CN108588040B - Recombinant MtMetRS, crystals thereof and application of recombinant MtMetRS and crystals thereof in preparation of anti-tuberculosis drugs - Google Patents
Recombinant MtMetRS, crystals thereof and application of recombinant MtMetRS and crystals thereof in preparation of anti-tuberculosis drugs Download PDFInfo
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- CN108588040B CN108588040B CN201810585417.7A CN201810585417A CN108588040B CN 108588040 B CN108588040 B CN 108588040B CN 201810585417 A CN201810585417 A CN 201810585417A CN 108588040 B CN108588040 B CN 108588040B
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
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- C12Y—ENZYMES
- C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
- C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
- C12Y601/0101—Methionine-tRNA ligase (6.1.1.10)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
Abstract
The invention discloses a recombinant MtMetRS, a crystal thereof and application thereof in preparation of anti-tuberculosis drugs. The amino acid sequence of the recombinant MtMetRS is shown as a sequence 2 in a sequence table. The method for preparing the recombinant MtMetRS crystal comprises the steps of crystallizing the recombinant MtMetRS in a recombinant MtMetRS solution by adopting a crystallization solution to obtain the recombinant MtMetRS crystal; the crystallization liquid contains calcium acetate, sodium cacodylate and PEG 8000. The three-dimensional structure of the recombinant MtMetRS ligand-free crystal is greatly different from the published crystal structure (PDB code: 6AX8) of WWW.PDB.ORG in conformation on structures such as a substrate binding pocket, a catalytic domain and the like related to enzyme activity. Structural information that differs significantly from 6AX8 in these critical regions is the basis for designing specific drugs. The recombinant MtMetRS provided by the invention is the basis for obtaining the recombinant MtMetRS ligand-free crystal and is also an essential technology for screening and designing the MtMetRS inhibitor. The invention has great application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant MtMetRS, a crystal thereof and application thereof in preparation of an anti-tuberculosis drug, in particular to preparation of the recombinant MtMetRS crystal and application thereof in design of the anti-tuberculosis drug.
Background
Tuberculosis (TB) is a serious infectious disease caused by mycobacterium tuberculosis. The long-term use of antibiotics in the treatment of tuberculosis causes the increase of drug-resistant tubercle bacillus infection cases, and brings unprecedented serious challenges for the prevention and treatment of tuberculosis. Therefore, the discovery of new drug targets and the design of new antitubercular drugs is urgent.
aminoacyl-tRNA synthetases (aaRS) as a highly conserved protease family in cells participate in the process of translating biological genetic information, are responsible for transferring amino acids to corresponding tRNA and are enzymes for ensuring that the genetic information in cells is translated faithfully. The aminoacyl tRNA synthetase system consists of 20 enzymes. Theoretically, each enzyme is a target for the action of an antibiotic. Of these 20 enzymes, methionyl tRNA synthetase exerts the greatest influence on vital activities, and it recognizes not only the initial tRNA but also participates in the elongation of peptide chains of proteins, so that inhibition of methionyl tRNA synthetase can maximally inhibit vital activities. In addition, MetRS from different species have structural diversity. In recent aaRS inhibitor studies, several aminoacyl-tRNA synthetase inhibitors have been found, which have high selectivity and strong inhibitory ability. aaRS has proven to be an ideal target for antibiotic action. Because the action targets of the anti-tuberculosis drugs used clinically at present are not in the aminoacyl tRNA synthetase system, the inhibitor aiming at the aminoacyl tRNA synthetase system has no cross drug resistance with the current drugs.
