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, poor stability, pollution, etc., and achieve the effect of simple method and low cost

Inactive Publication Date: 2005-08-10
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, take fresh bovine or human duodenal tissue, and extract total RNA according to Qigen kit (Catalog NO.74104) operation manual.

[0017] 2. RT-PCR reaction

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

[0019] Take 3 μl of the above reaction solution as a PCR amplification template. When prokaryotic cells are used as the 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 primer is CTC GAG ATG TAG AAA ACT TTG TAT...

Embodiment 1

[0059] The BL21 engineered bacteria fermentation using enterokinase light chain variants. Taking a 10-liter fermenter as an example (fermentation time lasts 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 intestines are obtained by Zn-Sepharose affinity chromatography Kinase light chain variant protein, after renaturation, the enterokinase activity per liter is 2.19×10 5 u, after STI-Sepharose affinity chromatography, recover enterokinase activity 1.64×10 5 u, 1.64×10 per 10 liters of fermentation broth 6 u Enterokinase activity. Calculated by hydrolyzing 50ug of fusion protein per unit of enterokinase activity, the enterokinase obtained per 10 liters of fermentation broth can degrade 82 grams of fusion protein.

[0060] Using the above method to prepare wild-type enterokinase light chain protein, only 1.18×10 is obtained per 10 liters of fermentation broth...

Embodiment 2

[0062] Fermentation with yeast engineered bacteria using enterokinase light chain variants (fermentation time lasts 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, recover enterokinase activity 1.03×10 6 u, 10 liters of fermentation broth get 1.03×10 7 u Enterokinase activity, calculated by hydrolyzing 50μg of fusion protein per unit of enterokinase activity, enterokinase obtained from 10 liters of fermentation broth can degrade 515 grams of fusion protein.

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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 serine proteolytic enzyme, especially a light chain variant of enterokinase. Background technique [0002] Enterokinase (EK) is one of the most basic serine proteolytic enzymes in the digestive system of mammals. Under physiological conditions, EK anchored on the cell membrane activates trypsinogen to trypsin, which then activates other zymogens in the pancreas to form a series of proteolytic enzyme mixtures. [0003] Enterokinase is composed of two polypeptide chains, a heavy chain and a light chain, which are connected by a pair of interchain disulfide bonds. The Cys in the light chain 112 Participated in the connection with the heavy chain. The light chain of enterokinase has a complete catalytic function, and its recognition sequence is Asp-Asp-Asp-Asp-Lys↓. Enterokinase has a high degree of specificity for its recognition sequence. It is currently widely used to cut the peptide bond between the carrier protein and the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50C12N15/57
Inventor 孙自勇刘建宁
Owner SUZHOU LANDING BIOPHARM CO LTD
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