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Preparation method and application of novel magnetic bead for separating, purifying and immobilizing histidine tag protein and bovine hemoglobin

A technology of histidine tag and bovine hemoglobin, which is applied in the field of new magnetic beads and its preparation, can solve the problems of ineffective separation of target protein, difficulty in large-scale application, and high equipment requirements, so as to improve preparation and extraction efficiency, improve Resistance and acid and alkali resistance, and the effect of improving adsorption specificity

Active Publication Date: 2020-04-21
WUHAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this type of method is easy to operate and has a large processing capacity, the resolution is usually too low to effectively separate the target protein
Other protein fine separation techniques include gel chromatography, affinity chromatography, ion exchange chromatography, etc. These methods generally have problems such as high price, low processing volume, high equipment requirements, and difficulty in large-scale application.

Method used

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  • Preparation method and application of novel magnetic bead for separating, purifying and immobilizing histidine tag protein and bovine hemoglobin
  • Preparation method and application of novel magnetic bead for separating, purifying and immobilizing histidine tag protein and bovine hemoglobin
  • Preparation method and application of novel magnetic bead for separating, purifying and immobilizing histidine tag protein and bovine hemoglobin

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Experimental program
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Effect test

Embodiment 1

[0030] 1) Preparation of nano-Fe3O4 magnetic core: Boil deionized water in a microwave oven for 10 minutes to obtain deoxygenated deionized water, seal and cool to room temperature for later use. 1.90g Fe(NH 4 ) 2 ·(SO 4 ) 2 ·6H 2 O, 2.33g FeCl 3 ·6H 2 O was placed in a three-necked flask, and then 50 mL of deoxygenated deionized water was added to fully dissolve it. Under nitrogen protection, 200-300r / min stirring rate, and 60°C water bath, heat the mixture for 30min, then add 45mL of ammonia water to adjust the pH of the solution to 11, heat the mixture to 80°C, age at this temperature for 1h, and then magnetically Adsorption separation, the obtained solid is washed with deoxygenated and deionized water to neutrality, and magnetically adsorbed again to obtain nano-ferroferric oxide magnetic core. Analysis results show that the average particle diameter of the magnetic core is 111.5nm.

[0031] Add the nanometer ferroferric oxide magnetic core into 100mL deoxygenated ...

Embodiment 2

[0035] 1) Preparation of nano-Fe3O4 magnetic core: Boil deionized water in a microwave oven for 10 minutes to obtain deoxygenated deionized water, seal and cool to room temperature for later use. 1.90g Fe(NH 4 ) 2 ·(SO 4 ) 2 ·6H 2 O, 2.33g FeCl 3 ·6H 2 O was placed in a three-necked flask, and then 50 mL of deoxygenated deionized water was added to fully dissolve it. Under nitrogen protection, 200-300r / min stirring rate, and 60°C water bath, heat the mixture for 30min, then add 45mL of ammonia water to adjust the pH of the solution to 11, heat the mixture to 80°C, age at this temperature for 1h, and then magnetically Adsorption separation, the obtained solid is washed with deoxygenated and deionized water to neutrality, and magnetically adsorbed again to obtain nano-ferroferric oxide magnetic core. Analysis results show that the average particle diameter of the magnetic core is 111.8nm.

[0036] Add the nanometer ferroferric oxide magnetic core into 100mL deoxygenated ...

Embodiment 3

[0040] 1) Preparation of nano-Fe3O4 magnetic core: Boil deionized water in a microwave oven for 10 minutes to obtain deoxygenated deionized water, seal and cool to room temperature for later use. 1.90g Fe(NH 4 ) 2 ·(SO 4 ) 2 ·6H 2 O, 2.33g FeCl 3 ·6H 2 O was placed in a three-necked flask, and then 50 mL of deoxygenated deionized water was added to fully dissolve it. Under nitrogen protection, 200-300r / min stirring rate, and 60°C water bath, heat the mixture for 30min, then add 45mL of ammonia water to adjust the pH of the solution to 11, heat the mixture to 80°C, age at this temperature for 1h, and then magnetically Adsorption separation, the obtained solid is washed with deoxygenated and deionized water to neutrality, and magnetically adsorbed again to obtain nano-ferroferric oxide magnetic core. Analysis results show that the average particle diameter of the magnetic core is 111.8nm.

[0041] Add the nanometer ferroferric oxide magnetic nucleus solution into 100mL d...

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Abstract

The invention relates to a novel magnetic bead capable of being used for separating, purifying and immobilizing histidine tag protein and bovine hemoglobin as well as a preparation method and application of the novel magnetic bead. The preparation method comprises the following steps: firstly, a nano ferroferric oxide magnetic core is prepared by utilizing a co-precipitation method; and then, thesurface of the magnetic core is directly modified with a plurality of different carboxylated silane coupling agents as ligands, so that the magnetic core can chelate metal ions. The magnetic bead prepared by the method is good in magnetism, stable in property and easy to disperse, and has the advantage that the synthesis method is simple and easy to control and implement. Target proteins such as His-tagged protein and bovine hemoglobin can be separated and purified with high selectivity and high efficiency through stable combination of strong coordination between metal ions on the surface of the magnetic bead and histidine residues on the surface of a recombinant protein.

Description

technical field [0001] The invention relates to the technical field of biological functional materials, in particular to a novel magnetic bead which can be used for separating, purifying and immobilizing histidine-tagged protein and bovine hemoglobin, and its preparation method and application. Background technique [0002] Protein is an important class of biomacromolecules, and it is the main bearer and material basis of all life activities. The premise and foundation of the research on the structure and function of proteins and the ultimate application is the efficient separation and purification of proteins. Conventional protein separation and extraction techniques usually rely on the differences in solubility, hydrophobicity, molecular size, surface charge and specific biological affinity of proteins, thus developing salting-out method, isoelectric point precipitation method, Coarse separation methods such as organic solvent precipitation. Although such methods are eas...

Claims

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Application Information

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IPC IPC(8): C07K14/805C07K14/00C07K17/14H01F1/00H01F41/00
CPCC07K14/00C07K14/805C07K17/14H01F1/0054H01F41/00
Inventor 孙恩杰王宇胡亚悦曾凯李呈祥谢浩
Owner WUHAN UNIV OF TECH
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