) used as the target for the search of a novel antibacterial agent. In order to solve this problem, the inventors focused on SA0775 as a sequence number in the putative gene ubiquitously found in any S. aureus genome, particularly the N315 strain.

Based on the results of general bioinformatics analysis using various public databases, the polypeptide that is the product of the gene is a kind of SUF machinery that is responsible for iron-sulfur cluster formation in E. coli (Non-Patent Document 4: Takahashi). , Y. and Tokumoto, U., J. Biol. Chem., 277: 28380-28383, 2002), and was considered to be a homologue of SufB. That is, this gene exists on the genome adjacent to several genes considered to be orthologs of protein genes (sufA, sufB, sufC, sufD, sufS, and sufE) constituting the SUF machinery of E. coli. The same rate of 25% as SufB is recognized. It has been suggested that SufB is necessary for the formation of iron-sulfur clusters in Escherichia coli, but it has not been clarified yet how it specifically functions or what kind of activity SufB itself has ( Non-patent document 4).

Iron-sulfur clusters are essential for the expression of activities of enzymes involved in various redox reactions, and are essential for bacterial growth and proliferation. E. coli (and various proteobacteria) have ISC machinery as the main mechanism responsible for iron-sulfur cluster formation. Therefore, it is known that proteins constituting the SUF machinery are not necessarily essential for the growth of E. coli (Non-patent Document 4). However, it is estimated from the genome information of various bacteria that Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, enterococci and pneumococci do not have ISC machinery unlike Escherichia coli (Gram-negative bacteria).

On the other hand, Bacillus subtilis which is a Gram-positive bacterium cannot inactivate (knock out) several orthologs (yurU, yurV, yurX, and yurY) corresponding to the gene of the protein constituting the SUF machinery of E. coli. Recently, it was reported to be essential for the growth or proliferation of fungi (Non-Patent Document 5: Kobayashi, K. et. Al., Proc. Natl. Acad. Sci. USA, 100: 4678-4683, 2003). Before that, in S. aureus, the gene having the highest homology with YurX of Bacillus subtilis (SA0775 in N315 strain) has been shown to be essential for the growth or proliferation of the fungus (Non-patent Document 6: Ji, Y. et. Al., Science, 293: 2266-2269, 2001) and another report (Non-patent document 7: Forsyth, RA et. Al., Mol. Micobiol., 43: 1387-1400, 2002) ) Does not show the essentiality of the gene (Non-patent Document 7: Forsyth, RA et. Al., Mol. Micobiol., 43: 1387-1400, 2002; both reports are analyzed by the antisense method). That is, the essentiality of the genes sufB or yurX orthologs in Staphylococcus aureus has not been established.

In addition, sulfC existing adjacent to sulfB has a known ATPase motif in the amino acid sequence of the encoded protein SufC. SufC homologues in certain bacteria (Erwinia chrysanthemi) show ATPase activity and interaction with their SufB homologues (Non-patent document 8: Nachin, L., et. Al., EMBO J., 22 : 427-437, 2003), a functional relationship between sufB and sufC was suggested. However, this suggestion also does not indicate the activity and essentiality of SufB and its homologue itself, and the function of the gene or the activity of the encoded protein remained unknown.

The present inventors revealed that the SA0775 gene in the N315 strain is essential for the growth or proliferation of Staphylococcus aureus, and the polypeptide corresponding to the product was first genetically engineered to produce the polypeptide. Was found to have ATP-degrading activity. The present invention has been completed based on this finding.

That is, according to the present invention, a polypeptide specified by SEQ ID NO: 1 in the sequence listing is provided. In addition, according to the present invention, the above-mentioned polypeptide essential for bacterial growth or proliferation; a polypeptide essential for bacterial growth or proliferation, wherein 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 1 Provided is a polypeptide consisting of a deleted, substituted, or inserted amino acid sequence; and any of the aforementioned polypeptides having ATP-degrading activity. According to a preferred embodiment of the present invention, any one of the above polypeptides wherein the bacterium is a Gram-positive bacterium, and according to a more preferred embodiment, any one of the above polypeptides wherein the bacterium is a bacterium of the genus Staphylococcus, further preferred embodiments Thus, any one of the above polypeptides wherein the bacterium is Staphylococcus aureus is provided.

