A method for purifying a rubber elongation factor protein

Abstract

The present disclosure provides a method for purifying rubber elongation factor protein, which comprises: obtaining rubber particles from Hevea brasiliensis latex and dispersing the rubber particles in a buffer solution; extracting rubber particle protein using an extraction solution containing a non-ionic detergent; adding ammonium sulfate and an alcohol reagent to the extraction solution containing the rubber particle protein to precipitate the rubber particle protein (mainly rubber elongation factor protein); and separating the extracted rubber particle protein by gel filtration chromatography to obtain purified rubber elongation factor protein. The method for purifying rubber elongation factor protein can expand the purification scale and obtain milligram-level high-purity rubber elongation factor protein.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of proteomics, and in particular to a method for purifying rubber elongation factor protein. BACKGROUND

[0002] Latex is the white milk flowing from the latex vessels (tissue formed by latex vessel cells) of Hevea brasiliensis after tapping, and is the main source of natural rubber. Rubber particles are a special organelle in latex vessel cells of Hevea brasiliensis, and are the site of natural rubber biosynthesis and storage. Rubber elongation factor (REF) is a class of proteins with high expression in rubber particles, which may interact with rubber transferase, rubber transferase activating protein, small rubber particle protein (SRPP) and other types of protein factors to form a protein complex and catalyze natural rubber biosynthesis. How to extract and purify rubber elongation factor protein from Hevea brasiliensis latex is a technical challenge faced by many researchers.

[0003] The only reported method for purifying rubber elongation factor protein from Hevea brasiliensis latex at present is mainly based on gel electrophoresis and electroelution technology, and the method is as follows: separating rubber particles from latex, extracting proteins from rubber particles, using SDS-PAGE electrophoresis to separate total proteins of rubber particles, and cutting the rubber elongation factor protein band (14.7 kDa) for electroelution to obtain purified rubber elongation factor protein (Alenius, H., et al. (1996). Int Arch Allergy Immunol 109(4): 362-368.).

[0004] The main defect of the above-mentioned method for purifying rubber elongation factor protein based on gel electrophoresis and electroelution technology is that the processing capacity of SDS-PAGE electrophoresis is limited, the purification scale is not easy to expand, and it is difficult to obtain milligram-level protein. SUMMARY

[0005] The present disclosure provides a method for purifying rubber elongation factor protein to at least solve the above technical problems existing in the prior art.

[0006] The present disclosure provides a method for purifying rubber elongation factor protein, which comprises:

[0007] obtaining rubber particles from Hevea brasiliensis latex and dispersing the rubber particles in a buffer solution;

[0008] extracting rubber particle proteins using an extraction solution containing a non-ionic detergent;

[0009] adding ammonium sulfate and an alcohol reagent to the extraction solution containing rubber particle proteins to precipitate rubber particle proteins, wherein the rubber elongation factor protein is the main component;

[0010] The purified rubber elongation factor protein is obtained by separating the extracted rubber particle protein through gel filtration chromatography.

[0011] In an embodiment, after obtaining the rubber particles, the method further comprises:

[0012] An alkaline solution is added to the rubber particles dispersed in the buffer solution to remove proteins not tightly bound to the rubber particles.

[0013] In an embodiment, the obtaining of the rubber particles and dispersing them in the buffer solution comprises:

[0014] The latex is collected and placed on ice;

[0015] The latex is centrifuged, and the upper layer of rubber particles is removed to a new centrifuge tube, added with the buffer solution and dispersed, and the rubber particles are collected by centrifugation, and the collected rubber particles are transferred to a new centrifuge tube and dispersed in the buffer solution.

[0016] The collected rubber particles are dispersed with the buffer solution, and the rubber particles are collected again by centrifugation for one washing process, and the rubber particles are washed multiple times.

[0017] In an embodiment, the buffer solution comprises a buffer salt, an antioxidant, a protease inhibitor, and an osmotic pressure regulator.

[0018] In an embodiment, the extraction solution for extracting the rubber particle protein comprises sodium chloride, imidazole, 6-aminohexanoic acid, a protease inhibitor, and a non-ionic detergent; and the volume ratio or weight ratio of the rubber particles to the extraction solution is 1:1 to 1:5.

