CN113201067A - Rapid purification cytochrome b6Method of f - Google Patents

Abstract

The invention discloses a method for quickly purifying cytochrome b₆The method of f, using cyanobacteria as raw material, breaking cell to obtain thylakoid membrane, washing to remove impurity protein, adding detergent undecyl maltoside or dodecyl-beta-D-maltoside, adopting three-step ammonium sulfate precipitation method to obtain cytochrome b₆f, obtaining cytochrome b by sucrose density gradient centrifugation and anion exchange column₆f, finishing the product. Compared with the prior art, the method takes cyanobacteria with high growth speed and simple culture as raw materials to purify cytochrome b₆f, high extraction rate, few operation steps, short time consumption and the obtained cytochrome b₆High purity and good homogeneity.

Description

Rapid purification cytochrome b6Method of f

Technical Field

The invention relates to a method for extracting cytochrome, in particular to a method for quickly extracting cyanobacteria cyt b with high purity₆f, the process of (a).

Background

The cytochrome is a protein taking a ferriporphyrin ring as a prosthetic group, is widely present in mitochondria of eukaryotes, chloroplasts of plants, photosynthetic microorganisms and bacteria, plays an extremely important role in the processes of cell respiration and energy transfer, and is a high-efficiency biological electron transporter. A plurality of cytochromes can be used as medicines to increase cell oxidation and improve oxygen utilization. The cytochrome mainly comprises cytochrome c and cytochrome P450 which are commercially available at present, and are extracted from animals such as hearts of cattle, horses and pigeons. However, the preparation of plant-derived cytochromes is difficult, and no commercial product is available.

Cytochrome b₆f complex (Cytochrome b)₆f,cyt b₆f) Is one of the main complexes of the photosynthetic electron transport chain, mediates the transfer of electrons from photosystem II to photosystem I, and facilitates the formation of transmembrane proton gradients on both sides of the thylakoid membrane. Cytochrome b₆f regulation of the relevant bonds in important processes such as plant state transition and respiration, and also participates in respiration of plants. Obtaining high purity cytochrome b₆f is a necessary condition for researching photosynthesis and respiration of plant cells in vitro. Photosynthesis provides a material source and an energy source for almost all organisms, and in recent years, researches on plant photosynthesis are very hot, and promote the development of agricultural economy and human society. Therefore, a commercial high-purity cytochrome b was developed₆f has great commercial value and scientific research value and is beneficial to solving the food problem in China.

Cytochrome b in plants at present₆The extraction of f is mostly concentrated in spinach and chlamydomonas, the required biomass is large, the extraction method is complicated, and the time and the labor are wasted.

Disclosure of Invention

Aiming at the current plant cytochrome b₆f difficult extraction, low yield and long time consumption, and the invention aims to provide a fast high-purity cyanobacterium b₆f, the method for extracting, so as to reduce the production cost and save the production time.

With spinach andextraction of cytochrome b from cyanobacteria as compared with Chlamydomonas₆f has the following advantages: firstly, the growth speed of cyanobacteria is high, the growth period is short, the culture mode is simple, and the cyanobacteria culture method is suitable for large-scale culture; secondly, the cyanobacteria is taken as a precursor of chloroplast, and the cells contain a large amount of thylakoid membranes after being crushed, so that the step of preparing the thylakoid membranes in the first step of extraction in spinach and chlamydomonas is saved. Therefore, a large amount of cytochrome b is extracted by cyanobacteria₆f has wide development and utilization prospect and is suitable for research and application.

In order to achieve the purpose, the invention uses cyanobacteria (such as Anabaena sp.PCC 7120 which is a freshwater blue algae) as a raw material, changes the types of detergents and protein purification columns by improving the precipitation times of ammonium sulfate, and obtains the high-purity cytochrome b by a simple and rapid method₆f。

Specifically, the invention can rapidly purify cytochrome b₆The method of f comprises the following steps:

(1) collecting broken bacteria: collecting cyanobacteria, crushing at 4 ℃, centrifuging and then retaining supernatant;

(2) preparing a thylakoid membrane: centrifuging the supernatant at high speed to obtain a thylakoid membrane, and washing with a high-salt solution to remove impurity proteins;

(3) extraction of cytochrome b₆f: adding detergent undecyl maltoside (UDM) or Dodecyl-beta-D-maltoside (DDM) into thylakoid membrane, and precipitating with ammonium sulfate to obtain cytochrome b₆f, crude product;

(4) purification of cytochrome b₆f: cytochrome b₆f, obtaining cytochrome b from the crude product by sucrose density gradient centrifugation and anion exchange column₆f, finishing the product.