Furthermore, according to the present invention, an isolated polynucleotide encoding any one of the above polypeptides; the above polynucleotide which is a polynucleotide specified by SEQ ID NO: 2 in the Sequence Listing; a polynucleotide comprising any one of the above polynucleotides A peptide expression vector; and a microorganism transformed with the vector.

From another point of view, according to the present invention, there is provided a method for screening a substance that inhibits bacterial growth or proliferation, which comprises the following steps:
(A) A step of measuring the ATP degradation activity and / or ATP binding ability of any of the above polypeptides in the presence of a test substance (B) ATP degradation activity and / or ATP binding of the polypeptide in the above step (A) A method comprising the step of determining that the test substance is a substance that inhibits the growth or proliferation of bacteria when the ability is inhibited is provided.

The present invention also provides a kit for screening for a substance that inhibits the growth or proliferation of bacteria, which comprises any of the above polypeptides and ATP.

From another viewpoint, the present invention provides a substance that inhibits the growth or proliferation of bacteria selected by the screening method described above. Further, according to the present invention, there is provided a medicament for inhibiting the growth or proliferation of bacteria, wherein the medicament comprises the substance as an active ingredient, the aforementioned medicament wherein the bacteria are Gram-positive bacteria, and the bacteria are Staphylococcus bacteria. There is provided the above medicament and the above medicament, wherein the bacterium is Staphylococcus aureus.
From another aspect, a method for preventing and / or treating a bacterial infection, the method comprising a step of administering an effective amount of the above substance to a mammal including a human; and inhibiting the growth or proliferation of the bacteria There is provided a method comprising the step of contacting an effective amount of the substance with the bacterium.

The gene encoding the polypeptide of the present invention is essential for the growth or proliferation of bacteria such as Staphylococcus aureus, and by searching for substances that inhibit the function of the polypeptide, a gene for inhibiting the growth or proliferation of bacteria Development is possible.

In general, as a method for analyzing the essentiality of a certain gene (and encoded protein), it is possible to destroy or inactivate a gene as applied to the above-mentioned research targeting Bacillus subtilis rather than the antisense method. The way to consider is a more direct and reliable approach. The putative gene on the chromosome of S. aureus containing the polynucleotide of SEQ ID NO: 2 encoding the polypeptide of SEQ ID NO: 1 of the present invention cannot be inactivated (destroyed) as shown in Examples described later. From this fact, the gene containing the polynucleotide of SEQ ID NO: 2 functions reliably, and the polypeptide identified by SEQ ID NO: 1 encoded by the gene is essential for the growth or proliferation of bacteria including S. aureus I can understand.

The bacterium is preferably a Gram-positive bacterium, more preferably a bacterium belonging to the genus Staphylococcus. Preferred examples of Staphylococcus bacteria include Staphylococcus aureus and Staphylococcus epidermidis, staphylococci (staphylococcus saprophyticus) and staphylococcus hemolyticus. Most preferred is S. aureus.

A gene presumed to encode a protein having high homology with the polypeptide of SEQ ID NO: 1 of the present invention is also found and encoded in Staphylococcus epidermides belonging to the genus Staphylococcus. The amino acid sequence of the protein had 87% identity. Therefore, it can be considered that the polypeptide of SEQ ID NO: 1 in the sequence listing or a homologous polypeptide having the same function (generally referred to as a homolog) exists universally in bacteria belonging to the genus Staphylococcus, for example.

Also from such a viewpoint, the scope of the present invention includes a polypeptide that is essential for bacterial growth or proliferation and has ATP-degrading activity, and is one to several or several in the amino acid sequence shown in SEQ ID NO: 1. A polymorphism comprising an amino acid sequence in which ten (for example, 1 to 60, more preferably 1 to 40, even more preferably 1 to 20, particularly preferably 1 to 10) amino acids have been deleted, substituted, or inserted. Peptides (hereinafter sometimes referred to as “modified polypeptides”) are included (hereinafter, the polypeptide shown in SEQ ID NO: 1 and the modified polypeptides in the sequence listing may be referred to as “polypeptides of the present invention”). ).

It is desirable that the amino acid sequence of the modified polypeptide has at least 30%, preferably 50%, more preferably 70%, particularly preferably 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1. Whether or not the modified polypeptide has a desired activity is determined by preparing an expression vector containing a polynucleotide encoding the modified polypeptide according to the method specifically described in the examples of the present specification, and After transformation, it can be confirmed by purifying the expressed polypeptide and examining its activity.

