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Recombinant human pro-urokinase purifying method

A purification method, the technology of prourokinase, applied in the direction of biochemical equipment and methods, enzymes, peptidases, etc., can solve the problems of low safety, low protein purity, DNA exogenous pollution, human irritation, etc.

Active Publication Date: 2017-06-20
TASLY BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, this method still has the problems of low protein purity and exogenous contamination such as DNA, host cell residual proteins, and viruses.
The safety of the method of CN1680550A is not high, the main reason is that after completing the D step, it is the product stock solution, and the stock solution will be prepared as the raw material of recombinant human urokinase for injection, but the Tris-HCl buffer solution adopted in the D step will enter the Finished product, directly into the human body through intravenous injection
Tris-HCl buffer is generally only used for research, it is irritating to the human body, and directly entering the human body will cause certain harm to the human body, so it is not suitable as a buffer system for the original solution

Method used

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  • Recombinant human pro-urokinase purifying method
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  • Recombinant human pro-urokinase purifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Purification Step 1: Protein Capture

[0098] Exchange chromatography was carried out with Streamline SP cationic packing as the medium. The chromatographic column is equilibrated with 0.01mol / L phosphate buffer (pH 6.0 containing 0.1mol / L NaCl), and the supernatant of the fermentation broth is adjusted to pH 6.0 before loading the sample, and 0.01mol / L phosphate buffer is used (pH6.0 containing 0.5mol / L NaCl) elution, according to the UV 280 ultraviolet absorption curve to collect the eluted protein peak, which is the intermediate of Purification 1.

[0099] Purification Step 2: Gel Chromatography

[0100] Gel chromatography was carried out with Sephacryl S-200HR gel molecular sieve filler as the medium. The chromatographic column was equilibrated with 0.01mol / L phosphate buffer (pH6.0 containing 0.09mol / L NaCl), and the purified 1 intermediate was directly loaded with 0.01mol / L phosphate buffer (pH6.0 containing 0.09 mol / L NaCl), and collect the target protein peak...

Embodiment 2

[0110] Purification Step 1: Protein Capture

[0111] Exchange chromatography was carried out with Streamline SP cationic packing as the medium. The chromatographic column is equilibrated with 0.005mol / L phosphate buffer (pH5.5 containing 0.05mol / L NaCl), and the supernatant of the fermentation broth is adjusted to pH 5.5 before loading the sample, and 0.005mol / L phosphate buffer is used (pH 5.5 containing 0.3mol / L NaCl elution, according to the UV 280 ultraviolet absorption curve to collect the eluted protein peak, which is the intermediate of Purification 1.

[0112] Purification Step 2: Gel Chromatography

[0113]Gel chromatography was carried out with Sephacryl S-200HR gel molecular sieve filler as the medium. The chromatographic column was equilibrated with 0.005mol / L phosphate buffer (pH5.5 containing 0.05mol / L NaCl), and the purified 1 intermediate was directly loaded with 0.005mol / L phosphate buffer (pH5.5 containing mol / LNaCl), and collect the target protein peak ac...

Embodiment 3

[0123] Purification Step 1: Protein Capture

[0124] Exchange chromatography was carried out with Streamline SP cationic packing as the medium. The chromatographic column was equilibrated with 0.015mol / L phosphate buffer (pH6.5 containing 0.15mol / L NaCl), and the supernatant of the fermentation broth was adjusted to pH 6.5 before loading the sample, and 0.015mol / L phosphate buffer was used (pH 6.5 containing 0.7mol / L NaCl elution, according to the UV 280 ultraviolet absorption curve to collect the eluted protein peak, which is the intermediate of Purification 1.

[0125] Purification Step 2: Gel Chromatography

[0126] Gel chromatography was carried out with Sephacryl S-200HR gel molecular sieve filler as the medium. The chromatographic column was equilibrated with 0.015mol / L phosphate buffer (pH6.5 containing 0.12mol / L NaCl), and the purified 1 intermediate was directly loaded with 0.015mol / L phosphate buffer (pH6.5 containing 0.12 mol / L) elution, according to the UV 280 u...

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Abstract

The invention provides a recombinant human pro-urokinase purifying method. The purifying comprises steps of protein capturing, gel chromatography, cation-exchange chromatography, affinity chromatography, anion-exchange chromatography and cation-exchange chromatography. With the application of the method, by adding a purifying step and optimizing purifying conditions, protein purity is improved to be greater than 98%.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for purifying recombinant human urokinase. Background technique [0002] Prourokinase (Prourokinase, referred to as pro-UK) is a kind of serine protease, which can activate plasminogen in the body and convert it into active plasmin, thereby dissolving fibrin in thrombus. Wide range of applications. The peptide bond between Lys158-Ile159 in pro-UK is easily hydrolyzed by plasmin and other enzymes to generate urokinase (UK for short). UK consists of two peptide chains, A chain and B chain, connected by interchain disulfide bonds. Compared with UK, pro-UK has a higher affinity with fibrin, and the site of activating the fibrinolytic system is often at the site of thrombus formation, so the side effects of systemic hemorrhage tendency are smaller than that of urokinase, and it is a superior fibrinolytic agent. Enzyme activator. There is often only one polypeptide bond differ...

Claims

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Application Information

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IPC IPC(8): C12N9/72
CPCC12N9/6456C12Y304/21031
Inventor 李雪峰赵国焓莫英王明林韩进
Owner TASLY BIOPHARMACEUTICALS CO LTD