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Method for purifying firefly luciferase

A luciferase, firefly technology

Inactive Publication Date: 2015-09-30
BEIJING ZHONGKEZIXIN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing luciferase purification technology, the purity of the obtained product is low, which easily affects the accuracy of the sequencing reaction and cannot meet the needs of genome sequencing

Method used

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  • Method for purifying firefly luciferase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Take 1L luciferase crude enzyme solution, add 10mmol of magnesium chloride, 15mmol of magnesium sulfate and 16mmol of calcium chloride;

[0030] (2) Add 6g of soybean polysaccharide and 3g of dimercaptothreitol;

[0031] (3) Organic solvent precipitation: slowly add 15ml acetone and 20ml acetonitrile, stir, let stand for 10 minutes, centrifuge at 13000rpm, discard the supernatant, add 10ml Tris-HCl buffer, stir, centrifuge at 13000rpm, collect the supernatant;

[0032] (4) Use a 0.22μm filter membrane for sterile filtration;

[0033] (5) Perform nickel column purification, use pH 7.4 Tris-HCl buffer for column equilibration and elution, and use 20-500mM imidazole for gradient elution during elution;

[0034] (6) Use GE molecular exclusion prepacked column superdex 200 for molecular exclusion, and use pH 7.4 Tris-HCl buffer for column equilibration and elution to obtain 100ml of enzyme solution, which is dried by rotary evaporation;

[0035] The above steps we...

Embodiment 2

[0037] (1) Take 1L luciferase crude enzyme solution, add 20mmol of magnesium nitrate, 12mmol of calcium sulfate, and 8mmol of calcium chloride;

[0038] (2) Add 5g of glycerin;

[0039] (3) Organic solvent precipitation: slowly add 20ml methanol and 12ml acetonitrile, stir, let stand for 10 minutes, centrifuge at 13000rpm, discard the supernatant, add 10ml Tris-HCl buffer, stir, centrifuge at 13000rpm, collect the supernatant;

[0040] (4) Use a 0.22μm filter membrane for sterile filtration;

[0041] (5) Perform nickel column purification, use pH 7.4 Tris-HCl buffer for column equilibration and elution, and use 20-500mM imidazole for gradient elution during elution;

[0042] (6) Use GE molecular exclusion prepacked column superdex 200 for molecular exclusion, and use pH 7.4 Tris-HCl buffer for column equilibration and elution to obtain 100ml of enzyme solution, which is dried by rotary evaporation;

[0043] The above steps (5) and (6) were carried out at 4°C, and other ...

Embodiment 3

[0045] (1) Take 1L luciferase crude enzyme solution, add 30mmol of magnesium chloride, 2mmol of calcium sulfate, 4mmol of calcium chloride and 4mmol of calcium nitrate;

[0046] (2) Add glycerin 8g, ethylene glycol 2g and soybean polysaccharide 5g;

[0047] (3) Organic solvent precipitation: slowly add 30ml of acetonitrile, stir, let stand for 10 minutes, centrifuge at 13000rpm, discard the supernatant, add 10ml Tris-HCl buffer, stir, centrifuge at 13000rpm, collect the supernatant;

[0048] (4) Use a 0.22μm filter membrane for sterile filtration;

[0049] (5) Perform nickel column purification, use pH 7.4 Tris-HCl buffer for column balance and elution, and use 20-500mM imidazole for gradient elution during elution;

[0050] (6) Use GE molecular exclusion prepacked column superdex 200 for molecular exclusion, and use pH 7.4 Tris-HCl buffer for column equilibration and elution to obtain 100ml of enzyme solution, which is dried by rotary evaporation;

[0051] The above st...

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Abstract

The invention belongs to the biotechnology field, and particularly relates to a method for purifying firefly luciferase. The method comprises the following steps that metal salt and stabilizers are added, precipitation and filtering of an organic solvent are conducted, and a nickel column and a molecular sieve are adopted. The method is reasonable in design. The firefly luciferase obtained by purification of the method is large in vitality and high in catalytic efficiency and can be widely applied to genomic sequencing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for purifying firefly luciferase. Background technique [0002] Luciferase is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or aliphatic aldehydes in organisms, and can be extracted from bacteria, fireflies and other organisms. The luminescent reaction catalyzed by firefly luciferase must involve the participation of ATP and firefly luciferin. Luciferase can be divided into two categories: firefly luciferase and bacterial luciferase. Because luciferase detection is simple, sensitive and rapid, luciferase gene has become a widely used reporter gene in the field of genetic engineering. Its endogenous content is low, the detection is not easily disturbed, the sensitivity is high, and the detection range is wide. Firefly luciferase and bacterial luciferase can be directly extracted from firefly and luminescent bact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02
CPCC12N9/0069C12Y113/12007
Inventor 高静蔡亦梅徐潇吴超张睿王者馥王绪敏殷金龙任鲁风
Owner BEIJING ZHONGKEZIXIN TECH
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