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A metal chelating nano medium and its preparation method, and its application to strengthen the renaturation and integrated purification of inclusion body protein

A metal chelation and nanotechnology, applied in the field of protein renaturation and separation, can solve the problems of low charge density and unsatisfactory effect of inclusion body assisted renaturation, so as to facilitate renaturation, enhance electrostatic repulsion, and inhibit aggregation Effect

Inactive Publication Date: 2017-09-29
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, limited by the IDA modification method on the surface of the medium, the charge density on the surface of the medium is low (about 108 μmol / g), so the effect of assisted renaturation for higher concentrations of inclusion bodies is not ideal

Method used

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  • A metal chelating nano medium and its preparation method, and its application to strengthen the renaturation and integrated purification of inclusion body protein
  • A metal chelating nano medium and its preparation method, and its application to strengthen the renaturation and integrated purification of inclusion body protein
  • A metal chelating nano medium and its preparation method, and its application to strengthen the renaturation and integrated purification of inclusion body protein

Examples

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Effect test

preparation example Construction

[0052] The preparation method of the metal chelating nano medium of high charge density of the present invention, its steps are as follows:

[0053] 1) Synthesizing the connection product GMA-IDA of GMA and IDA;

[0054] 2) Immobilizing the initiator 2-bromoisobutyryl bromide (BIBB) on the surface of silica nanoparticles with an average particle size of 20-30nm to obtain SNPs-Br of silica nanoparticles immobilizing BIBB;

[0055] 3) Add the BIBB-immobilized silica nanoparticles SNPs-Br in the above step 2) to the mixed liquid of N,N-dimethylformamide (DMF) and water, and make the concentration 0.1-0.2g / mL of suspension, the volume ratio of DMF to water is 1:2;

[0056] 4) Add GMA-IDA monomer, CuBr catalyst, 2,2'-bipyridyl (Bpy) ligand and CuBr to the suspension of the above step 3). 2 Inert agent, the concentration of GMA-IDA is 0.2-1.0mol / L, the solution is calculated as a mixed liquid of N,N-dimethylformamide (DMF) and water, and the molar concentration ratio of each comp...

Embodiment 1

[0074] The preparation method of the metal chelating nano medium of high charge density in the present embodiment is as follows:

[0075] 1) Synthesis of GMA-IDA (Synthesis of Magnetic Chelator for High-Capacity Immobilized Metal Affinity Adsorption of Protein by Cerium Initiated Graft Polymerization, Langmuir, 2005, 21, 6987-6994), the connection product of GMA and IDA;

[0076] 2) Immobilize the initiator α-bromoisobutyryl bromide (BIBB) on the surface of silica nanoparticles with an average particle size of 20-30nm to obtain the silica nanoparticles SNPs-Br with immobilized BIBB. For specific steps, see the literature ( Reversible immobilization of glucoamylase onto metal-ligand functionalized magnetic FeSBA-15, Biochemical Engineering Journal, 2012, 68, 159-166);

[0077] 3) Add 3.0 g of the BIBB-immobilized silica nanoparticles SNPs-Br in the above step 2) to the mixed system of 30 mL DMF and water to form a suspension with a concentration of 0.1 g / mL, the volume of DMF a...

Embodiment 2

[0080] The preparation method of the metal chelating nano medium of high charge density in the present embodiment is as follows:

[0081] 1) same as step 1) in embodiment 1;

[0082] 2) same as step 2) in embodiment 1;

[0083] 3) Add 4.5 g of the BIBB-immobilized silica nanoparticles SNPs-Br in the above step 2) to the mixed system of 30 mL of DMF and water to form a suspension with a concentration of 0.15 g / mL, the volume of DMF and water The ratio is 1:2;

[0084] 4) GMA-IDA monomer, CuBr catalyst, 2,2'-bipyridyl (Bpy) ligand, CuBr 2 Inert agent, the concentration of GMA-IDA is 0.6mol / L, the solution is calculated as a mixed liquid of N,N-dimethylformamide (DMF) and water, and the molar concentration ratio of each component is GMA-IDA:CuBr:CuBr 2 :Bpy=100:1:0.2:2, before the reaction, the mixed system was ventilated with nitrogen to deoxygenate for half an hour, and then polymerized for 1.5h to prepare the metal chelating nano-media SNPs-P (GMA-IDA). The metal chelating...

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Abstract

The invention relates to a metal chelating nano medium, a preparation method and a method applied to strengthen inclusion body protein renaturation and integrated purification, wherein connection polymers P (GMA-IDA) of iminodiacetic acid and glycidyl methacrylate are grafted on the surfaces of silicon dioxide nanoparticles which are 20-30nm in mean grain sizes, and the electric density of medium is 2520-5680Mu mol / g. Extremely high electric density enhances electrostatic repulsion between the medium and homocharge protein molecules, thereby more effectively restraining gathering of proteins. A large number of metal chelating groups which are arranged on the surface of the nano medium guarantee recombinant target proteins which are marked through poly-L-histidine to be effectively adsorbed and separated after completing renaturation. The metal chelating nano medium, the preparation method and the method applied to strengthen inclusion body protein renaturation and integrated purification do not need expensive large equipment such as chromatographic columns or chromatographic systems and the like, complete integration of inclusion body protein renaturation and purification outside the chromatographic columns, are simple, convenient and easy to operate and low in cost, simplify refinement and purification method, and are more suitable for large scale industrialization production.

Description

technical field [0001] The invention belongs to the protein renaturation and separation technology in the field of biotechnology, and relates to a metal chelating nanometer medium, a preparation method and a method for strengthening inclusion body protein renaturation and integrated purification. Background technique [0002] The rapid development of genetic engineering technology has made the large-scale production of genetically engineered drugs possible. The production of such drugs requires a suitable recombinant gene expression system. The bacterial expression system expresses the target protein quickly, in large quantities, and at low cost. However, the overexpression of foreign proteins often forms protein aggregates, that is, inclusion bodies. Inclusion bodies have no biological activity and need to be refolded to restore their natural structure and biological activity. The aggregation caused by the hydrophobic interaction between folding intermediates during renatu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F292/00C08F220/36C07K1/22C07K1/14
Inventor 董晓燕刘护余林玲孙彦
Owner TIANJIN UNIV
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