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Method for separating purified ferritins from biological tissues or engineering bacteria

A biological tissue, separation and purification technology, applied in the field of protein purification, can solve the problems of low yield, high instrument requirements, cumbersome steps, etc., and achieve the effects of low raw material cost, high loading, and fast and simple purification.

Inactive Publication Date: 2011-07-20
INST OF GEOLOGY & GEOPHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The above separation methods all have the disadvantages of cumbersome steps or high requirements for instruments.
Generally speaking, the more separation steps, the greater the loss of the target protein and the lower the yield; and some purification steps require high equipment requirements, for example: ultracentrifugation requires high-speed centrifuges, high-pressure liquid chromatography and other general laboratories are not equipped

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  • Method for separating purified ferritins from biological tissues or engineering bacteria
  • Method for separating purified ferritins from biological tissues or engineering bacteria
  • Method for separating purified ferritins from biological tissues or engineering bacteria

Examples

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Embodiment 1

[0055] 1) Raw materials: buy 1-2 fresh pig spleens in the market, dark red in color, rich in ferritin.

[0056] 2) Pretreatment: Use clean surgical scissors to remove connective tissue such as the white fascia on the ventral surface of the spleen, and then chop it into small pieces. Add deionized water in the ratio of mass 1:1.5-2. Under the protection of low-temperature ice water, use a tissue grinder to grind the spleen tissue into a homogenate at high speed for about 20-30 minutes. Use 10 layers of gauze to filter out unhomogenized tissue pieces, fascia and other connective tissue and adipose tissue to obtain a homogenate.

[0057] 3) Extraction of crude protein: put the homogenate in a constant temperature water bath at 70-75°C, stir and heat-denature it for 20 minutes, and most of the non-heat-resistant protein denatured precipitates can be seen. Quickly place the homogenate in an ice bath at 4°C to room temperature, centrifuge at 10,000g at 4°C for 20-30min, and collec...

Embodiment 2

[0061]The Escherichia coli BL21 strain containing the recombinant human ferritin H submatrix plasmid was fermented and cultured in LB medium, and the cells were collected by centrifugation. The cells were mixed with deionized water, ultrasonically disrupted, the cell lysate was centrifuged at 10,000 g for 30 min at 4°C, and the supernatant was collected. The supernatant was heated and denatured in a constant temperature water bath at 70-75°C for 20 minutes, and most of the non-heat-resistant proteins were denatured and precipitated. The homogenate was quickly cooled to room temperature at 4°C, centrifuged at 10,000g for 20-30min at 4°C, and the supernatant was collected for later use. Then ammonium sulfate was added to the supernatant to precipitate protein at a ratio of 52g / 100ml, and placed in a refrigerator at 4°C for 6-12 hours. After the precipitation is complete, centrifuge at 10,000 g for 30 min at 4°C to collect the precipitate. The precipitate was dissolved with 50 ...

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Abstract

The invention discloses a method for separating purified ferritins from biological tissues or engineering bacteria. The method comprises the following steps of: performing mechanical lapping or ultrasonication by taking ferritin-enriched animal tissues or organs or engineering bacteria which are collected by fermenting and contain recombination ferritin genes as a raw material for extracting ferritins to obtain uniform serum, performing heat denaturation treatment on the filtered uniform serum in water with the temperature between 70 DEG C and 75 DEG C for 20 minutes, and centrifugally extracting supernatant to obtain a crude protein solution; performing dialysis equilibrium on the obtain crude protein solution, performing nickel column affinity chromatography and eluting by using 250 mM of competitive imidazole buffer solution to obtain electrophoretically pure ferritins; and further performing sepHarose 6B molecular sieve exclusion chromatography on the ferritins to obtain monomer ferritins and polymer ferritins which are separated from each other. By adopting the method, electrophoretically pure ferritins can be obtained by directly performing nickel column affinity chromatography in the absence of tag, so that purifying steps of the ferritins are greatly eliminated.

Description

technical field [0001] The invention relates to a technology for separating and purifying ferritin from biological tissue samples or engineering bacteria, and belongs to the technical field of protein purification. Background technique [0002] Iron is one of the essential metal elements for biological organisms and plays an important role in maintaining the normal growth and metabolism of cells. Most of the iron ions in the body are tightly bound to other proteins, and at physiological pH, free Fe 2+ Concentration not more than 10 -8 M, Fe 3+ The concentration is not higher than 10 -18 M. Excessive free iron will react with peroxides in the body to generate active free radicals, which can cause fatal toxicity to cell lipid membranes, DNA chains and other biological macromolecules. Therefore, organisms temporarily store excess iron ions in the body through ferritin for reuse when the body needs iron; at the same time, it can also relieve the toxicity of free iron ions. ...

Claims

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Application Information

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IPC IPC(8): C07K14/79C07K1/36C07K1/30C07K1/22C07K1/16
Inventor 曹长乾田兰香潘永信
Owner INST OF GEOLOGY & GEOPHYSICS CHINESE ACAD OF SCI
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