Expression purification, crystal structure and application of mycobacterium tuberculosis ribosomal protein S7

A technology of mycobacterium tuberculosis and ribosomal protein, applied in the biological field, can solve the problems of no good treatment, large side effects, and long treatment cycle

Active Publication Date: 2019-09-20
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the treatment of tuberculosis mainly relies on antibiotics. The most commonly used anti-tuberculosis drugs on the market are rifampicin and isoniazid, but there is a common disadvantage of long treatment cycle, which usually takes more than 6 months. stabilize the patient
In recent years, due to the abuse of antibiotics, many pathogenic bacteria have multi-drug resistance. This type of multi-drug

Method used

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  • Expression purification, crystal structure and application of mycobacterium tuberculosis ribosomal protein S7
  • Expression purification, crystal structure and application of mycobacterium tuberculosis ribosomal protein S7
  • Expression purification, crystal structure and application of mycobacterium tuberculosis ribosomal protein S7

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1. Expression and purification of ribosomal protein S7

[0028] From tuberculosis pathogenic bacteria Mycobacterium tuberculosis H37Rv T Ribosomal protein S7, its amino acid sequence is shown in SEQ.ID.NO1, and its nucleic acid sequence is shown in SEQ.ID, NO2.

[0029] The full-length gene of mtb_rpsG was cloned into the pSMT3 vector by molecular cloning technology to express the fusion protein with 6 His and SUMO tags fused at the amino terminus, and the recombinant vector was transformed into Escherichia coli Rosetta (DE3), and cultured with shaking at 37°C When the OD600 reaches 0.6-1.0 (preferably 0.8), add IPTG with a final concentration of 0.1-1.0mM (preferably 0.4mM) to induce expression. The expression conditions are: 16°C shaking culture for 20h or 20°C shaking culture for 16h or 25°C Shake culture 8h. Preference is given to culturing at 16°C with shaking at 200rpm. Bacteria were collected by centrifugation after 20 hours for purification.

[0030]...

Embodiment 2

[0031] Example 2. Crystallization of ribosomal protein S7

[0032] Concentrate the ribosomal protein S7 expressed and purified by the above method to a concentration of about 17 mg / ml, and use a crystallization kit (Crystal Screen Kit I / II, Index, Salt, PEG / Ion, etc. from companies such as Hampton Research) as crystal growth The initial screening conditions were carried out by hanging drop (or sitting drop) gas phase diffusion method for crystallization. The present invention obtains primary crystals under a plurality of different crystallization reagent conditions. Through the optimization and adjustment in the later stage, preferably 20% PEG3350, the buffer solution of 8% Tacsimate (pH6.0) is used as the crystal growth condition, and a set of resolution is collected. X-ray diffraction data.

Embodiment 3

[0033] Example 3. Diffraction data collection and structural analysis of ribosomal protein S7

[0034] In the present invention, the prepared crystals are treated with 25% glycerin as an antifreezing agent, and then stored in liquid nitrogen. The crystallographic X-ray diffraction data were collected at the Shanghai Light Source (SSRF) Biomacromolecular Crystallography Beamline Station (5th line and 6th station BL17U1). HKL2000 was used for data processing, and the structural analysis was based on the S7 protein (PDB ID: 1HUS) derived from the bacterium Geobacillus stearothermophilus as a model. The molecular replacement method was used, and the Refmac program and Coot software were used to make further corrections, and finally the ribosomal protein S7 was analyzed. Protein crystal structure. Those of ordinary skill in the art know that the atoms in the three-dimensional crystal structure have at least 40% of the atomic coordinates listed in Table 1, or at least 40% of the ma...

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Abstract

The invention belongs to the field of biotechnology, and specifically relates to expression purification, a crystal structure and an application of a mycobacterium tuberculosis ribosomal protein S7. The protein S7 contains 156 amino acids, has the molecular weight of about 17.6 kDa, and has the isoelectric point of about 10.56; the protein S7 has high soluble expression in an expression strain escherichia coli rosetta (DE3), and has good purity and homogeneity. A three-dimensional structure of the ribosomal protein S7 is obtained by an X-ray diffraction method; the resolution ratio reaches up to 1.80 Angstroms; the space group is P2[1]2[1]2[1]. The structure is integrally composed of 6 alpha spirals and 2 beta spirals, wherein an alpha spiral beam structure composed of alpha spiral 1, alpha spiral 2, alpha spiral 3, alpha spiral 4 and alpha spiral 5 has interaction with other proteins on other ribosomes; a beta hairpin structure connected between alpha 3 and alpha 4 has an RNA recognition function. The three-dimensional structure of the ribosomal protein S7 has broad application prospects in screening of novel anti-tuberculosis drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the expression and purification of a ribosomal protein derived from tuberculosis pathogenic bacteria Mycobacterium tuberculosis, its crystal structure and its application in screening new anti-tuberculosis drugs. Background technique [0002] Tuberculosis (Tuberculosis) is called scrofula in traditional Chinese medicine. It is a kind of infectious disease caused by Mycobacterium tuberculosis. It is the most common infection in the lungs, and other parts of the body can also be infected. Most TB infections are latent tuberculosis, in which people with only Mycobacterium tuberculosis carry no symptoms. About 5-10% of latent tuberculosis will be transformed into active tuberculosis (active tuberculosis). Tuberculosis is widely distributed all over the world. According to the statistics of the World Health Organization, about a quarter of the world's population suffers from ...

Claims

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Application Information

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IPC IPC(8): C07K14/35C07K1/30C12N15/70
CPCC07K14/35C12N15/70C07K2299/00
Inventor 李继喜李正阳战博闻高宝才
Owner FUDAN UNIV
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