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Method for purifying recombinant mussel-like mucin

A technique for mussel mucin and a purification method, which is applied in the field of purification of recombinant mussel mucin, and can solve the problems of low purification yield, low content and high endotoxin content of the recombinant mussel mucin

Pending Publication Date: 2022-08-05
西安德诺海思医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to solve the low purification yield of Escherichia coli engineering bacteria to ferment and express recombinant mussel-like mucin, and the obtained recombinant mussel-like mucin does not contain L-3,4 dihydroxyphenylalanine (L-DOPA) Or the content is low, and the high content of endotoxin in the product affects the application

Method used

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  • Method for purifying recombinant mussel-like mucin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1) After the fermentation in the fermenter, the volume of the fermented liquid is 60 L, 12000 g of lactose and 600 mL of Triton X-100 are added to the fermented liquid, and the stirring is continued at 200 rpm for 1 h.

[0039] 2) discharge the feed liquid, centrifuge at 4000rpm for 15min through a high-speed centrifuge, remove the supernatant and collect the precipitation, the precipitation weighs 8kg, and add 80L of the 10mM citric acid-sodium citrate buffer that contains 1mM EDTA to the precipitation and is pH 6.5, Stir overnight (10 h) at 200 rpm.

[0040] 3) The feed liquid was discharged, centrifuged at 4000rpm for 15min through a high-speed centrifuge, removed the supernatant and collected the precipitation, the precipitation weighed 7kg, and added 56L of the 10mM citric acid-sodium citrate buffer with a pH of 3.7 to the precipitation, and stirred at 200rpm. dissolve.

[0041] 4) Centrifuge the feed liquid at 4000rpm for 15min by a high-speed centrifuge, remove ...

Embodiment 2

[0049] 1) After the fermentation in the fermenter, the volume of the fermentation broth was 60.5 L, 10000 g of lactose and 1200 mL of SDS were added to the fermentation broth, and the stirring was continued at 200 rpm for 1 h.

[0050] 2) The feed liquid was discharged, centrifuged at 4000rpm for 15min through a high-speed centrifuge, the supernatant was removed to collect the precipitate, the precipitate weighed 8.4kg, and 96L of 10mM citric acid-sodium citrate buffer containing 1.5mM EDTA with a pH of 6.5 was added to the precipitate. The solution was stirred at 200 rpm overnight (10 h).

[0051] 3) The feed liquid was discharged, centrifuged at 4000rpm for 15min through a high-speed centrifuge, the supernatant was removed to collect the precipitate, the precipitate weighed 7.2kg, and 72L of pH was added to the precipitate. In the 10mM citric acid-sodium citrate buffer, 200rpm Stir to dissolve.

[0052] 4) Centrifuge the feed liquid at 4000rpm for 15min through a high-speed...

Embodiment 3

[0060] 1) After the fermentation in the fermenter, the volume of the fermented liquid is 59 L, 9000 g of lactose and 600 mL of Triton X-100 are added to the fermented liquid, and the stirring is continued at 200 rpm for 1 h.

[0061] 2) The feed liquid was discharged, centrifuged at 4000rpm for 15min through a high-speed centrifuge, the supernatant was removed to collect the precipitate, the precipitate weighed 8.1kg, and 90L of 10mM citric acid-sodium citrate buffer containing 1mM EDTA at pH 6.5 was added to the precipitate. , stirred at 200 rpm overnight (10 h).

[0062] 3) The feed liquid was discharged, centrifuged at 4000rpm for 15min through a high-speed centrifuge, removed the supernatant and collected the precipitation, the precipitation weighed 6.8kg, and added 36L of the 10mM citric acid-sodium citrate buffer with a pH of 3.7 to the precipitation. Stir to dissolve.

[0063] 4) The feed liquid was centrifuged at 4000rpm for 15min through a high-speed centrifuge, and ...

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Abstract

The invention discloses a method for purifying recombinant mussel-like mucoprotein, and relates to the technical field of protein purification methods. According to the method for purifying the recombinant mussel-like mucin, the recombinant mussel-like mucin fermentation liquor which is subjected to fermentation induced expression of escherichia coli engineering bacteria is adopted as a raw material for the first time, and the method of removing impurities through cell disruption, enzyme catalytic oxidation, chromatographic purification and endotoxin removal through chromatography is adopted, so that the recombinant mussel-like mucin which the yield of each liter of fermentation liquor pure product can reach 0.7 g / L, the purity is greater than 95%, and L-3, 5-dihydroxy-2, 5-dihydroxy-2, 5-dihydroxy-2, 5-dihydroxy-2, 5-dihydroxy-2, 5-dihydroxy-2, 5- The method for purifying the recombinant mussel-like mucoprotein (Mlfp-151 with theoretical molecular weight of 22.61 KD) with the content of 2, 4-dihydroxyphenylalanine (L-DOPA) of more than 0.7% and the content of endotoxin of less than 20 EU / mg is simple in purification process and suitable for industrial production.

Description

technical field [0001] The present invention relates to the technical field of recombinant mussel-like mucin purification methods, in particular to a method for separating and purifying Escherichia coli to induce expression of recombinant mussel-like mucin Mlfp-151. Background technique [0002] Mussel adhesive protein (MAP) is a protein complex secreted by the secretory glands of the mussel foot. It has been vigorously promoted and applied in biomedicine. [0003] Mussel mucins have L-3,4-dihydroxyphenylalanine (L-DOPA), which is formed by the action of tyrosinase on tyrosine residues. The L-DOPA residues were cross-linked to each other due to an oxidation reaction, which resulted in strong and durable glues within the mussels and special surface fibers. Mussel mucin adhesion has no toxicity and immunogenicity to humans, and its binding ability and lifespan are not affected by water. It has a very broad application prospect in medicine. [0004] In the prior art, the acq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12P21/06C07K1/30C07K1/18C07K1/36C07K1/34
CPCC07K14/43509C12P21/06Y02A50/30
Inventor 郝东赵硕文魏文培乔莎周浩张立峰侯增淼
Owner 西安德诺海思医疗科技有限公司
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