Within the scope of the present invention, in addition to the polypeptide having the amino acid sequence specified by SEQ ID NO: 1 in the Sequence Listing and the above-mentioned modified polypeptide, polypeptides containing these polypeptides as partial sequences are also included. Such a polypeptide preferably has a total length (number of amino acid residues) of about 400 to 2000, more preferably about 400 to 1000. Methods for expressing a protein having a specific amino acid sequence as a partial sequence are well known and commonly used by those skilled in the art, and those skilled in the art can easily obtain a polypeptide containing the polypeptide of the present invention as a partial sequence.

An example of a polynucleotide encoding a polypeptide having the amino acid sequence specified by SEQ ID NO: 1 in the sequence listing is shown in SEQ ID NO: 2 in the sequence listing. However, the polynucleotide encoding the above polypeptide is limited to this specific embodiment. It is to be understood that any polynucleotide encoding the polypeptide is encompassed within the scope of the invention. In addition, a polynucleotide encoding the above-described modified polypeptide is also encompassed in the scope of the present invention (hereinafter, a polynucleotide encoding the polypeptide shown in SEQ ID NO: 1 in the sequence listing and a polynucleotide encoding the modified polypeptide). May be referred to as “polynucleotides of the invention”). The polynucleotide of the present invention is a concept including DNA and RNA. Furthermore, a polynucleotide containing the polynucleotide of the present invention as a partial sequence is also included in the scope of the present invention. A preferred example thereof is a polypeptide expression vector containing the polynucleotide of the present invention.

The method for producing a polypeptide expression vector containing the polynucleotide of the present invention is specifically described in the examples of the present specification, and those skilled in the art can easily produce a desired polypeptide expression vector. A preferred example of a vector that can be used in the present invention is pMAL-c2, but the type of vector is not limited to this, and a vector that is usually used in this field may be appropriately selected. Also, methods for transforming microorganisms with expression vectors are well known and commonly used by those skilled in the art. For example, transformation can be performed by any method that can be used as a gene transfer method such as electroporation, ultrasonic method, or surfactant method. The body may be manufactured. An example of these methods is specifically shown in the examples of the present specification, and those skilled in the art can easily produce the transformant of the present invention. Moreover, the microorganism transformed with the polypeptide expression vector may be any microorganism as long as it can express the polypeptide of the present invention, and any of bacteria, yeasts and fungi may be used. A method for obtaining and purifying a polypeptide expressed based on the polynucleotide sequence of the vector from a reaction solution of a cell such as a microorganism transformed with a polypeptide expression vector or a cell-free protein expression system is not particularly limited. Instead, conventional protein isolation and purification methods may be used.

The polypeptide of the present invention has ATP-degrading activity as shown in Examples described later. Therefore, using this ATP degrading activity as an index, it is possible to screen for substances that inhibit the growth or proliferation of bacteria involving the gene containing the polynucleotide of the present invention. Specifically, the following steps:
(A) A step of measuring the ATP degradation activity and / or ATP binding ability of the polypeptide of the present invention in the presence of a test substance (B) The ATP degradation activity and / or ATP binding ability of the polypeptide in the above step (A) A method of determining that the test substance is a substance that inhibits the growth or proliferation of bacteria in the case where is inhibited. Specific examples of the method for measuring ATP-degrading activity or ATP-binding ability in the method include a measuring method using as an index the amount of phosphate released with hydrolysis of ATP as shown in the Examples of the present specification. However, it is not limited to this.

It is also possible to screen for substances that inhibit the growth or proliferation of bacteria by screening substances that have a physicochemical interaction with the polypeptide of the present invention. The substance selected by such screening is a substance that has the polypeptide of the present invention as a direct action point, and is a candidate for a substance that inhibits a function essential for the growth or proliferation of bacteria possessed by the polypeptide of the present invention. Useful as. As a method for confirming the physicochemical interaction between the polypeptide of the present invention and a nucleic acid, protein, or low molecular weight compound, a fluorescence depolarization method or a surface plasmon resonance method that can be used in the industry is appropriately used. (For example, an interaction confirmation method described in International Publication WO01 / 16600 can be used).

