KR20050111454A - Polyphosphate kinase isolated from mycobacteruim tuberculosis, its mutants and genes coding the same - Google Patents

Abstract

The present invention relates to a polyphosphate kinase (hereinafter referred to as "ppk") derived from Mycobacterium tuberculosis , a Mycobacterium tuberculosis bacterium , and a variant protein thereof and a gene encoding the protein. A polyphosphate kinase of SEQ ID NO: 1 prepared by finding a polyphosphate kinase gene involved in phosphorylation of polyphosphate from Mycobacterium tuberculosis, cloning it, checking the nucleotide sequence, and overexpressing it in a transformant And a polyphosphate kinase variant protein having increased expression level in a transformant by mutating a part of the polyphosphate kinase gene and a gene encoding the protein. Mycobacterium tuberculosis-derived polyphosphate kinase of the present invention is involved in the synthesis of polyphosphate, which plays an important role in the infection process of the strain. It can be used to develop a new concept of antibiotic that solves resistance and to search for and develop a therapeutic agent for microbial diseases.

Description

Polyphosphate Kinase protein derived from Mycobacterium tuberculosis and its mutant protein and gene encoding the same {Polyphosphate Kinase isolated from Mycobacteruim tuberculosis, its mutants and genes coding the same}

The present invention relates to a polyphosphate kinase derived from Mycobacterium tuberculosis , a mutant protein thereof, and a gene encoding the protein, and specifically, a polyphosphate from Mycobacterium tuberculosis. Polyphosphate having increased expression in the transformant by mutating a polyphosphate kinase produced by overexpressing the phosphate kinase gene, identifying the nucleotide sequence, and overexpressing the transformant and a part of the polyphosphate kinase gene. Kinase variant proteins and genes encoding said proteins.

Tuberculosis is a chronic infectious disease caused by infection by Mycobacterium tuberculosis . Its severity is increasing in developing countries as well as in developing countries, with about 8 million new cases and about 3 million deaths every year.

Tuberculosis can generally be controlled using antituberculosis and antibiotic therapy, but unfortunately the misuse and abuse of antibiotics has resulted in antibiotic resistance and resistance of Mycobacterium tuberculosis. In particular, in developing countries, chronic carriers with antibiotic-resistant bacteria have increased due to inadequate treatment due to the lack of anti-tuberculosis drugs. In the last five years, a study of antibiotic-resistant tuberculosis bacteria in 35 countries found that 13% of them were resistant to one or more drugs, and that two or more antibiotics, including isoniazid (INH) and rifampin (RMP), The resistance to multidr ug-resistant M. tuberculosis (MDR-TB) is about 7.5%, reaching a severe level. As mentioned above, tuberculosis, especially antibiotic-resistant tuberculosis, poses a major threat to humans' healthy lives (Musser JM, Clin Microbiol Rev. 8, 496-514, 1995; 1998).

In practice, the rate at which bacteria are resistant to new antibiotics occurs much faster than the rate at which new antibiotic analogs are developed (Stuart B. Levy, Scientific American, 46-53, 1998). Tolerance to antibiotics was first discovered in Pneumococcus sp . In the 1970s and provided important clues to the mechanism of action of penicillin (Tomasz et al., Nature 227, 138-140, 1970). Tolerant species stop growing in the presence of the usual concentration of antibiotics but do not die as a result. Resistance is due to the absence of the activity of bacterial autolytic enzymes such as autolysin when antibiotics inhibit cell wall synthase, which in the case of penicillin activates endogenous hydrolytic enzymes. By killing bacteria, bacteria also inhibit the activity of these enzymes, resulting in survival during antibiotic therapy.

It is clinically very important for bacteria to be resistant to antibiotics because the inability to eradicate resistant bacteria makes antibiotic treatment less effective in clinical infections (Handwerger and Tomasz, Rev. Infec. Dis. 7 , 368-386, 1985). In addition, resistance becomes a prerequisite to develop bacterial resistance to antibiotics, resulting in a surviving strain despite antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow even in the presence of antibiotics.

As described above, in order to eliminate bacteria that exhibit resistance and resistance to antibiotics, development of new antibiotics is needed, and development of new antibiotics that functions independently of the activity of the autolysine enzyme is required.

There are many kinds of antibiotics currently in use, but one of the following five mechanisms of action shows their antibiotic action.

(1) cell wall synthesis inhibition

Bacterial cells, unlike animal cells, have a rigid cell wall that maintains high osmotic pressure inside. Therefore, when the bacterial cell wall synthesis is impaired, the high plasma osmotic pressure causes the bacterial plasma to escape and destroy.

(2) protein synthesis inhibition and disorder

Most antibiotics belong here and include drugs that cause misreading of mRNA; translocation inhibitory drugs; peptidyl transferase inhibitory drugs; binding inhibitor of aminoacyl tRNA; And drugs that result in alkylation of proteins.

(3) cell membrane permeability fluctuation

It is a mechanism that kills by changing the permeability of the cell membrane to lose balance with the inside and outside.

(4) inhibition of nucleic acid synthesis

In order to synthesize RNA, the information of cells recorded in DNA must be copied in the form of transcription.

(5) metabolic inhibition

Competitively antagonizes with the essential metabolites of bacteria to show antibacterial activity.

New antibiotics synthesized in recent years are based on the above mechanisms, reducing the likelihood of developing resistance. However, considering resistance, which is a big problem in modern medicine, it is urgent to develop antibiotics that exhibit antimicrobial activity by other methods besides the above-described mechanism, and this will be a next-generation antibiotic having strong antibacterial ability without problems of resistance.

Polyphosphate kinases, on the other hand, are proteins that synthesize polyphosphates, which have a close effect on the long-term survival of bacterial pathogens. The inpolymer refers to the presence of ten to hundreds of single phosphates as linear polymers by high energy phosphatehydride bonds. Such inpolymers are known to have a great influence on the long-term survival of pathogenic bacteria, especially in E. coli, the expression of more than 50 essential genes.

Polyphosphate kinases are involved in synthesizing such inpolymers from ATP. Polyphosphate kinases are very highly conserved in pathogenic bacteria, in particular Gram-negative bacteria, their genetic homology is very high, and are currently found in more than 20 Gram-negative bacteria. Mutant pathogens containing genes in which the activating site of the polyphosphate kinase is deleted are reported to have a very low survival rate. In particular, they show very weak activity and survival rate under conditions of growth inhibition, stress and feeding. In addition, the ability of these mutant pathogens to infect the host is known to be very low.

Based on the above facts, the present inventors have attempted to develop new antibiotics by solving the problems related to the resistance of existing antibiotics and by studying mechanisms that inhibit the invasion and pathological expression of pathogens in an effective new approach. In view of the fact that polyphophate plays an important role in the infection of bacterial bacteria into a host, the development of antibiotics targeting polyphosphate kinase, an enzyme involved in synthesizing the inpolymer, is unlike conventional antibiotics. We thought we could develop a new concept of antibiotic.

Accordingly, the present inventors find and clone a polyphosphate kinase gene derived from Mycobacterium tuberculosis , Mycobacterium tuberculosis , Mycobacterium tuberculosis , confirm the sequencing, and overexpress the recombinant polyphosphate kinase by overexpressing the transformant. The polyphosphate kinase mutant protein and the gene encoding the protein were increased and the present invention was completed by mutating a part of the polyphosphate kinase gene.

SUMMARY OF THE INVENTION An object of the present invention is to provide a polyphosphate kinase, a variant protein thereof, and a gene encoding the protein, which can be used for the search and development of a novel concept of antibiotics and microbial therapeutics for Mycobacterium tuberculosis, Mycobacterium tuberculosis. will be.

The present invention provides a polyphosphate kinase derived from Mycobacterium tuberculosis or a variant protein thereof.

The present invention also provides a gene encoding the polyphosphate kinase or a variant protein thereof.

The present invention also provides the use of polyphosphate kinases or variant proteins thereof for the development of new antibiotics and the search and development of therapeutic agents for microbial related diseases.

Hereinafter, the present invention will be described in detail.

The present invention provides a polyphosphate kinase derived from Mycobacterium tuberculosis or a variant protein thereof.

The polyphosphate kinase has an amino acid sequence represented by SEQ ID NO: 1, variants of proteins, preferably a protein, characterized in that that the amino acid residues of one or more of the protein described in SEQ ID NO: 1, deletion, addition, insertion or substitution Serine, the 137th amino acid of polyphosphate kinase, has an amino acid sequence as set forth in SEQ ID NO: 2 substituted with alanine.

The present invention also provides a gene encoding the polyphosphate kinase or a variant protein thereof.

The gene encoding the polyphosphate kinase has a nucleotide sequence of SEQ ID NO: 3 , and the gene encoding the mutant protein is the nucleotide sequence of SEQ ID NO: 4 where adenine, which is the 409 base of the gene encoding the polyphosphate kinase, is substituted with guanine Has

The present inventors cloned the gene of the polyphosphate kinase or its mutant protein to prepare an expression vector and E. coli transformants thereof. As a result of analyzing the protein expression level through SDS-PAGE and Western blotting, the polyphosphate kinase It was confirmed that the expression level of the mutant protein increased more than 1.5 times compared to the wild type. This is believed to be due to a decrease in the toxicity that can be caused in the expression of proteins in E. coli transformants due to mutations in specific portions of phosphate kinases.

The present invention also provides the use of polyphosphate kinases or variant proteins thereof for the development of new antibiotics and the search and development of therapeutic agents for microbial related diseases.

