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Expression and purification of mycobacterium tuberculosis trehalose-6-phosphate phosphatase otsb2 and the three-dimensional crystal structure of its complex with phosphate

A Mycobacterium tuberculosis, phosphate phosphatase technology, applied in the directions of hydrolase, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of complex and difficult disease control activities, increasing the susceptible population of Mycobacterium tuberculosis, etc.

Inactive Publication Date: 2018-01-19
TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The co-infection of M. tuberculosis and HIV (tuberculosis / HIV), especially in Africa, has increased the susceptibility of M. tuberculosis, and the emergence of multidrug resistance (MDR) and extensive drug resistance ( XDR) tuberculosis, complicating and arduous disease control activities
[0003] At present, anti-tuberculosis drugs mainly target active Mycobacterium tuberculosis by inhibiting the synthesis of its cell wall, energy ATP and DNA, and there is little research on drug targets targeting the dormancy period

Method used

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  • Expression and purification of mycobacterium tuberculosis trehalose-6-phosphate phosphatase otsb2 and the three-dimensional crystal structure of its complex with phosphate
  • Expression and purification of mycobacterium tuberculosis trehalose-6-phosphate phosphatase otsb2 and the three-dimensional crystal structure of its complex with phosphate
  • Expression and purification of mycobacterium tuberculosis trehalose-6-phosphate phosphatase otsb2 and the three-dimensional crystal structure of its complex with phosphate

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Experimental program
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Effect test

Embodiment 1

[0039] Construction of mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB 2 The soluble expression vector of the method that obtains the bacterial strain of a large amount of expression

[0040] The inventors initially tried to convert OtsB 2 Full-length protein expression, trying to construct different expression vectors pGEX, pET, etc., and different expression strains, such as BL21(DE3), BL21(DE3)pLyss, Transetta(DE3), etc., to obtain a protein that can express a large amount of soluble OtB 2 Protein recombinant vector pET-28a, and strain BL21(DE3).

[0041] OtsB of Mycobacterium tuberculosis H37Rv 2 expression purification method

[0042] The above-mentioned construct containing OtsB 2 The recombinant expression vector of the gene was transformed into Escherichia coli BL21 (DE3) to express the target protein, and the Escherichia coli prokaryotic system was used to express the fusion protein with 6 His tags connected to the N-terminus (amino-terminus), a...

Embodiment 2

[0046] OtsB of Mycobacterium tuberculosis H37Rv 2 crystallization of

[0047] Concentrate the OtsB2 protein of Mycobacterium tuberculosis H37Rv expressed and purified by the above method to a concentration of about 10-20mg / mL, and add DTT to a final concentration of 10mM, using a crystallization reagent (Crystal Screen Kit I / II, Index, Salt, PEG / ion, Additive Screen and other kits), and crystallized by the gas-phase hanging drop method at 4, 16 and 25 degrees Celsius, respectively, for preliminary screening of crystal growth conditions. After preliminary screening, the inventors obtained initial crystals under a plurality of different crystallization reagent conditions. By adjusting the reagent concentration in the pool solution, wherein the conditions are: 11.5% PEG20000, 0.12MMes (pH=6.5), needle-shaped crystals were obtained, and a set of X-ray parent diffraction data with a resolution of 3.5 angstroms was collected.

Embodiment 3

[0049] OtsB of Mycobacterium tuberculosis H37Rv 2 The method of selenoprotein carrier construction

[0050] The inventor initially tried to mutate the leucine at the 29th, 133rd, 172nd, 285th and 379th positions of OtsB2 into methionine by point mutation technology, and then respectively mutated the The recombinant vector was expressed and purified, and the results of SDS-PAGE showed that only the recombinant vector containing two mutations at 29 and 133 could express a large amount of protein.

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Abstract

The present invention relates to the expression, purification and crystallization method of mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2; and the vector construction, expression and purification method of trehalose-6-phosphate phosphatase OtsB2 selenoprotein, specifically The leucine at the 29th and 133rd positions of OtsB2 was mutated to methionine, and the selenoprotein OtsB2 was massively expressed in Escherichia coli B121(DE3) using a special medium; The crystallization method of the complex of root; and the three-dimensional crystal structure of the complex of trehalose-6-phosphate phosphatase OtsB2 and phosphate and its application in drug screening and drug design of Mycobacterium tuberculosis.

Description

technical field [0001] The present invention relates to mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB 2 Expression, purification, crystallization optimization method and trehalose-6-phosphate phosphatase OtsB in Escherichia coli 2 The three-dimensional crystal structure of its complex with phosphate and its application in drug screening and drug design of Mycobacterium tuberculosis. Background technique [0002] The global control of Mycobacterium tuberculosis is facing a major challenge today. According to WHO reports, more than two billion people in the world have been infected with Mycobacterium tuberculosis, causing nearly 2 million deaths per year. The co-infection of M. tuberculosis and HIV (tuberculosis / HIV), especially in Africa, has increased the susceptibility of M. tuberculosis, and the emergence of multidrug resistance (MDR) and extensive drug resistance ( XDR) tuberculosis, making disease control activities more complex and more arduous. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/63C12N15/70G06F19/16C12R1/32
Inventor 饶子和单珊杨诚刘祥娄智勇孙玉娜刘婷姚利娜
Owner TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE
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