Method for rapidly purifying cytochrome b6f

A cytochrome and rapid technology, applied in the preparation methods of cytochromes and peptides, chemical instruments and methods, etc., can solve the problems of long time, low yield, difficult extraction, etc., and achieve long time consumption, fast growth, and ensure protein activity Effect

Inactive Publication Date: 2021-08-03
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] For the current plant cytochrome b 6 F extraction is difficult, yield is low, time-consuming long problem, the purpose of the pres...

Method used

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  • Method for rapidly purifying cytochrome b6f
  • Method for rapidly purifying cytochrome b6f
  • Method for rapidly purifying cytochrome b6f

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Cultivation of strains: inoculate the cyanobacteria Anabaena sp.PCC 7120 in the culture medium with a 5% inoculum, at a temperature of 25°C and a light intensity of 30μE / m 2 / S, CO 2 The aerated culture was carried out under the condition of 1% aeration, and the bacterial liquid was collected after about 7 days.

[0040] Collect broken bacteria: Centrifuge at 4°C in a high-speed centrifuge at 8000 rpm for 5 minutes to enrich the bacteria. Resuspend 1 g of bacteria: 8 mL of buffer A, and add protease inhibitors (PMSF (phenylmethylsulfonyl fluoride), benzamidine, and 6-aminocaproic acid at a final concentration of 0.1 mM) to the resuspension. It was crushed 5 times in a high-pressure homogenizer under 1000 bar pressure at 4°C in the dark. The broken bacterial liquid was centrifuged at 4°C in a high-speed centrifuge at a speed of 6150 rpm for 10 minutes to remove unbroken cells and cell debris (keep at 4°C and avoid light for subsequent experiments);

[0041] Thylakoid ...

Embodiment 2

[0048] Example 2: Purified Cytochrome b 6 fPurity identification

[0049] Purified cytochrome b 6 f Tested by 15% SDS-PAGE electrophoresis, there is obvious cytochrome b 6 f Four large subunit bands, the band sizes are cyt f 32kDa, cyt b 6 25kDa, Rieske ISP 19kDa and Subunit Ⅳ 17kDa (see figure 2 ).

[0050] The purified protein was observed by transmission electron microscope, and cytochrome b of uniform size can be seen 6 f protein particles, the particle size is about 10nm, consistent with cytochrome b 6 The f complex has a total size of 220 kDa (see image 3 ).

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Abstract

The invention discloses a method for rapidly purifying cytochrome b6f, wherein the method comprises the steps: by taking cyanobacteria as a raw material, breaking cells to obtain a thylakoid membrane, washing to remove impure protein, adding a detergent undecyl maltoside or dodecyl-beta-D-maltoside, and obtaining a crude product of cytochrome b6f by adopting a three-step ammonium sulfate precipitation method; and performing sucrose density gradient centrifugation and anion exchange column treatment to obtain the cytochrome b6f finished product. Compared with the prior art, the method has the advantages that the cytochrome b6f is purified by taking cyanobacteria which is high in growth speed and simple to culture as a raw material, the extraction rate is high, the operation steps are few, the consumed time is short, and the obtained cytochrome b6f is high in purity and good in uniformity.

Description

technical field [0001] The invention relates to a method for extracting cytochrome, in particular to a high-purity and rapid extraction of cyanobacteria cytb 6 method of f. Background technique [0002] Cytochrome is a kind of protein with iron porphyrin ring as the prosthetic group. It widely exists in the mitochondria of eukaryotes, chloroplasts of plants, photosynthetic microorganisms and bacteria, and plays an extremely important role in the process of cellular respiration and energy transfer. , is a highly efficient biological electron transporter. A variety of cytochromes can be used as drugs to increase cell oxidation and improve oxygen utilization. Currently commercially available cytochromes mainly include cytochrome c and cytochrome P450, all of which are extracted from animals, such as bovine, horse, and pigeon hearts. However, the preparation of plant-derived cytochromes is difficult, and no commercial products have been seen. [0003] cytochrome b 6 f compl...

Claims

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Application Information

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IPC IPC(8): C07K14/80C07K1/18C07K1/30C07K1/14
CPCC07K14/80
Inventor 李西莹张坤韦佩君董春霞赵进东
Owner PEKING UNIV
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