Since the polypeptide of the present invention is essential for the growth or proliferation of bacteria, it becomes a target for searching for new antibacterial drugs, and the substance selected by the above screening method targeting the polypeptide is an active ingredient of a medicine Can be used.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.
Example 1: Gene essentiality analysis (A) Strain Staphylococcus aureus used RN4220 strain. In addition, E. coli strain MC1061 was used for plasmid preparation.
(B) Medium
E. coli was cultured using 2 × YT medium (Difco Laboratories, Sparks, MD, USA) and Luria-Bertani agar medium (Difco Laboratories), Brain Heart Infusion medium (BHI; Difco Laboratories) and its agar medium (BHIA; Difco Staphylococcus aureus was cultured using Laboratories). If necessary, ampicillin (Amp; Sigma-Aldrich Chemical Co., St. Louis, MO, USA) may be added to a final concentration of 100 μg / ml, or tetracycline (Tc; Sigma-Aldrich Chemical Co.) may be used. It added so that it might become 5 microgram / ml.

(C) Plasmid E. coli and S. aureus shuttle vector plasmid pYT3 (Kuwahara-Arai, K. et al., Antimicrob. Agents Chemother., 40: 2680-2685, 1996) was used. This plasmid has a temperature-sensitive replication origin and replicates at 30 ° C. in S. aureus but does not replicate at 42 ° C. Moreover, this plasmid contains an Amp resistance gene and a Tc resistance gene.
(D) Primer for PCR (synthetic oligonucleotide) 5′- TAG ACAACTGATATTTTGAACATTT-3 ′ [F1 primer: from the sequence in which the protein translation initiation codon (ATG) of the putative gene targeted for recombination is replaced with a stop codon (underlined) ], 5′-ATCAAGTAGGAGGAAAAAGTTATG-3 ′ (F2 primer), 5′-CATCACCTTTTACATCGTGTTGTTT-3 ′ (R1 primer), and 5′-GAGCGGATAACAATTTCACACAGG-3 ′ (RV-M primer) were used. FIG. 1 (A) shows the approximate positions where the above-mentioned primers are complemented together with the plasmid for gene homologous recombination described below and the expected gene recombination (plasmid integration) on the genome.

(E) Preparation of S. aureus genomic DNA S. aureus was inoculated into BHI, cultured at 37 ° C. for 16 hours, and collected. After suspending the cells in buffer TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)], lysostaphin (Wako Pure Chemical Industries, Ltd., Osaka) to a final concentration of 10 μg / ml ) Was added and incubated at 37 ° C. for 2 hours. Furthermore, after adding an equal amount of 0.7% SDS-containing TE, proteinase K (Sigma-Aldrich Chemical Co.) was added to a final concentration of 30 μg / ml, and the mixture was incubated at 55 ° C. for 2 hours. Subsequently, phenol treatment, phenol / chloroform / isoamyl alcohol treatment were performed, and genomic DNA was purified by ethanol precipitation. The DNA was dissolved at 4 ° C. using TE to which RNase A (Sigma-Aldrich Chemical Co.) was added so that the final concentration was 500 ng / ml.

(F) Construction of plasmid for gene homologous recombination
Using F1 and R1 primers and PCR using the RN4220 strain genomic DNA (400 ng / reaction) as a template [Expand ^™ High-Fidelity PCR System (Roche Diagnostics, Basel, Switzerland); reaction at 94 ° C for 2 minutes, then at 94 ° C 25 cycles of a series of reactions of 30 seconds at 50 ° C for 30 seconds, 72 ° C for 30 seconds, and finally 2 minutes at 72 ° C for 2 minutes.] 5 '(protein translation start codon) And a 3 'deletion DNA fragment (about 200 bp) was amplified. The resulting DNA fragment was purified and subjected to blunting and phosphorylation treatment at the ends. Thereafter, the treated DNA fragment and pYT3 previously subjected to BamHI treatment, followed by blunting and dephosphorylation treatment, were ligated and then introduced into E. coli DH5α strain, and the target plasmid [see FIG. 1 (A)] was obtained. I got it. The direction of DNA fragment (deletion gene) insertion in the constructed plasmid was the direction amplified by PCR using RV-M and F1 primers (as described above except for 30 cycles) [see FIG. 1 (A)] .

