Magnetic microsphere as well as preparation and application method for purifying histidine tag protein

A histidine tag and magnetic microsphere technology, applied in the field of magnetic microspheres, can solve the problems of cumbersome application procedures, low response, difficult rapid adsorption and movement, etc., achieve efficient chelation of metal ions, and simple purification and separation process , Guarantee the effect of purification efficiency

Active Publication Date: 2013-01-16
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the use of magnetic particles with a particle size of only 10 nanometers, their response to an external magnetic field is low, and it is difficult to achieve rapid adsorption and movement under the control of an external magnetic field, so they are limited in practical application.
[0005] Further search found that Stephen Rimmer et al. reported in "Biomacromolecules" Volume 7, pages 1124-1130 (HighlyBranched Poly(N-isopropylacrylamide) for Use in Protein Purification Biomacromolecules, 2006, 7, 1124-1130) that a highly branched Polyisopropylacrylamide, a polymer molecule with temperature sensitivity, chelates copper ions to purify His6-BRCA1 protein. Using highly branched polymer molecules as a brand-new metal ion chelating ligand is an advanced technology in the field. An innovative work, but in the application process, the dissolution and agglomeration characteristics of polymer molecules in the separation medium must be controlled through processes such as raising and lowering the temperature, so as to achieve purification and separation operations, and the application program is relatively cumbersome.

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  • Magnetic microsphere as well as preparation and application method for purifying histidine tag protein
  • Magnetic microsphere as well as preparation and application method for purifying histidine tag protein

Examples

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Effect test

Embodiment 1

[0037] (1) The method proposed in the Chinese invention patent ZL 2006100279617 was used to prepare magnetic silica microspheres with a particle size of 200nm in the surface-coated silica shell. Freeze-dry the silica microspheres, weigh 6g of freeze-dried magnetic particles, add them to a 100mL three-necked flask, and add 60mL of toluene to the flask; start stirring, put them in an oil bath at 120°C, and add them after reflux for 30 minutes 5 g of γ-aminopropyltrimethoxysilane was refluxed for 2 h. After the reaction was finished, ultrasonicated for 30 seconds, poured into a beaker, and separated under the assistance of a strong magnetic field, then added 50mL of toluene, ultrasonicated for 1 minute, and separated and washed by a strong magnetic field. After repeated operations three times, the obtained amino-modified magnetic microspheres Put it into a vacuum drying oven at 120°C for a vacuum reaction for 12 hours, and finally place the obtained magnetic microspheres in a sam...

Embodiment 2

[0044] (1) The method proposed in Chinese invention patent ZL 2006100279617 was used to prepare magnetic silica microspheres coated with a silica shell. The difference is that the inner core of the magnetic particle aggregates in the microspheres is 900nm, and the silicon oxide shell coating method is the same as that of the Chinese invention patent ZL 2006100279617, so the particle size of the finally obtained silicon oxide magnetic microspheres is 1000nm. Freeze-dry the silica microspheres, weigh 6g of freeze-dried magnetic particles, add them to a 100mL three-necked flask, and add 20mL of toluene to the flask; stir, put them in an oil bath at 120°C, reflux for 30 minutes, and then add γ - Aminopropyltriethoxysilane 1g, reflux reaction for 2h. After the reaction was finished, ultrasonicated for 30 seconds, poured into a beaker, and separated under the assistance of a strong magnetic field, then added 50mL of toluene, ultrasonicated for 1 minute, and separated and washed by a...

Embodiment 3

[0051] (1) The method proposed in Chinese invention patent ZL 2006100279617 was used to prepare magnetic silica microspheres coated with a silica shell. The difference is that the inner core of the magnetic particle aggregates in the microspheres is 900nm, and the silicon oxide shell coating method is the same as that of the Chinese invention patent ZL 2006100279617, so the particle size of the finally obtained silicon oxide magnetic microspheres is 1000nm. Freeze-dry the silicon oxide microspheres, weigh 6g of freeze-dried magnetic particles, add them to a 100mL three-necked flask, and add 20mL of toluene to the flask; stir, put them in an oil bath at 120°C, reflux for 30 minutes, and then add N -0.5 g of β(aminoethyl)-γ-aminopropyltrimethoxysilane, reflux for 2 hours. After the reaction was finished, ultrasonicated for 30 seconds, poured into a beaker, and separated under the assistance of a strong magnetic field, then added 50mL of toluene, ultrasonicated for 1 minute, and ...

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Abstract

The invention discloses a magnetic microsphere as well as preparation and preparation method thereof used for purifying histidine tag protein in the technical field of biological materials; wherein the magnetic microsphere includes 50wt percent to 80wt percent of ferroferric oxide nanoparticles, 40wt percent to 15wt percent of monox shell as well as 5wt percent to 10wt percent of hyperbranched polymer molecule with a poly carboxyl structure. The molecular structure of the hyperbranched polymer molecule with a poly carboxyl structure is as follows: wherein R refers to - (CH2CH2O-) 3-CH2CH2-. The invention adopts the magnetic microsphere with a quick magnetic response as a solid support, adopts the hyperbranched polymer molecule with a poly carboxyl structure to decorate on the surface of the magnetic microsphere to obtain a magnetic carrier material which can be effectively chelated with transition metal ions, and finally realizes the general goal of quickly, effectively and simply purifying and separating histidine tag protein.

Description

technical field [0001] The invention relates to a carrier in the technical field of biomaterials and its preparation and application, in particular to a magnetic microsphere used for the purification of histidine-tagged proteins and its preparation and application method. Background technique [0002] IMAC (Immobilized Metal-ion Affinity Chromatography) is that Porath et al. used iminodiacetic acid (IDA) resin on the surface as a filler in 1975, and used IDA as a ligand to chelate copper, nickel, cobalt, iron or zinc plasma. Using the principle that some amino acid residues on the protein surface, such as histidine, tryptophan, cysteine, etc., can have a strong coordination with these metal ions, the surface with histidine, tryptophan or cysteine A novel affinity purification method for the purification of proteins has been gradually developed for the separation and purification of proteins with amino acid residues. IMAC technology has the characteristics of relatively stab...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F2/04B01J13/02C07K1/22
Inventor 徐宏朱新远古宏晨王明启李国林
Owner SHANGHAI JIAOTONG UNIV
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