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, which can generally be controlled using antituberculosis and antibiotic therapy, but currently causes antibiotic resistance and resistance of Mycobacterium tuberculosis due to misuse and abuse of antibiotics. It became. Therefore, targeting a polyphosphate kinase, an enzyme involved in synthesizing a polyphophate, which plays an important role in the infection of bacterial bacteria into a host, it is possible to develop a new concept of antibiotic, which is completely different from conventional antibiotics. Provides a polyphosphate kinase or variant protein thereof for this purpose.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Expression Plasmids and Transformants of Mycobacterium tuberculosis-derived Polyphosphate Kinase and its Mutant Proteins

<1-1> Isolation and Amplification of Polyphosphate Kinase Gene

To clone a gene expressing Mycobacterium tuberculosis derived polyphosphate kinase, First, the polymerase chain was prepared using Mycobacterium tuberculosis -H37Rv chromosomal DNA obtained from Yonsei University College of Medicine Respiratory Infection Group Genome Research Center using primer pairs of SEQ ID NO: 5 and SEQ ID NO: 6 according to the conditions described in Table 1 . reaction) to amplify the genes expressing polyphosphate kinase.

Template ( Mycobacterium tuberculosis chromosomal DNA) 4 ng primer 50 pmole / ul 2.5mM dNTP 0.25mM 10X buffer 5ul Ex Taq Polymerase (TakaRa) 5 units / ul dH ₂ O Add to final volume of 50ul

The reaction of PCR was carried out for a total of 30 cycles in one cycle of 45 seconds at 95 ℃, 45 seconds at 68 ℃, 2 minutes at 72 ℃.

After the completion of PCR, the amplified PCR product was confirmed by electrophoresis on 1% agarose gel.

<1-2> Expression vector preparation of polyphosphate kinase and its mutant protein

To prepare an expression vector of Mycobacterium tuberculosis-derived polyphosphate kinase, 3 μl of the PCR product amplified in Example 1-1 , 1 μl of salt solution (0.2 M NaCl, 0.017 M MgCl ₂ ), and Add 1 μl of dH ₂ O to a final volume of 5 μl and add 1 μl (0.1 ug / ul) to the pCRT7 / CT-TOPO vector (Invitrogen) with the cleavage map of FIG. 1 for 5 minutes at room temperature. The expression vector pCRT7 / CT-TOPO (ppk) of polyphosphate kinase was prepared.

After the reaction, the prepared expression vector was gently shaken by mixing with 2 μl of TOP10F Escherichia coli strain, and then placed in ice and left for 30 minutes. Subsequently, the reaction was carried out at 42 ° C. for 30 seconds, immediately transferred to ice, and 250 μl of SOC culture solution was added thereto, followed by shaking culture at 200 rpm and 37 ° C. for 30 minutes. After shaking, 250 μl of the cells were inoculated into LB solid medium containing ampicillin (70 μg / ml), followed by incubation at 37 ° C. overnight.

The cultured strains were inoculated in LB medium and incubated at 37 ° C. for 12 to 14 hours, followed by purification of expression plasmid using QIAGEN miniprep kit, which was digested with Hind III restriction enzyme and the result was analyzed on agarose gel. Primary confirmation was made .

The purified expression plasmid pCRT7 / CT-TOPO (ppk) was again confirmed the gene sequence of polyphosphate kinase through sequencing. As a result, it was confirmed that the polyphosphate kinase has the amino acid sequence of SEQ ID NO: 1 and the gene sequence of SEQ ID NO: 3 encoding the same.

The inventors have found a mutant having the gene sequence of SEQ ID NO: 4 in which the adenine, the 409 base on the gene sequence of the polyphosphate kinase, was substituted with guanine, and the variant was substituted with alanine for the 137th amino acid. It was confirmed that the amino acid sequence of SEQ ID NO: 3 was encoded. The expression vector containing the mutated gene was named pCRT7 / CT-TOPO (ppk-1).

<1-4> Preparation of E. coli transformants

The expression vectors pCRT7 / CT-TOPO (ppk) and pCRT7 / CT-TOPO (ppk-1) prepared above were introduced into the E. coli BL21 (DE3) pLysS strain in the same manner as in Example <1-2> to form ampicillin. By selecting colonies resistant to E. coli transformants expressing the polyphosphate kinase of Mycobacterium tuberculosis and its mutant proteins were prepared.

Example 3 Expression of Polyphosphate Kinase and Its Mutant Protein

<3-1> Expression Analysis of Polyphosphate Kinase and Its Mutant Protein by SDS-PAGE

In order to express the phosphate kinase and its mutant protein prepared above, each E. coli transformant was first cultured overnight, and a portion of the culture was inoculated into a culture medium (LB medium, 100 mg / l ampicillin), Shake culture at 200 rpm at 37 ℃. When the OD _A600 measurement of the culture reached 0.5 to 0.6, IPTG (Sigma) was added at a concentration of 1 mM to induce the expression of recombinant genes. After addition of IPTG, the cells were further incubated for 4 hours under the same conditions.