(G) Plasmid introduction into S. aureus S. aureus RN4220 strain was inoculated into BHI and cultured at 37 ° C. until OD600 was 1.0. After collection, the cells were washed twice with a 10% sucrose solution, then once with a 10% sucrose / 10% glycerol solution, and finally resuspended in the same solution as a competent cell. The competent cell (80 μl) and the plasmid for gene homologous recombination (1 μg) are mixed, and electroporation is performed with a pulse controller (Bio-Rad, Hercules, CA, USA) under the conditions of 25 μF, 2.5 kV, and 100 Ω. went. Next, add 1 ml of 10% sucrose-containing BHI and incubate at 37 ° C for 1 hour. Seed part of the bacterial solution on a Tc-containing BHIA plate and incubate at 30 ° C for about 20 hours. Obtained as a body (transformant).

(H) Induction and detection of plasmid integration When a plasmid having the same sequence as that on the chromosome is present in the bacterial body, homologous recombination may occur between the same sequences, and the plasmid may be integrated into the chromosome. . This phenomenon is called plasmid integration (PI). In this example, this phenomenon was utilized, and when the above gene homologous recombination plasmid sensitive to replication temperature caused PI, the recombination gene was designed to be inactivated (disrupted). That is, the original protein coding region is deficient in the 3 ′ side by PI; at the same time, a gene in which the protein translation start codon (ATG) becomes a stop codon (TAG) remains downstream of the plasmid inserted into the genome. It is a base sequence incapable of protein expression [see FIG. 1 (B)].

As an execution procedure, first, a plasmid-introduced RN4220 strain for gene homologous recombination was seeded again on a Tc-containing BHIA plate and cultured at 30 ° C. for 20 hours. Thereafter, the formed colonies were inoculated into BHI, and cultured at 42 ° C., a non-permissive temperature for plasmid replication, for about 20 hours (culture <1>: PI was induced). Next, the bacterial solution was inoculated into Tc-containing BHI at a 1:10 dilution and cultured at 42 ° C. for 20 hours (culture <2>: if PI occurred in a non-essential gene, the PI body would grow) . Furthermore, the bacterial solution was appropriately diluted and seeded on a Tc-containing BHIA plate and cultured at 42 ° C. for 16 hours (culture <3>: acquisition of PI colonies). Detection of specific PI based on homologous recombination uses genomic DNA (400 ng / reaction) extracted from cells derived from culture <1> and <2> or colonies formed from culture <3>, F2 primer and PCR was carried out by PCR using RV-M primers specific to the plasmid [see FIG. 1 (B)] [30 cycles under the conditions described in (F)].

As a result, although specific PI was detected in the genomic DNA extracted from the cells derived from the culture <1>, when the genomic DNA extracted from the cells derived from the culture <2> was used, the amount of PCR product, that is, the amount of PI The amount was clearly small. In addition, specific PI could not be detected in colonies formed in culture <3>. That is, it was determined that a bacterium that caused a specific PI could not proliferate, and the putative gene targeted for homologous recombination functioned reliably and was essential for the growth or proliferation of the bacterium. (The reason why colonies were obtained in culture <3> is that plasmids may be randomly incorporated into the genome even if not based on homologous recombination, or, at a very low frequency, the bacteria may become Tc resistant by mutation. It can be understood that it may be.)

Example 2: Gene isolation and protein acquisition (A) Strain and medium S. aureus used RN4220 strain. For plasmid preparation, Escherichia coli DH5α strain (Toyobo Co., Ltd., Osaka) was used. In addition, E. coli BL21 (DE3) strain (Invitrogen Corp., Carlsbad, CA, USA) was used for protein production. The medium used was in accordance with Example 1 above.
(B) Plasmid pMAL-c2 for plasmid maltose binding protein (MBP) fusion protein expression (New Englad Biolabs, Beverly, MA, USA), plasmid pGEX-5X-1 for glutathione S-transferase (GST) fusion protein expression ( Amersham Biosciences, Piscataway, NJ, USA) and His-tagged protein expression plasmid pET28a (Novagen brand, EMD Biosciences, Inc., Darmstadt, Germany). pMAL-c2 and pGEX-5X-1 contain the Amp resistance gene, and pET28a contains the kanamycin resistance gene.

