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Toxin protein purifying method

A technology of toxin protein and purification method, which is applied in the field of purification of toxin protein, can solve the problems of low protein purity, time-consuming, poor activity, etc., and achieve the effect of good activity, short cycle and low cost

Active Publication Date: 2017-07-25
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] The purpose of the present invention is to overcome the problems of low protein purity, poor activity, and long time-consuming when separating and purifying existing toxin proteins, and to provide a purification method of toxin proteins that is efficient and can maintain protein activity

Method used

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Embodiment 1

[0054] Take the copGA-copGT toxin-antitoxin system in Pseudoalteromonas as an example:

[0055] The toxin protein CopGT in the copGA-copGT toxin-antitoxin system was found through NCBI and multiple sequence alignment. It belongs to the ParE family protein, and its activity is a DNA gyrase inhibitor.

[0056] 1. Preparation of whole cell lysate of pseudoalteromonas SCSIO 6842:

[0057] (1) Strain SCSIO 6842 stored at -80°C, streaked on a 2216E plate, and placed in a 25°C incubator overnight;

[0058] (2) Pick a single colony and place it in a 250ml Erlenmeyer flask containing 25ml of fresh 2216E medium, in an incubator at 25°C, and shake at 220rpm for 12 hours;

[0059] (3) Transfer to a 1000ml Erlenmeyer flask containing 300ml fresh 2216E medium at a ratio of 1:100, and culture with shaking at 220rpm for 6 hours (to the logarithmic phase);

[0060] (4) Centrifuge at 12,000 rpm for 10 minutes at low temperature, collect the bacteria, and remove the supernatant;

[0061] (5) ...

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Abstract

The invention discloses a toxin protein purifying method. The method comprises the steps of 1, breaking thalli of pseudoalteromonas SCSIO 6842 containing protease capable of specifically degrading antitoxin protein, so as to obtain whole cell lysate; 2, conducting toxin gene and antitoxin gene amplification, and conducting cloning into an expression vector to induce the expression of toxin genes and antitoxin genes, so as to obtain a toxin protein and antitoxin protein composition; 3, purifying the toxin protein and antitoxin protein composition; and 4, mixing the whole cell lysate obtained in step 1 with the composition obtained in step 3, so as to separate out toxin protein, and conducting purification and desalination to obtain toxin protein. The purifying method has the advantages that protein activity is high, operation is easy and cost is low, and has broad application prospects. The invention further discloses pseudoalteromonas SCSIO 6842, preserved in the China General Microbiological Culture Collection Center in Beijing, China on Feb 23, 2016, with the preservation number of CGMCC 1.15540.

Description

technical field [0001] The invention belongs to the field of protein extraction and purification, and more specifically relates to a high-efficiency purification method of toxin protein capable of maintaining protein activity. Background technique [0002] The toxin-antitoxin system widely exists in the genomes of environmental microorganisms and pathogenic microorganisms, and is also found in intestinal probiotics, and the frequency of the toxin-antitoxin system in mobile genetic elements is higher than other positions in the genome, which has attracted international attention. attention of the microbiology community. The toxin-antitoxin system consists of a stable toxin protein and an unstable antitoxin. The toxin-antitoxin system was first discovered to exist on low-copy plasmids and played a key role in maintaining the stability of the plasmids. At present, experimental results have confirmed that the existence of pairs can effectively regulate the stability of the plas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/06C07K14/195C07K1/36C07K1/22C07K1/14C12R1/01
CPCC07K14/195C12N1/06C12N1/205C12R2001/01
Inventor 王晓雪李百元
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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