A method for extracting and purifying small rubber particle proteins from hevea brasiliensis latex
By using alkaline buffer solutions, strong anionic detergents, and organic solvents for precipitation in Brazilian rubber tree latex, combined with ion exchange and gel filtration chromatography, the tedious and time-consuming process of extracting and purifying small rubber particle proteins in existing technologies has been solved, achieving efficient and time-saving protein separation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
- Filing Date
- 2022-10-24
- Publication Date
- 2026-06-26
AI Technical Summary
Existing techniques for extracting and purifying small rubber particle proteins from Brazilian rubber tree latex are cumbersome, time-consuming, and prone to sample loss and protein self-aggregation and precipitation.
After stabilizing the latex with an alkaline buffer solution, a strong anionic detergent and organic solvent were added to precipitate and remove most of the protein. Small rubber particle proteins were separated and purified by ion exchange chromatography and gel filtration chromatography. Salt was removed by ammonium sulfate precipitation and a three-phase method.
It simplifies the operation steps, reduces sample complexity, improves extraction and purification efficiency, shortens the time, and achieves efficient separation of target proteins from non-target proteins.
Smart Images
Figure CN115651060B_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of proteomics technology, and more particularly to a method for extracting and purifying small rubber particle proteins from Brazilian rubber tree latex. Background Technology
[0002] Latex is the white sap that flows from the latex ducts (a network of cells forming the latex) of the Brazilian rubber tree after tapping, and it is the main source of natural rubber. Rubber particles are subcellular structures present in latex, responsible for the synthesis and storage of natural rubber; small rubber particles specifically refer to those with a diameter of less than 200 nm, requiring considerable centrifugal force to collect. Small rubber particle protein is the most abundant protein in small rubber particles, with a molecular weight of approximately 24 kDa, accounting for more than 60% of the total protein content of small rubber particles.
[0003] Currently, the only reported method (Wititsuwannakul et al., 2008) for purifying small rubber particle proteins from rubber tree latex involves: firstly, separating small rubber particles (containing a large amount of small rubber particle protein) from the latex, and then extracting and purifying the small rubber particle protein from these particles. Specifically: small rubber particles are separated from the latex by differential centrifugation; total protein is extracted from the small rubber particles (using a solution containing 0.2% Triton X100 overnight); the extracted protein is precipitated with acetone; the total protein is then dissolved in 50 mM Tris–HCl solution; heating at 70°C for 5 min precipitates most of the non-target protein; the supernatant (containing the target protein and rubber elongation factor) is collected; the protein in the supernatant is concentrated and subjected to gel filtration chromatography and DEAE ion exchange chromatography, ultimately obtaining purified small rubber particle protein.
[0004] The above extraction and purification methods have the following drawbacks: (1) Differential centrifugation is required to collect small rubber particles, and the small rubber particles usually need to be washed multiple times. Since the small rubber particles need to be centrifuged multiple times at a high centrifugal force, they are prone to coagulation and sample loss. Moreover, these steps are cumbersome and time-consuming; (2) Using a low concentration of detergent to extract proteins for a long time (0.2% Triton X100 extraction overnight) is inefficient and time-consuming; (3) The authors of the original method claimed that heating the precipitated protein at 70°C could selectively precipitate other proteins, while the small rubber particle protein and rubber elongation factor protein remained dissolved. Other researchers pointed out that the small rubber particle protein and rubber elongation factor protein (another high-abundance protein of rubber particles) have a tendency to self-aggregate and precipitate. That is, these two proteins will spontaneously precipitate even without heating. Therefore, heating cannot selectively separate the target protein from most non-target proteins. There is a need for an efficient and time-saving method to separate the target protein from non-target proteins. Summary of the Invention
[0005] This disclosure provides a method for extracting and purifying small rubber particle proteins from Brazilian rubber tree latex, thereby at least solving the problem of overly cumbersome and time-consuming operation steps in the prior art.