The culture medium of the E. coli transformant was centrifuged at 6000 rpm to recover the cell precipitate, suspended in lysis buffer (10 mM Tris-HCl (pH 8.0), 0.2% Triton X-100, 2 mM PMSF), and then ultrasonic crusher ( Crushed using Bandelin Heinrichstrasse 3-4 D-12207 Berlin). The lysate was electrophoresed on 10% polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie Brilliant Blue R-250 solution, and analyzed for its expression ( FIG. 2 ).

As a result, it was confirmed that both phosphate kinase and its mutant protein were expressed at the position of 81.3 kDa, and in particular, the mutant protein increased its expression amount by 1.5 times or more. This is thought to be due to the reduced toxicity that can be caused in protein expression in E. coli transformants due to mutations in specific portions of phosphate kinases.

<3-2> Expression analysis through Western blot of polyphosphate kinase and its mutant protein

In addition, Western blotting was performed to more accurately identify the polyphosphate kinase and its mutated protein expressed above. First, sample buffer was added to the crushing solution, denatured by heat for 5 minutes, and then electrophoresed with the 10% polyacrylamide gel electrophoresis (SDS-PAGE) used above, followed by polyvinylidene difluoride (PVDF) membrane (Millipore, pore size 0.45). Moved again. The membrane was blocked by adding 5% (W / V) of Bovine Serum Albumin (BSA) to Tri-buffered saline (TBST) containing 10 mM Tris (pH 7.6), 150 mM NaCl and 0.1% Tween-20. Soaked in a blocking solution and left at room temperature for 1 hour, and the anti-His tag (mouse monoclonal; abcom Inc) antibody was added to the blocking solution diluted in a ratio of 1 / 1,000 and left for 1 hour at room temperature. . Then, the membrane was washed with TBST, and then, the anti-mouse IgG (abcom Inc) antibody was immersed in a blocking solution diluted at a ratio of 1 / 2,000 and left at room temperature for 1 hour. The membrane was washed again with TBST for 30 minutes and then treated with chemiluminescent solution (ECL Kit, Amersham Bioscience Inc.) for 1 minute and exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) to confirm the results. .

As a result of Western blot, it was confirmed that the expression protein expressed at the position of 81.3 kDa in the result of SDS-PAGE was polyphosphate kinase and its mutant protein, and also the protein expression of the mutant was increased by 1.5 times or more ( FIG. 3). ).

As described above, the mycobacterium tuberculosis-derived polyphosphate kinase, its mutant protein, and the gene encoding the protein are phosphors that play an important role in the infection process of the strain. As it is involved in the synthesis of polyphosphate, this mechanism can be used to develop new concepts of antibiotics that solve the toxicity and resistance of existing antibiotics, and to search for and develop therapeutics for microbial diseases.

1 is a cleavage map of the cloning vector pCRT7 / CT-TOPO used to clone the polyphosphate kinase gene amplified by PCR,

2 is a photograph showing expression of the protein through Coomassie blue staining by expressing the protein from E. coli transformed with an expression vector including the polyphosphate kinase gene and its variants, followed by electrophoresis by SDS-PAGE.

Figure 3 is a photograph confirmed by Western blotting analysis for the expressed protein.