[0019] In an embodiment, the extracting of the rubber particle protein using the extraction solution containing the non-ionic detergent comprises:

[0020] The rubber particles dispersed in the buffer solution are centrifuged, and the upper layer of rubber particles is collected and placed in a new centrifuge tube;

[0021] The extraction solution is added to the new centrifuge tube and the rubber particles are dispersed, and the centrifuge tube is placed on ice and shaken at a low speed on a shaker to obtain an extraction mixture;

[0022] The extraction mixture is centrifuged, and the lower layer of supernatant is transferred to a new centrifuge tube;

[0023] Centrifugation at a higher speed is performed, and after removing the upper floating matter in the centrifuge tube, a clear protein extraction solution is obtained.

[0024] In an embodiment, the extracting of the rubber particle protein comprises:

[0025] The extraction solution containing rubber particle protein, ammonium sulfate and alcohol reagent are mixed and centrifuged, and the protein precipitate between the organic phase and the aqueous phase is taken out;

[0026] The protein precipitate is dissolved and desalted to obtain rubber particle protein.

[0027] In an embodiment, the volume ratio of the extraction solution containing rubber particle protein, ammonium sulfate and alcohol reagent is 1:(0.1-0.5):(0.3-2).

[0028] In an embodiment, the separation of the extracted rubber particle protein by gel filtration chromatography comprises:

[0029] The desalted rubber elongation factor protein is dissolved or diluted in a mobile phase, subjected to gel filtration chromatography and the elution fractions are collected;

[0030] The main elution fraction collected is precipitated and desalted to obtain purified rubber elongation factor protein.

[0031] In an embodiment, the mobile phase comprises: 1) 50-250 mM sodium chloride; 2) 3-9 M urea or 0.2-2% of a non-UV absorbing detergent at 280 nm wavelength.

[0032] The method for purifying rubber elongation factor protein of the present disclosure comprises the following steps: obtaining rubber particles and dispersing the rubber particles in a buffer solution; using an extraction solution containing a non-ionic detergent to extract protein from the rubber particles; adding ammonium sulfate and an alcohol reagent to the extraction solution containing rubber particle protein to precipitate the rubber particle protein (rubber elongation factor protein is the main component); and subjecting the extracted rubber particle protein to gel filtration chromatography separation. The present disclosure uses an extraction solution containing a non-ionic detergent to selectively extract rubber particle protein, especially to ignore the protein with a molecular weight and isoelectric point close to that of rubber elongation factor and an abundance second only to that of rubber elongation factor. Removing this protein is the key to purifying rubber elongation factor protein, because it is difficult to separate two proteins with such close molecular weight and isoelectric point by gel filtration or ion exchange chromatography. The rubber particle protein in the extraction solution is further precipitated by the three-phase method, and by using a suitable reagent ratio, the rubber elongation factor protein with the highest abundance is preferentially precipitated. The present disclosure only uses gel filtration chromatography once in the purification of rubber elongation factor protein. The existing rubber elongation factor purification method (based on gel electrophoresis and electroelution technology) mainly relies on SDS gel electrophoresis (with higher resolution) to separate proteins, but its preparation scale is limited and is not easy to expand. The protein three-phase precipitation method and gel filtration chromatography used in the present disclosure are both easy to scale up: the three-phase precipitation method only needs to simply scale up the volume of the precipitation reaction, and the gel filtration chromatography only needs to use a larger diameter chromatography column and use a higher flow rate. Therefore, by using the purification method of the present disclosure, the purification scale can be expanded, and milligram-level high-purity rubber elongation factor protein can be obtained.

[0033] It is to be understood that the details described in this section are not intended to identify key or critical elements of the embodiments of the present disclosure or to limit the scope of the present disclosure. Other features of the present disclosure will become apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0034] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will be more apparent from the following detailed description read in conjunction with the accompanying drawings, in which:

[0035] In the drawings, identical or corresponding reference signs refer to identical or corresponding parts.

[0036] Figure 1 A flowchart of a method for purifying a rubber elongation factor protein according to an embodiment of the present disclosure is shown;

[0037] Figure 2 A result of SDS gel electrophoresis of a protein sample during extraction and purification of a rubber elongation factor protein according to an embodiment of the present disclosure is shown;

[0038] Figure 3 A separation process diagram of gel filtration chromatography according to an embodiment of the present disclosure is shown. DETAILED DESCRIPTION

[0039] In order to make the purposes, features and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below with reference to the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, but not all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present disclosure.

[0040] Unless otherwise specified, the reagents involved in the present disclosure are conventional reagents.