[0006] This disclosure provides a method for extracting and purifying small rubber particle proteins from Brazilian rubber tree latex, the method comprising:
[0007] An alkaline buffer solution is added to the rubber latex to stabilize it;
[0008] Strong anionic detergent and organic solvent were added successively to precipitate most of the proteins in the latex, except for small rubber particle proteins, and solid substances mainly composed of rubber hydrocarbons; the solids were collected by centrifugation, and the clear liquid portion, i.e., crude protein extract, was transferred.
[0009] Ammonium sulfate was added to the crude protein extract and the mixture was shaken to precipitate the protein. The precipitate was the crude protein extract.
[0010] The crude protein extract was separated by ion exchange chromatography, and the main elution peak was collected.
[0011] The proteins contained in the main elution peak of the precipitated ion exchange are mainly composed of small rubber particle proteins.
[0012] The proteins contained in the main elution peak of ion exchange were further separated by gel filtration chromatography to obtain purified small rubber particle proteins.
[0013] In one embodiment, a strong anionic detergent and an organic solvent are added sequentially to precipitate most of the proteins (excluding small rubber particle proteins) and solid substances mainly composed of rubber hydrocarbons in the latex, including:
[0014] Add a strong anionic detergent to the rubber latex to a concentration of 0.5-2%, and incubate on ice with shaking for 10-60 minutes;
[0015] Add an organic solvent and shake for 10-60 minutes to precipitate most of the proteins, except for small rubber particle proteins, and solid substances mainly composed of rubber hydrocarbons in the latex.
[0016] In one embodiment, the volume ratio of the rubber latex to the organic solvent is 1:0.3 to 1:2.
[0017] In one embodiment, the crude protein extract contains small rubber particle proteins. Protein components are precipitated from the crude protein extract, and the precipitated crude protein extract is obtained, comprising:
[0018] Add ammonium sulfate to the crude protein extract and shake. Centrifuge to collect and transfer the protein precipitate between the upper organic phase and the lower aqueous phase.
[0019] Add ultrapure water to disperse and wash the protein precipitate to remove salts;
[0020] The desalted protein precipitate is then dissolved to obtain crude protein extract.
[0021] In one embodiment, ion exchange chromatography is either anion exchange or cation exchange.
[0022] In one embodiment, the crude protein extract is separated by anion exchange chromatography, wherein the mobile phase comprises: A, 3-9 M urea, 10-50 mM Tris-HCl, pH 6-9; B, 3-9 M urea, 10-50 mM Tris-HCl, 1 M sodium chloride, pH 6-9.
[0023] In one embodiment, the crude protein extract is separated by cation exchange chromatography, wherein the mobile phase comprises: A: 3-9 M urea, 10-50 mM acetic acid or succinic acid, citric acid, lactic acid, pH 2-4.2; B: 3-9 M urea, 10-50 mM acetic acid or succinic acid, citric acid, lactic acid, 1 M sodium chloride, pH 2-4.2.
[0024] In one embodiment, the proteins contained in the precipitated ion exchange main elution peak include:
[0025] Mix the ion-exchange main elution peak components, ammonium sulfate, and alcohol reagent, centrifuge, and then transfer the protein precipitate from the intermediate layer.
[0026] The protein precipitate was dispersed in ultrapure water, the salt was washed away, and the protein precipitate was collected by centrifugation again.
[0027] In one embodiment, the volume ratio of the ion-exchange main elution peak component, 4 M ammonium sulfate, and alcohol reagent is 1:(0.1-0.5):(0.2-2).
[0028] In one embodiment, the proteins contained in the ion-exchange main elution peak are further separated by gel filtration chromatography to obtain purified small rubber particle proteins, including:
[0029] The protein precipitate was dissolved in the mobile phase, and gel filtration chromatography was performed to collect the eluent. The mobile phase included: 1) 50-250 mM sodium chloride, 2) 3-9M urea or 0.2-2% detergent that has no UV absorption at 280 nm.
[0030] The protein in the main elution fraction was precipitated and desalted to obtain purified small rubber particle protein.