<110> Helix Pharms. Inc. <120> Polyphosphate kinase isolated from Mycobacterium tuberculosis, its mutants and genes coding the same <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 742 <212> PRT <213> Mycobacterium tuberculosis <400> 1Met Ser Asn Asp Arg Lys Val Thr Glu Ile Glu Asn Ser Pro Val 1 5 10 15 Glu Val Arg Pro Glu Glu His Ala Trp Tyr Pro Asp Asp Ser Ala 20 25 30 Ala Ala Pro Pro Ala Ala Thr Pro Ala Ala Ile Ser Asp Gln Leu 35 40 45 Ser Asp Arg Tyr Leu Asn Arg Glu Leu Ser Trp Leu Asp Phe Asn 50 55 60 Arg Val Leu Ala Leu Ala Ala Asp Lys Ser Met Pro Leu Leu Glu 65 70 75 80 Ala Lys Phe Leu Ala Ile Phe Ala Ser Asn Leu Asp Glu Phe Tyr 85 90 95 Val Arg Val Ala Gly Leu Lys Arg Arg Asp Glu Met Gly Leu Ser 100 105 110 Arg Ser Ala Asp Gly Leu Thr Pro Arg Glu Gln Leu Gly Arg Ile 115 120 125 Glu Gln Thr Gln Gln Leu Ala Ser Arg His Ala Arg Val Phe Leu 130 135 140 Ser Val Leu Pro Ala Leu Gly Glu Glu Gly Ile Tyr Ile Val Thr 150 155 160 Ala Asp Leu Asp Gln Ala Glu Arg Asp Arg Leu Ser Thr Tyr Phe 165 170 175 Glu Gln Val Phe Pro Val Leu Thr Pro Leu Ala Val Asp Pro Ala 180 185 190 Pro Phe Pro Phe Val Ser Gly Leu Ser Leu Asn Leu Ala Val Thr 195 200 205 Arg Gln Pro Glu Asp Gly Thr Gln His Phe Ala Arg Val Lys Val 210 215 220 Asp Asn Val Asp Arg Phe Val Glu Leu Ala Ala Arg Glu Ala Ser 230 235 240 Glu Ala Ala Gly Thr Glu Gly Arg Thr Ala Leu Arg Phe Leu Pro 245 250 255 Glu Glu Leu Ile Ala Ala Phe Leu Pro Val Leu Phe Pro Gly Met 260 265 270 Ile Val Glu His His Ala Phe Arg Ile Thr Arg Asn Ala Asp Phe 275 280 285 Val Glu Glu Asp Arg Asp Glu Asp Leu Leu Gln Ala Leu Glu Arg 290 295 300 Leu Ala Arg Arg Arg Phe Gly Ser Pro Val Arg Leu Glu Ile Ala 310 315 320 Asp Met Thr Glu Ser Met Leu Glu Leu Leu Leu Arg Glu Leu Asp 325 330 335 His Pro Gly Asp Val Ile Glu Val Pro Gly Leu Leu Asp Leu Ser 340 345 350 Leu Trp Gln Ile Tyr Ala Val Asp Arg Pro Thr Leu Lys Asp Arg 355 360 365 Phe Val Pro Ala Thr His Pro Ala Phe Ala Glu Arg Glu Thr Pro 370 375 380 Ser Ile Phe Ala Thr Leu Arg Glu Gly Asp Val Leu Val His His 390 395 400 Tyr Asp Ser Phe Ser Thr Ser Val Gln Arg Phe Ile Glu Gln Ala 405 410 415 Ala Asp Pro Asn Val Leu Ala Ile Lys Gln Thr Leu Tyr Arg Thr 420 425 430 Gly Asp Ser Pro Ile Val Arg Ala Leu Ile Asp Ala Ala Glu Ala 435 440 445 Lys Gln Val Val Ala Leu Val Glu Ile Lys Ala Arg Phe Asp Glu 450 455 460 Ala Asn Ile Ala Trp Ala Arg Ala Leu Glu Gln Ala Gly Val His 470 475 480 Ala Tyr Gly Leu Val Gly Leu Lys Thr His Cys Lys Thr Ala Leu 485 490 495 Val Arg Arg Glu Gly Pro Thr Ile Arg Arg Tyr Cys His Val Gly 500 505 510 Gly Asn Tyr Asn Ser Lys Thr Ala Arg Leu Tyr Glu Asp Val Gly 515 520 525 Leu Thr Ala Ala Pro Asp Ile Gly Ala Asp Leu Thr Asp Leu Phe 530 535 540 Ser Leu Thr Gly Tyr Ser Arg Lys Leu Ser Tyr Arg Asn Leu Leu 550 555 560 Ala Pro His Gly Ile Arg Ala Gly Ile Ile Asp Arg Val Glu Arg 565 570 575 Val Ala Ala His Arg Ala Glu Gly Ala His Asn Gly Lys Gly Arg 580 585 590 Arg Leu Lys Met Asn Ala Leu Val Asp Glu Gln Val Ile Asp Ala 595 600 605 Tyr Arg Ala Ser Arg Ala Gly Val Arg Ile Glu Val Val Val Arg 610 615 620 Ile Cys Ala Leu Arg Pro Gly Ala Gln Gly Ile Ser Glu Asn Ile 630 635 640 Val Arg Ser Ile Leu Gly Arg Phe Leu Glu His Ser Arg Ile Leu 645 650 655 Phe Arg Ala Ile Asp Glu Phe Trp Ile Gly Ser Ala Asp Met Met 660 665 670 Arg Asn Leu Asp Arg Arg Val Glu Val Met Ala Gln Val Lys Asn 675 680 685 Arg Leu Thr Ala Gln Leu Asp Glu Leu Phe Glu Ser Ala Leu Asp 690 695 700 Cys Thr Arg Cys Trp Glu Leu Gly Pro Asp Gly Gln Trp Thr Ala 710 715 720 Pro Gln Glu Gly His Ser Val Arg Asp His Gln Glu Ser Leu Met 725 730 735 Arg His Arg Ser Pro 740 <210> 2 <211> 742 <212> PRT <213> Mycobacterium tuberculosis <400> 2Met Ser Asn Asp Arg Lys Val Thr Glu Ile Glu Asn Ser Pro Val 1 5 10 15 Glu Val Arg Pro Glu Glu His Ala Trp Tyr Pro Asp Asp Ser Ala 20 25 30 Ala Ala Pro Pro Ala Ala Thr Pro Ala Ala Ile Ser Asp Gln Leu 35 40 45 Ser Asp Arg Tyr Leu Asn Arg Glu