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Method for purifying enterokinase

A purification method and enterokinase technology, applied in the field of purified enterokinase, can solve the problems of large product loss, complex purification steps, and high purification difficulty, and achieve the effect of avoiding product loss and cost loss

Inactive Publication Date: 2016-07-27
JIANGSU WANBANG BIOPHARMLS +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention provides a method for purifying enterokinase, aiming at problems such as high difficulty in purification, complicated purification steps, and large product loss in the purification process, which exist in the field of enterokinase purification at present. .5 in the UniPS30-300 chromatographic column obtained by elution of an equal volume of mixed elution buffer A and elution buffer B, the elution buffer A is made of 10% ethanol and 5mmolT-ris-HCl buffer The formed pH is 8.5 buffer solution, elution buffer B is the buffer solution with pH 8.5 formed by 50% ethanol and 5mmol T-ris-HCl buffer

Method used

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  • Method for purifying enterokinase
  • Method for purifying enterokinase

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Embodiment 1

[0012] Obtaining of the enterokinase crude enzyme solution to be separated in embodiment 1

[0013] The mixed solution containing enterokinase was obtained according to relevant content in the prior art.

[0014] After the fermentation, the cells were collected by centrifugation, and the crushing buffer (25mmol / LTris-HCL+5mmol / LEDTA, pH 7.5) was added at a weight-to-volume ratio of 1:10, crushed twice under the condition of a high-pressure homogenizer at 700bar, and collected by centrifugation. The inclusion body precipitated, and the wet weight of the inclusion body was 30g / L fermentation broth. The precipitate was added to the washing buffer (2mol / L urea + 1.5% Triton) at a weight-to-volume ratio of 1:10, stirred magnetically at room temperature for 1 hour, and the precipitate collected by centrifugation was washed twice with the washing buffer. The inclusion body dissolution buffer (8 mol / L urea + 10 mmol / LEDTA + 25 mmol / LTris-HCL, pH 7.5) was used to dissolve overnight at...

Embodiment 2

[0015] The purification of embodiment 2 enterokinase

[0016] Preparation of buffer A: Mix 10% ethanol with 5 mmol T-ris-HCl buffer to form buffer A with a pH of 8.5.

[0017] Preparation of buffer B: Mix 50% ethanol with 5 mmol T-ris-HCl buffer to form a buffer with a pH of 8.5.

[0018] After mixing equal volumes of buffer A and buffer B, an elution buffer is formed.

[0019] Take the UniPS30-300 reversed-phase chromatography filler, soak it in the elution buffer of pH 8.5, and pack the treated filler into a column to form a ready-to-use UniPS30-300 reversed-phase chromatography column.

[0020] The flow rate of the buffer used for elution is: the linear flow rate is 120 cm / h.

[0021] The chromatography equipment is: Saipu High Pressure Chromatography System.

[0022] Chromatograms are obtained as figure 1 shown.

[0023] The eluents at different elution peaks were collected respectively and subjected to electrophoresis analysis. The analysis results were as follows: ...

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Abstract

The invention relates to the fields of pharmaceutical synthesis and chemical industry, and especially relates to a method for purifying enterokinase. The invention aims at the problem which exists in the field of enterokinase purification at present, and provides the method for purifying enterokinase. The method comprises the following steps: a separating medium of enterokinase is obtained by elution with an elution buffer A and an elution buffer B which are mixed with the same volume in a UniPS30-300 chromatography column whose pH value is 8.5, wherein 10% ethanol and a 5mmol T-ris-HCl buffer form the elution buffer A whose pH value is 8.5, and 50% ethanol and a 5mmol T-ris-HCl buffer form the elution buffer B whose pH value is 8.5. Only once purification elution is needed in order to obtain the electrophoretically pure enterokinase sample, and product loss and cost consumption brought by multitime repeat elution are avoided.

Description

technical field [0001] The invention relates to the fields of drug synthesis and chemical industry, in particular to a method for purifying enterokinase. Background technique [0002] Enterokinase exists in the duodenal mucosa of higher animals and is an endo-peptidase that hydrolyzes trypsinogen to become active trypsin. This reaction is dozens of times faster than the activation caused by the trypsin self-catalyst, EC3.4.21.9. It is a sugar-containing protein with a molecular weight of about 100,000. [0003] At present, the research on the application and recombination of enterokinase has gradually been valued by people in the industry. [0004] The purification of active crude enzyme liquid is the last step in the entire preparation field, and it is also a very critical step. How to effectively obtain purified enterokinase is crucial for obtaining high-purity recombinant enterokinase. [0005] At present, in the field of enterokinase purification, it is generally ca...

Claims

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Application Information

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IPC IPC(8): C12N9/64
Inventor 文良柱朱亮赵梅
Owner JIANGSU WANBANG BIOPHARMLS
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