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A method for purifying fusion protein of tumor blood vessel blocking agent

A tumor blood vessel and fusion protein technology, which is applied in the field of protein purification, can solve the problems of affecting protein renaturation efficiency, easy loss of target protein, protein aggregation, etc., and achieves excellent purification effect, simple purification method, and increased ionic strength.

Active Publication Date: 2021-05-14
BEIJING HUAAN INNOVATION BIOTECH
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages are: due to repeated washing, it takes a long time, and the target protein is often easily lost, resulting in low recovery rate and even low purity.
The former promotes the correct folding of the protein, while the latter causes the protein to aggregate without activity. The two compete with each other and affect the protein refolding efficiency.

Method used

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  • A method for purifying fusion protein of tumor blood vessel blocking agent
  • A method for purifying fusion protein of tumor blood vessel blocking agent

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Embodiment 1

[0053] 1. Bacteria destruction: the fermentation broth obtained by engineering strains was fermented, and the lysate was added to the E. coli cells obtained after centrifugation at 4°C and 8000g for 30 minutes. After resuspension and magnetic stirring to dissolve, the high-pressure homogenizer was crushed three times to obtain The crushed solution was centrifuged at high speed again, the supernatant was discarded, and the precipitate was collected.

[0054] 2. Washing: The precipitate collected after crushing and centrifugation was washed in three steps to obtain crude inclusion bodies.

[0055] Washing 1: Break up the precipitate collected after centrifugation, resuspend in washing solution A to completely redissolve, and stir magnetically at 4°C for 2 hours. Then centrifuge at 4°C and 9000g for 20min to collect the precipitate.

[0056] Washing 2: The precipitate obtained in washing 1 was resuspended in washing solution B to completely redissolve it, and then magnetically s...

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Abstract

The invention provides a method for purifying a fusion protein of a tumor blood vessel blocking agent. The method of the present invention is to crush the fermented cells of the fusion protein of a tumor blood vessel blocking agent expressed in Escherichia coli and in the form of inclusion bodies through a high-pressure homogenizer, Centrifuge at high speed to obtain crude inclusion bodies. Wash with different washing solutions to obtain relatively clean inclusion bodies, and then denature and dissolve the inclusion bodies with 8M urea, centrifuge to get the supernatant, and then renature with dialysis. After renaturation, the samples undergo ion exchange and molecular sieve column purification, and the purity of the target protein obtained by SDS-PAGE electrophoresis and RP-HPLC can reach more than 95%. The purification method provided by the invention is simple, low in cost, easy to operate and easy to industrialized production.

Description

technical field [0001] The invention relates to the technical field of protein purification, in particular to a method for purifying a tumor blood vessel blocking agent fusion protein expressed from Escherichia coli in the form of inclusion bodies. Background technique [0002] Under certain growth conditions, Escherichia coli can accumulate certain special biomacromolecules in host cells expressing foreign genes, which are densely gathered in the cells, or are wrapped by membranes or form naked structures without membranes, thus forming inclusion bodies. This expression method has the advantages of low cost, high yield, not easy to be degraded, easy to purify, etc., and has become the main expression method of exogenous proteins today. The prokaryotic expression system represented by Escherichia coli strains can obtain a large amount of expressed protein at low cost in a short period of time. The expression vector has a strong promoter and can be expressed efficiently under...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/14A61K38/17A61K47/64A61P35/00
CPCA61K38/00C07K14/4705C07K2319/33
Inventor 史权威潘向辉李延虎杨子义
Owner BEIJING HUAAN INNOVATION BIOTECH
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