The study of M.tuberculosis methionyl tRNA synthetase (MtMetRS) reported from the end of the last century that there was no new progress after its aminoacylation, which is related to the difficulty in recombinant protein production. The MetRS inhibitor has the problems of strong specificity and narrow coverage of antibacterial spectrum. MetRS inhibitors for the treatment of tuberculosis need to be designed and screened according to the MtMetRS structure. Obtaining an accurate molecular model of MtMetRS is the basis for the rational design of inhibitors based on structure. Currently, the crystal structure of mycobacterium tuberculosis MetRS containing ligands in the protein structure database PDB, but ligand-free MtMetRS structures are not obtained. Therefore, the structural research aiming at the mycobacterium tuberculosis methionyl tRNA synthetase firstly needs to solve the problems of preparation and crystallization of recombinant protein.
Disclosure of Invention
The invention aims to prepare the MtMetRS ligand-free crystal and analyze the structure of the MtMetRS ligand-free crystal, and provide an action target for preparing antituberculosis drugs.
The invention firstly protects the recombinant MtMetRS, and the amino acid sequence of the recombinant MtMetRS can be shown as a sequence 2 in a sequence table.
Nucleic acid molecules encoding the recombinant MtMetRS are also within the scope of the invention.
The nucleic acid molecule may be b1) or b 2):
b1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;
b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table.
Expression cassettes, recombinant vectors or recombinant microorganisms containing the nucleic acid molecules also belong to the scope of protection of the present invention.
The invention also protects the application of the recombinant MtMetRS, or any one of the nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any one of the nucleic acid molecules, which can be at least one of A1) to A5);
A1) preparing a ligand-free crystal of the recombinant MtMetRS;
A2) preparing antituberculosis drugs;
A3) preparing a MetRS inhibitor;
A4) designing and/or screening small molecule compounds;
A5) and can be used for treating tuberculosis.
In the above application, the small molecule compound may be specifically an antibiotic.
The invention also protects a ligand-free crystal of the recombinant MtMetRS.
The invention also protects a method for preparing the ligand-free crystal of the recombinant MtMetRS, which is to crystallize the recombinant MtMetRS in the recombinant MtMetRS solution by adopting a crystallization solution to obtain the ligand-free crystal of the recombinant MtMetRS;
the crystallization liquid contains calcium acetate, sodium cacodylate and PEG 8000.
In the above method, the crystal liquid may contain 0.08-0.12M calcium acetate, 0.08-0.12M sodium cacodylate and 4-6mg/100mLPEG8000, and its pH value is 6.0-6.2. The crystallization liquid can be an aqueous solution containing 0.08-0.12M calcium acetate, 0.08-0.12M sodium cacodylate and 4-6mg/100mLPEG8000, and the pH value is 6.0-6.2. The crystallization liquid can be specifically an aqueous solution containing 0.1M calcium acetate, 0.1M sodium cacodylate and 5mg/100mLPEG8000, and the pH value is 6.1.
In the above method, the crystallization condition may be 17-23 deg.C (such as 17-20 deg.C, 20-23 deg.C, 17 deg.C, 20 deg.C or 23 deg.C) and standing for more than 48h (such as 48h, 3d, 4d, 6d, 10d, 15d, 20d or 25 d).
In the above method, the "crystallizing the recombinant MtMetRS in the recombinant MtMetRS solution using a crystallization solution" may specifically be a pendant drop method.
In the method, the recombinant MtMetRS solution can be a protein solution obtained by crushing recombinant Escherichia coli, purifying by a Ni affinity chromatography column, and concentrating by an ultrafiltration concentration tube and a molecular sieve column when the recombinant MtMetRS is expressed by the Escherichia coli. The ultrafiltration concentration tube may have a molecular weight cut-off of 10 kD. The molecular sieve column can be a superdex200 molecular sieve column, and the molecular sieve buffer can be an aqueous solution containing 100mM NaCl and 50mM Tris with the pH value of 8.5.
The invention also provides a ligand-free crystal of the recombinant MtMetRS prepared by any one of the methods.
The invention also provides a model for screening the MtMetRS inhibitor, or screening mycobacterium tuberculosis inhibitor, or screening anti-tuberculosis drugs, which can be the ligand-free crystal of the recombinant MtMetRS.
