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Method for separating human urinary kallidinogenase and thrombin regulatory protein

A technology of urokininogenase and protein regulation, which is applied in the biological field, can solve the problems of unreachable affinity chromatography, high resolution, poor use effect, etc., achieve convenient purification and detection, simplify operation steps, and improve the purity of crude products Effect

Pending Publication Date: 2022-01-11
YANGZHOU AIDEA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] After investigation, there is no one-step chromatographic separation study for KN and TM in the literature. Because the isoelectric point and molecular weight of the two proteins are very close, they cannot be completely separated by conventional ion exchange, gel filtration and other methods. to open
In addition, according to the fact that KN and TM have different affinity characteristics for different substrates / reactants, they can be separated theoretically by affinity chromatography, but this method is costly and the types of starting material protein components Complicated, affinity chromatography often cannot achieve the highest resolution, and the use effect is poor

Method used

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  • Method for separating human urinary kallidinogenase and thrombin regulatory protein
  • Method for separating human urinary kallidinogenase and thrombin regulatory protein
  • Method for separating human urinary kallidinogenase and thrombin regulatory protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) Equilibrium solution: the balance solution is 0.02mol / L acetate buffer containing 1.5mol / L (NH 4 ) 2 SO 4 , pH is 4.0, conductivity is 180ms / cm;

[0038] (2) Pretreatment of crude protein urine: take 5.00g crude protein urine (KN content is 7.0PNA / / g, TM content is 0.4ug / g), add 8 times of pure water, stir and dissolve for 10min, centrifuge at 4000rpm for 10min, leave Clear, add ammonium sulfate to adjust conductance and pH, pH is 4.0, conductance is adjusted to 183mS / cm, makes sample solution;

[0039] (3) Loading samples and flushing with equilibrium solution: 50 mL of phenyl-sepharose hydrophobic chromatography column is pre-installed, and equilibrated with 5 times column volume with equilibrium solution, which is 0.02mol / L acetate buffer containing 1.5mol / L(NH 4 ) 2 SO 4 , the pH is 4.0, and the conductivity is 180ms / cm, and then the sample solution prepared in step (2) is loaded on the chromatographic column. Wash the column with volume washing solution...

Embodiment 2

[0045] (1) Equilibrium solution: the balance solution is 0.02mol / L phosphate buffer, containing 1.5mol / L (NH 4 ) 2 SO 4 , pH is 7.0, conductivity is 182ms / cm;

[0046] (2) Pretreatment of crude protein urine: take 5.00g crude protein urine (KN content is 5.0PNA / / g, TM content is 0.3ug / g), add 10 times of pure water, stir and dissolve for 10min, centrifuge at 4000rpm for 10min, leave Clear, add ammonium sulfate to adjust conductance and pH, pH is 7.0, conductance is adjusted to 184mS / cm, makes sample solution;

[0047] (3) Loading samples and flushing with equilibrium solution: 50 mL of butyl-agarose hydrophobic chromatography column is pre-installed, and equilibrated with 5 times the column volume with equilibrium solution. The equilibrium solution is 0.02mol / L phosphate buffer, containing 1.5mol / L (NH 4 ) 2 SO 4 , the pH is 7.0, and the conductivity is 182ms / cm. Then, the sample solution prepared in step (2) is loaded into the chromatographic column. Wash the column wi...

Embodiment 3

[0053] (1) Equilibrium solution: the balance solution is 0.2mol / L sodium bicarbonate buffer containing 1.3mol / L (NH 4 ) 2 SO 4 , pH is 10.0, conductivity is 153ms / cm;

[0054] (2) Pretreatment of crude protein urine: take 100.00g crude protein urine (wherein KN content is 5.0PNA / / g, TM content 3.0ug / g), add 5 times of pure water, stir and dissolve for 30min, then centrifuge at 4000rpm for 10min, keep the supernatant, Add ammonium sulfate to adjust the conductivity and pH, the pH is 10.0, and the conductivity is adjusted to 158mS / cm to prepare the sample solution;

[0055] (3) Sample loading and equilibration liquid washing: 1000mL phenyl-sepharose hydrophobic chromatography column is pre-installed, and the equilibration liquid is used to equilibrate 5 times the column volume. The equilibration liquid is 0.2mol / L sodium bicarbonate buffer solution, containing / L(NH 4 ) 2 SO 4 , the pH is 10.0, the conductivity is 153ms / cm, and then the sample solution prepared in step (2)...

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Abstract

The invention discloses a method for separating human urinary kallidinogenase and thrombin regulatory protein in the technical field of biology. The method adopts hydrophobic chromatography to separate and obtain a human urinary kallidinogenase crude product and a thrombin regulatory protein crude product in one step for the first time, wherein a hydrophobic ligand of a hydrophobic chromatography column is one of phenyl, butyl sulfur and octyl; and a filler main body framework of the hydrophobic chromatography column is one of agarose, cellulose or polymer. The method has the advantages of simple operation, low cost, easy industrialized amplification, high yield of the two separated products and obvious impurity removal effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating human urinary kininogenase and thrombin regulatory protein. Background technique [0002] Human urine contains hundreds of proteins. Currently, urokinase, human urinary trypsin inhibitor and human urinary kininogenase have been developed and extracted. These components have important therapeutic value in clinic. [0003] Among them, human urinary kallidinogenase (Urinary Kallidinogenase, hereinafter referred to as KN), is a glycoprotein composed of 238 amino acids isolated from human urine, with an isoelectric point of about 4.0 and a molecular weight of about 54,000D. Serine protease. It activates the conversion of human plasma kininogen to kinin. At present, it has been developed as a drug for the treatment of acute cerebral infarction. [0004] Soluble thrombin modulin (Thrombomodulin, hereinafter referred to as TM) mainly exists in human plasma and huma...

Claims

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Application Information

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IPC IPC(8): C12N9/72C07K14/47C07K1/20C07K1/14
CPCC12N9/6462C12Y304/21073C07K14/47
Inventor 侯晓彦贾小刚苏古方薛云杰许冬志袁玉傅和亮
Owner YANGZHOU AIDEA BIOTECH
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