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Method for purifying and directionally immobilizing histidine tag protein and application

A technique of histidine tagging and directional immobilization, which is applied in the preparation of purified and directional immobilized histidine tagged protein materials, and in the field of surface modification of biological materials, which can solve the problems of easy dissociation of immobilized proteins, high product prices, and complicated preparation processes and other problems, to achieve the effect of easy reuse, convenient purification and simple operation

Active Publication Date: 2017-12-08
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a preparation method and application of polyphenolic compounds, metal ions, and anti-protein adhesion hybrid materials for purifying and directional immobilizing histidine-tagged proteins, thereby solving the problems in the prior art. The preparation process of amino acid tagged protein immobilization materials is complicated, the product price is high, and the immobilized protein is easy to dissociate.

Method used

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  • Method for purifying and directionally immobilizing histidine tag protein and application
  • Method for purifying and directionally immobilizing histidine tag protein and application
  • Method for purifying and directionally immobilizing histidine tag protein and application

Examples

Experimental program
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Embodiment 1

[0040] Example 1 Histidine-tagged protein purification and immobilization experiments

[0041] 1) With deionized water as solvent, prepare 6.4mg / ml tannic acid solution and 1.6mg / ml FeCl respectively 3 ·6H 2 O solution, 1 mg / ml polysulfobetaine solution, 2.5 mg / ml CoCl 2 solution;

[0042] 2) Using silicon wafers as the carrier material, place 1×1cm silicon wafers in a 5ml centrifuge tube, add 1ml tannic acid solution and 1ml FeCl in sequence 3 Solution, oscillate evenly, place at room temperature for 3mins, take out the silicon wafer with tweezers, and wash it three times with deionized water;

[0043] 3) Put the silicon chip in 2) back into the 5ml centrifuge tube, and add 1ml FeCl in turn 3 solution and 1ml tannic acid solution, shake evenly, place at room temperature for 3mins, take out the silicon wafer, and wash it three times with deionized water;

[0044] 4) Repeat step 3) 5 times to obtain multilayer modified TA / Fe 3+ material surface ( figure 1 a)

[0045] 5)...

Embodiment 2

[0049] Example 2 Histidine-tagged protein purification experiment

[0050] 1) With deionized water as solvent, prepare 6.4mg / ml tannic acid solution and 1.6mg / ml FeCl respectively 3 ·6H 2 O solution, 1 mg / ml polysulfobetaine solution, 2.5 mg / ml CoCl 2 solution;

[0051] 2) Using magnetic microspheres with a diameter of 300nm as the carrier material, put 0.1g of magnetic microspheres into a 5ml centrifuge tube, add 2ml of tannic acid solution and 2ml of FeCl in turn 3 Solution, shake and disperse evenly, stir at room temperature for 5mins, magnetically separate, wash with deionized water three times;

[0052] 3) Put the magnetic microspheres in 2) back into the 5ml centrifuge tube, and add 2ml FeCl in turn 3 solution and 2ml tannic acid solution, shake and disperse evenly, stir at room temperature for 5mins, take out by magnetic separation, and wash with deionized water three times;

[0053] 4) Repeat step 3) 3 times;

[0054] 5) Place the silicon chip in 4) in a 5ml cent...

Embodiment 3 2

[0059] Example 3 The experiment of the influence on the stability of immobilized protein after divalent cobalt is oxidized to trivalent

[0060] 1) With deionized water as solvent, prepare 6.4mg / ml tannic acid solution and 1.6mg / ml FeCl respectively 3 ·6H 2 O solution, 1 mg / ml polysulfobetaine solution, 2.5 mg / ml CoCl 2 solution;

[0061] 2) Using silicon wafers as the carrier material, place 1×1cm silicon wafers in a 5ml centrifuge tube, add 1ml tannic acid solution and 1ml FeCl in sequence 3 Solution, oscillate evenly, place at room temperature for 3mins, take out the silicon wafer with tweezers, and wash it three times with deionized water;

[0062] 3) Put the silicon chip in 2) back into the 5ml centrifuge tube, and add 1ml FeCl in turn 3 solution and 1ml tannic acid solution, shake evenly, place at room temperature for 3mins, take out the silicon wafer, and wash it three times with deionized water;

[0063] 4) Repeat step 3) 5 times to obtain multilayer modified TA / F...

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Abstract

The invention discloses a method for purifying and directionally immobilizing histidine tag protein and application. According to the method disclosed by the invention, tannic acid, metal ions and a hydrophilic anti-protein adhesion polymer are self-assembled and modified on the surface of a carrier to obtain a material surface which is efficiently chelated with cobalt ions, so that one-step purification and immobilization of the histidine tag protein is realized; a ligand of trivalent cobalt has inertia and divalent cobalt ions are oxidized into trivalent cobalt ions so that the stability of the immobilized protein can be improved. The method disclosed by the invention has the advantages of simple process and low price; a separation and purification or immobilization operation process for the histidine tag protein of the method is simple and effective, so that the technology has a wide application prospect.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically to a biomaterial surface modification technology and a preparation method and application for purifying and directional immobilizing histidine-tagged protein materials Background technique [0002] Protein immobilization refers to the process of using physical, chemical or biological methods to immobilize proteins on the surface of materials and maintain their activity. The immobilization technology of proteins on the surface of materials has a wide range of applications, such as protein analysis, drug screening, and biocatalysis. Due to the inherent instability and variability of protein molecules, the immobilization of proteins is particularly important. [0003] There are many methods to achieve protein immobilization, which can be divided into two categories: directional immobilization and non-directional immobilization. Traditional protein immobilization methods mostl...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/08C12N9/42C07K19/00B01J20/22B01J20/30
CPCB01J20/223C07K2319/21C12N9/2442C12N11/08C12N11/14C12Y302/01014
Inventor 贾凌云韩璐璐刘起杨立为叶通何知恩
Owner DALIAN UNIV OF TECH
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