The invention also protects the application of the ligand-free crystal of any one of the recombinant MtMetRSs, which can be at least one of A2) to A5);
A2) preparing antituberculosis drugs;
A3) preparing a MetRS inhibitor;
A4) designing and/or screening small molecule compounds;
A5) and can be used for treating tuberculosis.
In the above application, the small molecule compound may be specifically an antibiotic.
Any of the above-mentioned crystallization solutions also falls within the scope of the present invention.
The application of any one of the crystallization liquids in the preparation of the ligand-free crystal of the recombinant MtMetRS also belongs to the protection scope of the invention.
The three-dimensional structure of any of the recombinant MtMetRS crystals described above can be represented as a in fig. 1.
Any of the MetRS inhibitors described above can specifically be MtMetRS inhibitors.
In order to obtain a crystal structure of the MtMetRS ligand-free, the inventor designs the crystal structure on the basis of an amino acid sequence of natural MtMetRS, adds a crystallization promoting sequence, obtains a coding gene of the recombinant MtMetRS shown in a sequence 1 in a sequence table, and codes the recombinant MtMetRS shown in a sequence 2 in the sequence table. The recombinant MtMetRS ligand-free crystal is obtained by optimizing the expression and purification process flow of the recombinant MtMetRS and designing and optimizing the preparation process flow of the recombinant MtMetRS ligand-free crystal, and finally the three-dimensional structure information of the recombinant MtMetRS ligand-free crystal is obtained. This structure is significantly different from the crystal structure (PDB code: 6AX8) disclosed in WWW.PDB.ORG in the conformation of the enzyme activity-related structures such as the substrate binding pocket and the catalytic domain. Structural information that differs significantly from 6AX8 in these critical regions is the basis for designing specific drugs. The recombinant MtMetRS has different configuration changes from other species in catalytic reaction, and the inhibition of the configuration changes can screen and design high-selection antibiotics. In the screening of small molecular compounds, the assistance of recombinant MtMetRS ligand-free crystals is required, and the optimization of the later-stage compound structure also requires that combination mode data is obtained by soaking the recombinant MtMetRS ligand-free crystals and the small molecular compounds. The recombinant MtMetRS and the crystallization liquid provided by the invention are the basis for obtaining the recombinant MtMetRS ligand-free crystal and are also necessary technologies for screening and designing the MtMetRS inhibitor. The invention has great application value.
Drawings
FIG. 1 is a three-dimensional structure of ligand-free MtMetRS and binding ligand MtMetRS (6AX 8).
FIG. 2 is an analysis of the recombinant MtMetRS ligand-free crystal structure and the human cytoplasmic MetRS (HcMetRS) crystal structure.
FIG. 3 is a graph of the prepared recombinant MtMetRS ligand-free crystal and its X-ray diffraction pattern.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 preparation of recombinant MtMetRS
Through a large number of experiments, the inventor adds a plurality of basic amino acids at two ends of a natural protein MtMetRS to obtain a recombinant MtMetRS which can be used for preparing a ligand-free crystal. Therefore, the combination of a cationic polypeptide chain formed by adding a plurality of basic amino acids at two ends of the natural protein MtMetRS and anion regions on the surfaces of other molecules can help to form protein crystals by stacking, and is a necessary condition for preparing ligand-free crystals. The recombinant MtMetRS is a non-natural protein and is obtained by modifying the natural protein MtMetRS.
Firstly, construction of recombinant plasmid pQE60-MtMetRS
The DNA molecule between the NcoI recognition sequence and the BglII recognition sequence of pQE60 vector (QIAGEN) was replaced with the double-stranded DNA molecule shown in sequence 1 of the sequence Listing, and the other sequences of pQE60 vector were kept unchanged to obtain the recombinant plasmid pQE 60-MtMetRS.
The recombinant plasmid pQE60-MtMetRS expresses a recombinant MtMetRS shown in a sequence 2 in a sequence table.