(C) PCR primer (synthetic oligonucleotide)
5'-ACACAC GGATCC ATGACAACTGATATTTTG-3 '(F3 primer; underlined BamHI recognition sequence), 5'-ATGACAACTGATATTTTGAACATTTC-3' (F4 primer), 5'-ACACAC GGATCC TTATGACAACTGATATTT-3 '(F5 primer; underlined BamHI Recognition sequence), and 5′-ACACAC GTCGAC TTATTTAGAAACTTTGCG-3 ′ (R3 primer; underlined SalI recognition sequence).

(D) Isolation of gene and construction of protein expression vector
PCR using F4 and R3 primers and RN4220 strain genomic DNA (400 ng / reaction) as a template (Expand ^™ High-Fidelity PCR System (Roche Diagnostics)); reaction at 94 ° C for 2 minutes, then at 94 ° C for 30 seconds, 50 A series of reactions at 30 ° C. for 30 seconds and 72 ° C. for 1 minute and 30 seconds was performed 25 cycles, and finally, reaction was performed at 72 ° C. for 2 minutes] to amplify a DNA fragment (about 1.3 kbp). The DNA fragment was purified, blunted and phosphorylated at the ends, and then treated with SalI. This was ligated with pMAL-c2 previously treated with XmnI and SalI, and then introduced into E. coli DH5α strain to obtain an MBP fusion protein expression vector.
In addition, a DNA fragment (about 1.3 kbp) obtained by PCR as described above using F5 and R3 primers was purified and treated with BamHI and SalI. This was ligated with pGEX-5X-1 previously treated with BamHI and SalI, and then introduced into E. coli DH5α strain to obtain a GST fusion protein expression vector.

Furthermore, a DNA fragment (about 1.3 kbp) obtained by PCR as described above using F3 and R3 primers was purified and treated with BamHI and SalI. This was ligated with pET28a previously treated with BamHI and SalI, and then introduced into Escherichia coli DH5α strain to obtain a His-tagged protein expression vector.
For any protein expression vector, the base sequence of the inserted DNA fragment was analyzed and determined, and the amino acid sequence of the expressed protein was confirmed to be SEQ ID NO: 1 in the sequence listing from the determined base sequence.

(E) Acquisition of protein Each of the above protein expression vectors was introduced into Escherichia coli BL21 (DE3) strain. E. coli strains with MBP fusion protein expression vector or GST fusion protein expression vector introduced are in Amp (100 μg / ml) containing 2 × YT medium, and E. coli strains with His-tagged protein expression vector are introduced with kanamycin (50 μg / ml) After culturing overnight at 37 ° C. in 2 × YT medium, a portion was reinoculated into each of the same medium (1: 100 dilution) and cultured at 37 ° C. until OD600 was 0.8. Thereafter, in order to induce protein expression, isopropyl-(-D-thiogalactopyranoside (Sigma-Aldrich Chemical Co.) was added to a final concentration of 0.4 mM, and further cultured at 37 ° C. for 2 hours. Bacteria were suspended in buffer TGED (10 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 10% glycerol) and then sonicated, and the lysate was centrifuged (15,000 rpm, 2 minutes). The obtained supernatant and precipitate were used as a soluble fraction and an insoluble fraction, respectively.
At this point, the proteins contained in both fractions derived from each protein expression vector-introduced Escherichia coli strain were analyzed by SDS-PAGE. As a result, it was found that only the MBP fusion protein was mainly present in the soluble fraction.

Therefore, the protein was purified and obtained from the soluble fraction derived from the E. coli strain introduced with the MBP fusion protein expression vector. That is, after mixing the soluble fraction and amylose resin (New Englad Biolabs), the resin was centrifuged and washed with TGED (three times), and the resin-adsorbed protein was eluted with TGED containing 10 mM maltose.
The pMAL-c2 plasmid is designed such that a proteolytic enzyme Factor Xa recognition sequence is added to a ligation part of a protein expressed by fusion with MBP. Therefore, the fusion protein obtained as described above was treated with Factor Xa (New Englad Biolabs) and analyzed by SDS-PAGE. As a result, it was confirmed that cleavage of the polypeptide occurred in one place as expected, and MBP was removed by the treatment (FIG. 2).