Leu Ser Trp Leu Asp Phe Asn 50 55 60 Arg Val Leu Ala Leu Ala Ala Asp Lys Ser Met Pro Leu Leu Glu 65 70 75 80 Ala Lys Phe Leu Ala Ile Phe Ala Ser Asn Leu Asp Glu Phe Tyr 85 90 95 Val Arg Val Ala Gly Leu Lys Arg Arg Asp Glu Met Gly Leu Ser 100 105 110 Arg Ser Ala Asp Gly Leu Thr Pro Arg Glu Gln Leu Gly Arg Ile 115 120 125 Glu Gln Thr Gln Gln Leu Ala Ala Arg His Ala Arg Val Phe Leu 130 135 140 Ser Val Leu Pro Ala Leu Gly Glu Glu Gly Ile Tyr Ile Val Thr 150 155 160 Ala Asp Leu Asp Gln Ala Glu Arg Asp Arg Leu Ser Thr Tyr Phe 165 170 175 Glu Gln Val Phe Pro Val Leu Thr Pro Leu Ala Val Asp Pro Ala 180 185 190 Pro Phe Pro Phe Val Ser Gly Leu Ser Leu Asn Leu Ala Val Thr 195 200 205 Arg Gln Pro Glu Asp Gly Thr Gln His Phe Ala Arg Val Lys Val 210 215 220 Asp Asn Val Asp Arg Phe Val Glu Leu Ala Ala Arg Glu Ala Ser 230 235 240 Glu Ala Ala Gly Thr Glu Gly Arg Thr Ala Leu Arg Phe Leu Pro 245 250 255 Glu Glu Leu Ile Ala Ala Phe Leu Pro Val Leu Phe Pro Gly Met 260 265 270 Ile Val Glu His His Ala Phe Arg Ile Thr Arg Asn Ala Asp Phe 275 280 285 Val Glu Glu Asp Arg Asp Glu Asp Leu Leu Gln Ala Leu Glu Arg 290 295 300 Leu Ala Arg Arg Arg Phe Gly Ser Pro Val Arg Leu Glu Ile Ala 310 315 320 Asp Met Thr Glu Ser Met Leu Glu Leu Leu Leu Arg Glu Leu Asp 325 330 335 His Pro Gly Asp Val Ile Glu Val Pro Gly Leu Leu Asp Leu Ser 340 345 350 Leu Trp Gln Ile Tyr Ala Val Asp Arg Pro Thr Leu Lys Asp Arg 355 360 365 Phe Val Pro Ala Thr His Pro Ala Phe Ala Glu Arg Glu Thr Pro 370 375 380 Ser Ile Phe Ala Thr Leu Arg Glu Gly Asp Val Leu Val His His 390 395 400 Tyr Asp Ser Phe Ser Thr Ser Val Gln Arg Phe Ile Glu Gln Ala 405 410 415 Ala Asp Pro Asn Val Leu Ala Ile Lys Gln Thr Leu Tyr Arg Thr 420 425 430 Gly Asp Ser Pro Ile Val Arg Ala Leu Ile Asp Ala Ala Glu Ala 435 440 445 Lys Gln Val Val Ala Leu Val Glu Ile Lys Ala Arg Phe Asp Glu 450 455 460 Ala Asn Ile Ala Trp Ala Arg Ala Leu Glu Gln Ala Gly Val His 470 475 480 Ala Tyr Gly Leu Val Gly Leu Lys Thr His Cys Lys Thr Ala Leu 485 490 495 Val Arg Arg Glu Gly Pro Thr Ile Arg Arg Tyr Cys His Val Gly 500 505 510 Gly Asn Tyr Asn Ser Lys Thr Ala Arg Leu Tyr Glu Asp Val Gly 515 520 525 Leu Thr Ala Ala Pro Asp Ile Gly Ala Asp Leu Thr Asp Leu Phe 530 535 540 Ser Leu Thr Gly Tyr Ser Arg Lys Leu Ser Tyr Arg Asn Leu Leu 550 555 560 Ala Pro His Gly Ile Arg Ala Gly Ile Ile Asp Arg Val Glu Arg 565 570 575 Val Ala Ala His Arg Ala Glu Gly Ala His Asn Gly Lys Gly Arg 580 585 590 Arg Leu Lys Met Asn Ala Leu Val Asp Glu Gln Val Ile Asp Ala 595 600 605 Tyr Arg Ala Ser Arg Ala Gly Val Arg Ile Glu Val Val Val Arg 610 615 620 Ile Cys Ala Leu Arg Pro Gly Ala Gln Gly Ile Ser Glu Asn Ile 630 635 640 Val Arg Ser Ile Leu Gly Arg Phe Leu Glu His Ser Arg Ile Leu 645 650 655 Phe Arg Ala Ile Asp Glu Phe Trp Ile Gly Ser Ala Asp Met Met 660 665 670 Arg Asn Leu Asp Arg Arg Val Glu Val Met Ala Gln Val Lys Asn 675 680 685 Arg Leu Thr Ala Gln Leu Asp Glu Leu Phe Glu Ser Ala Leu Asp 690 695 700 Cys Thr Arg Cys Trp Glu Leu Gly Pro Asp Gly Gln Trp Thr Ala 710 715 720 Pro Gln Glu Gly His Ser Val Arg Asp His Gln Glu Ser Leu Met 725 730 735 Arg His Arg Ser Pro 740 <210> 3 <211> 2241 <212> DNA <213> Mycobacterium tuberculosis <400> 3atgatcgcaa ggtgaccgaa atcgaaaaca gtcccgtcac agaggtgcgg 60 atgcgtggta tccagacgac tcggcgctgg cggcaccgcc cgctgccacc 120 ttagcgacca gctaccctcg gatcgctacc tgaaccggga gctgagttgg 180 acgcgcgcgt gcttgccctg gccgccgata agtcgatgcc attgctcgag 240 ttctggcaat cttcgcgtcc aatctcgacg agttctacat ggtccgggtg 300 aacgccgcga cgagatgggg ttgtcggtgc gctccgccga cggtctaaca 360 aactaggccg gatcggcgag cagactcaac agctcgccag ccggcatgcc 420 tcgattcggt gctacccgcg ctcggcgagg aaggcatcta catcgtcacc 480 tggatcaggc tgagcgcgac cgattgtcga cctatttcaa cgaacaggtc 540 tgaccccgct ggccgtcgat cccgcccacc cgttcccgtt tgtcagcggg 600 acctggcggt cacggtacgc caacctgaag acggcaccca gcatttcgcg 660 tgcccgacaa cgtcgaccgc ttcgtcgaac tcgctgcacg tgaggccagc 720 cggggaccga