[0041] The present disclosure provides a method for purifying a rubber elongation factor protein, as shown in Figure 1 A flowchart of a method for purifying a rubber elongation factor protein according to an embodiment of the present disclosure is shown, which comprises the following steps:

[0042] Step S1, obtaining rubber particles from latex of Hevea brasiliensis and dispersing the rubber particles in a buffer solution;

[0043] In one example, obtaining rubber particles and dispersing the rubber particles in a buffer solution comprises:

[0044] S11, collecting latex and placing it on ice;

[0045] S12, centrifuging the latex, taking the upper rubber particle layer to a new centrifuge tube, adding a buffer solution and dispersing, centrifuging the rubber particles, and transferring the collected rubber particles to a new centrifuge tube and dispersing in the buffer solution;

[0046] The collected rubber particles are dispersed with the buffer solution, and the rubber particles are collected again by centrifugation for one washing process. The centrifugation parameters are 500-25000 rpm for 10 min at 4°C.

[0047] The washing buffer solution includes a buffer salt, an antioxidant, a protease inhibitor, and an osmotic pressure regulator, for example, the osmotic pressure regulator can be sucrose or mannitol. The buffer salt is 10-50 mM Tris or Hepes, etc. The present application does not limit this; the antioxidant can be 1-10 mM DTT or β-mercaptoethanol for antioxidant; the protease inhibitor is 1-10 mM EDTA; the preferred concentration of the osmotic pressure regulator is 250 mM sucrose or 400 mM mannitol.

[0048] In one example, after obtaining the rubber particles, the method further includes:

[0049] Step S2, adding an alkali solution to the rubber particles dispersed in the buffer solution to remove proteins not tightly combined with the rubber particles.

[0050] The alkali solution can be a Na2CO3 solution or an alkaline buffer solution, such as a Tris solution. Washing the rubber particles with an alkaline solution removes some proteins not tightly combined with the rubber particles, reducing the complexity of the total protein sample before purification. For the present application, the step of washing the rubber particles with alkali can be weakened or cancelled, and pure rubber elongation factor protein can still be obtained.

[0051] Step S3, extracting the rubber particle protein using an extraction solution containing a non-ionic detergent, and dispersing the extracted rubber particle protein in the extraction solution;

[0052] In one example, the extraction of the rubber particle protein using the extraction solution containing the non-ionic detergent includes:

[0053] S31, centrifuging the collected rubber particles dispersed in the buffer solution, and collecting the upper rubber particles into a new centrifuge tube;

[0054] S32, adding the extraction solution to the centrifuge tube and dispersing the rubber particles, placing the centrifuge tube in an ice bath and extracting at a low speed on a shaker to obtain a mixture; wherein the extraction solution comprises: 50 mM sodium chloride, 50 mM imidazole, 2 mM 6-aminohexanoic acid, a protease inhibitor (for example, 1 mM EDTA, or other protease inhibitor cocktails), and 0.05-1% non-ionic detergent, which can be Triton X-100 or Brij35, Triton X-114, NP40, etc.; the volume ratio or weight ratio of the rubber particles to the extraction solution is 1:1 to 1:5.

[0055] S33, centrifuging the mixture to separate the mixture into layers (the upper layer is solid residues such as rubber hydrocarbon, and the lower layer is the protein extract), and transferring the lower layer to a new centrifuge tube. For example, this step is preferably centrifuged at 18000 rpm for 20-60 min at 4°C.

[0056] S34, transferring the protein extract of S33 to an ultracentrifuge tube and centrifuging at a higher speed to obtain a clear protein extract after removing the upper floating material in the centrifuge tube. For example, centrifuging at 40000 rpm for 40 min at 4°C, and removing the solid material in the upper layer with a syringe. If the solid material has been disturbed and dispersed, the floating solid material is removed again after centrifugation until the liquid in the centrifuge tube is completely clear.

[0057] Step S4, adding ammonium sulfate and an alcohol reagent to the extraction solution containing rubber particle proteins to precipitate the rubber particle proteins;

[0058] In one example, the extraction of rubber particle proteins comprises:

[0059] S41, mixing the extraction solution containing rubber particle proteins with ammonium sulfate and an alcohol reagent, and then centrifuging to remove the upper and lower layers of liquid and retain the middle layer, i.e., the protein precipitate (or directly removing the middle layer with a pipette);

[0060] S42, dissolving the protein precipitate and desalting after dissolution.