In the step (1), the cyanobacteria is preferably Anabaena, Nostoc, or the like, for example, Anabaena sp.pcc 7120, Anabaena Variabilis ATCC 29413.

As a preferred scheme, the step (1) is to enrich the thallus by centrifuging the cyanobacteria liquid, then to re-suspend the thallus by using a buffer solution, to add a protease inhibitor, and to crush the thallus at 4 ℃ under the condition of keeping out of the sun; and centrifuging the crushed bacteria liquid to remove the uncrushed cells and cell fragments, and keeping the supernatant. Wherein the protease inhibitors added are preferably PMSF (phenylmethylsulfonyl fluoride), benzamidine and 6-aminocaproic acid, all at a final concentration of 0.1 mM.

And (3) in the step (2), centrifuging the supernatant at a high speed to obtain a coarse thylakoid membrane precipitate, re-suspending the coarse thylakoid membrane precipitate by using a high-salt buffer solution, stirring, and centrifuging at a high speed again to obtain the thylakoid membrane precipitate with the foreign proteins washed.

As a preferable scheme, the rotation speed of each high-speed centrifugation in the step (2) is 80000-100000g, and the time is 40-60 min. Re-suspending the thylakoid membrane coarse precipitate by using a high-salt buffer solution containing 2M NaBr, adjusting the chlorophyll concentration to be 1-1.5 mg/mL, stirring for a period of time, and adding ddH according to the volume ratio of 1:1₂And O, performing high-speed centrifugation to obtain the thylakoid membrane precipitate washed with the foreign protein.

In the step (3), the thylakoid membrane precipitate is resuspended to the chlorophyll concentration of 1.5-2 mg/mL by using a buffer solution containing a protease inhibitor, UDM or DDM is slowly added to the final concentration of 8-10 mM while stirring, and ammonium sulfate is slowly added to the mixture after the mixture is stirred for a period of time for precipitation.

As a preferable mode, the three-step ammonium sulfate precipitation method in the step (3) comprises: firstly, slowly adding ammonium sulfate to a final concentration of 30-35% (g/mL, namely 35g of ammonium sulfate is contained in 100mL of solution, the same is applied below), stirring, centrifuging for 10-30 min at a rotation speed of 180000-210000g, and taking a supernatant; then slowly adding ammonium sulfate to a final concentration of 40-45%, stirring, centrifuging for 10-30 min at a rotation speed of 180000-210000g, and taking a supernatant; slowly adding ammonium sulfate again to a final concentration of 55-60%, stirring, centrifuging at a rotation speed of 180000-210000g for 10-30 min, and precipitating to obtain cytochrome b₆f, crude product.

In the above step (4), cytochrome b is added₆And f, dialyzing the crude product to remove ammonium sulfate salt, and then performing sucrose density gradient centrifugation, so that the yield can be improved.

As a preferable mode, cytochrome b is added in step (4)₆After the crude product is resuspended by buffer solution, dialyzed overnight by a 100kDa dialysis bag, and the solution is changed one or more times midwayNext, the dialyzate was a buffer supplemented with 0.1mM PMSF, 0.2mM UDM or DDM, and 25mM sucrose. The buffer is preferably TNE buffer (50mM Tris-HCl, pH 7.5, 50mM NaCl, 1mM EDTA). After dialysis the samples were subjected to sucrose density gradient centrifugation and in one embodiment of the invention a continuous sucrose gradient of 5% -30% (m/v, in g/mL) was prepared with TNE buffer plus sucrose and 1mM UDM or DDM final concentration. The rotation speed of the density gradient centrifugation of the sample cane sugar after dialysis is 32000 and 36000rpm, and the centrifugation time is 14-16 hr. The orange cytochrome b was then aspirated in a sucrose gradient₆The f bands are put on an anion exchange column for ion exchange chromatography, and high-purity cytochrome b is obtained by elution₆f. The anion exchange column is preferably a Q-sepharose column.

The invention can purify cytochrome b quickly₆Compared with the prior art, the method of f has the following advantages:

1. saving the culture time of the strains: the protein with the tag is directly purified without being expressed into a strain and then purified, so that the conversion time is saved, and more natural cytochrome b is obtained₆f. In addition, the growth speed of the cyanobacteria is high, the culture is simple, and the method is suitable for large-scale protein extraction.