Secondly, expression and purification of recombinant plasmid pQE60-MtMetRS
Balance liquid: an aqueous solution containing 100mM NaCl, 50mM Tris, 10mM imidazole and 0.1% (v/v) beta-mercaptoethanol, pH 8.5.
Washing liquid: an aqueous solution containing 100mM NaCl, 50mM Tris, 10mM imidazole and 0.1% (v/v) beta-mercaptoethanol, pH 8.5.
Eluent: an aqueous solution containing 100mM NaCl, 50mM Tris, 350mM imidazole and 0.1% (v/v) beta-mercaptoethanol, pH 8.5.
Lysis solution: an aqueous solution containing 200mM NaCl, 50mM Tris-HCl, 10mM imidazole, 0.1% (v/v) beta-mercaptoethanol, and 1mM PMSF, at pH 8.5.
1. The recombinant plasmid pQE60-MtMetRS is introduced into Escherichia coli M15 to obtain recombinant Escherichia coli.
2. Inoculating the recombinant Escherichia coli obtained in step 1 into LB liquid medium containing 200mg/L ampicillin, and performing shake culture at 37 deg.C and 2000rpm to obtain OD600nmThe value of the culture broth was about 0.8.
3. Taking the culture solution obtained in the step 2, placing the culture solution in an ice bath for 30min, and then adding IPTG (isopropyl-beta-thiogalactoside) to obtain an induction system; in the induction system, the concentration of IPTG was 0.5 mmol/L.
4. Taking the induction system obtained in the step 3, and carrying out shaking culture at the temperature of 14-18 ℃ and the rpm of 200 for 12-18 h.
5. And (4) taking the induction system which finishes the step (4), centrifuging at 4000rpm for 15min, and collecting thalli.
6. The cells collected in step 5 were resuspended in an ice-cold lysate, and then disrupted by sonication on ice (sonication power 400W, 5s disruption time interval, total 99 times) to obtain a disrupted cell solution.
7. And (4) taking the thallus crushing liquid obtained in the step (6), centrifuging for 30min at 15000rpm, and collecting supernatant.
8. And (4) filtering the supernatant collected in the step (7) by using a 0.45-micron filter membrane, and collecting filtrate.
9. Taking a Ni affinity chromatographic column (a product of QIAGEN company), eluting with 25mL of a balance solution (aiming at balancing the column), adding the filtrate collected in the step 8, eluting with 25mL of a washing solution (aiming at removing foreign proteins), eluting with 20mL of an eluent, and collecting a post-column solution obtained by eluting with the eluent.
10. And (4) putting the post-column solution collected in the step (9) into an ultrafiltration concentration tube with the molecular weight cutoff of 10KD for ultrafiltration concentration to obtain a protein concentrated solution.
11. The protein concentrate obtained in step 10 was purified using a superdex200 molecular sieve column (molecular sieve buffer: aqueous solution containing 100mM NaCl and 50mM Tris, pH 8.5), and the purified protein was concentrated to a protein solution having a concentration of 7.8 mg/mL.
Example 2 preparation of recombinant MtMetRS ligand-free crystals and uniqueness of their structure
Preparation of recombinant MtMetRS ligand-free crystal
The method adopts a pendant drop method to carry out crystallization of the recombinant MtMetRS, and comprises the following specific steps:
1. 200. mu.L of the crystallization solution was added to the well.
Crystallization liquid: an aqueous solution containing 0.1M calcium acetate, 0.1M sodium cacodylate and 5mg/100mLPEG8000, and having a pH of 6.1.
2. 1 part by volume of the protein solution having a concentration of 7.8mg/mL obtained in step 11 of example 1 and 1 part by volume of the crystallization solution were mixed on a glass slide, and then covered on the well where step 1 was completed, and sealed with vaseline.
3. And (3) after the step 2 is completed, standing for 15-25 days at 20 ℃ to obtain the recombinant MtMetRS ligand-free crystal.
