Separation purification method of urokinase

A technology for separation and purification of urokinase, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve problems such as inactivation of urokinase and infeasibility of urokinase, and achieve effective separation

Active Publication Date: 2016-01-13
JIANGSU YOULIKA BIOTECHNOLOYG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It will not be destroyed by heating at a high temperature of 100°C for 1 hour, and its biological activity can only be destroyed by heating at a temperature of 160°C for 2 to 4 hours, or heating and boiling with strong alkali, strong acid or strong oxidant for 30 minutes. Under the above conditions, urokinase will inevitably lead to inactivation, and the above methods are not feasible in the purification of urokinase

Method used

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  • Separation purification method of urokinase
  • Separation purification method of urokinase
  • Separation purification method of urokinase

Examples

Experimental program
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Effect test

Embodiment 1

[0037] The preparation of embodiment 1 urokinase crude product

[0038] a) Male urine, adjust the pH to 8.5 at a temperature below 10°C, let it stand for 2 hours, discard the precipitate, and obtain the supernatant;

[0039] b) Take the supernatant, adjust the pH to 5.0-5.5 and acidify to obtain acidified urine, which is adsorbed through a diatomite column;

[0040] c) Wash the column with cold water at 5°C, and elute with 1% ammonia water and sodium chloride to obtain an eluent;

[0041] d) The eluate was salted out with saturated ammonium sulfate and 4% ammonia water to obtain crude urokinase;

[0042] e) Add a certain amount of ammonia water to the precipitate to fully dissociate the urokinase in the precipitate to obtain a urokinase solution;

Embodiment 2

[0044] 1) To regenerate the medium, wash the QAE-Sephadex gel medium with 6 times the column volume of 1% sodium deoxycholate, and then wash the gel medium with 8 times the column volume of water;

[0045] 2) Gel medium pretreatment, called gel medium, suspended in distilled water, poured off the upper layer of fine particles after 1 hour, and soaked the gel medium in 0.5NNaOH solution at a ratio of 15ml of 0.5NNaOH solution per gram of gel medium medium, stir well, let stand for 30 minutes, filter with suction, wash with distilled water until the pH is neutral; then treat with 0.5N HCl solution as above, and finally treat with 0.5N NaOH solution again, after treatment, soak the gel medium Overnight in 0.1M pH7.4 phosphate buffer;

[0046] 3) Pack the column, suspend the gel medium, take an appropriate amount of gel and fill it in the chromatography column, wash the gel with 3 to 5 times the column volume of water or buffer; pack the column;

[0047] 4) Equilibrium medium, eq...

Embodiment 3

[0053] 1) To regenerate the medium, wash the DEAE-Sephadex gel medium with 6 column volumes of 1% sodium deoxycholate, and then wash the gel medium with 8 column volumes of water;

[0054] 2) Gel medium pretreatment, called gel medium, suspended in distilled water, poured off the upper layer of fine particles after 1 hour, and soaked the gel medium in 0.5NNaOH solution at a ratio of 15ml of 0.5NNaOH solution per gram of gel medium medium, stir well, let stand for 30 minutes, filter with suction, wash with distilled water until the pH is neutral; then treat with 0.5N HCl solution as above, and finally treat with 0.5N NaOH solution again, after treatment, soak the gel medium In 0.1M pH7.4 phosphate buffer overnight.

[0055] 3) Pack the column, suspend the gel medium, take an appropriate amount of gel and fill it in the chromatography column, wash the gel with 3 to 5 times the column volume of water or buffer; pack the column;

[0056] 4) Equilibrate the medium, equilibrate the...

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Abstract

The invention relates to a separation purification method of urokinase. The separation purification method is characterized by comprising the following steps: (1) separating the urokinase; and (2) purifying the urokinase. The preparation method specifically comprises the following steps: (a) collecting urine of men, adjusting the pH value of the urine to 8.5 at the temperature of 10 DEG C or lower, standing for 2h, and removing precipitates, thus obtaining supernatant; (b) taking the supernatant, adjusting the pH to 5.0 to 5.5, acidifying to obtain acidified urine, and adsorbing the acidified urine by virtue of a diatomite column; (c) washing the diatomite column by cold water of 5 DEG C, eluting by utilizing 1% ammonia water and sodium chloride to obtain an elution solution; (d) salting out the elution solution by utilizing saturated ammonium sulfate and 4% ammonia water to obtain a urokinase crude product; and (e) adding a given amount of ammonia water into the precipitates, sufficiently dissociating the urokinase in the precipitates to obtain a urokinase solution.

Description

technical field [0001] The invention belongs to the field of separation and extraction of natural products, and in particular relates to a urokinase separation method of urokinase. Background technique [0002] Urokinase is an enzyme protein isolated from healthy human urine or obtained from human kidney tissue culture. Urokinase is a thrombolytic drug, but it does not directly use the blood clot itself, but first activates the plasmin in the body to become active plasmin, and the plasmin acts on the plasmin clot to decompose it into Soluble polycarbonate. Urokinase can be stable for several years in a freeze-dried state, and a sterile solution of 1mg / ml can be stored in the refrigerator for several months. When the salt concentration is lower than 0.03MNaCl, the stability decreases. Inactivation produces a precipitate, and human blood albumin or gelatin prevents surface denaturation of the enzyme. [0003] Urokinase usually exists in two molecular forms, namely molecular...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/72
CPCC12N9/6462C12Y304/21073
Inventor 李尔华顾京陈爱秀
Owner JIANGSU YOULIKA BIOTECHNOLOYG CO LTD
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