2. The extraction speed is high: extracting cytochrome b from spinach₆Compared with the prior art, a large number of thylakoid membranes can be obtained by directly breaking cells in cyanobacteria, and a special method is not needed, so that the method is an ideal source for obtaining stable photosynthetic membrane protein. In the extraction process, the invention does not remove the foreign protein by cleaning the thylakoid membrane for many times, but only cleans the foreign protein by high salt once, thereby shortening the extraction time. In the extraction process, the detergent extraction and the first-step ammonium sulfate precipitation are synchronously performed, so that the extraction time is shortened under the condition of not influencing the extraction effect. In the purification process, the invention adopts an anion exchange column, and compared with a hydroxyapatite-cellulose column which is commonly used for extracting plants, the anion exchange column has a commercial finished product (such as a Q-sepharose column), thereby ensuring the extraction purity. Hydroxyapatite chromatography columns do not have a pre-packed column and require personnel to fill the column. The column filling method is complicated, the column bed is easy to collapse, only can bear low flow rate, the consumed time is long, and the requirement on personnel is high.

3. Saving the detergent: in the prior art, a plurality of detergents are commonly adopted to extract cytochrome b together₆f, not only has great damage to the activity of the protein, but also causes economic waste. The invention adopts mild nonionic detergent, which can ensure protein activity more mildly and to the maximum extent. And the use of a nonionic detergent does not interfere with the purification process of the subsequent ion exchange column chromatography.

4. The yield and purity are high: 5mg b can be obtained by 5g dry weight of algae by the method of the invention₆f protein. The invention improves the ammonium sulfate precipitation step, and changes the commonly adopted one-step ammonium sulfate precipitation into three steps. The strength of precipitating the foreign protein by ammonium sulfate in one step is small, the separation difficulty in the subsequent steps is increased, and the yield of the final product is influenced. The times of precipitating the hybrid protein are increased, and the hybrid protein can be removed to the maximum extent. The purification effect of the molecular sieve can be achieved by utilizing sucrose density gradient centrifugation and anion exchange chromatography in the purification process.

Compared with the prior art, the cytochrome b in the cyanobacteria of the invention₆The preparation method has high extraction rate, saves extraction steps, shortens extraction time, and obtains cytochrome b₆High purity and good homogeneity.

Drawings

FIG. 1 shows crude cytochrome b of example 1₆f banding pattern after sucrose density gradient centrifugation.

FIG. 2 is a graph showing the identification of purified cytochrome b in example 2₆And (f) SDS-PAGE protein electrophoresis pattern.

FIG. 3 is a graph showing the identification of purified cytochrome b in example 2₆f transmission electron micrograph of sample.

Detailed Description

The process according to the invention is further illustrated by the following examples, without restricting the scope of protection of the invention in any way.

The strain used in this example was the cyanobacterium Anabaena sp.PCC 7120, purchased from ATCC (American Type Culture Collection) strain Bank, accession number 27893. The culture medium used was Bg11 liquid medium: 1.5g of sodium nitrate and 0.04g of dipotassium hydrogen phosphate0.075g of magnesium sulfate 7H₂O, 0.036g CaClo7H₂O, 0.02g of sodium carbonate, 0.006g of citric acid, 0.006g of ferric citrate, 1ml of trace element solution A5 and distilled water to 1 liter. Wherein the trace element solution a5 comprises: 2.86g of boric acid, 1.81g of Mn4H chloride₂O, 0.222g zinc sulfate, 0.39g sodium molybdate, 0.079g bluestone 5H₂O, 49.4g Cobaltosulfate.6H₂O, adding distilled water to 1 liter.

The formula of the buffer solution (buffer) related by the invention is as follows:

and (3) buffer solution A: 50mM Tris-HCl, pH 8.0, 10mM NaCl, 0.4M sucrose;

and (3) buffer solution B: 10mM Tricine-NaOH pH 8.0, 2M NaBr, 0.4M sucrose;

and (3) buffer C: 30mM Tris-HCl, pH 7.5, 50mM NaCl, 1mM EDTA, 0.3M sucrose;

TNE buffer solution: 50mM Tris-HCl, pH 7.5, 50mM NaCl, 1mM EDTA;

ion exchange column elution buffer: 50mM Tris-HCl, pH 7.5, 0/500mM NaCl.

Example 1:

culturing the strain: inoculating cyanobacteria Anabaena sp.PCC 7120 into the culture solution with the inoculation amount of 5 percent, and controlling the temperature at 25 ℃ and the light intensity at 30 mu E/m²/S，CO₂Aeration culture was carried out at an aeration rate of 1%, and the bacterial suspension was collected after about 7 days.

Collecting broken bacteria: centrifuging at 4 deg.C with a high speed centrifuge at 8000rpm for 5min to enrich thallus. 1g of the cells were resuspended in 8mL of buffer A, and protease inhibitors (PMSF (phenylmethylsulfonyl fluoride), benzamidine and 6-aminocaproic acid, all at a final concentration of 0.1 mM) were added to the resuspended solution. Crushing 5 times in a high pressure homogenizer at 1000bar pressure and in the absence of light at 4 ℃. Centrifuging the crushed bacterial liquid at 4 ℃ by using a high-speed centrifuge at the rotating speed of 6150rpm for 10min, and removing uncrushed cells and cell fragments (the subsequent experiments are kept at 4 ℃ and protected from light);

preparing a thylakoid membrane: centrifuging the supernatant with an ultracentrifuge SW70Ti rotor at 90000g for 45min to obtain crude thylakoid membrane. Resuspending the thylakoid membrane precipitate with buffer B, adjusting chlorophyll concentration to 1mg/mL, addingPut on a magnetic stirrer to be stirred for 30 min. After 30min, ddH is added according to the volume ratio of 1:1₂And after O, centrifuging by using an ultracentrifuge SW70Ti rotor at the rotating speed of 90000g for 45min to obtain the thylakoid membrane sediment for washing the foreign proteins.

Extraction of cytochrome b₆f: the thylakoid membrane precipitate was resuspended to a chlorophyll concentration of 2mg/mL using protease inhibitor-added buffer C, placed on a magnetic stirrer with the addition of a rotor, and UDM was added slowly to a final concentration of 10 mM. Stirring for 10min, slowly adding ammonium sulfate with final concentration of 35%, stirring for 10min, centrifuging with ultracentrifuge SW70Ti rotor at 200000g for 10 min. Centrifuging, taking the supernatant, and performing ammonium sulfate precipitation in the second step. Slowly adding ammonium sulfate with final concentration of 45%, stirring for 10min, centrifuging with ultracentrifuge SW70Ti rotor at 200000g for 10 min. Centrifuging, taking the supernatant, and performing ammonium sulfate precipitation in the third step. Slowly adding ammonium sulfate with final concentration of 55%, stirring for 10min, centrifuging with ultracentrifuge SW70Ti rotor at 200000g for 30min to obtain crude cytochrome b₆f。

Purification of cytochrome b₆f: crude cytochrome b₆The precipitate was resuspended in about 1mL TNE buffer, dialyzed overnight against a 100kDa dialysis bag and replaced once in the middle, the dialysis solution being TNE buffer plus 0.1mM PMSF, 0.01% UDM (0.2mM) and 25mM sucrose. After dialysis, the samples were centrifuged through a sucrose density gradient and a continuous sucrose gradient from 5% (m/v) to 30% (m/v) was prepared by adding sucrose to the TNE buffer and 1mM UDM as the final concentration. After the addition of the sample, the mixture was centrifuged in an ultracentrifuge SW 41Ti rotor at 35000rpm for 16hr (see FIG. 1). After equilibration of the Q-sepharose column, orange cytochrome b was extracted from the sucrose gradient₆f, loading the sample on the strip, eluting with 0-500mM salt concentration elution buffer solution of an ion exchange column at the rate of 1mL/min, collecting the eluted sample, and obtaining the high-purity cytochrome b₆f。

In the prior art, different plant species (spinach and Verbena) are selected to extract cytochrome b₆Method of f and extraction of cytochrome b of this example₆The process comparison of f is shown in Table 1, and it can be seen that the method of the present invention can effectively reduce the production cost and save the production time。

TABLE 1 different cytochromes b₆Comparison of preparation methods

Example 2: purified cytochrome b₆f purity determination

Purified cytochrome b₆f, the clear cytochrome b is detected by 15% SDS-PAGE electrophoresis₆f four large subunit bands with cyt f 32kDa and cyt b size₆25kDa, Rieske ISP 19kDa and Subunit IV 17kDa (see FIG. 2).

The purified protein was observed by transmission electron microscopy to reveal uniform size of cytochrome b₆Protein particles with a particle size of about 10nm, which conform to cytochrome b₆The total size of the f complex at 220kDa (see FIG. 3).

Claims (10)

1. Rapid purification cytochrome b₆f, comprising the steps of:

1) collecting cyanobacteria, crushing at 4 ℃, centrifuging and then retaining supernatant;

2) centrifuging the supernatant at high speed to obtain a thylakoid membrane, and washing with a high-salt solution to remove impurity proteins;

3) adding detergent undecyl maltoside or dodecyl-beta-D-maltoside into thylakoid membrane, and adopting three-step ammonium sulfate precipitation method to obtain cytochrome b₆f, crude product;

4) mixing cytochrome b₆f, purifying the crude product by sucrose density gradient centrifugation and anion exchange column to obtain cytochrome b₆f, finishing the product.

2. The method of claim 1, wherein in step 1), the cyanobacteria are of the genus anabaena or nostoc.

3. The method as claimed in claim 1, wherein the step 1) is carried out by enriching the bacteria from the cyanobacteria liquid by centrifugation, then suspending the bacteria in buffer solution, adding protease inhibitor, and crushing the bacteria at 4 ℃ in a dark condition; and centrifuging the crushed bacteria liquid to remove the uncrushed cells and cell fragments, and keeping the supernatant.

4. The method of claim 3, wherein the protease inhibitor added in step 1) is phenylmethylsulfonyl fluoride, benzamidine and 6-aminocaproic acid, all at a final concentration of 0.1 mM.

5. The method as claimed in claim 1, wherein the step 2) is to centrifuge the supernatant at high speed to obtain a crude thylakoid membrane precipitate, resuspend the crude thylakoid membrane precipitate with a high salt buffer solution containing 2M NaBr, adjust the chlorophyll concentration to 1-1.5 mg/mL, stir for a period of time, and add ddH at a volume ratio of 1:1₂And O, performing high-speed centrifugation to obtain the thylakoid membrane precipitate washed with the foreign protein.

6. The method according to claim 1, wherein in step 3), the thylakoid membrane precipitate is resuspended in a buffer solution containing protease inhibitors to a chlorophyll concentration of 1.5-2 mg/mL, the detergent is slowly added to a final concentration of 8-10 mM while stirring, and after stirring for a while, ammonium sulfate is slowly added for precipitation.

7. The method of claim 1, wherein the three-step ammonium sulfate precipitation process in step 3) comprises: slowly adding ammonium sulfate to a final concentration of 30-35%, stirring, centrifuging at a rotation speed of 180000-210000g for 10-30 min, and taking a supernatant; then slowly adding ammonium sulfate to a final concentration of 40-45%, stirring, centrifuging for 10-30 min at a rotation speed of 180000-210000g, and taking a supernatant; slowly adding ammonium sulfate again to a final concentration of 55-60%, stirring, centrifuging at a rotation speed of 180000-210000g for 10-30 min, and precipitating to obtain cytochrome b₆f, crude product.

8. The method of claim 1, wherein step 4) comprises subjecting cytochrome b to₆And f, dialyzing the crude product to remove ammonium sulfate salt, and then performing sucrose density gradient centrifugation.

9. The method of claim 8, wherein step 4) comprises subjecting cytochrome b to₆After the crude product is resuspended by buffer solution, dialyzing by a 100kDa dialysis bag overnight, and changing the solution for one or more times in the midway, wherein the dialyzate is TNE buffer solution added with 0.1mM phenylmethylsulfonyl fluoride, 0.2mM undecyl maltoside or dodecyl-beta-D-maltoside and 25mM sucrose; adding sucrose and undecyl maltoside with the final concentration of 1mM or dodecyl-beta-D-maltoside into TNE buffer solution to prepare a 5% -30% continuous sucrose gradient, and performing sucrose density gradient centrifugation on the dialyzed sample at the centrifugation speed of 32000 and 36000rpm for 14-16 hours; the orange cytochrome b was then aspirated in a sucrose gradient₆The f bands are put on an anion exchange column for ion exchange chromatography, and high-purity cytochrome b is obtained by elution₆f。

10. The method of claim 1, wherein the anion exchange column in step 4) is a Q-sepharose column.

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