Example 3: Measurement of protein activity The protein used for activity measurement was basically expressed and purified and obtained in E. coli as described in Example 2 above, but culture of an E. coli strain introduced with an MBP fusion protein expression vector was performed. The temperature is 30 ° C .; the condition is that the bacterial suspension buffer during bacterial disruption (sonication) is 20 mM Tris-HCl containing 200 mM NaCl, 1 mM EDTA, and 10 mM 2-mercaptoethanol. However, this difference has no essential effect on the protein. As a control, MBP obtained from pMAL-c2-introduced Escherichia coli BL21 (DE3) strain in the same manner as described above was also used.
ATP-degrading activity was measured using as an index the amount of phosphoric acid liberated with ATP hydrolysis. Purified protein or MBP 42 pmol, Factor Xa 250 ng, and ATP 100 nmol were added to 25 mM Tris-HCl (pH 7.5) containing 5 mM MgCl 2 (reaction volume 0.2 ml) and reacted at 37 ° C. for 1 hour. Then, malachite green reagent (6% HCl containing 5.7% ammonium molybdate, 2.3% polyvinyl alcohol, 0.081% malachite green, and water in a 1: 1: 2: 2 volume and left for 30 minutes) 1 ml was added, and after standing for 15 minutes, the OD630 value was measured. The amount of free phosphoric acid was calculated based on a calibration curve (primary regression line) created from the relationship between the concentration and the final measured OD630 value using various concentrations of KH2PO4 instead of purified protein in the above reaction system. .

As a result, about 3 nmol of free phosphate was detected in the presence of the purified protein (and Factor Xa) obtained as an MBP fusion protein. On the other hand, in the presence of MBP (and Factor Xa), it was 0.2 nmol or less. Based on the above results and the fact that the MBP fusion protein can be separated by Factor Xa (see Example 2), it is not MBP but the protein / polypeptide expressed by fusion with ATP-degrading activity. Understandable.

The outline of the plasmid used for recombination of the putative S. aureus gene and the state of the gene on the chromosome after recombination [plasmid integration (PI)] are shown together with the outline of the position where the primers used for PCR complement. It is a figure. In the figure, (A) shows the outline of the plasmid for PI and the position where the PCR primer complements, and (B) shows the state of the gene on the chromosome after PI and the position where the primer used for the specific PI detection PCR complements. It is the figure which showed the SDS-PAGE analysis image of the protein produced and acquired using colon_bacillus | E._coli.

Claims (18)

A polypeptide identified by SEQ ID NO: 1 in the sequence listing. The polypeptide according to claim 1, which is essential for the growth or proliferation of bacteria. A polypeptide that is essential for the growth or proliferation of bacteria and comprises an amino acid sequence in which one to several amino acids are deleted, substituted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1. Furthermore, the polypeptide of Claim 2 or 3 which has ATP degradation activity. The polypeptide according to any one of claims 2 to 4, wherein the bacterium is a Gram-positive bacterium. The polypeptide according to any one of claims 2 to 4, wherein the bacterium is a bacterium of the genus Staphylococcus. The polypeptide according to any one of claims 2 to 4, wherein the bacterium is Staphylococcus aureus. An isolated polynucleotide encoding the polypeptide of any one of claims 1-7. The polynucleotide according to claim 8, which is a polynucleotide specified by SEQ ID NO: 2 in the sequence listing. A polypeptide expression vector comprising the polynucleotide according to claim 8 or 9. A microorganism transformed with the vector according to claim 10. A method for screening a substance that inhibits the growth or proliferation of bacteria, comprising the following steps:
(A) A step of measuring the ATP degrading activity and / or ATP binding ability of the polypeptide according to any one of claims 1 to 7 in the presence of a test substance (B) The polypeptide in the step (A) A method comprising the step of determining that the test substance is a substance that inhibits the growth or proliferation of bacteria when ATP-degrading activity and / or ATP-binding ability is inhibited. A kit for screening for a substance that inhibits bacterial growth or proliferation, comprising the polypeptide according to any one of claims 1 to 7 and ATP. A substance that inhibits the growth or proliferation of bacteria selected by the screening method according to claim 12. A medicament for inhibiting the growth or proliferation of bacteria, comprising the substance according to claim 14 as an active ingredient. The medicine according to claim 15, wherein the bacterium is a Gram-positive bacterium. The medicine according to claim 15, wherein the bacterium is a bacterium of the genus Staphylococcus. The medicine according to claim 15, wherein the bacterium is Staphylococcus aureus.