aggccggacc gcgctgcggt tcctgccgat ggaggagctg 780 tccttccggt gcttttcccg ggtatggaaa tcgtcgagca ccacgcattt 840 gcaacgctga cttcgaggtt gaagaggatc gcgacgagga cctactgcag 900 gagaactggc ccgccgccgg ttcggttcac cggtgcgact cgagatcgca 960 ccgagagcat gctggagttg ctgcttcgcg aactcgacgt gcatcccggt 1020 aagtgcccgg gctgctcgac ctatcgtcgt tgtggcagat ctacgccgtg 1080 cgcttaagga tcggacattc gtcccagcta cccatcccgc cttcgccgag 1140 ccaaaagcat cttcgcgacg ctgcgcgaag gcgatgtgct ggttcaccat 1200 cgttctccac cagcgtgcag cgattcatcg aacaggccgc ggccgacccc 1260 cgatcaaaca gacgctgtac cgcacctccg gcgactcgcc gatcgtccgg 1320 acgccgccga agccggaaag caagtggtgg cactggtcga gatcaaggca 1380 aacaggccaa catcgcctgg gcgcgcgcac tagaacaagc cggcgtgcat 1440 ggctcgtcgg gctcaagacg cactgcaaga ccgccttggt ggtgcgccgc 1500 caatccggcg gtactgccat gtcggcaccg gcaattacaa cagcaagaca 1560 acgaggacgt cggactgctg accgctgcac ccgatatcgg cgccgacttg 1620 tcaattcgct caccggctac tcacgcaagt tgtcctaccg caacttgttg 1680 acggaatccg cgccggcatc attgaccgcg tcgagcggga ggtcgcggcg 1740 agggtgccca caacggcaaa ggccgcatcc gactcaagat gaatgccctt 1800 aggtcatcga tgcgctgtac cgcgcgtcgc gagccggtgt gcggatcgag 1860 gcggcatctg cgcgctgcgt ccaggtgcgc agggcatttc ggaaaacatc 1920 cgattctcgg ccgcttcctc gagcactcgc ggatcctcca tttccgtgcc 1980 tctggatcgg cagcgccgac atgatgcacc gcaacctcga ccggcgagtc 2040 ctcaagtcaa aaacccgagg ctgaccgcgc agctggacga attgttcgaa 2100 acccgtgcac ccggtgctgg gagctcgggc ccgacgggca gtggaccgcg 2160 aaggccatag cgtgcgcgac catcaggaat cgctgatgga acggcaccgc 2220 ccaagcttgg g 2241 <210> 4 <211> 2241 <212> DNA <213> Mycobacterium tuberculosis <400> 4atgatcgcaa ggtgaccgaa atcgaaaaca gtcccgtcac agaggtgcgg 60 atgcgtggta tccagacgac tcggcgctgg cggcaccgcc cgctgccacc 120 ttagcgacca gctaccctcg gatcgctacc tgaaccggga gctgagttgg 180 acgcgcgcgt gcttgccctg gccgccgata agtcgatgcc attgctcgag 240 ttctggcaat cttcgcgtcc aatctcgacg agttctacat ggtccgggtg 300 aacgccgcga cgagatgggg ttgtcggtgc gctccgccga cggtctaaca 360 aactaggccg gatcggcgag cagactcaac agctcgccgg ccggcatgcc 420 tcgattcggt gctacccgcg ctcggcgagg aaggcatcta catcgtcacc 480 tggatcaggc tgagcgcgac cgattgtcga cctatttcaa cgaacaggtc 540 tgaccccgct ggccgtcgat cccgcccacc cgttcccgtt tgtcagcggg 600 acctggcggt cacggtacgc caacctgaag acggcaccca gcatttcgcg 660 tgcccgacaa cgtcgaccgc ttcgtcgaac tcgctgcacg tgaggccagc 720 cggggaccga aggccggacc gcgctgcggt tcctgccgat ggaggagctg 780 tccttccggt gcttttcccg ggtatggaaa tcgtcgagca ccacgcattt 840 gcaacgctga cttcgaggtt gaagaggatc gcgacgagga cctactgcag 900 gagaactggc ccgccgccgg ttcggttcac cggtgcgact cgagatcgca 960 ccgagagcat gctggagttg ctgcttcgcg aactcgacgt gcatcccggt 1020 aagtgcccgg gctgctcgac ctatcgtcgt tgtggcagat ctacgccgtg 1080 cgcttaagga tcggacattc gtcccagcta cccatcccgc cttcgccgag 1140 ccaaaagcat cttcgcgacg ctgcgcgaag gcgatgtgct ggttcaccat 1200 cgttctccac cagcgtgcag cgattcatcg aacaggccgc ggccgacccc 1260 cgatcaaaca gacgctgtac cgcacctccg gcgactcgcc gatcgtccgg 1320 acgccgccga agccggaaag caagtggtgg cactggtcga gatcaaggca 1380 aacaggccaa catcgcctgg gcgcgcgcac tagaacaagc cggcgtgcat 1440 ggctcgtcgg gctcaagacg cactgcaaga ccgccttggt ggtgcgccgc 1500 caatccggcg gtactgccat gtcggcaccg gcaattacaa cagcaagaca 1560 acgaggacgt cggactgctg accgctgcac ccgatatcgg cgccgacttg 1620 tcaattcgct caccggctac tcacgcaagt tgtcctaccg caacttgttg 1680 acggaatccg cgccggcatc attgaccgcg tcgagcggga ggtcgcggcg 1740 agggtgccca caacggcaaa ggccgcatcc gactcaagat gaatgccctt 1800 aggtcatcga tgcgctgtac cgcgcgtcgc gagccggtgt gcggatcgag 1860 gcggcatctg cgcgctgcgt ccaggtgcgc agggcatttc ggaaaacatc 1920 cgattctcgg ccgcttcctc gagcactcgc ggatcctcca tttccgtgcc 1980 tctggatcgg cagcgccgac atgatgcacc gcaacctcga ccggcgagtc 2040 ctcaagtcaa aaacccgagg ctgaccgcgc agctggacga attgttcgaa 2100 acccgtgcac ccggtgctgg gagctcgggc ccgacgggca gtggaccgcg 2160 aaggccatag cgtgcgcgac catcaggaat cgctgatgga acggcaccgc 2220 ccaagcttgg g 2241 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5atgatcgcaa ggtgaccg 28

