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Preparation and application of super tolerance metal chelating affinity packing

A metal chelation and tolerance technology, applied in the field of preparation of protein purification fillers, can solve the problems of free metal ion environmental pollution, physical denaturation damage, reduced use efficiency, etc., achieve strong component compatibility, long service life, and easy to use low cost effect

Active Publication Date: 2016-10-12
CHANGZHOU SMART LIFESCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are two main problems in the current purification of proteins with metal chelate affinity fillers: 1) Proteins are a class of biologically active substances that may lose their activity due to proteolysis, physical denaturation, and other damage during the purification process. Buffer solution to stabilize pH and add reducing agents such as DTT to stabilize redox potential, and sometimes protease inhibitors such as EDTA
2) The purification packing needs to be cleaned after each purification. The current metal chelating affinity packing cannot tolerate sodium hydroxide with a concentration above 0.1M. When cleaning in place, the metal ions must be stripped first, and then chelated after alkali washing Metal ion
This process takes a lot of time, reduces the use efficiency, and produces a large amount of free metal ions to cause environmental pollution

Method used

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  • Preparation and application of super tolerance metal chelating affinity packing
  • Preparation and application of super tolerance metal chelating affinity packing
  • Preparation and application of super tolerance metal chelating affinity packing

Examples

Experimental program
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Effect test

Embodiment 1

[0021] A preparation method of super-tolerant metal chelate affinity filler Ni Smart Beads 6FF, the preparation method comprises the following steps:

[0022] (1) Amino reaction: Add epichlorohydrin and NaOH solution to the agarose microspheres, and activate with DMSO at 35°C for 2 hours. After activation, clean the agarose microspheres until no epichlorohydrin is present; The pH is 10, and the ligand coupling filler is obtained by reacting at 40°C for 24 hours; the NaOH solution is 2M, the ratio of epichlorohydrin to microspheres is 0.2mg:1g, and the ratio of ligand to microspheres is 100mg: 1g;

[0023] (2) Metal ion chelation

[0024] Add the above coupling ligand filler to 50-100mg / ml NiSO 4 Solution, react at 25°C for 2 hours to obtain super-tolerant metal chelate affinity filler; wherein the ligand structural formula is

Embodiment 2

[0026] A preparation method of super-tolerant metal chelate affinity filler Co Smart Beads 6FF, the preparation method comprises the following steps:

[0027] (1) Ligand coupling

[0028] Utilize carboxyl reaction: Add epichlorohydrin and NaOH solution to agarose microspheres, shake and activate DMSO at 30°C for 2 hours. After activation, clean the microspheres until there is no epichlorohydrin; , reacted at 30°C for 24 hours to obtain amino-activated fillers; the NaOH solution was 1M, the ratio of epichlorohydrin to microspheres was 0.4mg:1g, and the ratio of amine to microspheres was 0.1mg:1g;

[0029] Dissolve the ligand in 0.1M MES, add the above-mentioned amino-activated filler, add 0.1M EDC when the pH is 5.0, react at 20-30°C for 1 hour, and continue to react for 24 hours when the pH is 5.0 to obtain the coupled ligand filler; among them, The amount of ligand added is 50mg / ml filler;

[0030] (2) Metal ion chelation

[0031] Add the above coupling ligand filler to 10...

Embodiment 3

[0033] A preparation method of ultra-tolerant metal chelate affinity filler Cu Smart Beads 6FF, the preparation method comprises the following steps:

[0034] (1) Ligand coupling

[0035] Carboxyl reaction: Dissolve the ligand in 0.1M MES, add amino magnetic microspheres, add 0.1MEDC at pH 5.0, react at 20°C for 1 hour, and continue to react at pH 5.0 for 24 hours to obtain a coupled ligand filler; , the amount of ligand added is 100mg / ml filler;

[0036] (2) Metal ion chelation

[0037] Add the above coupling ligand filler to 75mg / ml CuSO 4 React at 10°C for 8 hours to obtain the super-tolerant metal chelate affinity filler; the structural formula of the ligand is:

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Abstract

The invention relates to the technical field of preparation of purified packing, in particular to preparation and application of super tolerance metal chelating affinity packing. The preparation method comprises the steps of ligand coupling and metal ion chelating. The obtained metal chelating affinity packing (IMAC) chelates quite firm metal ions, and can be applied to capturing and purifying protein with His-tag. The tolerance packing of the type can efficiently purify target protein on the condition that EDTA and DTT are contained, and the tedious treatment of switching buffer liquid before purification is avoided; in the using process, few metal ions are disengaged, 1M sodium hydroxide in-place cleaning can be carried out without stripping of metal ions, the regeneration treatment step for packing can be effectively simplified, and the packing has strong component compatibility, is applicable to buffer conditions of wide substrate and salinity, has the advantages of being high in capacity, high in purity, long in service life and low in using cost, and is suitable for being further applied and popularized.

Description

technical field [0001] The invention relates to the technical field of preparation of protein purification packing, in particular to the preparation and application of a super-tolerant metal chelating affinity packing. Background technique [0002] Metal chelate affinity packing (IMAC) is a key technology in the field of protein purification, and it has been widely used in the study of protein function and structure and the purification of recombinant protein drugs. Its basic principle is to use amino acid residues such as histidine and cysteine ​​on natural or recombinant proteins to form coordination bonds with metal ions chelated on solid-phase microspheres to adsorb the target protein from the solution to the solid phase. The surface of the microspheres achieves the purpose of separation and purification. The number of histidine, cysteine ​​and other residues contained in natural proteins is limited and rarely distributed continuously, so the binding force with metal ch...

Claims

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Application Information

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IPC IPC(8): B01J20/22B01J20/30C07K1/22
CPCB01J20/223C07K1/22
Inventor 单玉飞曹飞婷
Owner CHANGZHOU SMART LIFESCI CO LTD
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