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A kind of protection method of ADP matching base chromatography medium

A medium and base-layer technology, applied in the field of affinity chromatography, can solve the problems of decreased separation efficiency, ADP ligand damage, and failure to maintain high yield reproducibility, etc., achieving strong practicability, low economic cost, and significant protective effect Effect

Active Publication Date: 2019-06-21
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the ADP ligand damage in the process of using 2'5' ADP Sepharose affinity chromatography to purify thioredoxin reductase TrxR in the prior art, resulting in a decrease in separation efficiency and failure to maintain high yield reproducibility, etc. The problem is to provide a protection method for ADP ligand-based chromatography medium, improve the purification performance of 2'5'ADP Sepharose affinity chromatography column, and prolong its service life

Method used

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  • A kind of protection method of ADP matching base chromatography medium
  • A kind of protection method of ADP matching base chromatography medium

Examples

Experimental program
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Effect test

Embodiment 2

[0022] Take 10ml TrxR crude enzyme solution, add 200μl 0.5M EDTA to make the concentration of EDTA in the crude enzyme solution be 10mM. Equilibrate the column with 15ml TE buffer, then add the TrxR crude enzyme solution to the 2'5'ADP Sepharose affinity chromatography column with a column volume of 5ml, then wash the column with 100ml TE buffer, and then wash with 15ml TE+0.5M NaCl Remove the TrxR specifically bound to the 2'5' ADP ligand, and then use 15ml regeneration buffer 1 (0.1M Tris-HCl, 0.5M NaCl, pH 8.5) and 15ml regeneration buffer 2 (0.1M sodium acetate, 0.5M NaCl , pH 4.5) alternately regenerated the column three times, followed by equilibrating the column with 15 ml of TE buffer. The DTNB method was used to measure the total activity of TrxR before and after the affinity chromatography, and the yield of the affinity chromatography was 66%.

[0023] Analyze Comparative Example 1 and Example 2. In the thioredoxin reductase TrxR crude enzyme solution in Comparativ...

Embodiment 3

[0025] Take 10ml TrxR crude enzyme solution, add 400μl 0.5M EDTA to the concentration of EDTA in the crude enzyme solution is 20mM. Equilibrate the column with 15ml TE buffer, then add the TrxR crude enzyme solution to the 2'5'ADP Sepharose affinity chromatography column with a column volume of 5ml, then wash the column with 100ml TE buffer, and then wash with 15ml TE+0.5M NaCl Remove the TrxR specifically bound to the ADP ligand, and then use 15ml regeneration buffer 1 (0.1M Tris-HCl, 0.5M NaCl, pH 8.5) and 15ml regeneration buffer 2 (0.1M sodium acetate, 0.5M NaCl, pH 4.5) Alternately regenerate the column three times, then equilibrate the column with 15 ml TE buffer. The DTNB method was used to measure the total activity of TrxR before and after the affinity chromatography, and the yield of the affinity chromatography was 85%.

Embodiment 4

[0027] Take 10ml of TrxR crude enzyme solution and add 1ml of 0.5M EDTA to the concentration of EDTA in the crude enzyme solution is 50mM. Equilibrate the column with 15ml TE buffer, then add the TrxR crude enzyme solution to the 2'5'ADP Sepharose affinity chromatography column with a column volume of 5ml, then wash the column with 100ml TE buffer, and then wash with 15ml TE+0.5M NaCl Remove the TrxR specifically bound to the ADP ligand, and then use 15ml regeneration buffer 1 (0.1M Tris-HCl, 0.5M NaCl, pH 8.5) and 15ml regeneration buffer 2 (0.1M sodium acetate, 0.5M NaCl, pH 4.5) Alternately regenerate the column three times, then equilibrate the column with 15 ml TE buffer. The DTNB method was used to measure the total activity of TrxR before and after the affinity chromatography, and the yield of the affinity chromatography was 69%.

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Abstract

The invention belongs to the technical field of affinity chromatography, and provides an efficient and simple protection method of an ADP aglucon chromatography media. The method comprises the following steps: adding a chelating agent into flavoprotein TrxR crude enzyme, so as to chelate Mg<2+> in the crude enzyme, inhibit DNase activity and protect ADP aglucon; balancing a 2'5'ADP Sepharose affinity column through a TE buffer solution, adding the TrxR crude enzyme containing the chelating agent intothe column to ensure that TrxR is combined with the ADP aglucon, washing miscellaneous proteinnonspecifically combined on the column with the TE buffer solution, eluting the TrxR combined with the ADP aglucon with TE+0.5M NaCl, treating the column with a regulation buffer solution to perform regeneration on the column, and then immediately balancing the column subjected to regeneration with the TE buffer solution. The method has the advantages that an operation method is simple, the chelating agent is cheap and east to get, the economic cost is low, the protection effect on the ADP aglucon is obvious, and the practicability is high.

Description

technical field [0001] The invention belongs to the technical field of affinity chromatography and relates to a method for protecting an ADP ligand chromatography medium. Background technique [0002] Sepharose's versatility and high mechanical stability make it an excellent matrix for high-performance chromatographic procedures in affinity, ion-exchange, and other modes of separation. The agarose-based Sepharose chromatography media family can achieve stable binding of ligands for the purification of different enzymes, antibodies and other proteins and peptides, greatly expanding the range of potential applications and widely used in laboratory and industrial applications. [0003] 2'5'ADP is a structural analogue of NADP immobilized on Sepharose 4B by cyanogen bromide activation method. It has affinity for enzymes that require NADP cofactor, so 2'5'ADP Sepharose 4B is mainly used for purification and requires NADP as a cofactor enzymes. 2'5'ADP Sepharose 4B has a strong ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/08B01D15/20
CPCB01D15/08B01D15/20
Inventor 许建强张竞争徐卫平孙世博关水马昆魏宁宁王晓晓张楠
Owner DALIAN UNIV OF TECH
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