The recombinant MtMetRS ligand-free crystal obtained in the step (3) and an X-ray diffraction pattern thereof are shown in FIG. 3 (diffraction conditions: X-ray wavelength: 1 angstrom, distance: 200mM, rotation angle: 0.2 degree). The three-dimensional structure of the recombinant MtMetRS ligand-free crystal is shown in figure 1 a.
Uniqueness of recombinant MtMetRS ligand-free crystal structure
There are important differences in the published crystal structure (PDB code: 6AX8) (see B in fig. 1) of www.pdb.org and the structure of the recombinant MtMetRS ligand-free crystal, the differences being mainly found in the region of the catalytic domain associated with the enzymatic activity. PDB org discloses a crystal structure (PDB code: 6AX8) in which the catalytic domain is in a catalytic state. The substrate binding pocket of the recombinant MtMetRS ligand-free crystal structure does not form a closed catalytic state, and the amino acid polypeptide involved in substrate ATP binding also differs in shape from 6AX8, and the difference in shape allows multiple specific hydrophobic pockets on the surface of the ligand-free MtMetRS molecule to be associated with the enzymatic activity of MtMetRS. These pockets are targets for designing specific inhibition.
By analyzing the recombinant MtMetRS ligand-free crystal structure and the human cytoplasmic MetRS (HcMetRS) crystal structure (see fig. 2, mycobacterium tuberculosis ligand-free MetRS is the recombinant MtMetRS ligand-free crystal), it was found that HcMetRS in the tRNA amino acid acceptor binding domain is a binding face, while the MtMetRS molecule is a hydrophobic pocket. This provides structural biological evidence for the design of specific inhibitors. In addition, specific hydrophobic pockets existing between a nucleic acid binding loop of the recombinant MtMetRS and alpha 10 and between beta 2 and beta 3 are found to be related to the conformational change of the recombinant MtMetRS, and the occupation of the two hydrophobic pockets can inhibit the substrate-induced conformational change of the recombinant MtMetRS and is also a good inhibitor design target.
<110> institute of pathogenic biology of Chinese academy of medical sciences
<120> recombinant MtMetRS, crystals thereof and application thereof in preparation of anti-tuberculosis drugs
<160> 2
<170> PatentIn version 3.5
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atgggccacc atcatcatca tcatatgaag ccctattacg tcaccaccgc gatcgcatat 60
cccaacgctg caccccacgt aggtcacgcc tacgaataca tcgccaccga cgcgatcgcc 120
cggttcaaac ggctggatcg ctatgacgtg cgcttcctga ccgggaccga cgagcatggc 180
ctgaaggtcg cacaagccgc cgcggcagcg ggcgtgccca ccgcggcgct tgcccggcgc 240
aattccgacg tgtttcagcg catgcaggag gcgctgaaca tctccttcga ccgattcatc 300
cgcactaccg atgccgacca ccacgaggcg tccaaggaac tctggcgacg gatgtcggcg 360
gccggcgaca tctatctgga caactattcc gggtggtact cggtgcgcga cgagcggttc 420
ttcgtcgaat cggagaccca acttgtcgac ggcacgcgcc tgacggtaga gaccggcacg 480
ccggtgacct ggaccgagga gcagacctac ttcttccggc tgtcggccta taccgacaag 540
ctgctggccc actatcacgc caaccccgac ttcatcgcgc cggagacgcg gcgcaacgaa 600
