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Method for purifying human urinary trypsin inhibitor

A technique for urinary aprotinin and purification method, which is applied in the directions of biochemical equipment and methods, enzymes, digestive systems, etc., can solve the problems of complex process, low total yield, and many steps, etc.

Active Publication Date: 2009-09-09
SHANGHAI TECHWELL BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] The current hUTI preparation process mainly uses the combination of anion exchange chromatography, hydrophobic chromatography, metal chelation chromatography, and affinity chromatography to purify hUTI from a solution containing human urinary aprotinin. There are many steps, the process is complicated, and the total yield is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Take 20 grams of hUTI raw material (potency 690IU / mg), with a total titer of 13.8 million IU, dissolve it in water to 260mL, adjust the pH to about 3.2, collect the supernatant by centrifugation, adjust the conductance for 3-4ms, and pass through the diameter at a flow rate of about 10cm / h. The sulfenyl agarose resin S-Sepharose F.F. (200 mL, provided by Amersham) with a size of 5 cm and a height of 10 cm has been equilibrated with 0.1 M citric acid and pH 3.2 buffer in advance. After loading the samples, wash with 2 liters of balance solution at a flow rate of 10 cm / h. Then use the eluent (0.1M citric acid + 0.4MNaCl, pH3.2) to elute with a flow rate of 10cm / h, monitor the 280nm with a UV detector, collect the distillate peak, combine the active ingredients to about 0.25L, add 3 times Precipitate in absolute ethanol at -10°C overnight, collect the precipitate by centrifugation the next day, dehydrate with absolute ethanol, and dry in vacuo to obtain 3.3 grams of purifi...

Embodiment 2

[0072] Take 30 grams of hUTI raw material (potency 690IU / mg), with a total titer of 20.7 million IU, dissolve it in water to 390mL, adjust the pH to about 3.5, collect the supernatant by centrifugation, adjust the conductance for 3-4ms, and pass through the diameter at a flow rate of about 15cm / h. Sulfonate propyl agarose resin SP-Sepharose F.F. (300 mL, provided by Amersham) with a size of 5 cm and a height of 15 cm has been equilibrated with 0.1 M citric acid and pH 3.5 buffer beforehand. After loading the samples, wash with 3 liters of balance solution at a flow rate of 15 cm / h. Then use the eluent (0.06M NaAc, pH4.2) to elute with a flow rate of 15cm / h, monitor the 280nm place with an ultraviolet detector, collect the distillate peak, combine the active ingredients to about 0.35L, and add 3 times -10°C Precipitate in absolute ethanol overnight, collect the precipitate by centrifugation the next day, dehydrate with absolute ethanol, and dry in vacuum to obtain 4.8 grams of ...

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PUM

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Abstract

The invention discloses a method for purifying a human urinary trypsin inhibitor (hUTI). The method comprises the following steps that: a, a solution containing the urinary trypsin inhibitor and cation exchange resin are contacted, so that the urinary trypsin inhibitor is adsorbed on the cation exchange resin; and b, a pH1-7 buffering liquid is used to elute the urinary trypsin inhibitor adsorbed on the cation exchange resin to obtain purified urinary trypsin inhibitor. The method is simple and high-efficient; and an obtained product has high purity.

Description

technical field [0001] The invention relates to a method for purifying protein, in particular to a method for purifying human urinary aprotinin. Background technique [0002] Human urinary trypsin inhibitor (Human urinary trypsin inhibitor, also known as urinary trypsin inhibitor, hereinafter referred to as hUTI) is a protease inhibitor present in human urine, which has a broad-spectrum enzyme inhibitory effect on trypsin, α- Chymotrypsin, elastase, hyaluronidase, sulfhydrylase, plasmin, etc. all have inhibitory effects. At the same time, hUTI can stabilize lysosomal enzymes and inhibit the production of cardioinhibitory factor. As early as 1909, Bauer and Reich first reported the existence of trypsin preparations in urine. It was not until the 1950s and 1960s that several methods were reported to separate and purify several small-molecular-weight hUTIs under acidic conditions until 1977. Sumi et al. successfully separated and purified the complete hUTI molecule (molecular...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00A61K38/57A61P1/18A61P7/08A61P9/00A61P15/00A61P35/00
Inventor 季晓铭高霄梁庞骏谢莽李勇洪云海李晓东
Owner SHANGHAI TECHWELL BIOPHARMACEUTICALS CO LTD
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