[0061] Specifically, according to the volume ratio of the extraction solution containing rubber particle proteins: 4M ammonium sulfate: t-butanol (or n-butanol or amyl alcohol) = 1: (0.1-0.5): (0.3-2), the above reagents are added, mixed for 1-60 min, and then centrifuged at a speed of 400-15000 rpm. The obtained middle layer is the protein precipitate. Carefully remove the upper and lower layers of liquid and retain the protein precipitate between the two phases (or directly remove the protein precipitate with a pipette), dissolve the protein precipitate (use a solution containing urea or a detergent), and then desalt and concentrate the protein sample (use chloroform-methanol precipitation method, desalting column, ultrafiltration centrifugation, etc.).

[0062] Step S5, separating the extracted rubber particle protein (mainly rubber elongation factor) by gel filtration chromatography.

[0063] In one example, the separation of the extracted rubber particle protein (mainly rubber elongation factor) by gel filtration chromatography comprises:

[0064] S51, dissolving or diluting the desalted rubber particle protein in a mobile phase, and performing gel filtration chromatography and collecting elution components;

[0065] S52, precipitating and desalting the collected main elution components to obtain purified rubber elongation factor protein.

[0066] The mobile phase contains sodium chloride and urea or a non-UV-absorbing detergent at 280 nm wavelength, for example, the specific components of the mobile phase are: 3-9 M urea or 0.2-2% Brij35, 50-250 mM sodium chloride, and the pH of the mobile phase is 7-10. Since the rubber elongation factor protein is prone to self-aggregation and precipitation, urea or a non-UV-absorbing detergent at 280 nm wavelength is required to assist dissolution in the mobile phase. Gel filtration chromatography is performed and elution components are collected, wherein the elution peak with the maximum light absorption is the rubber elongation factor protein, which is precipitated using the above-mentioned three-phase method, and the desalting is the purified rubber elongation factor protein.

[0067] The present application selectively extracts rubber particle protein using an extraction solution containing non-ionic detergent, especially ignoring the protein with molecular weight and isoelectric point close to that of rubber elongation factor and abundance second only to rubber elongation factor during extraction. Removing this protein is the key to purifying rubber elongation factor protein, because it is difficult to separate two proteins with such close molecular weight and isoelectric point by gel filtration or ion exchange chromatography. Further, the rubber elongation factor protein in the extraction solution is precipitated by the three-phase method, and by using appropriate reagent ratios, the rubber elongation factor protein with the highest abundance is preferentially precipitated. The present application only uses gel filtration chromatography once in the purification of rubber elongation factor protein. Since the existing rubber elongation factor purification method (based on gel electrophoresis and electric elution technology) mainly relies on SDS gel electrophoresis (with higher resolution) to separate proteins, but the preparation scale of SDS gel electrophoresis is limited and not easy to expand. The three-phase precipitation method and gel filtration chromatography used in the present application are both easy to expand the scale: the three-phase precipitation method only needs to expand the volume of the precipitation reaction; the gel filtration chromatography only needs to use a larger diameter chromatography column and a higher flow rate. Therefore, by using the preparation and purification method of the present application, the purification scale can be expanded to obtain milligram-level rubber elongation factor protein.

[0068] The purification method of the present application will be described in detail below in combination with specific operation steps.

[0069] 1. Tapping and collecting latex in the rubber garden, and placing the centrifuge tube receiving the latex on ice to prevent protein degradation during collection.

[0070] 2. Washing of rubber particles: Add buffer solution to the centrifuge tube and centrifuge the rubber particles (which are in the upper layer) at 500-25000 rpm. Transfer the rubber particle layer to a new centrifuge tube and repeat the washing procedure 2-3 times by adding buffer solution and dispersing the rubber particles.

[0071] 3. Removal of loosely bound proteins from rubber particles: Add an appropriate volume of a concentrated Na2CO3 solution (e.g. 1 M) to the rubber particles dispersed in buffer solution to adjust the Na2CO3 concentration of the rubber particle dispersion to 5-100 mM. Place the centrifuge tube containing the rubber particle dispersion on ice and slowly shake on a shaker for 10-60 min to remove loosely bound proteins from the rubber particles and thus reduce the complexity of the total protein sample prior to purification.

