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Immobilized metal ion affinity chromatograph (IMAC) material, and preparation and application thereof

A technology for immobilizing metals and chromatographic materials, applied in release and purification, functional biological materials and their preparation, and in the field of protein-specific capture, can solve the problems of large steric resistance of intact proteins and inability to enter the polymer network, and achieve the realization of Specific capture, reduction of metal ion leakage, and reduction of non-specific adsorption

Active Publication Date: 2017-05-10
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the sieving effect of the polymer network, the exposed histidine tag at the end of the histidine-tagged protein can enter the polymer network and be captured by the IMAC material, while other proteins lack the terminal histidine tag, and the steric resistance of the intact protein is too large. Can't access polymer network

Method used

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  • Immobilized metal ion affinity chromatograph (IMAC) material, and preparation and application thereof
  • Immobilized metal ion affinity chromatograph (IMAC) material, and preparation and application thereof
  • Immobilized metal ion affinity chromatograph (IMAC) material, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Based on NTA and Ni 2+ Preparation of Chelating Silica IMAC Surface Coating Material (SC-IMAC1)

[0022] 3 g of amino-modified silica nanoparticles (particle size: 350 nm) were dispersed in 60 mL of PB buffer, 2 mL of glutaraldehyde was added, reacted at room temperature for 6 h, and washed twice with water to remove unreacted glutaraldehyde. Resuspend the material with 90mL PB buffer, add 200mg NTA, adjust the pH to 8.0, react overnight at room temperature, wash twice with water to remove unreacted NTA. Disperse the resulting material in 30 mL of 10 mg / mL NiSO 4 In aqueous solution, Ni was immobilized on the surface 2+ IMAC material. Subsequently, 100 mg of particles were redispersed in 20 mL of acetonitrile, 10 mg of acrylamide (AAm), 10 mg of methylenebisacrylamide (MBA), 0.5 mg of AIBN, and N 2 After 20 min, 65 ° C magnetic stirring reaction for 12 h, centrifuged to collect the product, washed 3 times with ultra-pure water, vacuum dried to obtain NTA and Ni-base...

Embodiment 2

[0026] Based on NTA and Ni 2+ Chelating Silica IMAC Surface Coating Material (SC-IMAC1) for Purification of Histidine-Tagged Proteins

[0027] 4 mg of SC-IMAC was washed twice with 80 μL of loading buffer containing 10 mM imidazole at 4°C to keep SC-IMAC1 in the loading environment. Dilute the recombinant protein extract to 4 mg / mL with loading buffer, add 200 μL to the pre-balanced SC-IMAC1, and incubate at 4°C for 30 min with shaking. The material was collected by centrifugation and washed once with 100 μL of loading buffer to remove unbound protein. Wash twice with 30 μL of washing buffer containing 25 mM imidazole, 30 min each time, to remove weakly bound foreign proteins. 30 μL of elution buffer containing 250 mM imidazole was eluted four times, 30 min each time, to elute the histidine-tagged protein, and the purified product was analyzed by SDS-PAGE.

[0028] As a reference group, under the same operating conditions, IMAC was used as the affinity purification material...

Embodiment 3

[0031] Based on IDA and Ni 2+ Preparation of Chelating Magnetic IMAC Surface Coating Material (SC-IMAC2)

[0032] Add 5g of silica nanoparticles into 100mL of GLYMO-IDA solution, react at 65°C for 24 hours, and modify the IDA group on the surface of the silica material. Add 5 g of the above materials into 150 mL of methanol, then add 10 mL of γ-MAPS, reflux at 90° C. for 15 hours, and then modify γ-MAPS on the surface of the matrix material by methanol reflux. With embodiment 1 method, can make based on IDA and Ni 2+ Chelating magnetic IMAC surface coating material (SC-IMAC2), such as image 3 shown. Same as Example 2, using SC-IMAC2 to purify histidine-tagged protein, the purification result is as follows Figure 4 shown.

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Abstract

The invention relates to a novel immobilized metal ion affinity chromatograph (IMAC) material, and a preparation and application thereof. According to the novel IMAC material, the surface of the traditional IMAC material is subjected to coating modification, the naked tail end of protein enters the coating through the selectivity of the surface coating pore sieving action to be captured by metal ions, and other integrated protein has high mass transfer resistance and enters the coating difficultly. The material combines the strong affinity of the IMAC material and the high selectivity of the surface coating pore sieving action, and is further applied to specific capture, release and purification of histidine label protein in the technical fields of biological analysis, biological analysis and biology.

Description

technical field [0001] The invention belongs to functional biological materials and their preparation and application in protein specific capture, release and purification, specifically a novel surface coating-modified immobilized metal ion affinity chromatography material preparation and its use as Affinity purification materials are used for the specific capture, release and purification of histidine-tagged proteins. Background technique [0002] Recombinant protein technology plays a very important role in protein crystallization, protein drug, protein interaction and structural proteomics research (T.Hyeon, et al. Adv. Mater., 2010, 22, 57–60; A.S.Robinson, et al. al. Biotechnol. J., 2012, 7, 620–634). From the study of protein structure and function to the development of functional protein expression and purification processes, affinity tags have become a very important and effective tool in the purification of recombinant proteins. Because the histidine tag has the a...

Claims

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Application Information

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IPC IPC(8): B01J20/286B01J20/30B01D15/38C07K1/22
Inventor 张丽华李森武杨开广赵宝锋李潇刘路宽陈远波张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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