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Method for preparing chitosan separation medium suitable for protein purification

A separation medium and protein purification technology, which is applied in the field of natural polymer materials, can solve the problems of low adsorption capacity and unstable conditions of chitosan separation medium, and achieve the solution of metal ion leakage, process stability and reproducibility Good, simple operation effect

Active Publication Date: 2011-05-25
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In recent years, the research on porous chitosan films and microspheres has been reported in the literature. The research on chitosan has developed rapidly, but most of the research has focused on the use of chitosan to modify drug delivery systems such as liposomes, microspheres, and microcapsules. There are not many reports about it as a separation medium, and the conditions of the prepared chitosan separation medium are not stable, and the adsorption capacity is not high.

Method used

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  • Method for preparing chitosan separation medium suitable for protein purification
  • Method for preparing chitosan separation medium suitable for protein purification
  • Method for preparing chitosan separation medium suitable for protein purification

Examples

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Embodiment 1

[0023] The preparation of embodiment 1 chitosan skeleton

[0024] 3.0 g of chitosan (molecular weight 30000 Da, deacetylation degree ≥ 95%) was dissolved in 100 mL of acetic acid solution with a mass fraction of 2.0%, and left standing overnight at room temperature for later use. In a 500mL three-necked bottle equipped with a mechanical stirrer and a thermometer, add 100mL of liquid paraffin, 15mL of cyclohexane, and 6 drops of Span 80 in sequence, and after stirring for 0.5h, add the above chitosan solution, and heat the system to 55°C with a water bath. ℃, stirred for 1 h, added 3 mL of glutaraldehyde with a mass fraction of 25%; adjusted the pH value to 10 with 10% NaOH solution, then raised the temperature to 65 °C, continued the reaction for 3 h, and filtered the micro The ball is filtered out, washed repeatedly with distilled water, then washed with petroleum ether and absolute ethanol, and vacuum-dried to constant weight to obtain a chitosan skeleton, 90% of which have ...

Embodiment 2

[0025] Embodiment 2 chitosan grafting

[0026] Take by weighing the product that 0.5g embodiment 1 obtains, after water fully swells, wash with the DMSO aqueous solution of 20%, 50%, 70% successively; Add 27mL DMSO / NaOH solution (DMSO volume fraction is 0.4 , the concentration of NaOH aqueous solution is 0.4mol / L) and epichlorohydrin, so that the volume fraction of epichlorohydrin is 10%, the shaking reaction is 4h, and the reaction temperature is 50°C. After the reaction, rinse with a large amount of distilled water until no epoxy group is detected in the cleaning solution. Chitosan epoxy group modification density was measured by sodium thiosulfate titration method, and the epoxy group modification density reached 0.1mmol / g.

Embodiment 3

[0027] Embodiment 3 chitosan grafting

[0028] Take by weighing the product that 0.5g embodiment 1 obtains, after water fully swells, wash with the DMSO aqueous solution of 20%, 50%, 70% successively; Add 27mL DMSO / NaOH solution (DMSO volume fraction is 0.5 , the concentration of NaOH aqueous solution is 0.4mol / L) and epichlorohydrin, so that the volume fraction of epichlorohydrin is 6%, the shaking reaction is 3h, and the reaction temperature is 40°C. After the reaction, rinse with a large amount of distilled water until no epoxy group is detected in the cleaning solution. Chitosan epoxy-modified density was measured by sodium thiosulfate titration method, and the epoxy-modified density reached 0.089mmol / g.

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Abstract

The invention relates to a method for preparing a chitosan separation medium suitable for protein purification, which comprises the following steps of: (1) dispersing acetic acid solution of chitosan into liquid paraffin with stirring to form chitosan particles in the presence of cyclohexane serving as a porogen and a small amount of span80, and further cross-linking the chitosan particles to form a chitosan skeleton under the action of glutaraldehyde serving as a cross-linking agent; (2) swelling the chitosan skeleton, and reacting a hydroxyl group on the chitosan skeleton with epoxy chloropropane in dimethyl sulfoxide (DMSO) / NaOH mixed solution to introduce an epoxy group into the chitosan skeleton so as to obtain a grafted chitosan skeleton; and (3) adding iminodiacetic acid (IDA) / NaOHmixed solution into the grafted chitosan skeleton, reacting at the temperature of between 20 and 80 DEG C for 1 to 10 hours, and performing suction-filtration to obtain the chitosan separation medium. The chitosan separation medium solves the leakage problem of metal ions to a large extent, has the advantages of process stability, high repeatability and the like, and is suitable for mass production.

Description

1. Technical field [0001] The invention belongs to the technical field of natural polymer materials, and in particular relates to a preparation method of a chitosan separation medium suitable for protein purification. 2. Background technology [0002] Although there are many methods for protein separation at present, metal chelation chromatography (IMAC) has the advantages of simple and convenient preparation of chelation medium, large exchange capacity, mild separation conditions, strong versatility, and easy to scale up, especially in the purification process of proteins. Among them, its mild elution conditions can better maintain the biological activity of proteins and other advantages, making its application more and more attention, and will become the most potential chromatography method in protein separation and purification. [0003] However, most of the conventional metal chelate chromatography (IMAC) chromatography columns use soft matrices such as dextran or agaros...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/30C07K1/22
Inventor 应国清崔国艳易喻王鸿梅建凤陈建澍
Owner ZHEJIANG UNIV OF TECH
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