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Preparation method and application of affinity chromatography porous medium with glutathione ligand

A technology of glutathione and porous media, applied in the direction of transferase, etc., to achieve the effects of high protein purity, cost reduction, and simple preparation method

Inactive Publication Date: 2013-03-20
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the current affinity chromatography medium has become a huge bottleneck restricting the industrialization of genetic engineering, and there is an urgent need for a GST protein affinity chromatography medium with simple preparation methods, low cost, simple operation and high efficiency.

Method used

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  • Preparation method and application of affinity chromatography porous medium with glutathione ligand
  • Preparation method and application of affinity chromatography porous medium with glutathione ligand
  • Preparation method and application of affinity chromatography porous medium with glutathione ligand

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: configure the aqueous solution of glutathione

[0032] (1) Prepare glutathione aqueous solution: prepare 100mL glutathione aqueous solution according to the glutathione concentration in Table 1 and the ratio of dithiothreitol (DTT) to glutathione, and add the proportioning Amount of DTT, room temperature mechanical stirring reaction time as listed in Table 1, to obtain glutathione aqueous solution.

[0033] Table 1 Configuration of glutathione aqueous solution

[0034]

Embodiment 2

[0035] Example 2: Emulsion Template Synthesis of Affinity Chromatography Porous Media with Glutathione Ligands

[0036]Preparation of double bond modified dextran emulsion: get 5 mL of glutathione-1 aqueous solution prepared in Example 1, weigh 1.0 g of double bond modified dextran (molecular weight 200,000, 100 sugar rings contain 23 double key) and 0.5g emulsifier Triton X-405 were added into the three-necked flask together, and mechanically stirred at a speed of 400rpm for 20min;

[0037] Preparation of toluene solution of azobisisobutyronitrile (AIBN): add 0.15 g of azobisisobutyronitrile (AIBN) into 15 ml of toluene and stir until dissolved, the concentration is 1% (w / v);

[0038] Preparation of oil-water emulsion: Add AIBN toluene solution dropwise to the double bond modified dextran emulsion in the three-necked bottle under stirring. The total volume of AIBN toluene solution is 3 times the volume of the double bond modified dextran solution. Continue stirring for 5 minu...

Embodiment 3

[0044] Example 3: Emulsion Template Synthesis of Affinity Chromatography Porous Media with Glutathione Ligands

[0045] Prepare the emulsion of double bond modified dextran: get 20 mL of newly prepared glutathione-4 aqueous solution in Example 1, weigh 4.0 g of double bond modified dextran (molecular weight 500,000, relative to the double bond of sugar ring The molar content is 27%) and 2.0 g of emulsifier Tween-80 are added into the three-necked flask together, and dissolved by mechanical stirring at a speed of 400 rpm for 20 min;

[0046] Prepare the toluene solution of azobisisobutyronitrile (AIBN): add 0.6g of azobisisobutyronitrile (AIBN) into 60ml of toluene and stir until dissolved, the concentration is 1% (w / v);

[0047] Preparation of oil-water emulsion: Add AIBN toluene solution dropwise to the double bond modified dextran emulsion in the three-necked bottle under stirring. The total volume of AIBN toluene solution is 3 times the volume of the double bond modified de...

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Abstract

The invention relates to a preparation method and application of an affinity chromatography porous medium with glutathione ligand. The affinity chromatography porous medium with the glutathione ligand is skillfully synthesized by taking ligand glutathione as a radical transfer agent, and preparation of matrix, processing and shaping of the medium, and loading of the ligand are finished by one-step reaction. A matrix material which is low in price is adopted, so cost is greatly reduced. The affinity chromatography porous medium material has an opening structure, the aperture is 100 to 200 mu m; the bonding quantity of the ligand glutathione can reach 75.3 mg / g, the bonding efficiency is high and purity is high; and results show that the affinity chromatography porous medium has good protein separation performance, and the combination efficiency of glutathione-s- transferase(GSF) reaches 1.5 mg / g. The prepared affinity chromatography porous medium with the glutathione ligand is used for filling a chromatographic column and can be used for the separation and purification of GSF and fusion protein in which GSF is taken as a label.

Description

technical field [0001] The invention relates to a preparation method and application of an affinity chromatography porous medium with a glutathione (hereinafter referred to as glutathione) ligand, belonging to the field of biomedical polymer materials. technical background [0002] Glutathione-s-transferase (GST) is a protein with a molecular weight of about 26,000. Because of its good solubility in water, it is expressed at a high level in E. coli and maintains the enzyme activity that specifically binds to glutathione. It is widely used as tag protein in genetic engineering fusion protein system. By immobilizing glutathione on an affinity medium, the protein with the GST tag in the crushed bacterial liquid is separated, eluted with free glutathione, and then the target protein can be obtained by cutting off the GST tag. [0003] At present, the affinity medium technology used for the separation of GST-tagged proteins is monopolized by a few companies. Usually, agarose gel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10
Inventor 景遐斌董晓庆黄宇彬
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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