Glycosylated worm kinase purified by m-aminobenzoic boric acid affinity chromatography and purifying method thereof

A technology of aminophenylboronic acid and lumbrokinase, applied in biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of complex methods, long half-life, high cost, etc., and achieve the effect of simple equipment, increased half-life, and low cost

Inactive Publication Date: 2009-07-01
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to: overcome the complex method, high cost, low yield and other defects in the existing lumbrokinase separation technology; in order to improve the efficacy of lumbrokinase, create conditions for further development of injections; the present invention uses boron on the ligand -The hydroxyl group is combined with the carbonyl group on the sugar chain of glycosylated lumbrokinase to obtain a glycosylated single component with stronger stability, longer half-life in vivo, and lower toxicity than the non-glycosylated component; in order to achieve Rapid and effective large-scale separation of glycosylated lumbrokinase components to achieve industrial production; thereby providing a method for purifying earthworm protease with simple operation and low-cost m-aminophenylboronic acid affinity chromatography; also in other carbohydrates Make useful explorations in the separation and purification of proteins

Method used

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  • Glycosylated worm kinase purified by m-aminobenzoic boric acid affinity chromatography and purifying method thereof
  • Glycosylated worm kinase purified by m-aminobenzoic boric acid affinity chromatography and purifying method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Carbonyldiimidazole was used to activate the Sepharose filler Sepharose-CL-6B, and m-aminophenylboronic acid was used as the ligand of the affinity chromatography resin: the m-aminophenylboronic acid was dissolved in 0.2M NaHCO 3 In the buffer (pH=8.5, 0.15M NaCl), the concentration is 10g / L, and then mixed with the activated agarose filler at a ratio of 5:1 (V / V), stirred for 2 hours (4°C) to connect the ligand onto the agarose packing, and packed into the column. Equilibrate with a mixed solution of 0.01M asparagine, 0.01M magnesium chloride and 0.01% sodium azide at a final concentration of pH=6.0.

[0023] Weigh the freeze-dried powder of the crude extract of Eisenia chinensis, and dissolve it in Tris-HCl buffer solution with pH = 5.0 to a final concentration of 0.5 mg / mL. The loading volume was 1 / 3 of the column volume. Use pH=6.0, 0.001M Tris-HCl, 0.001M asparagine, 0.05M magnesium chloride and 0.01% sodium azide as the eluent to remove non-specifically bound fo...

Embodiment 2

[0027] Activation of Sepharose-CL-4B with cyanogen bromide, using m-aminophenylboronic acid as ligand for affinity chromatography resin: dissolve m-aminophenylboronic acid in 0.2M NaHCO 3 In the buffer (pH=8.5, 0.15M NaCl), the concentration is 100g / L, and then mixed with the activated agarose filler at a ratio of 1:5 (V / V), stirred for 0.5 hours (40°C) to connect the ligand onto the agarose packing, and packed into the column. Equilibrate with a mixed solution of pH 10.2 and final concentrations of 0.02M asparagine, 0.02M magnesium chloride and 0.02% sodium azide respectively.

[0028] Weigh the freeze-dried powder of the crude extract of Umbilicus spp., and dissolve it in Tris-HCl buffer solution of Ph14 to a final concentration of 5 mg / mL. The loading volume was 1 / 10 of the column volume. Use pH 12, the final concentration of 0.01M Tris-HCl, 0.005M asparagine, 0.5M magnesium chloride and 0.05% sodium azide mixture as the eluent to remove non-specifically bound impurities ....

Embodiment 3

[0032] Activate Sepharose-6B with carbonyldiimidazole, and use m-aminophenylboronic acid as the ligand of affinity chromatography resin: dissolve m-aminophenylboronic acid in 0.2M NaHCO 3 In the buffer (pH=8.5, 0.15M NaCl), the concentration is 10-100g / L, and then mixed with the activated agarose filler at a ratio of 1:1 (V / V), stirred for 1 hour (at room temperature), and the ligand Connect to agarose packing and pack the column. Equilibrate with a mixed solution of pH 8.75, final concentration of 0.02M asparagine, 0.02M magnesium chloride and 0.02% sodium azide respectively.

[0033] Weigh the spray-dried powder of the crude extract of A. serrata, and dissolve it in Tris-HCl buffer solution with pH 9.0 to a final concentration of 1 mg / mL. The loading volume was 1 / 5 of the column volume. Use a mixture of Tris-HCl with a final concentration of 0.001M, 0.002M asparagine, 0.1M magnesium chloride and 0.02% sodium azide at pH 11.0 as the eluent to remove non-specifically bound i...

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Abstract

The method adopts agarose gel-m-aminophenylboronic acid affinity chromatography to separate and purify a single glycosylation component with plasmin activity from earthworm homogenate. In the process of affinity chromatography, agarose gel is used as a filler, m-aminophenylboronic acid is used as a ligand, and cis-monosaccharide is used as a specific eluent, and desalted by dialysis or molecular sieve to obtain glycosylated components; dry store. The component is detected as a single component by SDS-PAGE, and the N-terminal sequence is N-Ala-Glu-Val-Cys-Cys-Pro-Asp-Ile-, which is a new protein. Chromogenic substrate activity test showed that it has high plasminase activity. The method is simple and convenient, low in cost and suitable for large-scale production.

Description

technical field [0001] The invention relates to a method for separating and purifying earthworm fibrinolytic protease (lumbrokinase), in particular to a method for separating and purifying a single stable glycosyl from earthworms by using agarose gel-m-aminophenylboronic acid affinity chromatography Protein components, and have a higher plasminase activity method. Background technique [0002] In recent years, a series of proteases have been isolated from earthworms by using separation methods such as salting out, ultrafiltration, molecular sieve, ion exchange, and high-pressure liquid chromatography. The in vitro plate method shows that these proteases can both activate plasminogen and directly degrade fibrin, so they are named lumbrokinase (earthworm plasmin). Clinical experiments have shown that oral administration of lumbrokinase has a significant preventive effect on cardiovascular and cerebrovascular embolism. However, the oral lumbrokinase preparation commonly used ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/68C07K1/14C07K1/22
Inventor 李莉赫荣乔
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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