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Method for separating and purifying recombinant human ferritin

A technology for separation and purification of ferritin, applied in the field of separation and purification of recombinant human ferritin, can solve the problems of low recovery rate, complicated desalting and dilution operations, unfavorable large-scale production, etc., and achieves increased reliability, high purity, and high recovery rate. Easy-to-use effects

Inactive Publication Date: 2019-03-22
PINGHU YOUPU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, some existing chromatography methods have a low recovery rate, each step of chromatography requires desalting and dilution operations, which is cumbersome and unfavorable for large-scale production, and the purity of ferritin cannot reach the level of medical supplies

Method used

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  • Method for separating and purifying recombinant human ferritin
  • Method for separating and purifying recombinant human ferritin
  • Method for separating and purifying recombinant human ferritin

Examples

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Embodiment 1

[0031] Embodiment 1, a method for separating and purifying recombinant human ferritin of the present invention, comprising the following steps:

[0032] 1) Centrifuge 10g of Escherichia coli fermentation liquid to obtain the sludge. Add 20mM PBpH7.0 to the sludge according to the mass volume ratio of 1:10. After stirring well, the high-pressure homogenate is crushed twice under the environment of 800bar, heated to 72°C and kept constant. Heating for 5 minutes, centrifuging at 12000g for 10 minutes, then removing the supernatant, filtering with a 0.22um filter membrane to obtain the filtrate, and performing gel filtration detection and ion chromatography detection on the filtrate;

[0033] 2) Anion chromatography treatment: Take 30ml of anion chromatography medium Q Bestarose High Performance and put it into a BXK16 / 20 chromatography column. The following chromatography processes are all carried out on the AKTA protein purification instrument, and the anion chromatography medium...

Embodiment 2

[0040] Embodiment 2, a method for separating and purifying recombinant human ferritin of the present invention, comprising the following steps:

[0041] 1) Centrifuge 10g of Escherichia coli fermentation liquid to obtain the sludge. Add 20mM PBpH7.0 to the sludge according to the mass volume ratio of 1:10. After stirring well, the high-pressure homogenate is crushed twice under the environment of 800bar, heated to 72°C and kept constant. Heating for 5 minutes, centrifuging at 12000g for 10 minutes, then removing the supernatant, filtering with a 0.22um filter membrane to obtain the filtrate, and performing gel filtration detection and ion chromatography detection on the filtrate;

[0042] 2) Anion chromatography treatment: Take 40ml of anion chromatography medium DEAE Bestarose High Performance and put it into a BXK16 / 20 chromatography column. The following chromatography processes are all carried out on the AKTA protein purification instrument, and the anion chromatography med...

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Abstract

The invention discloses a method for separating and purifying recombinant human ferritin. The method comprises the following steps of: centrifuging fermented Escherichia coli bacterial solution to obtain bacteria sludge, and dissolving the bacterial sludge in buffer solution for heating, centrifugation or filtration to obtain filtrate; separating the filtrate obtained in the step 1) by utilizing anion chromatography, and collecting elution buffer containing target protein; carrying out secondary separation on the elution buffer containing the target protein in the step 2) and collecting the elution buffer containing the target protein; and carrying out third separation on the elution buffer containing the target ferritin in the step 3), and collecting the elution buffer containing the target protein. The method has the advantages of being simple to operate, high in recovery rate of the target protein and high in purity.

Description

【Technical field】 [0001] The invention relates to the technical field of protein separation and purification, in particular to the technical field of a recombinant human ferritin separation and purification method. 【Background technique】 [0002] Ferritin (Ferritin) widely exists in various organisms, composed of 24 subunits spontaneously, and has a cage-like protein complex structure. It is a natural polymer biomaterial. It has the functions of storing iron element and anti-oxidation in the living body, but as a natural polymer material, it can also be widely used as a nanocarrier for drug delivery, medical imaging, etc. At the same time, ferritin can efficiently interact with tumor cells The expressed transferrin receptor is used for the diagnosis and treatment of tumors. Ferritin can be highly expressed in Escherichia coli by transgenic technology, and then the bacterial cells are broken to separate and purify the protein. Most of the traditional separation and purifica...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/36C07K1/34C07K1/20C07K1/18
CPCC07K14/47
Inventor 崔晴晴张洪时鑫倪春杰
Owner PINGHU YOUPU BIOTECH CO LTD
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