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Enterpeptidase light chain variant with high activity and high stability

A high-stability, enterokinase technology, applied in the field of serine proteolytic enzymes, can solve the problems of decreased enzyme activity, decreased enzyme activity, pollution, etc., and achieves the effects of low cost and simple method

Inactive Publication Date: 2004-01-28
SUZHOU LANDING BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the currently commercially available enterokinase is very expensive, and the enterokinase isolated and purified from tissues often has other protease pollution, while the wild-type enterokinase light chain (EKLC) prepared by genetic engineering has poor stability and can be used for a long time. Preservation is prone to aggregates or precipitation, resulting in a decrease in enzyme activity
We believe that the free Cys in the enterokinase light chain molecule 112 Able to cause intermolecular polymerization of the light chain of enterokinase, resulting in a decrease in enzyme activity

Method used

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  • Enterpeptidase light chain variant with high activity and high stability
  • Enterpeptidase light chain variant with high activity and high stability
  • Enterpeptidase light chain variant with high activity and high stability

Examples

Experimental program
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Effect test

preparation example Construction

[0016] 1. Preparation of duodenal total RNA, fresh bovine or human duodenal tissue was taken, and total RNA was extracted according to the operation manual of Qigen kit (Catalog NO.74104).

[0017] 2. RT-PCR reaction

[0018] The RT-PCR kit was purchased from Invitrogen Corporation (Catalog No. L1310-01). The experimental process refers to the operation manual. Take 3 μg of total RNA, add water to a final volume of 11.5 μl, then add 1 μl thymine oligonucleotide primer, mix well, incubate at 65°C for 10 minutes, room temperature for 2 minutes, then add 1 μl RNase inhibitor, 4 μl 5x reaction buffer, 1 μl 100mM dNTP, 1μl 80mM sodium pyrophosphate, 0.5μl AMV reverse transcriptase, mix well and set it at 42°C for 60min.

[0019] Take 3 μl of the above reaction solution as a template for PCR amplification. When prokaryotic cells are used as expression host bacteria, the 5' end primers for PCR amplification are CAT ATG GAC GAC GAT GAC AAG ATT GTC GGA GGA AGT GAC TCC , the 3′ end...

Embodiment 1

[0058] Fermentation with BL21 engineered bacteria of enterokinase light chain variant. Taking a 10-liter fermenter as an example (fermentation lasts for 2 days), 27.8 grams of bacteria can be obtained per liter of fermentation broth, containing about 578 mg of enterokinase light chain variant protein, and 415 mg of denatured enterokinase protein is obtained by Zn-Sepharose affinity chromatography. Kinase light chain variant protein, enterokinase activity 2.19×10 per liter after renaturation treatment 5 u, after STI-Sepharose affinity chromatography, the recovery of enterokinase activity was 1.64×10 5 u, get 1.64×10 per 10 liters of fermentation broth 6 uEnterokinase activity. Calculated by hydrolyzing 50ug of the fusion protein per unit of enterokinase activity, the enterokinase obtained per 10 liters of fermentation broth can degrade 82 grams of the fusion protein.

[0059] Using the above method to prepare wild-type enterokinase light chain protein, only 1.18×10 per 10 li...

Embodiment 2

[0061] Fermentation with yeast engineered strains of enterokinase light chain variants (fermentation time lasted 96 hours). Taking a 10-liter fermenter as an example, the enterokinase activity contained in the supernatant of each liter of fermentation broth is 1.45×10 6 u, after Zn-Sepharose affinity chromatography, the recovery of enterokinase activity was 1.03×10 6 u, 10 liters of fermentation broth obtained 1.03×10 7 uEnterokinase activity, calculated by hydrolyzing 50 μg of fusion protein per unit of enterokinase activity, the enterokinase obtained from 10 liters of fermentation broth can degrade 515 grams of fusion protein.

[0062] References: Grant, D.A.W. & Hermon-Taylar, J. Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum. Biochim. Biophys. Acta, (1979); 567, 207-215.

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Abstract

The present invention is characterized by that making the cysteine of 112th site of enterokinase light chain gene produce site-specific mutation, using colibacillus and microzyme to express enterokinase light chain variant, and utilizing Zn-Sepharose and STI-Sepharose affinity chromatography so as to obtain high-purity enterokinase light chain variant protein. As compared with wild enterokinase light chain said varient has higher stability and enzyme activity.

Description

technical field [0001] The present invention relates to a light chain variant of a serine proteolytic enzyme, in particular enterokinase. Background technique [0002] Enterokinase (Enterokinase, EK) is one of the most basic serine proteolytic enzymes in the mammalian digestive system. Under physiological conditions, EK anchored on the cell membrane activates trypsinogen to trypsin, which in turn activates other zymogens in the pancreas to form a series of proteolytic enzyme mixtures. [0003] Enterokinase consists of two polypeptide chains, a heavy chain and a light chain, connected by a pair of interchain disulfide bonds, in which Cys in the light chain 112 Involved in attachment to the heavy chain. The light chain of enterokinase has a complete catalytic function, and its recognition sequence is Enterokinase has a high degree of specificity for its recognition sequence, and is currently widely used to cleave the peptide bond between the carrier protein and the target ...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N15/57
Inventor 孙自勇刘建宁
Owner SUZHOU LANDING BIOPHARM CO LTD
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