Nickel-chelated magnetic nanoparticle and preparation method thereof, and application of nickel-chelated magnetic nanoparticle in separation and purification of histidine tag protein

A technology of magnetic nanoparticles and histidine tags, which is applied in the field of materials, can solve the problems of cumbersome operation, falling off of nickel ions, and expensive modified ligands, and achieves the advantages of simple separation and purification process, simple modification reaction, and high reuse rate. Effect

Active Publication Date: 2021-07-27
ANHUI UNIVERSITY OF TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When magnetic nanomaterials are used for the purification of histidine-tagged proteins, it is necessary to chemically modify the surface of the magnetic nanomaterials to chelate transition metal ions to achieve the purpose of separating and purifying histidine-tagged proteins. The surface-modified magnetic Nanoparticles can achieve effective separation of histidine-tagged proteins, but the surface modification process of magnetic nanoparticles is complicated and cumbersome, and after multi-step modification, the modified small molecules or chelated nickel ions may fall off
In addition, commonly used modified ligands are usually expensive, so there is an urgent need to develop new simple and efficient purification methods for histidine-tagged proteins based on magnetic nanomaterials

Method used

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  • Nickel-chelated magnetic nanoparticle and preparation method thereof, and application of nickel-chelated magnetic nanoparticle in separation and purification of histidine tag protein
  • Nickel-chelated magnetic nanoparticle and preparation method thereof, and application of nickel-chelated magnetic nanoparticle in separation and purification of histidine tag protein
  • Nickel-chelated magnetic nanoparticle and preparation method thereof, and application of nickel-chelated magnetic nanoparticle in separation and purification of histidine tag protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 nickel-SB magnetic nanoparticles

[0031] The preparation method comprises the following steps:

[0032] (1) The ferroferric oxide magnetic nanoparticles are prepared by co-precipitation method, and are dispersed in deionized aqueous solution for storage. Take 10mL of this solution (about 30mg of ferroferric oxide magnetic nanoparticles) in a beaker, add 0.50g of sodium hydrogen tartrate powder, stir to dissolve it gradually, ultrasonic at room temperature for more than 4h, then carry out magnetic adsorption separation, remove the supernatant, add Washing with deionized water was repeated 5 times to remove uncoated sodium hydrogen tartrate, and finally obtained ferric iron tetroxide magnetic nanoparticles wrapped by sodium hydrogen tartrate.

[0033] (2) The above magnetic nanoparticles are dispersed in 10mL deionized water, and 0.10mol L -1 NaOH solution, so that the pH of the solution reaches between 9.0-10.0 to ensure that the carbox...

Embodiment 2

[0037] The application of embodiment 2 nickel-SB magnetic nanoparticles

[0038] The method for separating and purifying histidine-tagged recombinant protein from complex Escherichia coli lysate with nickel-SB magnetic nanoparticles, the specific steps are as follows:

[0039] (1) Escherichia coli expressing six histidine-tagged recombinant proteins were induced to express by isopropyl-β-D-1-thiogalactoside, and the bacterial liquid was collected, and the protein extraction and lysate were added, followed by ultrasonic disruption , after high-speed centrifugation, the supernatant was obtained as the total protein solution of E. coli.

[0040] (2) Take 200 μL of nickel-SB magnetic nanoparticles (2 mg / mL) for magnetic separation on a magnetic stand, discard the supernatant, add 300 μL of the above bacterial total protein solution, resuspend the nanoparticles, rotate at 4 °C for 1 h, and magnetically Separation, the supernatant is the supernatant solution after magnetic material...

Embodiment 3

[0046] The determination of the maximum binding capacity of embodiment 3 nickel-SB magnetic nanoparticles

[0047] Specific steps are as follows:

[0048] Take 50 μg of nickel-SB magnetic nanoparticles and different concentrations of purified histidine-tagged carbonic anhydrase protein solutions and incubate at 4°C for 1 hour with rotation. After magnetic separation, the unbound carbonic anhydrase in the supernatant solution is determined by Bradford method protein concentration.

[0049] The adsorption capacity of Ni-SB magnetic nanoparticles (Q e , mg g -1 ) is calculated by formula (1).

[0050]

[0051] where C 0 (mg mL -1 ) is the initial concentration of carbonic anhydrase, C s (mg mL -1 ) is the protein concentration in the supernatant solution after adsorption, V (mL) is the volume of the protein solution, and m (g) is the mass of the magnetic material.

[0052] The adsorption isotherm conforms to the Langmuir model, and the saturated adsorption capacity Q i...

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Abstract

The invention discloses nickel-chelated magnetic nanoparticle and a preparation method thereof, and application of the nickel-chelated magnetic nanoparticle to separation and purification of histidine tag protein. The nickel-chelated magnetic nanoparticle is a nickel-chelated sodium hydrogen tartrate-modified ferroferric oxide magnetic nanoparticle, and is simple in preparation steps and capable of effectively fixing and chelating nickel ions and realizing specific and efficient separation and purification of histidine tag recombinant protein. The nickel-chelated magnetic nanoparticle has strong binding capacity to histidine tag recombinant protein, and is obviously superior to the prior art. In addition, nickel fixed on the surface of the nickel-chelated magnetic nanoparticle is not easy to fall off, and the repeated utilization rate of the nanoparticle is high.

Description

technical field [0001] The invention belongs to the field of materials and relates to the preparation and application of new materials, in particular to a nickel chelated magnetic nanoparticle, a preparation method and an application for separating and purifying a histidine-tagged protein. Background technique [0002] The use of genetic engineering technology to produce various recombinant proteins has become an important method for the preparation of protein drugs. The acquisition of high-purity recombinant proteins is inseparable from effective separation and purification techniques, which generally rely on various protein labels and metal ion chelates on solid resins. together to achieve. Among them, the most common affinity tag is histidine tag, which can be coordinated with immobilized nickel ions or cobalt ions to achieve protein separation. The conventional separation method of histidine-tagged recombinant protein is to use nickel-containing complex resin material a...

Claims

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Application Information

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IPC IPC(8): B01J20/22B01J20/28B01J20/30C07K1/22
CPCB01J20/22B01J20/06B01J20/0225B01J20/28009B01J20/28016C07K1/22
Inventor 刘姿陈玉茹马亮刘祥陈学明颜庭轩葛阿雪
Owner ANHUI UNIVERSITY OF TECHNOLOGY
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