[0031] This invention provides a method for extracting and purifying small rubber particle proteins from Brazilian rubber tree latex. First, latex from the rubber tree is collected. An alkaline buffer solution is added to the latex to stabilize it. A strong anionic detergent and an organic solvent are added to selectively precipitate most proteins in the latex, excluding small rubber particle proteins, while simultaneously precipitating solid substances mainly composed of rubber hydrocarbons. The solid substances are collected by centrifugation, and the clarified liquid portion, i.e., the crude protein extract, is transferred. Ammonium sulfate is added to the crude protein extract, and the mixture is shaken to precipitate the proteins. The precipitate is the crude protein extract. The crude protein extract is separated by ion exchange chromatography, and the main elution peak is collected. The proteins contained in the main ion exchange elution peak are then precipitated, with small rubber particle proteins being the main component. Finally, the proteins contained in the main ion exchange elution peak are further separated by gel filtration chromatography to obtain purified small rubber particle proteins. This invention directly extracts and purifies small rubber particle proteins from rubber tree latex. By using a strong anionic detergent (low concentration) and organic solvents to precipitate the latex and remove most of the latex proteins, only a few proteins, primarily small rubber particle proteins, are retained, reducing the complexity of the protein sample before column chromatography purification. A three-phase precipitation method is used for both protein extraction and chromatographic fraction concentration, simplifying the operation and significantly saving time. Therefore, the extraction and purification method of this invention efficiently and time-savingly separates target proteins from non-target proteins.
[0032] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description
[0033] The above and other objects, features, and advantages of this disclosure will become readily apparent from the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings. Several embodiments of this disclosure are illustrated in the drawings by way of example and not limitation, in which:
[0034] In the accompanying drawings, the same or corresponding reference numerals indicate the same or corresponding parts.
[0035] Figure 1 A schematic flowchart of a method for extracting and purifying small rubber particle proteins from rubber tree latex according to an embodiment of the present disclosure is shown.
[0036] Figure 2 The image shows an SDS gel electrophoresis diagram of protein samples during the extraction of latex protein and purification of small rubber particle protein according to embodiments of this disclosure;
[0037] Figure 3 The column chromatography purification process of small rubber particle proteins according to embodiments of this disclosure is shown in Figure A, which shows the anion exchange separation process; and Figure B, which shows the gel filtration chromatography separation process. Detailed Implementation
[0038] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.
[0039] This invention provides a method for extracting and purifying small rubber particle proteins from Brazilian rubber tree latex, such as... Figure 1 A flowchart of the method is shown, which includes the following steps:
[0040] S1. Add an alkaline buffer solution to the rubber latex to stabilize it;
[0041] The present invention uses the Brazilian rubber tree as an example to illustrate the process of tapping rubber trees in a rubber plantation and collecting latex. The centrifuge tubes used to collect the latex are placed on ice, and an alkaline buffer solution, such as Tris-HCl buffer (pH>7), is added to the latex to make it alkaline and maintain its stability.
[0042] S2. Strong anionic detergent and organic solvent are added successively to precipitate most of the protein in the latex except for small rubber particle protein (small rubber particle protein did not precipitate) and solid substances mainly composed of rubber hydrocarbons; the solid substances are collected by centrifugation, and the clear liquid part is transferred, which is the crude protein extract.
[0043] Specifically, strong anionic detergents and organic solvents are added sequentially to precipitate most of the proteins and solid substances, mainly rubber hydrocarbons, in the latex, except for small rubber particle proteins.
[0044] S21. Add a strong anionic detergent to the rubber latex to a concentration of 0.5-2%, and incubate on ice with shaking for 10-60 min.
[0045] S22. Add organic solvent and shake for 10-60 min to precipitate most of the proteins and solid substances, mainly rubber hydrocarbons, in the latex, except for small rubber particle proteins.
