A kind of purification process of cytochrome c

A cytochrome and process technology, applied in the purification process of cytochrome C and the field of cytochrome C purification by cutting-edge hydrophobic chromatography, can solve the problems of low production efficiency, low utilization rate of chromatographic fillers, and many purification steps, and achieve high production efficiency. , the purification effect is good, the effect of reducing the purification cost

Inactive Publication Date: 2011-12-28
NORTHWEST UNIV(CN)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a purification process of cytochrome C, to overcome the deficiencies of the prior art, such as many purification steps, low utilization rate of chromatographic filler, low production efficiency, etc.

Method used

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  • A kind of purification process of cytochrome c
  • A kind of purification process of cytochrome c

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Separation and purification of cytochrome C in porcine heart by frontier hydrophobic chromatography

[0022] (1) Extraction of cytochrome C from pig heart

[0023] Take 1 kg of fresh pig heart, remove the fatty connective tissue, mince it, add 400 mL of water, adjust the pH to 4.0 with 1.0 mol / L sulfuric acid, stir at room temperature for 2 h, filter with gauze to remove the residue, in order to fully extract the residue Re-extract for 1 h under the above conditions. Remove the residue and combine the two extracts, then adjust the pH to 7.0 with 1.0 mol / L ammonia water, let it stand at 4°C, and finally centrifuge, collect the supernatant, and filter to obtain about 760 mL of crude extraction solution. attached figure 1 The middle band b is the SDS-PAGE picture of the cytochrome C crude extract.

[0024] (2) Ammonium sulfate precipitation crude purification of cytochrome C in pig heart

[0025] Add solid ammonium sulfate to the obtained crude extract to ma...

Embodiment 2

[0028] Example 2: Separation and purification of cytochrome C from porcine heart by hydrophobic chromatography in elution mode

[0029] With 3 mol / L (NH 4 ) 2 SO 4 + 50 mmol / L PBS (pH 7.0) to equilibrate a hydrophobic chromatographic column (10 × 0.46 cm I.D.) equipped with PEG600 stationary phase, inject 2 mL of the cytochrome C solution obtained in (2) of Example 1 into the equilibrated In a hydrophobic column, the protein is eluted with a linear gradient such that (NH 4 ) 2 SO 4The concentration was reduced to 0 mol / L within 30 min, and the cytochrome C fraction was collected. The flow rate throughout the chromatographic process was 1.0 mL / min, and the detection wavelength was 280 nm. After testing, the purity of the purified cytochrome C was 99.4%, and only 0.8 mg of high-purity cytochrome C could be obtained in one purification. The yield of purified cytochrome C in this process is only 0.8 mg / (hour.g filler).

Embodiment 3

[0030] Example 3: Purification of Cytochrome C from Porcine Heart by Classical Adsorption Chromatography

[0031] (1) Artificial zeolite column adsorption and elution

[0032] At a flow rate of 1.0 mL / min, 70 mL of the cytochrome C solution obtained in (1) in Example 1 was loaded onto a chromatographic column filled with 5 g of artificial zeolite, so that the artificial zeolite fully adsorbed the cytochrome C extract and became For bright red. Then the artificial zeolite was taken out from the column and washed three times with distilled water, 0.2% NaCl and distilled water, each time for 20 min. Repack the washed artificial zeolite column with 25% (NH4) at a flow rate of 2.0 mL / min 2 SO 4 The solution was eluted, and the red cytochrome C fraction was collected.

[0033] (2) Purification of cytochrome C by precipitation with ammonium sulfate and trichloroacetic acid

[0034] Add a certain amount of solid ammonium sulfate to the cytochrome C fraction collected in (1) so ...

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Abstract

The invention discloses a new process for purifying cytochrome C, which comprises the following steps: (1) extracting cytochrome C coarse extract from animal organs or yeasts; (2) adding an inorganic salt into the coarse extract to perform salting, standing, centrifuging and removing settled proteins, and collecting supernate; (3) adding buffer solution to regulate the inorganic salt concentration in the supernate to 20 to 35 percent, and purifying the supernate by using front-end hydrophobic chromatography, namely continuously loading the obtained solution onto a hydrophobic chromatographic column which is balanced by inorganic salt solution at a concentration of 20 to 35 percent in advance and collecting penetration solution; and (4) washing the chromatographic column by using the inorganic salt solution at a concentration of 20 to 35 percent till a stable light absorbance, collecting the efflux of the chromatographic column, mixing the efflux of the chromatographic column with penetration solution, and purifying to obtain cytochrome C. In the invention, the operation is simple and convenient, the separation and purification effect is good, the yield is high and cost is low.

Description

technical field [0001] The invention relates to a process for purifying cytochrome C, in particular to a process for purifying cytochrome C by frontier hydrophobic chromatography, and belongs to the technical field of biochemical separation. Background technique [0002] Cytochrome (cytochrome) is a protein that is very common in various organisms. It is a type of electron transfer protein containing an iron porphyrin group. It only exists in aerobic cells and widely exists in the mitochondrial inner membrane of eukaryotes. and in the endoplasmic reticulum, in the chloroplasts of plants, and in photosynthetic microorganisms and bacteria. Cytochrome plays the role of transferring electrons on the inner mitochondrial membrane. Cytochrome C (cytochrome c) is a kind of cytochrome, which is located between cytochrome B and cytochrome oxidase in the mitochondrial respiratory chain, and is an important part of the respiratory chain. Each molecule of cytochrome C contains one heme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/80C07K1/20
Inventor 王超展樊花陈湘琴
Owner NORTHWEST UNIV(CN)
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