[0072] 4. Extraction of rubber particle proteins: Centrifuge the mixture from step 3 at 500-25000 rpm to collect the rubber particles. Transfer the collected rubber particles to a new centrifuge tube and add an extraction solution containing a non-ionic detergent. The rubber particles should be present in a 1 : 1 to 1 :5 ratio by weight or volume to the extraction solution. Place the centrifuge tube on ice and extract the rubber particles at low speed on a shaker for 10-60 min or overnight. Centrifuge the extraction mixture at 18000 rpm for 30-60 min at 4°C to separate the layers and transfer the lower layer to a new centrifuge tube. Further clarify the protein extract in the new centrifuge tube by centrifugation at 40000 rpm for 40 min at 4°C. Remove the uppermost layer of floating solids using a syringe. If the solids are not well dispersed, repeat the centrifugation and remove the floating solids until the liquid in the centrifuge tube is clear.

[0073] 5. Precipitation of rubber particle proteins by the three-phase method: Add the sample and reagents in the following ratio: protein extract from rubber particles (i.e. the liquid in the centrifuge tube after step 4): 4 M ammonium sulphate: t-butanol (or n-butanol or amyl alcohol) = 1 : (0.1-0.5) : (0.3-2) by volume. Mix the solution for 1-60 min and centrifuge (400-15000 rpm) to obtain a protein precipitate in the middle layer (the upper layer is the organic phase and the lower layer is the aqueous phase, plus the middle layer, which is the three-phase system). Remove the upper and lower layers and retain the protein precipitate between the two phases (or directly transfer the protein precipitate using a pipette). Dissolve the protein precipitate (using a solution containing urea or a detergent) and then desalt and concentrate the protein sample (using the chloroform-methanol precipitation method or a desalting column or ultrafiltration centrifugation, etc.).

[0074] 6. Purification of desalted proteins by gel filtration chromatography: Dissolve or dilute the desalted protein sample (i.e. the sample obtained in step 5) in the mobile phase and perform gel filtration chromatography and collect the eluted fractions. Precipitate the main eluted fractions using the three-phase method described above and desalt to obtain the purified rubber elongation factor protein.

[0075] As shown in Figure 2 Figure 1 is the electrophoresis result of the protein sample extracted from rubber particles by the conventional method, the protein sample extracted from rubber particles by the method of the present application, and the main elution peak of the protein sample extracted from rubber particles by the method of the present application after gel filtration chromatography. Figure 2 In Figure 1, M is a protein molecular weight standard. Lane 1 is the protein sample extracted from rubber particles by the conventional method, i.e. the rubber particles are washed with a near neutral buffer solution and then extracted with an extraction solution containing 2% SDS. The protein bands are numerous and the thickest band is the rubber elongation factor protein. Lane 2 is the protein sample extracted from rubber particles by the method of the present application, i.e. the rubber particles are washed with a Na2CO3 solution and then extracted with an extraction solution containing a non-ionic detergent Triton X-100. The protein bands are fewer and the second most abundant protein in Lane 1 is almost completely removed. Lane 3 is the protein sample extracted from rubber particles by the method of the present application, i.e. the rubber particles are washed with a Na2CO3 solution and then extracted with an extraction solution containing a non-ionic detergent Triton X-100. After gel filtration purification, there are almost no other proteins except the rubber elongation factor band.

[0076] As shown in Figure 3 Figure 2 is the gel filtration chromatography separation result of the protein sample obtained by washing the rubber particles with a Na2CO3 solution and extracting the rubber particle proteins with an extraction solution containing a non-ionic detergent Triton X-100. After separating the rubber particle proteins using a gel filtration chromatography column with higher resolution (e.g. Sephacryl S200 16 / 60), the main peak is collected, desalted by three-phase precipitation and chloroform-methanol precipitation, and the purity of the obtained protein is shown in Figure 2 Lane 3.

[0077] Therefore, Figure 2 and Figure 3 The experimental results shown in

[0078] (1) Washing the rubber particles with an alkaline solution can remove some proteins that are not tightly bound to the rubber particles, thereby reducing the complexity of the total protein sample before column chromatography purification.

[0079] (2) Selectively extracting the rubber particle proteins with a non-ionic detergent, such as Triton X-100, especially ignoring the protein with molecular weight and isoelectric point close to that of the rubber elongation factor and abundance second only to the rubber elongation factor, further reduces the complexity of the total protein sample before column chromatography purification.

[0080] (3) Three-phase precipitation of proteins in the extraction solution, by using a suitable reagent ratio, preferentially precipitates the rubber elongation factor protein with the highest abundance, thereby increasing the relative content of the rubber elongation factor protein.