gtgatcagct tcgtctccgg cggcctggac gacctgtcga tctcgcgcac ctcgtttgac 660
tggggtgtgc aggtgcccga gcaccccgac cacgtcatgt acgtctgggt cgacgcgctg 720
accaattacc tgaccggggc gggcttcccg gataccgact cggagttgtt ccgccgctac 780
tggcccgccg atttgcacat gatcggcaag gacatcatca ggtttcatgc cgtctattgg 840
ccggcgtttt tgatgtcagc cggaatcgag ttgccgcgaa ggatcttcgc gcacgggttc 900
ttgcacaacc gcggcgagaa gatgagtaaa tcggtgggca acatcgtcga cccggttgcc 960
ctggcggaag cgctcggggt ggaccaggtc cgctacttcc tgttgcggga ggtcccgttc 1020
ggccaggacg gcagttacag cgacgaggcc atcgtcactc ggatcaacac cgatctggcc 1080
aacgagctcg gcaacttggc ccaacgctcg ttgtcgatgg tggccaaaaa ccttgacggc 1140
agggtgccca acccgggtga gttcgccgac gccgacgccg cgctgcttgc gaccgccgat 1200
ggcttgttgg agcgagtgcg cggtcacttc gacgcacagg cgatgcacct ggcgctggag 1260
gcgatctggc tgatgctcgg cgacgcgaac aagtactttt cggtgcagca gccgtgggta 1320
ctgcgcaaga gcgagtccga agccgatcag gcccggttcc gcaccacgct ctacgtcacc 1380
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Met Gly His His His His His His Met Lys Pro Tyr Tyr Val Thr Thr
1 5 10 15
Ala Ile Ala Tyr Pro Asn Ala Ala Pro His Val Gly His Ala Tyr Glu
20 25 30
Tyr Ile Ala Thr Asp Ala Ile Ala Arg Phe Lys Arg Leu Asp Arg Tyr
35 40 45
Asp Val Arg Phe Leu Thr Gly Thr Asp Glu His Gly Leu Lys Val Ala
50 55 60
Gln Ala Ala Ala Ala Ala Gly Val Pro Thr Ala Ala Leu Ala Arg Arg
65 70 75 80
Asn Ser Asp Val Phe Gln Arg Met Gln Glu Ala Leu Asn Ile Ser Phe
85 90 95
Asp Arg Phe Ile Arg Thr Thr Asp Ala Asp His His Glu Ala Ser Lys
100 105 110
Glu Leu Trp Arg Arg Met Ser Ala Ala Gly Asp Ile Tyr Leu Asp Asn
115 120 125
Tyr Ser Gly Trp Tyr Ser Val Arg Asp Glu Arg Phe Phe Val Glu Ser
130 135 140
Glu Thr Gln Leu Val Asp Gly Thr Arg Leu Thr Val Glu Thr Gly Thr
145 150 155 160
Pro Val Thr Trp Thr Glu Glu Gln Thr Tyr Phe Phe Arg Leu Ser Ala
165 170 175
Tyr Thr Asp Lys Leu Leu Ala His Tyr His Ala Asn Pro Asp Phe Ile
180 185 190
Ala Pro Glu Thr Arg Arg Asn Glu Val Ile Ser Phe Val Ser Gly Gly
195 200 205
Leu Asp Asp Leu Ser Ile Ser Arg Thr Ser Phe Asp Trp Gly Val Gln
210 215 220
Val Pro Glu His Pro Asp His Val Met Tyr Val Trp Val Asp Ala Leu
225 230 235 240
Thr Asn Tyr Leu Thr Gly Ala Gly Phe Pro Asp Thr Asp Ser Glu Leu
245 250 255
Phe Arg Arg Tyr Trp Pro Ala Asp Leu His Met Ile Gly Lys Asp Ile
260 265 270
Ile Arg Phe His Ala Val Tyr Trp Pro Ala Phe Leu Met Ser Ala Gly
275 280 285
Ile Glu Leu Pro Arg Arg Ile Phe Ala His Gly Phe Leu His Asn Arg
290 295 300
Gly Glu Lys Met Ser Lys Ser Val Gly Asn Ile Val Asp Pro Val Ala
305 310 315 320
Leu Ala Glu Ala Leu Gly Val Asp Gln Val Arg Tyr Phe Leu Leu Arg
325 330 335
Glu Val Pro Phe Gly Gln Asp Gly Ser Tyr Ser Asp Glu Ala Ile Val
340 345 350
Thr Arg Ile Asn Thr Asp Leu Ala Asn Glu Leu Gly Asn Leu Ala Gln
355 360 365
Arg Ser Leu Ser Met Val Ala Lys Asn Leu Asp Gly Arg Val Pro Asn
370 375 380
Pro Gly Glu Phe Ala Asp Ala Asp Ala Ala Leu Leu Ala Thr Ala Asp
385 390 395 400
Gly Leu Leu Glu Arg Val Arg Gly His Phe Asp Ala Gln Ala Met His
405 410 415
Leu Ala Leu Glu Ala Ile Trp Leu Met Leu Gly Asp Ala Asn Lys Tyr
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Phe Ser Val Gln Gln Pro Trp Val Leu Arg Lys Ser Glu Ser Glu Ala
