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A method of extracting and purifying thioredoxin reductase from chicken livers

A liver thioredoxin, separation and purification technology, applied in the field of separation and purification of thioredoxin reductase, can solve the problems of difference in activity, high requirements, difficulty in obtaining materials from cattle liver, etc. Get difficult effects

Active Publication Date: 2013-09-25
UNIVERSITY OF CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, thioredoxin reductases from Escherichia coli, spinach, Arabidopsis thaliana, cattle, and rats have been purified. Generally, the liver of cattle or rats is used as raw material to extract TrxR for human disease-related research, but bovine The liver is difficult to obtain and requires high requirements (fresh male calf liver), and there is a large difference in activity between different batches

Method used

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  • A method of extracting and purifying thioredoxin reductase from chicken livers
  • A method of extracting and purifying thioredoxin reductase from chicken livers
  • A method of extracting and purifying thioredoxin reductase from chicken livers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Extraction and purification of chicken liver thioredoxin reductase

[0029] Material

[0030] Fresh chicken liver, anion exchange chromatography filler (DEAE-Cellulose DE52, Whatman), ADP affinity chromatography filler (2,5-ADP Sepharose TM 4B, Amersham), and other reagents were of analytical grade.

[0031] main instrument

[0032] High-speed tissue masher (DS200, Jiangsu Jiangyin Zhouzhuang Scientific Research Equipment Factory), super constant temperature water tank (DKB-501A, Shanghai Precision Experimental Equipment Co., Ltd.), spectrophotometer (UV-VIS8500, KEDA), low-temperature chromatography cabinet (DW -1. Shengxian Refrigerator Factory in Zhejiang Province), fully automatic high-speed refrigerated centrifuge (GL-20, Xiangxi Instrument Factory Mechanical Instrument Factory), constant temperature magnetic stirrer (JB-3, Xinjing Branch of Shanghai Leici Instrument Factory) factory), circulating water multi-purpose vacuum pump (SHB-III, Zhengzhou G...

Embodiment 2

[0046] Embodiment 2: the concentration, activity, purity and sequence determination of chicken thioredoxin reductase

[0047] main instrument

[0048] Stabilized voltage and current electrophoresis instrument (DYY-6B, Beijing Liuyi Instrument Factory), decolorization shaker (TS-1, Jiangsu Haimen Qilin Medical Instrument Factory), pH meter (PHS-3D, SANXIN)

[0049] method:

[0050] The enzyme solution finally extracted in Example 1 was used to measure the protein concentration by Coomassie Brilliant Blue staining. Take 20 μl of 2-fold diluted enzyme solution and 20 μl of bovine serum albumin at standard concentrations of 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, and 0.50 mg / ml, respectively, and add them to a 96-well plate Add 180 μl of Coomassie Brilliant Blue staining solution, and monitor the light absorption value of each well at 595 nm after 5 to 10 minutes (because the complex formed by Coomassie Brilliant Blue and protein has specific absorption at 595 ...

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Abstract

The invention relates to an enzyme extraction and purification technology, specifically to a method of extracting and purifying thioredoxin reductase from chicken livers. The method comprises steps of obtaining a crude extract from fresh chicken livers; salting out; and extracting and purifying thioredoxin reductase from chicken livers by employing chromatography that uses two chromatography media comprising a DEAE anion exchange chromatography column and an ADP affinity chromatography column. The method provided by the invention is advantageous in that the raw material chicken livers have wide source and low cost; process of the extraction and purification technology is simple, and can be operated conveniently; finished products have high purity and the same capability of catalyzing human Trx reduction as cattle or mouse thioredoxin reductase, and therefore the finished products can be applied to scientific researches and practices, in place of cattle or mouse thioredoxin reductase, for human-related disease researches; and problems, of extraction and purification processes for cattle or mouse TrxR, comprising difficulty in obtaining raw materials, large individual differences, etc. are solved. The method provided by the invention fills in the blank of biological reagent preparations in our country and has high economic value.

Description

technical field [0001] The invention relates to an enzyme extraction and purification process, in particular to a method for separating and purifying thioredoxin reductase from fresh chicken liver. Background technique [0002] Thioredoxin system (thioredoxin system) is an important redox regulation system in organisms, involved in cell signal transduction, defense against oxidative stress, regulation of growth and other processes. The thioredoxin system is composed of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Thioredoxin reductase, which catalyzes the electron transfer from NADPH to Trx, plays a central role in this system. Thioredoxin reductase is a member of the pyridine nucleotide disulfide oxidoreductase family. Its structure varies greatly in different organisms, but they are all homodimers. Each subunit has a NADPH binding site point, FAD coenzyme and redox active site, while mammalian thioredoxin reduct...

Claims

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Application Information

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IPC IPC(8): C12N9/02
Inventor 梁巍杜澄钟良玮
Owner UNIVERSITY OF CHINESE ACADEMY OF SCIENCES
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