[0046] A strong anionic detergent, such as sodium dodecyl sulfate (SDS) or sodium dodecyl sulfonate, is preferred. The concentration of the anionic detergent is between 0.05% and 2%. After adding the anionic detergent, the centrifuge tube is placed on ice and shaken for 10-60 min. During the shaking extraction process, the negatively charged groups of the low-concentration strong anionic detergent bind to the positively charged groups of the protein, causing co-precipitation of the detergent and protein. Then, organic solvent (such as acetonitrile) is added at a volume ratio of latex to organic solvent (e.g., acetonitrile) of 1:0.3 to 1:2, and the mixture is shaken for 10-60 min to allow rubber hydrocarbons, membrane fragments, and proteins to coagulate and precipitate. After shaking, the mixture in the centrifuge tube is centrifuged until the liquid in the centrifuge tube is clear (small rubber particle proteins are mainly in the liquid fraction). By using a strong anionic detergent and organic solvent to precipitate most of the proteins in the latex, a few proteins, mainly small rubber particle proteins, are retained, thus reducing the complexity of the sample in subsequent purification processes.
[0047] S3. Add ammonium sulfate to the crude protein extract and shake to precipitate the protein. The precipitate is the crude protein extract.
[0048] In one example, the crude protein extract contained small rubber particle proteins. After precipitation, the crude protein extract was obtained, which included:
[0049] S31. Add ammonium sulfate to the crude protein extract and shake. Centrifuge to collect and transfer the protein precipitate between the upper organic phase and the lower aqueous phase.
[0050] S32. Add ultrapure water to disperse and wash the protein precipitate to remove salt.
[0051] S33. Dissolve the desalted protein precipitate to obtain crude protein extract.
[0052] Transfer the clarified crude protein extract to a new centrifuge tube. Then, add 4 M ammonium sulfate at 10-100% of the extract volume. Place the centrifuge tube on a shaker to precipitate the protein. After centrifugation, the protein precipitate will be located between the organic and aqueous phases. Transfer the protein precipitate (intermediate phase) at the interface to a new centrifuge tube and centrifuge again to collect the precipitate. Add ultrapure water at 4-20 times the precipitate volume to disperse and wash the precipitate. Then, centrifuge to collect the precipitate to remove salts. Alternatively, the protein precipitate can be dissolved in a solution containing urea or detergent first, then precipitated with an organic solvent to remove salts and other impurities. The desalted protein can then be dissolved in a suitable solvent.
[0053] S4. Separate the crude protein extract by ion exchange chromatography and collect the main elution peak;
[0054] Ion exchange chromatography can be used to separate crude protein extracts, either through anion exchange or cation exchange chromatography.
[0055] If separating crude protein extracts by anion exchange chromatography, use a strong anion exchange column (Q, quaternary ammonium resin) or a weak anion exchange column. The mobile phase includes: A, 3-9 M urea, 10-50 mM Tris-HCl, pH 6-9; B, 3-9 M urea, 10-50 mM Tris-HCl, 1 M sodium chloride, pH 6-9. After loading the sample, wash with 80 mM NaCl for two column volumes to remove some non-target proteins, then use a linear gradient elution. The elution range for the target protein (i.e., small rubber particle protein) is 80-200 mM NaCl.
[0056] If crude protein extract is separated by cation exchange chromatography, the mobile phase includes: A: 3-9 M urea, 10-50 mM acetic acid or succinic acid, citric acid, lactic acid, pH 2-4.2; B: 3-9 M urea, 10-50 mM acetic acid or succinic acid, citric acid, lactic acid, 1 M sodium chloride, pH 2-4.2.
[0057] S5, the main elution peak of the precipitation ion exchange contains proteins, with small rubber particle proteins as the main component;
[0058] In one example, precipitating the protein contained in the main elution peak of ion exchange involves the following steps:
[0059] S51. Mix the ion exchange main elution peak components, ammonium sulfate, and alcohol reagent, centrifuge, and then transfer the protein precipitate from the intermediate layer.