Claims (13)

Polyphosphate Kinase from Mycobacterium tuberculosis or a variant protein thereof. The protein of claim 1, wherein the polyphosphate kinase has an amino acid sequence set forth in SEQ ID NO: 1 . The protein of claim 1, wherein the variant protein comprises deletion, addition, insertion, or substitution of one or more amino acid residues of the protein set forth in SEQ ID NO: 1 . 4. The protein of claim 3, wherein the variant protein has an amino acid sequence as set forth in SEQ ID NO: 2 . A gene encoding the polyphosphate kinase of claim 1 or a variant protein thereof. The gene of claim 5, wherein the gene encoding the polyphosphate kinase has a nucleotide sequence as set forth in SEQ ID NO: 3. 7 . The gene of claim 5, wherein the gene encoding the variant protein has a nucleotide sequence set forth in SEQ ID NO: 4. 7 . An expression vector pCRT7 / CT-TOPO (ppk) comprising the polyphosphate kinase gene of SEQ ID NO: 3 . E. coli transformants transformed by the expression vector pCRT7 / CT-TOPO (ppk) of claim 8. An expression vector pCRT7 / CT-TOPO (ppk-1) comprising the polyphosphate kinase variant protein gene of SEQ ID NO: 4 . E. coli transformants transformed by the expression vector pCRT7 / CT-TOPO (ppk-1) of claim 10. A method for producing a polyphosphate kinase of claim 1 or a variant protein thereof. Use of polyphosphate kinase or variant proteins thereof for the search and development of antibiotics and therapeutic agents for microbial diseases.