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Magnetic affinitive nanomaterial for separating and purifying GST (glutathione S-transferase) tag fusion protein as well as preparation method and application thereof

A technology of nanomaterials and label fusion, applied in chemical instruments and methods, ion exchange, other chemical processes, etc., can solve the problems of high cost, complex synthesis and modification process, lack of GST label fusion protein, etc., to increase the binding rate and binding amount, wide application prospect and practical value, and the effect of simple preparation method

Inactive Publication Date: 2014-06-25
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a magnetic affinity nanomaterial for the separation and purification of GST tag fusion protein and its preparation method and application, thereby solving the synthesis of magnetic affinity nanomaterial for the purification of GST tag fusion protein in the prior art The modification process is complicated, the defect of high cost and price, and the lack of a simple separation and purification method for GST tag fusion protein in the prior art

Method used

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  • Magnetic affinitive nanomaterial for separating and purifying GST (glutathione S-transferase) tag fusion protein as well as preparation method and application thereof
  • Magnetic affinitive nanomaterial for separating and purifying GST (glutathione S-transferase) tag fusion protein as well as preparation method and application thereof
  • Magnetic affinitive nanomaterial for separating and purifying GST (glutathione S-transferase) tag fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Preparation of Magnetic Affinity Nanomaterial

[0024] (1) Weigh 2.7g FeCl 3 ·6H 2 O and 0.99 g FeCl 2 4H 2 O was dissolved in 250ml ultrapure water under the protection of nitrogen, the pH was adjusted to about 9 by adding 5M NaOH, stirred at 40°C for 2 hours, and ferric oxide nanoparticles were obtained by magnetic separation. Then it was dissolved in ultrapure water to prepare a 1mg / ml magnetic Fe3O4 nanoparticle solution.

[0025] (2) Disperse 50 mg of iron ferric oxide nanoparticles in 50 mL of Tris-HCl buffer (10 mM, pH 8.5), ultrasonically disperse for 15 minutes, and add 50 mg of dopamine hydrochloride. After stirring at room temperature for 1 hour, polydopamine-modified Fe3O4 nanoparticles were obtained by magnetic separation.

[0026] (3) Disperse the above-mentioned polydopamine-modified iron ferric oxide nanoparticles in 50 mL of ultrapure water, add 100 mg of reduced glutathione, stir and react at room temperature for 6 hours, and separate...

Embodiment 2

[0028] Example 2 Separation and purification of GST-fused green fluorescent protein (GST-GFP)

[0029]The GST-GFP-producing recombinant Escherichia coli selected in this example was constructed in this laboratory by using the original green fluorescent protein gene as a template and fusing GST tags by means of genetic engineering (for the original green fluorescent protein gene without GST tags added, See Kaisa Hakkila, Mikael Maksimow, Matti Karp, Marko Virta, Reporter Genes lucFF, luxCDABE, gfp, and dsred Have Different Characteristics in Whole-Cell Bacterial Sensors, Analytical Biochemistry, Volume 301, Issue 2, Pages 235–242). Wherein, the plasmid containing the green fluorescent protein gene can be purchased through existing commercial channels, and the green fluorescent protein gene is amplified by PCR, and the forward primer used is: 5'-GGACTAGTATGAGTAAAGGAGAAGAACTTTTCA-3'; The primer is: 5'-CGGGATCCTTATTTGTATAGTTCATCCATG-3'. The PCR products were separated by agarose ...

Embodiment 3

[0031] The separation and purification of the lipase (GST-Lip) of embodiment 3GST fusion

[0032] The GST-Lip-producing recombinant Escherichia coli selected in this example was constructed in this laboratory by fusion of GST tags with lipase genes as templates (for the original lipase genes without GST tags, please refer to Bei Gao , Erzheng Su, Jinping Lin, Dongzhi Wei. Development of recombinant Escherichia coli whole-cell biocatalyst expressing a novel alkali lipase-coding gene from Proteus sp. for biodiesel production. Journal of Biotechnology. 2009,139(2):169-175) . Wherein, the plasmid with the lipase LipK107 gene can be purchased through existing commercial channels and used as a template to amplify the lipase LipK107 gene by PCR. The forward primer used is: 5'-GGACTAGTATGTCAACGAAATATCCTATC-3 '; the reverse primer is: 5'-GGAATTCTTAAAGTTGCTTACTCGCTAAG-3'. The target gene fragment was recovered by gel recovery, and the fragment was double-digested with SpeI and EcoRI r...

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Abstract

The invention provides a magnetic affinitive nanomaterial for separating and purifying GST (glutathione S-transferase) tag fusion protein as well as a preparation method and application thereof. The magnetic affinitive nanomaterial comprises ferroferric oxide nanoparticles and a polydopamine coating which wraps the ferroferric oxide nanoparticles, wherein reduced glutathione is fixed on the surface of the polydopamine coating. According to the magnetic affinitive nanomaterial provided by the invention, the specific surface area is relatively large, the rate and quantity of bonding between the nanomaterial and protein molecules are increased, the superparamagnetic property of the nanoparticles simplifies the operation strength of a separation process, the preparation method is simple and low in cost, and the process for separating and purifying the GST fusion protein is simple, convenient, quick and effective. The nanomaterial has wide application prospect and practical value in the field of biotechnology.

Description

technical field [0001] The invention relates to the technical field of biomaterials, and more specifically relates to a magnetic affinity nanomaterial used for separating and purifying GST tag fusion proteins and its preparation method and application. Background technique [0002] In the field of biotechnology research, the separation and purification of proteins is a very important link, especially in the research of proteomics, the complex composition and extremely low protein content increase the difficulty of protein separation and purification. The development of fusion tag technology provides a more convenient way for protein separation and purification. The fusion tag technology mainly uses recombinant DNA technology to fuse the coding gene of a specific tag at the 3' or 5' end of the target protein coding gene and express the recombinant protein in a suitable expression host. Utilizing the affinity between the immobilized specific ligand and the fusion tag of the r...

Claims

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Application Information

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IPC IPC(8): B01J20/286B01J20/30B01D15/08C07K1/22
Inventor 任宇红魏东芝倪克奉杨剑冰
Owner EAST CHINA UNIV OF SCI & TECH
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