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Method for purifying and preparing anti-tumor factor-cathepsin beta inhibiting factor from soybean or bean dregs

A technology of cathepsin and inhibitors, applied in the field of protein purification, can solve the problems of no direct purification of cathepsin B inhibitors, no research reports on purified cathepsin B inhibitors, etc.

Inactive Publication Date: 2005-10-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research report on the purification of cathepsin B inhibitor from soybean, and there is no report on the technology of directly purifying cathepsin B inhibitor from soybean or soybean meal.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Grind soybeans or soybean meal into powder, dissolve them in Tris-HCl buffer (10mmol / L Tris-HCl pH7.5, or other buffers with the same function) and stir overnight, filter to remove the precipitate, and centrifuge the supernatant at 10,000rpm After 10 min, 50% ammonium sulfate was precipitated, and the precipitate was dissolved in a small amount of Tris-HCl buffer solution, and dialyzed overnight in 10 times the volume of Tris-HCl buffer solution. The dialyzed solution was centrifuged at 10,000rpm for 10min, and the supernatant was slowly added to a size-exclusion chromatography column (Sephadex G-75, or other functionally equivalent chromatography columns) equilibrated with Tris-HCl buffer solution. The HCl buffer was separated and the fraction with inhibitory activity was collected in fractions. Slowly add the collected liquid to the ion exchange column (such as DEAE-Toyopearl, CM-Sepharose column or other chromatography columns with the same function) equilibrated wit...

Embodiment 2

[0035] The buffer in Example 1 was replaced with phosphate buffer (or other suitable buffer) at pH 7.5, and the cathepsin B inhibitor and the obtained product were purified from soybean or soybean meal. Detailed technology: Grind soybeans or soybean meal into powder, dissolve them in phosphate buffer (20mmol / L pH7.5), stir overnight, filter to remove precipitates, centrifuge the supernatant at 12,000rpm for 8min, precipitate with 50% ammonium sulfate, and dissolve the precipitates Dialyze in 10 volumes of phosphate buffered solution for 22 hours in the phosphate buffered solution just submerged. The dialyzed solution was centrifuged at 10,000rpm for 13min, and the supernatant was slowly added to a size-exclusion chromatography column (Sephadex G-75) equilibrated with phosphate buffer, separated with phosphate buffer and collected in fractions where there was inhibitory activity. part. Then slowly add the collected solution to the ion exchange column (DEAE-Toyopearl) equilibra...

Embodiment 3

[0037] The chromatographic columns in Examples 1 and 2 were replaced with other chromatographic columns with the same function, and the cathepsin B inhibitor and the obtained product were purified from soybean or soybean meal. Detailed technique: Grind soybean or soybean meal into powder, dissolve in Tris-HCl buffer (10mmol / L Tris-HCl pH7.5), stir overnight, filter to remove precipitate, supernatant is centrifuged at 10,000rpm for 10min, 50% sulfuric acid For ammonium precipitation, the precipitate was dissolved in a small amount of Tris-HCl buffer, and dialyzed overnight in 10 times the volume of Tris-HCl buffer. The dialyzed solution was centrifuged at 10,000rpm for 10min, and the supernatant was slowly added to a size-exclusion chromatography column (Superdex) equilibrated with Tris-HCl buffer, separated with Tris-HCl buffer and collected in fractions, which had inhibitory activity part. Slowly add the collected solution to the ion exchange column (DEAE-Sepharose) equilibr...

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Abstract

The process of purifying and preparing cathepsin B inhibiting factor as one antitumor factor from soybean or soybean dregs includes grinding soybean or soybean dregs, stirring, precipitation, dialysis, exclusion chromatography and ion exchange column separation, elution, separate collection and other steps. The soybean cathepsin B inhibiting factor prepared through the process has obvious effect of inhibiting the growth of human cervical carcinoma cell (Hela) and neuroglioma cell (glioma). The present invention provides new way for developing new medicine and relevant health food for preventing and treating tumor and for deep processing of soybean product.

Description

technical field [0001] The invention relates to protein purification technology in the technical field of bioengineering, in particular to a method for purifying and preparing antitumor factor-cathepsin B inhibitor from soybean or soybean meal. Background technique [0002] Protease refers to the enzyme that hydrolyzes protein and is a key molecule to maintain normal physiological functions of the human body. According to the different parts of protein hydrolysis, protease can be divided into exopeptidase and endopeptidase, exopeptidase hydrolyzes protein from the carboxyl or amino terminal of protein, and endopeptidase hydrolyzes protein from the middle of protein. Endopeptidases are further divided into cysteine ​​proteases, aspartic acid proteases, serine proteases and metalloproteases according to the amino acid residues in the active center, among which cysteine ​​proteases include dozens of different proteases. In normal physiological metabolism, protease is mainly in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/56A61P35/00
Inventor 赵小凡王金星
Owner SHANDONG UNIV
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