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Process for preparing glutathion-5-transferase protein affinity chromatography medium and its use

A chromatographic medium and glutathione technology, applied in the field of genetic engineering, can solve the problems of high price, cost and price barriers, restrictions, etc., and achieve the effect of reducing practical costs

Inactive Publication Date: 2006-01-04
华子春
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AI Technical Summary

Problems solved by technology

However, at present, the preparation technology of GST protein affinity chromatography medium is only mastered by some large foreign biological products companies, and the price is expensive, which limits the further application of this system, especially the promotion and application of this expression system in China, especially in the genetic engineering industry The use of modernization poses cost and price barriers

Method used

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  • Process for preparing glutathion-5-transferase protein affinity chromatography medium and its use
  • Process for preparing glutathion-5-transferase protein affinity chromatography medium and its use
  • Process for preparing glutathion-5-transferase protein affinity chromatography medium and its use

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Embodiment Construction

[0029] 1. Activation of the medium: Take 10ml of Sepharose 4B and remove the protective solution in the G-3 funnel, rinse with 100ml of 0.5mol / L NaCl solution, wash with 100ml of distilled water for 4 times, and transfer to a 100ml Erlenmeyer flask. Add 7ml of 1mol / l NaOH solution (containing NaBH4 2mg / ml), a small amount of 1,4-butanediol diglycidyl ether, and slowly shake the reaction.

[0030] 2. Washing: filter the solution, wash it fully with distilled water until the pH is about 7.0, rinse it with 20ml 0.1mol / L pH9.5 NaHCO3-Na2CO3 buffer solution, and drain it for later use.

[0031] 3. Coupling reaction: Add 20ml of phosphate buffer and 10ml of glutathione solution (dissolve 0.5g of reduced glutathione in water, adjust the pH to 7.0 with KOH, and set the volume to 10ml), shake at 37°C.

[0032] 4. Post-treatment of the coupling reaction: filter the solution, wash with 100ml of distilled water, and add 100ml of 1mol / L ethanolamine to terminate the reaction. Then use 100...

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Abstract

The present invention belongs to the field of genetic engineering technology. GST protein affinity chromatography medium is prepared with glutathion as affinity arm and 1, 4-butanediol diglycidyl ether as activating cross-linking agent, and through activating and washing of chromatography medium, glutathion coupling reaction, terminating reaction and washing. The preparation conditions and technological process are optimized. Test shows that the affinity chromatography medium of the present invention has very high specificity and purifying efficiency and affinity adsorption performance mach higher than that of affinity medium with chloropropylene oxide as activator. The affinity chromatography medium may be used in separation and purification of GST and GST fusing protein in bioengineering, gene engineering, biochemistry, molecular biology and other fields.

Description

1. Technical field: [0001] The invention belongs to the technical field of genetic engineering. 2. Background technology: [0002] In the practice of genetic engineering, fusion expression is often used to express and produce target proteins, and the most widely used fusion protein is glutathione-S-transferase (GST). Glutathione-S-transferase itself has a very high expression level in E. coli, and its solubility is very good, so that the expression level of the target protein to be fused and expressed is high, and the solubility of the expression product is good, so it is more effective Avoid or solve the difficulty of recombinant expression product inclusion body. Moreover, with the help of the biochemical characteristics and properties of glutathione-S-transferase with high specific affinity for its substrate glutathione, researchers can conveniently perform GST using glutathione affinity media. Separation and purification of fusion expression products. However, at pres...

Claims

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Application Information

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IPC IPC(8): C12N9/10C07K1/14
Inventor 华子春
Owner 华子春
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