[0081] (4) The protein three-phase precipitation method and gel filtration chromatography used in the present application are both easy to scale up: the three-phase precipitation method only needs to expand the volume of the precipitation reaction; the gel filtration chromatography only needs to use a larger diameter chromatography column and use a higher flow rate.

[0082] It should be understood that the various forms of flow shown above can be used to reorder, add or delete steps. For example, the steps described in the present disclosure can be performed in parallel, sequentially or in a different order, as long as the desired results of the technical solutions disclosed in the present disclosure can be achieved, which is not limited herein.

[0083] In addition, the terms "first", "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "a plurality of" is two or more, unless otherwise specifically limited.

[0084] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present disclosure, which should be covered within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the protection scope of the claims.

Claims

1. A method for purifying a rubber elongation factor protein, characterized by, The method comprises: obtaining rubber particles from latex of Hevea brasiliensis and dispersing the rubber particles in a buffer solution; the buffer solution comprises buffer salt, antioxidant, protease inhibitor and osmotic pressure regulator, the buffer salt is Tris or Hepes buffer salt, the antioxidant is DTT or β-mercaptoethanol, the protease inhibitor is EDTA, and the osmotic pressure regulator is sucrose or mannitol; adding an alkali solution to the rubber particles dispersed in the buffer solution to remove proteins not tightly combined with the rubber particles; the alkali solution is a Na2CO3 solution or a Tris solution; extracting rubber particle proteins using an extraction solution containing a non-ionic detergent, and dispersing the extracted rubber particle proteins in the extraction solution; the extraction solution comprises sodium chloride, imidazole, 6-aminohexanoic acid, protease inhibitor and non-ionic detergent; the protease inhibitor is EDTA, and the non-ionic detergent is Triton X-100, Brij35, Triton X-114 or NP40; the volume ratio or weight ratio of the rubber particles to the extraction solution is 1:1 to 1:5; adding ammonium sulfate and an alcohol reagent to the extraction solution containing rubber particle proteins to precipitate rubber particle proteins, wherein the rubber elongation factor protein is the main component; the alcohol reagent is tert-butyl alcohol, n-butyl alcohol or amyl alcohol; separating the extracted rubber particle proteins by gel filtration chromatography to obtain purified rubber elongation factor proteins; the mobile phase used in the gel filtration chromatography comprises: 1) 50-250 mM sodium chloride; 2) 3-9 M urea or 0.2%-2% non-ultraviolet absorbing detergent at 280 nm wavelength, and the non-ultraviolet absorbing detergent is Brij35.

2. The method of claim 1, wherein, The method comprises: collecting latex and placing it on ice; centrifuging the latex, taking the upper rubber particle layer to a new centrifuge tube, adding a buffer solution and dispersing, centrifuging to gather the rubber particles, and transferring the gathered rubber particles to a new centrifuge tube and dispersing them in the buffer solution; dispersing the gathered rubber particles in the buffer solution and centrifuging to gather the rubber particles again as a one-time washing process, and washing the rubber particles multiple times.

3. The method of claim 1, wherein, The method comprises: centrifuging the rubber particles dispersed in the buffer solution, collecting the upper rubber particles and placing them in a new centrifuge tube; adding the extraction solution to the new centrifuge tube and dispersing the rubber particles, placing the centrifuge tube in an ice bath and extracting at a low speed on a shaker to obtain a mixture; centrifuging the mixture to separate the layers, and transferring the lower clear liquid to a new centrifuge tube; centrifuging at a higher speed, removing the upper floating matter in the centrifuge tube, and obtaining a clear protein extraction solution.

4. The method of claim 1, wherein, The method comprises: mixing the extraction solution containing rubber particle proteins, ammonium sulfate and an alcohol reagent, and centrifuging to take out the middle layer, which is a protein precipitate; dissolving the protein precipitate and desalting to obtain rubber particle proteins.

5. The method of claim 4, wherein, The volume ratio of the extraction solution containing rubber particle proteins, ammonium sulfate and alcohol reagent is 1:(0.1-0.5):(0.3-2).

6. The method of claim 4, wherein, The gel filtration chromatography separation of the extracted rubber particle proteins comprises: dissolving or diluting the desalted rubber particle proteins in a mobile phase, performing gel filtration chromatography and collecting elution fractions; precipitating and desalting the collected elution fractions to obtain purified rubber elongation factor proteins.

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