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Expression and purification of Mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2, and crystalline three-dimensional structure of complex of Mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2 and phosphoric acid

A technology of Mycobacterium tuberculosis and phosphatase, applied in hydrolase, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as little research on drug targets

Inactive Publication Date: 2013-06-26
TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, anti-tuberculosis drugs mainly target the active Mycobacterium tuberculosis by inhibiting the synthesis of its cell wall, energy ATP and DNA, and there are few studies 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 crystalline three-dimensional structure of complex of Mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2 and phosphoric acid
  • Expression and purification of Mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2, and crystalline three-dimensional structure of complex of Mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2 and phosphoric acid
  • Expression and purification of Mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2, and crystalline three-dimensional structure of complex of Mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2 and phosphoric acid

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Effect test

Embodiment 1

[0039] Construction of mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB 2 Soluble expression vectors, methods for obtaining large amounts of expressed strains

[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 large amount of expression 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), and the front of the tag contained Th...

Embodiment 2

[0046] OtsB of Mycobacterium tuberculosis H37Rv 2 crystallization of

[0047] The OtsB2 protein of Mycobacterium tuberculosis H37Rv expressed and purified by the above method was concentrated to a concentration of about 10-20mg / mL, and DTT was added 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 gas-phase hanging drop method at 4, 16 and 25 degrees Celsius respectively, and carried out preliminary screening of crystal growth conditions. After preliminary screening, the inventors obtained initial crystals under different conditions of crystallization reagents. By adjusting the reagent concentration in the pool solution, the conditions are: 11.5% PEG20000, 0.12MMes (pH=6.5), needle-like 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 inventors 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 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 invention relates to an expression, purification and crystallization method of Mycobacterium tuberculosis trehalose-6-phosphate phosphatase OtsB2, and a vector construction, expression and purification method of a trehalose-6-phosphate phosphatase OtsB2 selenoprotein. The vector construction, expression and purification method of the trehalose-6-phosphate phosphatase OtsB2 selenoprotein is characterized in that leucine at the 29th and 133th positions of the OtsB2 undergoes mutation to form methionine, and a special culture medium is used in Escherichia coli to massively express the selenoprotein OtsB2. The invention also relates to a crystallization method of a complex of trehalose-6-phosphate phosphatase OtsB2 and a phosphate radical, a crystalline three-dimensional structure of the complex of the trehalose-6-phosphate phosphatase OtsB2 and the phosphate radical, and applications of the structure in the Mycobacterium tuberculosis drug screening field and the drug designing field.

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 major challenges today. According to WHO reports, more than two billion people in the world have been infected with Mycobacterium tuberculosis, resulting in 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 Applications(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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