Method for enriching and purifying saccharide binding protein

A protein-binding and enrichment technology, applied in the field of enrichment and purification of sugar-binding proteins, can solve the problems of complex method, low recovery rate, inconvenient use, etc., and achieve the effect of simple connection method and low cost

Active Publication Date: 2007-05-09
SHANXI LIFEGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for separating, enriching and purifying sugar-binding proteins based on micro-nano magnetic technology, which solves the technical problems of complicated methods, inconvenient use and low recovery rate in the background technology

Method used

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  • Method for enriching and purifying saccharide binding protein
  • Method for enriching and purifying saccharide binding protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The implementation steps of embodiment 1 are as follows:

[0074] 1) Coupling of sugar and micro-nano magnetic particles (1) Activation of coupling agent:

[0075] ① Take the coupling agent 4-hydroxyphenylvaleric acid, N-hydroxysuccinimide and dicyclohexylcarbodiimide according to the ratio of 1:5:5, and dissolve them in organic solvent respectively. As the solvent, ethyl acetate, dimethylformamide (DMF) and the like can be used.

[0076] ② First mix 4-hydroxyphenylvaleric acid and N-hydroxysuccinimide and dissolve them all; then add dicyclohexylcarbodiimide and mix; use ethyl acetate to dissolve to 25ml, so that the concentrations of the three are respectively 20mM, 100mM and 100mM.

[0077] ③Protected with nitrogen gas, incubated at 37°C for 12 hours, a white precipitate formed.

[0078] (2) Pretreatment of micro-nano magnetic particles derivatized with amino groups: take 15.58mg / ml of micro-nano magnetic particles derivatized with amino groups in a 5ml centrifuge ...

Embodiment 2

[0097] The implementation steps of embodiment 2 are as follows:

[0098] 1) Coupling of sugar and micro-nano magnetic particles (1) Activation of coupling agent:

[0099] ① Dissolve the coupling agent 4-hydroxybenzamide or 4-hydroxybutyric acid hydrazine in an organic solvent to prepare a 20-50mol / L coupling agent solution. The organic solvent can adopt dimethylformamide (DMF) or ethyl acetate etc.

[0100] ② Adding micro-nano magnetic particles derivatized with oxirane groups into the coupling agent solution at a ratio of 1:10.

[0101] ③ Incubate at room temperature for 3 hours.

[0102] (2) Cleaning: Magnetic separation, discarding the supernatant. Wash 3 times with ethanol and then 3 times with sodium acetate wash buffer, pH 5.6.

[0103] (3) Coupling: add 2ml of 100mM D-mannose solution prepared with pH 5.6 sodium acetate buffer, incubate at 37°C for 12 hours, then the sugar is derivatized by linking to the oxirane group On the micro-nano magnetic particle, the micro-n...

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Abstract

This invention discloses a method for enriching and purifying glycoprotein. The coupling of sugar with magnetic micro- and nano-particles comprises: activating the coupling agent and coupling sugar onto amino-derived magnetic micro- and nano-particles or ethylene oxide derived magnetic micro- and nano-particles. The separation, enriching and purification of glycoprotein comprises: adding pretreated glycoprotein into sugar-coupled magnetic micro- and nano-particles for connection, washing and eluting to obtain enriched and purified glycoprotein. This invention solves the problems of complex process, inconvenient usage and low recovery rate faced by background technology. The method has such advantages as simple process, high speed, stable glycoprotein bioactivity, and high efficiency in separating, enriching and purifying glycoprotein.

Description

technical field [0001] The invention relates to a hemiacetal ROH at the reducing end of the reducing sugar, which is covalently coupled to hydroxyl (-OH) derivatized micro-nano magnetic particles in the form of glycosidic bonds, and is fixed on the micro-nano magnetic particles by using this The sugar of the present invention specifically binds to the sugar-binding protein, and the method for separating, enriching and purifying the sugar-binding protein. Background technique [0002] Carbohydrate-binding proteins are a class of proteins that can transfer non-covalently to carbohydrates, and their structures contain carbohydrate recognition domains without sugar groups. Carbohydrate-binding proteins have important biological functions, including communication and recognition between cells, interaction between cells and pathogenic microorganisms, intracellular protein transport, protein-protein interaction, specific binding between proteins and receptors, and so on. With the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 李铮陈超杨刚龙崔亚丽惠文利
Owner SHANXI LIFEGEN
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