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Regioselective bacterium nitroreductase gene and application thereof

A selective and reductase technology, applied in the field of enzyme engineering, can solve problems such as poor catalytic effect and lack of regioselectivity in reduction

Inactive Publication Date: 2014-10-15
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the poor catalytic effect in vivo has become the main factor inhibiting its wide application, and one of the reasons for this result is the lack of regioselectivity of NfsB for the reduction of the CB1954 nitro group.

Method used

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  • Regioselective bacterium nitroreductase gene and application thereof
  • Regioselective bacterium nitroreductase gene and application thereof
  • Regioselective bacterium nitroreductase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Gene acquisition and expression vector construction of mutant nitroreductase F124W, N71S / F124W, F123A / F124W and N71S / F123A / F124W

[0025] 1. Cloning of wild-type nfsB gene and construction of cloning vector

[0026] According to the nfsB gene sequence in the E.coli K12 strain (Invitrogen) provided by NCBI, primers (nfsB-WT-F: 5'-GGAATTC CATATG GATATCATTTCTGTCGCCTTAAAG-3',nfsB-WT-R:5'-G GAATTCTTACACTTCGGTTAAGGTGATGTT-3'), where the underlined part represents the gene sequence corresponding to the restriction site. Using the extracted E.coli K12 genomic DNA as a template, PCR amplification was performed. Take 10 μl of the PCR reaction product and run agarose gel electrophoresis to verify that the electrophoresis band is about 650bp in size consistent with the nfsB gene. Use TaKaRa Agarose Gel DNA Purification Kit Ver.3.0 to cut the gel to recover the above electrophoresis target band, then digest the recovered product with EcoR I / Nde I double digestion, use...

Embodiment 2

[0043] Example 2. Expression and purification of bacterial nitroreductase mutants

[0044] 1. Induced expression of nitroreductase mutants

[0045] The recombinant plasmids pET28a-nfsB-F124W, pET28a-nfsB-N71S / F124W, pET28a-nfsB-F123A / F124W and pET28a-nfsB-N71S / F123A / F124W were transformed into E. coli E. coli BL21 (DE3) competent Cells (Invitrogen), plated and incubated overnight at 37°C. Pick positive clones and name them as expression strains E.coli BL21(DE3)-nfsB-F124W, E.coli BL21(DE3)-nfsB-N71S / F124W, E.coli BL21(DE3)-nfsB-F123A / F124W and E.coli BL21(DE3)-nfsB-N71S / F123A / F124W were inoculated in LB liquid medium (containing 50 μg / ml Kana) and shaken overnight at 37°C. Transfer the seed culture solution to 100ml LB expansion medium (containing 50μg / ml Kana) at 1% inoculum size, and culture it on a shaker at 37°C until OD 600 at 0.4-0.6. Add final concentration of 0.5mM IPTG to induce expression at 30°C for 6h. After the cultivation, the fermentation broth was centrifu...

Embodiment 3

[0059] Example 3. Activity determination of bacterial nitroreductase mutants

[0060] The recombinant protein eluted with 250mM imidazole was mixed according to the ratio of molar concentration ratio protein:FMN=1:10, and placed in a 4°C refrigerator for incubation for 2 hours. After incubation, the recombinant protein was desalted into 20 mM Tris-HCl buffer (pH 7.0) for subsequent experiments such as protein activity detection.

[0061] 1. HPLC analysis of the mutant's regioselectivity for CB1954 reduction

[0062] The regioselectivity of mutants NfsB-F124W, NfsB-N71S / F124W, NfsB-F123A / F124W and NfsB-N71S / F123A / F124W for the reduction of substrate CB1954 was analyzed by high performance liquid chromatography (HPLC) Agilent 1200. The HPLC enzyme-catalyzed reaction system contains: 0.1mM CB1954, 0.2mM NADH, 200nM mutant enzyme, the reaction buffer is 20mM Tris-HCl pH7.0, and react at room temperature for 5-15 minutes. Since CB1954 and its reaction product were unstable when h...

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Abstract

The invention discloses a mutant bacterium nitroreductase gene and an application thereof and belongs to the field of enzyme engineering. On the basis of protein three-dimensional structures and molecular docking, phenylalanine in the 124 position of Escherichia coli nitroreductase NfsB interacts with side chain groups of a multi-nitro substrate, so that positioning of the substrate in an activity pocket is affected. Mutants F124W, N71S / F124W, F123A / F124W and N71S / F123A / F124W can catalyze the specificity of a prodrug CB1954 for treating cancers to produce a 4-NHOH product with high biotoxicity. Meanwhile, the catalytic activity of the mutants is improved by 7-24 times when being compared with that of wild type enzymes, and the N71S / F123A / F124W is the most significant. The nitroreductase mutants with specific reduction selectivity have the good application prospect in aspects of treatment of cancers and other diseases.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and specifically relates to a mutant nitroreductase gene, a recombinase containing the gene, and its application in nitro site-selective reduction. Background technique [0002] Nitroreductase is a class of cytoplasmic enzymes dependent on FMN or FAD, which can use NAD(P)H to catalyze the reduction of nitro groups, and finally generate hydroxylamine or amino products through nitroso intermediates. The nitro group is a common functional group in biomedicine, antibacterial agents and insecticides. Its reduction is a key step in the realization of drug biotherapy and in vivo degradation and metabolism. In this process, nitroreductase plays a key role. effect. Among them, the activation therapy system composed of Escherichia coli nitroreductase NfsB and cancer prodrug 5-(1-aziridine)-2,4-dinitrobenzamide (CB1954) has entered the phase III clinical trial stage. NfsB can convert CB1954 into a strong...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/63
Inventor 杨君白敬刘培瑜姜熙杨青
Owner DALIAN UNIV OF TECH
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