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GSH affinity chromatography medium for purifying GST-Tag fusion protein and preparation method and application thereof

A chromatographic medium and fusion protein technology, applied in the field of GST-tag fusion protein purification, can solve the problems of not being able to effectively reduce protein purification costs, unfavorable large-scale use in laboratories, and low protein purification capacity, achieving high capacity, Small steric hindrance, the effect of improving purification efficiency

Active Publication Date: 2019-08-06
武汉菲恩生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the domestic production of this medium mainly relies on imports, which are expensive, and the protein purification capacity is low, and the efficiency drops significantly after several uses, which cannot effectively reduce the cost of protein purification, and is not conducive to large-scale use in laboratories.

Method used

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  • GSH affinity chromatography medium for purifying GST-Tag fusion protein and preparation method and application thereof
  • GSH affinity chromatography medium for purifying GST-Tag fusion protein and preparation method and application thereof

Examples

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Embodiment 1

[0030] This embodiment provides a GSH affinity chromatography medium for purifying GST-tag fusion protein, which is composed of 4B agarose microspheres surface-modified with cyanogen bromide and reduced glutathione that can specifically bind to GST-tag fusion protein Peptide linking, the surface modified with cyanogen bromide 4B agarose microspheres and the reduced glutathione through ethylenediamine and 11-maleimido undecanoic acid succinimidyl ester Make connections for the connecting arms. In this example, cyanogen bromide is used to activate the 4B agarose microsphere matrix. Compared with the common epoxy activation method, it can provide more active groups that can be used for coupling, and the cyanogen bromide activation method is more convenient to operate. The time-consuming preparation of the affinity medium is greatly shortened; the activated microspheres are modified with ethylenediamine with a simple structure, which can provide primary amino groups with more sele...

Embodiment 2

[0053] This embodiment provides a GSH affinity chromatography medium for purifying GST-tag fusion protein, which is composed of 4B agarose microspheres surface-modified with cyanogen bromide and reduced glutathione that can specifically bind to GST-tag fusion protein Peptides are connected, the surface is modified with cyanogen bromide 4B agarose microspheres and the reduced glutathione is passed through urea and maleimide PEG N-hydroxysuccinimide MW: 20000 (MAL- PEG20000-NHS) for connecting arms. The GSH affinity chromatography medium for purifying the GST-tag fusion protein provided in this implementation can be prepared by the following preparation method.

[0054] This embodiment also provides a preparation method of GSH affinity chromatography medium for purifying GST-tag fusion protein, comprising the following steps:

[0055] 1) Mix 10% cyanogen bromide solution, 0.1mol / L sodium carbonate solution, and 4B agarose microspheres at a volume ratio of 0.5:2:4, and vortex at...

Embodiment 3

[0062] This embodiment provides a GSH affinity chromatography medium for purifying GST-tag fusion protein, which is composed of 4B agarose microspheres surface-modified with cyanogen bromide and reduced glutathione that can specifically bind to GST-tag fusion protein Peptides are linked, the surface is modified with cyanogen bromide 4B agarose microspheres and the reduced glutathione is passed through diaminodecane and maleimide PEGN-hydroxysuccinimide MW: 5000 ( MAL-PEG5000-NHS) for connecting arms. The GSH affinity chromatography medium for purifying the GST-tag fusion protein provided in this implementation can be prepared by the following preparation method.

[0063] This embodiment also provides a preparation method of GSH affinity chromatography medium for purifying GST-tag fusion protein, comprising the following steps:

[0064] 1) Mix 10% cyanogen bromide solution, 0.1mol / L sodium carbonate solution, and 4B agarose microspheres at a volume ratio of 0.1:0.5:2, and vort...

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Abstract

The invention belongs to the technical field of GST-Tag fusion protein purification, and relates to a GSH affinity chromatography medium for purifying GST-Tag fusion protein. The GSH affinity chromatography medium is formed by connecting a chromatography medium with hydrogen bromide modified on the surface and reduced glutathione by using a p-amino compound and a maleimide-succinimide linker as connecting arms. The invention also provides a preparation method of the GSH affinity chromatography medium for purifying the GST-TAG fusion protein, the preparation method comprises the following steps: modifying a chromatography medium activated by the hydrogen bromide with the p-amino compound, then modifying the chromatography medium with the maleimide-succinimide linker, and finally coupling the chromatography medium with the reduced glutathione to form the GSH affinity chromatography medium. The GSH affinity chromatography medium prepared by the preparation method is used for purifying theGST-Tag fusion protein. The GSH affinity chromatography medium with high loading capacity, high recovery rate and convenient preparation is synthesized by amidation modification and maleimide-succinimide linker coupling, and can be widely applied to the separation and purification engineering of the GST-Tag tag fusion protein.

Description

technical field [0001] The invention belongs to the technical field of purification of GST-tag fusion protein, and in particular relates to a GSH affinity chromatography medium for purifying GST-tag fusion protein and its preparation method and application. Background technique [0002] With the rapid development of the modern biomedical industry, the co-expression strategy of protein fusion tags has been widely used. Among them, the glutathione sulfhydryl transferase (GST) tag system is often used for the expression and dissolution of recombinant proteins because of its ability to promote protein expression and dissolution. Express. Correspondingly, in the subsequent purification, in order to obtain a higher purity target protein, reduce separation steps, and reduce production costs, it is usually necessary to modify the agarose. [0003] The glutathione affinity chromatography medium is prepared by using the characteristic that the recombinant protein containing the gluta...

Claims

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Application Information

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IPC IPC(8): B01J20/286C12N9/10B01D15/38B01J20/30
CPCB01D15/3804B01J20/286C12N9/13
Inventor 董垚
Owner 武汉菲恩生物科技有限公司
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