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Asp Gln Ala Arg Phe Arg Thr Thr Leu Tyr Val Thr Cys Glu Val Val
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Arg Ile Ala Ala Leu Leu Ile Gln Pro Val Met Pro Glu Ser Ala Gly
465 470 475 480
Lys Ile Leu Asp Leu Leu Gly Gln Ala Pro Asn Gln Arg Ser Phe Ala
485 490 495
Ala Val Gly Val Arg Leu Thr Pro Gly Thr Ala Leu Pro Pro Pro Thr
500 505 510
Gly Val Phe Pro Arg Tyr Gln Pro Pro Gln Pro Pro Glu Gly Lys Arg
515 520 525
Ser His His His His His His
530 535
Claims (11)
1. The amino acid sequence of the recombinant MtMetRS is shown as a sequence 2 in a sequence table.
2. A nucleic acid molecule encoding the recombinant MtMetRS of claim 1.
3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is b1) or b 2):
b1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;
b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table.
4. The ligand-free crystal of recombinant MtMetRS of claim 1.
5. The method for preparing the ligand-free crystal of the recombinant MtMetRS as claimed in claim 1 is to crystallize the recombinant MtMetRS in the recombinant MtMetRS solution by adopting a crystallization solution to obtain the ligand-free crystal of the recombinant MtMetRS;
the crystallization liquid contains calcium acetate, sodium cacodylate and PEG 8000.
6. The method of claim 5, wherein: the crystallization conditions are as follows: standing at 17-23 deg.C for more than 48 h.
7. A ligand-free crystal of recombinant MtMetRS produced by the method of claim 5 or 6.
8. A model for screening an inhibitor of MtMetRS or for screening a drug for inhibiting Mycobacterium tuberculosis or for screening an anti-tuberculosis drug, which is the ligand-free crystal of the recombinant MtMetRS according to claim 1.
9. Use of the recombinant MtMetRS of claim 1 in the preparation of ligand-free crystals of recombinant MtMetRS.
10. Use of the nucleic acid molecule of claim 2 or 3 for the preparation of ligand-free crystals of recombinant MtMetRS.
11. Use of the ligand-free crystals of recombinant MtMetRS according to claim 1, which are a2) or A3);
A2) preparing antituberculosis drugs;
A3) preparation of MetRS inhibitors.
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Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5798240A (en) * | 1994-09-13 | 1998-08-25 | Cubist Pharmaceuticals, Inc. | Recombinant mycobacterial methionyl-tRNA synthetase genes and methods of use therefore |
| WO2001000665A2 (en) * | 1999-06-28 | 2001-01-04 | Oklahoma Medical Research Foundation | Inhibitors of memapsin 2 and use thereof |
| PT1944322E (en) * | 2002-07-19 | 2015-07-01 | Abbvie Biotechnology Ltd | Treatment of tnf alpha related disorders |
| US8518666B2 (en) * | 2006-03-03 | 2013-08-27 | California Institute Of Technology | Site-specific incorporation of amino acids into molecules |
| CN101952428B (en) * | 2007-09-20 | 2013-10-23 | 独立行政法人理化学研究所 | Mutant pyrrolysyl-tRNA synthetase, and method for production of protein having non-natural amino acid integrated therein by using same |
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