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Method for purifying human urinary trypsin inhibitor by utilizing salt resistant mixed mode adsorbent

A technology of trypsin inhibition and mixed mode, which is applied in the direction of protease inhibitors, peptide preparation methods, chemical instruments and methods, etc., can solve the problems of low total yield, complicated process, and low efficiency, and achieve easy amplification and high purification efficiency , the effect of fewer process steps

Inactive Publication Date: 2013-03-27
江西浩然生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preparation process of HUTI is mainly obtained by anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, metal chelation chromatography, gel chromatography, affinity chromatography or these methods. Combined purification of HUTI, some of these methods are too complex and the total yield is very low, while others have simple processes, but have the disadvantages of instability or low purity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) Preparation of salt-tolerant mixed-mode adsorption chromatography medium, the steps are as follows:

[0022] Take 1L of methacrylic acid polymer (Toyo-peal HW65, product of TOSOH, Japan), drain to remove preservatives, wash 2-3 times with deionized water, and drain; prepare by dissolving 320 g of carbonyldiimidazole in 1L of acetone Form a carbonyldiimidazole (CDI) solution, add an equal volume of washed methacrylic acid polymer medium, react with electric stirring at 30°C, and cool to room temperature; wash the activated medium with acetone, and then wash it with deionized water 2-3 times After sucking to dry, a methacrylic acid polymer with imidazole carbamate active groups is obtained. Then add it to aminocaproic acid solution (1kg aminocaproic acid dissolved in 1L carbonic acid-sodium carbonate buffer, pH 9.3), react with electric stirring at 30°C for 15h, cool to room temperature, drain, and wash with 0.5mol / L NaCl 2-3 times, washed 2-3 times with deionized wa...

Embodiment 2

[0026] (1) Preparation of salt-tolerant mixed-mode adsorption chromatography medium, the steps are as follows:

[0027] Example 1 (1) was repeated, using agarose (Sepharose CL 4B, product of GE Company) instead of methacrylic acid polymer, and the same operation was performed to prepare a salt-tolerant mixed-mode adsorption chromatography medium with agarose as the carrier matrix.

[0028] (2) Prepare HUTI by using salt-tolerant mixed-mode adsorption chromatography medium, the steps are as follows:

[0029] Dissolve 500g of crude HUTI (titer 47IU / mg) in 1500ml of water at a ratio of 1:3, collect the filtrate after filtration, conductance 35ms / cm, and adjust the pH of the filtrate to 2-5 with glacial acetic acid. Fill the chromatographic column with the salt-tolerant mixed-mode adsorbent synthesized in (1), and equilibrate with 0.2-0.4mol / L acetate buffer solution with pH 2-5. The filtrate of HUTI was then passed through the adsorbent at a flow rate of 1-2 CV / h. After loading...

Embodiment 3

[0031] (1) Preparation of salt-tolerant mixed-mode adsorption chromatography medium, the steps are as follows:

[0032] Example 1 (1) was repeated, and the methacrylic acid polymer was replaced by cellulose (Cellufine GCL-2000, produced by CHISSO, Japan) in the same manner to obtain a salt-tolerant mixed-mode adsorption chromatography medium with cellulose as the carrier matrix.

[0033] (2) Prepare HUTI by using salt-tolerant mixed-mode adsorption chromatography medium, the steps are as follows:

[0034] Dissolve 500g of crude HUTI (potency 52IU / mg) in 1500ml of water at a ratio of 1:3, collect the filtrate after filtration, conductance 41ms / cm, and adjust the pH of the filtrate to 2-5 with glacial acetic acid. Fill the chromatographic column with the salt-tolerant mixed-mode adsorbent synthesized in (1), and equilibrate with 0.2-0.4mol / L acetate buffer solution with pH 2-5. The filtrate of HUTI was then passed through the adsorbent at a flow rate of 1-2 CV / h. After loading...

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Abstract

The invention provides a method for purifying human urinary trypsin inhibitor by utilizing a salt resistant mixed mode adsorbent. The method comprises the following steps: 1) dissolving crude human urinary trypsin inhibitor in water to ensure that the conductance is no less than 30ms / cm after filtration, adjusting the pH value of filtrate to 2-5, then using the salt resistant mixed mode adsorbent to selectively adsorb human urinary trypsin inhibitor; 2) using elution buffer solution of which pH value is 7-10 to elute the salt resistant mixed mode adsorbent, collecting the elution peak product of the human urinary trypsin inhibitor; and 3) performing freeze drying to the elution peak product to obtain high purity human urinary trypsin inhibitor. The invention has the following technical effects: the purification method provided by the invention is characterized in that the method has high purification efficiency and fewer technical steps, is easy to amplify, and the like; and the industrial application of the technology is convenient to realize.

Description

technical field [0001] The invention relates to a method for purifying human urinary trypsin inhibitor, in particular to a method for purifying human urinary trypsin inhibitor by using a salt-tolerant mixed-mode adsorbent. Background technique [0002] Human urinary trypsin inhibitor (Human urinary trypsin inhibitor, referred to as HUTI) is a medicinal protein isolated and purified from human urine, which has inhibitory effects on trypsin, plasmin, elastase and other enzymes. In 1909, Beurer et al. first discovered a protein that inhibits trypsin in human urine, and it was named human urinary trypsin inhibitor. Until 1977, scientists successfully isolated and purified the complete HUTI molecule from human urine for the first time. HUTI is a glycoprotein composed of 143 amino acids, with alanine and leucine at the N-terminal and C-terminal, respectively. The molecular weight of HUTI is 37000~43000 (SDS-PAGE electrophoresis method), and the isoelectric point is about 2.5. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C07K1/22C07K1/16
Inventor 杨华英万偲朱碧君高铁旦饶志伟
Owner 江西浩然生物制药有限公司
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