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Recombinant mtmetrs, its crystals and their application in the preparation of anti-tuberculosis drugs

A technology of crystallization and crystallization, applied in antibacterial drugs, recombinant DNA technology, pharmaceutical formulations, etc., can solve problems such as unobtained

Active Publication Date: 2021-06-15
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the protein structure database PDB contains the crystal structure of Mycobacterium tuberculosis MetRS with ligand, but the structure of MtMetRS without ligand is not available

Method used

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  • Recombinant mtmetrs, its crystals and their application in the preparation of anti-tuberculosis drugs
  • Recombinant mtmetrs, its crystals and their application in the preparation of anti-tuberculosis drugs
  • Recombinant mtmetrs, its crystals and their application in the preparation of anti-tuberculosis drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1, Preparation of recombinant MTMETRS

[0044]The inventors of the present invention have been subjected to a large number of experiments, and a number of alkaline amino acids can be prepared by adding a plurality of alkaline amino acids at both ends of the natural protein MtmeTRS to obtain recombinant MtmeTRs. It can be seen that the cationic polypeptide chains formed in the natural protein MtmeTRS are combined with the anionic region of the other molecule surface to the anionic region of the other molecule, which can help accumulate the formation of protein crystals, and is the preparation of the elimination of the ligand crystal. The recombinant MtmeTRS is a non-natural protein and is a protein obtained by modification based on natural protein MtmeTRS.

[0045] First, the construction of recombinant plasmid PQE60-MTMETRS

[0046] The NcoI identification sequence of the PQE60 vector (QIAGEN's product) and DNA molecules between the BGLII identification sequence rep...

Embodiment 2

[0064] Example 2, recombinant MTMETRS no ligand crystal preparation and uniqueness of its structure

[0065] First, the preparation of recombinant MTMETRS-free crystals

[0066] The crystallization of recombinant MtmeTRs is performed using hanging droplets. The specific steps are as follows:

[0067] 1, 200 μl of crystalline solution was added to the small hole.

[0068] Crystallization: The aqueous solution containing 0.1 M calcium acetate, sodium dimethyl sodium acetate and 5 mg / 100 mlpeg8000, and the pH is 6.1.

[0069] 2. A protein solution obtained from 1 body 7.8 mg / ml and 1 body volume crystal solution is mixed on the slide and then covered on the small hole of step 1, and is covered with Vaseline.

[0070] 3. After completing step 2, stand at 20 ° C for 15-25 days to obtain recombinant MtmeTRs without ligand crystals.

[0071] Recombinant MtmeTRs without ligand crystals and its X-ray diffraction map thereof image 3 (Diffraction conditions are: x-ray wavelength: 1 angst...

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Abstract

The invention discloses recombinant MtMetRS, its crystals and their application in preparing anti-tuberculosis drugs. The amino acid sequence of the recombinant MtMetRS is shown as sequence 2 in the sequence listing. The method for preparing the recombinant MtMetRS crystal is to crystallize the recombinant MtMetRS in the recombinant MtMetRS solution by using crystallization solution to obtain the recombinant MtMetRS crystal; the crystallization solution contains calcium acetate, sodium cacodylate and PEG8000. The three-dimensional structure of the recombinant MtMetRS ligand-free crystal and the published crystal structure of WWW.PDB.ORG (PDB code: 6AX8) have major conformational differences in structures related to enzyme activity such as the substrate binding pocket and catalytic domain. Structural information that differs significantly from 6AX8 in these key regions is the basis for designing specific drugs. The recombinant MtMetRS provided by the present invention is the basis for obtaining recombinant MtMetRS ligand-free crystals, and is also an essential technology for screening and designing MtMetRS inhibitors. The invention has great application value.

Description

Technical field [0001] The present invention belongs to the biotechnology field, and more particularly to recombinant MtmeTRs, whose crystals and their applications in the preparation of anti-tuberculosis drugs, and more particularly to the preparation of recombinant MTMETRS crystals and their applications in anti-tuberculosis drug design. Background technique [0002] Tuberculosis (TB) is a severe infectious disease caused by Mycobacterium. Antibiotics in the long-term use of tuberculosis treatment, causing increased drug tuberculosis infections, bringing unprecedented serious challenges for tuberculosis. Therefore, it is found that new drug targets and design new anti-tuberculosis drugs are imminent. [0003] AMINOACYL-TRNASYNTHETAS, AARS is involved in the intracellular height conserved protease family, participating in the biosensive information translation process, is responsible for transferring amino acids to the corresponding TRNA, is an enzyme that ensures that intracell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52A61K38/53A61P31/06
CPCA61K38/00A61P31/06C07K2299/00C12N9/93C12Y601/0101
Inventor 崔胜王炜高小攀秦博
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI