CN106939032B - Extracting solution for rapidly and efficiently extracting total plant protein and extracting method - Google Patents
Extracting solution for rapidly and efficiently extracting total plant protein and extracting method Download PDFInfo
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Abstract
The invention belongs to the field of extraction of total plant protein, and discloses an extracting solution for quickly and efficiently extracting the total plant protein, which is prepared from the following raw materials: 200mM Tris-Base, 200mM NaCl, 50mM sodium ascorbate, 1g/100mL deoxycholate, 1mL/100mL NP-40, 1g/100mL SDS, 1mL/100mL Triton X-100, 1mL/100mL Tween 20, 5mM EDTA, 10mL/100mL glycerol, 50mM sodium tetraborate, and 1g/100mL PVPP. The invention also discloses an extraction method, which comprises the steps of preparing an extracting solution, weighing and pretreating a sample, cracking and centrifuging, extracting with Tris-saturated phenol, precipitating the vegetable protein, resuspending the vegetable protein precipitate and the like. The invention has wide application range, short extraction time, high extraction rate and no interference of impurities.
Description
Technical Field
The invention belongs to the technical field of extraction of total plant protein, and relates to an extracting solution for quickly and efficiently extracting the total plant protein and an extracting method, which are particularly suitable for extracting the total protein of a recalcitrant species or a tissue.
Background
The extraction of the total protein of the plant, particularly the extraction of the total protein of the recalcitrant plant tissue has been a great problem. The methods for extracting proteins from different species and tissues are not consistent, but the basic principle and the basic process of the extraction method are similar, mainly comprising trichloroacetic acid-acetone method and phenol-acetone method. The trichloroacetic acid-acetone method has simple operation process and short time consumption, but has the problems of low protein yield and more impurities. The phenol-acetone method is that cell is first cracked with chemical detergent or physical ultrasonic crushing and other measures, protein and lipid are then extracted with phenol from water phase while nucleic acid, polysaccharide and other organic matter are left in the water phase, and the protein is finally precipitated with acetone to obtain high purity protein.
The main operation steps of the phenol-acetone process are: 1. grinding plant tissues in liquid nitrogen, and then adding a protein extracting solution; 2. adding equal volume of Tris-saturated phenol to extract protein, and then centrifuging at high speed; 3. absorbing the upper phenol phase, adding saturated ammonium sulfate methanol solution with five times of volume for precipitating overnight to obtain protein precipitate; 4. washing the protein precipitate with methanol and acetone respectively once to obtain dry vegetable protein powder; 5. the dry protein powder is dissolved overnight in 0.5% SDS solution, and if SDS interferes with downstream experiments, dialysis is also required to remove the SDS as much as possible.
Disclosure of Invention
The first purpose of the invention is to provide an extracting solution for quickly and efficiently extracting the total plant protein, in particular to the extraction of the total plant protein of a stiffness tissue, such as lignified roots, woody stem tips, fat-rich seeds and nuts, and the full cracking of the plant tissue can be realized by adjusting the components and the content of the extracting solution.
The second purpose of the invention is to provide an extraction method for rapidly and efficiently extracting the total plant protein by using the extracting solution, which optimizes the step of extracting the plant protein by using Tris-saturated phenol and the purification step, greatly shortens the extraction time, has simple purification step and has no interfering substances, and can be directly used for downstream experiments.
The technical scheme of the invention is as follows:
an extracting solution for rapidly and efficiently extracting total plant protein comprises the following raw materials: 200mM Tris-Base, 200mM NaCl, 50mM sodium ascorbate, 1g/100mL deoxycholate, 1mL/100mL NP-40, 1g/100mL SDS, 1mL/100mL Triton X-100, 1mL/100mL Tween 20, 5mM EDTA, 10mL/100mL glycerol, 50mM sodium tetraborate, and 1g/100mL PVPP.
The invention also discloses a method for quickly and efficiently extracting the total plant protein by using the extracting solution, which comprises the following steps:
(1) preparing extracting solution
Preparing an extracting solution according to the content composition of claim 1, and storing at 4 ℃;
(2) sample weighing and pretreatment
Weighing not less than 0.5g of plant tissues in a mortar, fully grinding the plant tissues in a liquid nitrogen environment to powder, and then adding the extracting solution;
(3) lysis and centrifugation
Ensuring that the plant tissue powder and the extracting solution are fully and uniformly mixed, cracking for 1-3 h at 4 ℃, and then centrifuging to obtain protein supernatant;
(4) tris-saturated phenol extraction of plant proteins
Adding Tris-saturated phenol into the protein supernatant for extraction, uniformly mixing and centrifuging to obtain a supernatant, adding the extracting solution into the supernatant for back extraction, uniformly mixing and centrifuging to obtain another supernatant;
(5) precipitation of vegetable proteins
Adding a supersaturated ammonium sulfate methanol solution into the other supernatant obtained in the step (4) for precipitation, precipitating for 2-3 h, centrifuging to obtain a protein precipitate, and washing with methanol and 80% acetone in sequence;
(6) resuspending the plant protein pellet
And (5) resuspending the protein precipitate obtained in the step (5) by using a PBS buffer solution to obtain a purified vegetable protein solution.
Preferably, in step (2), 1mM PMSF, 1mM Cocktail and 1g/100mL PVPP are also added to the plant tissue powder.
Preferably, in the step (5), ultrasonic treatment is performed for 3-5 times during resuspension, so that protein precipitates can be dissolved in a PBS buffer solution, and then the supernatant vegetable protein solution after purification is obtained after standing and centrifugation.
Preferably, step (5) further comprises a final protein content determination step to calculate the protein extraction efficiency.
Compared with the prior art, the invention has the following beneficial effects:
the invention is widely applicable to protein extraction of various different species and tissues by optimizing the formula of the extracting solution and improving the cell cracking process, and can ensure high protein extraction efficiency even for some stubborn tissues, such as old roots with serious lignification and stem tips of woody family, seeds with rich fat, harder nuts and other tissues;
secondly, in the prior art, the Tris-saturated phenol extraction process is rough, so that the obtained protein precipitate contains more impurities, and methanol precipitation is needed to stay overnight, so that the time is wasted;
thirdly, after the protein precipitate obtained by the prior art is washed by methanol and acetone, 0.5% SDS is needed to be used for dissolving overnight, if the protein precipitate is needed to be used for downstream experiments, the protein solution needs to be centrifuged firstly to obtain supernatant, then the supernatant can be used for the downstream experiments after being dialyzed, the process takes one day for a short time and two days for a long time, and some plant proteins in the protein precipitate can not be fully dissolved; the protein precipitate is washed by methanol and acetone, washed by 80% acetone once, dried, directly resuspended by PBS buffer solution, and ultrasonically treated for three to five times, the precipitate is fully dissolved, and the supernatant can be directly used for downstream experiments after standing and centrifugation, wherein the process can be completed within one day, and the obtained plant protein does not contain SDS and can be directly used for downstream experiments.
Drawings
FIG. 1 is a flow chart of a process for rapidly and efficiently extracting total plant protein according to the present invention;
FIG. 2 is a schematic strip diagram of the method of the present invention and the prior art method for extracting tomato fruit protein in example 2, wherein A is a schematic strip diagram of the method of the present invention for extracting tomato fruit protein, and B is a schematic strip diagram of the prior art method for extracting tomato fruit protein;
fig. 3 is a schematic diagram of bands obtained by extracting sugarcane stem apex protein by the method of the present invention and the existing method in example 3, wherein a is a schematic diagram of bands obtained by extracting sugarcane stem apex protein by the method of the present invention, and B is a schematic diagram of bands obtained by extracting sugarcane stem apex protein by the existing method.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In practice, the invention will be understood to cover all modifications and variations of this invention provided they come within the scope of the appended claims.
The invention relates to an extracting solution for quickly and efficiently extracting total plant protein, which is prepared from the following raw materials: 200mM Tris-Base, 200mM NaCl, 50mM sodium ascorbate, 1g/100mL deoxycholate, 1mL/100mL NP-40, 1g/100mL SDS, 1mL/100mL TritonX-100, 1mL/100mL Tween 20, 5mM EDTA, 10mL/100mL glycerol, 50mM sodium tetraborate, and 1g/100mL PVPP.
The action principle of the extracting solution of the invention is as follows: fully grinding the tissue sample in liquid nitrogen, adding an extracting solution, and destroying cell walls and membrane systems of cells by using ionic detergent and nonionic detergent in the extracting solution so as to fully release proteins in the cells; for some strongylous tissue samples, the cell walls of the strongylous tissue samples are damaged by means of ultrasonic or repeated freezing and thawing with liquid nitrogen after the extracting solution is added. Glycerol in the extract is used for protecting protein; the antioxidant mainly provides an antioxidant environment to prevent organic matters such as protein, polyphenol and the like from being oxidized; the protease inhibitor is mainly used for inhibiting protease released by cells and preventing the protein from being degraded; sodium tetraborate and PVPP are mainly used for removing organic matters such as polysaccharide, polyphenol and the like, and improving the purity of the extracted protein. After the protein is released and dissolved in the extracting solution, adding equal-volume Tris-saturated phenol and mixing uniformly, mainly using the principle that the protein and lipid are more easily dissolved in the phenol to extract the protein in the extracting solution into the Tris-saturated phenol, then using the principle that the protein is not dissolved in methanol and acetone, adding methanol to precipitate the protein, and using the methanol to wash the protein precipitate; washing the precipitate with eighty percent acetone to remove salt ions in the protein precipitate, re-suspending the precipitate with PBS, and ultrasonically breaking the denatured protein precipitate to dissolve the protein precipitate in PBS buffer solution; PBS buffer is a commonly used buffer, and the solution environment does not interfere with downstream experiments.
The specific composition, content and function of the corresponding components of the extract of the present invention are shown in table one, and the pH of the extract is 8.0.
Watch 1
The invention also discloses a method for quickly and efficiently extracting total plant protein by using the extracting solution, which specifically comprises the following steps as shown in figure 1:
(1) preparing extracting solution
Preparing an extracting solution according to the content composition of claim 1, and storing at 4 ℃; after 10ml solution is prepared by PMSF, subpackaging, and subpackaging 10 tubes by 1.5ml EP tube at one time; subpackaging, storing at-20 deg.C, and repeatedly freezing and thawing for several times; 1g of the Cocktail protease inhibitor is taken out and dissolved in 1ml of water each time, and then the Cocktail protease inhibitor is used according to the proportion of 50X; other specific types of protease inhibitors are added according to project requirements and purpose preparation;
(2) sample weighing and pretreatment
Taking out fresh tissue or frozen tissue, weighing on an electronic balance, and recording the weight of the fresh tissue or frozen tissue; inserting the container filled with the tissue into an ice box for later use, wherein the tissue weight is not less than 0.5 g;
precooling the grinding bowl by using liquid nitrogen, and then putting a sample into the grinding bowl; adding liquid nitrogen, grinding repeatedly until the sample is ground into powder, and adding the liquid nitrogen in time in the process to ensure that the sample is in a low-temperature state;
grinding a sample into powder, adding the powder into a precooling centrifugal tube filled with liquid nitrogen as soon as possible, then adding a prepared extracting solution, 1mM PMSF and 1mM Cocktail as protease inhibitors and 1g/100mL PVPP reagents, wherein the adding proportion of the extracting solution can be higher for a refractory tissue which is rich in protein or difficult to crack;
(3) lysis and centrifugation
Covering the cover of the centrifuge tube, sealing with a sealing film to prevent liquid leakage, and placing in an oscillator to vibrate for about 5 minutes to ensure that the powder is fully and uniformly mixed in the extracting solution; for the recalcitrant tissues, such as stem tips, old roots and seeds of woody plants, ultrasonic treatment can be carried out for thirty times to fully destroy cell walls, and the sample is preferably cracked in an extracting solution for two hours to ensure that the cells are fully cracked; this is preferably carried out at 4 ℃ to inhibit protease activity;
after the sample is fully cracked, centrifuging at 12500rpm for thirty minutes, taking protein supernatant, temporarily preventing cell lysate sediment from being frozen at 4 ℃, and discarding the cell lysate sediment after the successful extraction of the protein is determined;
(4) tris-saturated phenol extraction of plant proteins
Adding equal volume of Tris-saturated phenol into the protein supernatant for extraction, reversing and mixing uniformly, then rotating and mixing uniformly for 30 minutes at normal temperature, centrifuging for 15 minutes at 12000rpm/min, then sucking the supernatant into another clean centrifugal tube, adding equal volume of protein extract for back extraction, reversing and mixing uniformly, mixing uniformly for 5 minutes at normal temperature, then centrifuging at the same rotating speed, taking the supernatant to obtain another supernatant, and discarding the lower-layer water phase;
(5) precipitation of vegetable proteins
Adding a 5-time volume of supersaturated ammonium sulfate methanol solution into the other supernatant obtained in the step (4) for precipitation (the supersaturated ammonium sulfate methanol solution is pre-cooled at-20 ℃ in advance), uniformly mixing, standing at-20 ℃, and precipitating for 2-3 hours;
centrifuging the protein solution which is precipitated overnight at 13000rpm/min for 30 minutes, and centrifuging the protein solution at 4 ℃; then, the methanol supernatant is discarded, the protein precipitate is washed once by using methanol solution, and the precipitate can be transferred to a smaller centrifugal tube in the process, so that the protein precipitate can be conveniently collected and stored;
centrifuging, removing the supernatant of methanol, and washing the obtained protein precipitate with acetone precipitate; centrifuging, removing acetone supernatant, washing protein precipitate with 80% acetone once, and removing salt ions from the protein precipitate;
(6) resuspending the plant protein pellet
The resulting protein pellet was resuspended in an appropriate amount of PBS buffer (PBS buffer volume was added according to protein pellet amount), and then sonicated three to five times; care should not be taken to sonicate too many times, otherwise the protein is easily broken into smaller polypeptide fragments; if the ultrasonic treatment is carried out for five times, the protein precipitate can not be fully dissolved, which indicates that the protein precipitate contains more impurities, and methanol and acetone are not precooled at the temperature of minus 20 ℃ in advance in the washing process with the methanol and the acetone;
(7) protein quantification and quality detection
Measuring the protein concentration by adopting a BCA or amino black method, and then calculating the extracted protein amount according to the volume of the protein solution; the ratio of the amount of protein to the amount of sample used is the efficiency of protein extraction.
If the protein extraction efficiency is consistent with the conventional principle or is the same as that reported in the literature, the extracted protein is subjected to counterstaining to further determine the distribution and brightness of protein bands, so as to further determine the quality of protein extraction;
if the test result is in accordance with expectation, the quality of the extracted protein can be further detected according to research requirements; if the research requirement is met, the device can be provided for a downstream platform to use.
Example 1
The method for quickly and efficiently extracting the total protein of the plant and the method for extracting the total protein in advance in the prior art are respectively used for extracting the proteins of the anther tissues of rice, arabidopsis thaliana and wheat. The method improves the extraction efficiency to about four thousandths, the protein strips are uniformly distributed, the high-peak protein is not obviously distributed, and the whole protein extraction period is shortened from the original three days to one day; the extraction efficiency of the existing method is only four ten-thousandths, and the high-peak protein is obvious.
Example 2
The method for rapidly and efficiently extracting the total protein of the plant and the method for extracting the total protein in advance in the prior art are respectively used for extracting the protein of tomato, blueberry and grape fruit samples. The method improves the extraction efficiency to one fifth per thousand, has high protein purity and uniform strip distribution, and shortens the whole protein extraction period to one day; the efficiency of extracting the samples by the existing method is only two ten-thousandth, and the whole protein extraction process needs three days, wherein, the method for extracting the tomato fruit protein strip is shown as a figure 2(A), and the existing method for extracting the tomato fruit protein strip is shown as a figure 2(B), so the comparison shows that the tomato fruit protein extracted by the existing method has low total protein yield and less strip distribution; the total protein of the tomato fruit extracted by the method has high yield and uniform and clear stripe distribution.
Example 3
The method for rapidly and efficiently extracting the total protein of the plant and the method for extracting the total protein in advance in the prior art are respectively used for extracting the protein of the sugarcane stem apex sample. Sugarcane stem apex contains much water, polysaccharide impurities are abundant, and stem apex cells are seriously lignified. According to the method, the whole protein extraction process only needs one day, and the extraction efficiency is improved to five ten-thousandths; the conventional protein extraction method has the protein extraction efficiency of less than one ten-thousandth, and the whole period needs three days, wherein, the method is used for extracting the sugarcane stem apex protein strip, as shown in figure 3(A), and the conventional method is used for extracting the sugarcane stem apex protein strip, as shown in figure 3(B), so that the comparison shows that the sugarcane stem apex total protein extracted by the conventional method has low yield and less strip distribution; the total protein of the stem apex of the sugarcane extracted by the invention has high yield and uniform and clear distribution of strips.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (5)
1. An extracting solution for rapidly and efficiently extracting total plant protein is characterized by being prepared from the following components in parts by weight: 200mM Tris-Base, 200mM NaCl, 50mM sodium ascorbate, 1% (m/v) deoxycholate, 1% (v/v) NP-40, 1% (m/v) SDS, 1% (v/v) Triton X-100, 1 (v/v)% Tween 20, 5mM EDTA, 10 (v/v)% glycerol, 50mM sodium tetraborate, 1% (m/v) PVPP, said plant being selected from the group consisting of: tomato, blueberry, grape fruit or sugarcane.
2. A method for rapidly and efficiently extracting total plant protein by using the extracting solution of claim 1, which is characterized by comprising the following steps:
(1) preparing extracting solution
Preparing an extracting solution according to the content composition of claim 1, and storing at 4 ℃;
(2) sample weighing and pretreatment
Weighing not less than 0.5g of plant tissues in a mortar, fully grinding the plant tissues in a liquid nitrogen environment to powder, and then adding the extracting solution;
(3) lysis and centrifugation
Ensuring that the plant tissue powder and the extracting solution are fully and uniformly mixed, cracking for 1-3 h at 4 ℃, and then centrifuging to obtain protein supernatant;
(4) tris-saturated phenol extraction of plant proteins
Adding Tris-saturated phenol into the protein supernatant for extraction, uniformly mixing and centrifuging to obtain a supernatant, adding the extracting solution into the supernatant for back extraction, uniformly mixing and centrifuging to obtain another supernatant;
(5) precipitation of vegetable proteins
Adding a supersaturated ammonium sulfate methanol solution into the other supernatant obtained in the step (4) for precipitation, precipitating for 2-3 h, centrifuging to obtain a protein precipitate, and washing with methanol and 80% acetone in sequence;
(6) resuspending the plant protein pellet
And (5) resuspending the protein precipitate obtained in the step (5) by using a PBS buffer solution to obtain a purified vegetable protein solution.
3. The method for rapidly and efficiently extracting total plant protein according to claim 2, wherein in the step (2), 1mM PMSF, 1mM Cocktail and 1g/100mL PVPP are further added to the plant tissue powder.
4. The method for rapidly and efficiently extracting total plant protein according to claim 2, wherein in the step (5), the ultrasonic treatment is performed for 3-5 times during the resuspension, so that the protein precipitate is dissolved in the PBS buffer solution, and then the supernatant vegetable protein solution after purification is obtained after standing and centrifugation.
5. The method for rapidly and efficiently extracting total plant protein according to claim 2, wherein the step (5) further comprises a final protein content determination step for calculating the protein extraction efficiency.
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| CN111909234A (en) * | 2020-08-21 | 2020-11-10 | 南京康齐生物科技有限公司 | Preparation method of allium sativum fruit protein |
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