[0060] S52. Disperse the protein precipitate in ultrapure water, wash away the salt, and centrifuge again to collect the protein precipitate.
[0061] This invention precipitates proteins contained in the main elution peak of ion exchange using a three-phase method. The volume ratio of eluent, 4 M ammonium sulfate, and alcohol reagent is 1:(0.1-0.5):(0.2-2). After adding the eluent, ammonium sulfate, and alcohol reagent to a centrifuge tube according to the above ratio, mix on a shaker for 5-60 min, then centrifuge at 400-15000 rpm. The resulting intermediate layer is the protein precipitate. Transfer the protein precipitate (intermediate phase) at the two-phase interface to a smaller centrifuge tube using a pipette, centrifuge again to remove the liquid carried over during transfer, and obtain the protein precipitate. Add 4-20 times the volume of ultrapure water to the precipitate, disperse the precipitate by pipetting, wash away salts, and then centrifuge to collect the protein precipitate.
[0062] S6. The proteins contained in the main elution peak of ion exchange are further separated by gel filtration chromatography to obtain purified small rubber particle proteins.
[0063] In one example, the proteins contained in the ion-exchange main elution peak were further separated by gel filtration chromatography to obtain purified small rubber particle proteins, including:
[0064] S61. Dissolve the protein precipitate in the mobile phase, perform gel filtration chromatography and collect the eluent, wherein the mobile phase includes: 1) 50-250 mM sodium chloride, 2) 3-9M urea or 0.2-2% detergent that has no UV absorption at 280 nm wavelength;
[0065] S62. Precipitate the protein in the main elution fraction and desalt it to obtain purified small rubber particle protein.
[0066] The protein precipitate obtained in step S5 is dissolved in the mobile phase. Since small rubber particle proteins may self-aggregate and precipitate, 3-9M urea or detergent Brij35 (Brij35 has no UV absorption at 280 nm and is therefore compatible with the chromatography detector) is added to the mobile phase to ensure the protein remains dissolved and does not precipitate during chromatography. The eluent is collected; the main elution peak is small rubber particle protein. The collected eluent is precipitated using the aforementioned three-phase method. The collected eluent, 4M ammonium sulfate, and alcohol reagent are added to a centrifuge tube at a ratio of 1:(0.1-0.5):(0.2-2), mixed on a shaker for 5-60 min, and then centrifuged at 400-15000 rpm. The protein precipitate at the two-phase interface (intermediate phase) is transferred to a smaller centrifuge tube using a pipette, and centrifuged again to remove the liquid carried over during transfer. The resulting protein precipitate is washed with water to remove salt, yielding the purified single protein—small rubber particle protein.
[0067] This invention first collects latex from rubber trees, adds an alkaline buffer solution to stabilize the latex, adds a strong anionic detergent and an organic solvent to selectively precipitate most proteins in the latex except for small rubber particle proteins, and simultaneously precipitates solid substances mainly composed of rubber hydrocarbons; the solid substances are collected by centrifugation, and the clarified liquid portion, i.e., crude protein extract, is transferred. Ammonium sulfate is added to the crude protein extract and shaken to precipitate the proteins, and the precipitate is the crude protein extract; the crude protein extract is separated by ion exchange chromatography, and the main elution peak is collected; the proteins contained in the main elution peak are then precipitated, with small rubber particle proteins being the main component; finally, the proteins contained in the main elution peak are further separated by gel filtration chromatography to obtain purified small rubber particle proteins. This invention directly extracts and purifies small rubber particle proteins from rubber tree latex (without prior separation of the small rubber particles). It uses a strong anionic detergent (low concentration) and organic solvents to precipitate and remove most latex proteins, retaining only a few proteins, primarily composed of small rubber particle proteins, thus reducing the complexity of the protein sample before column chromatography purification. A three-phase precipitation method is used for both protein extraction and chromatographic fraction concentration, simplifying the operation and significantly saving time. Therefore, the extraction and purification method of this invention efficiently and time-savingly separates target and non-target proteins.
[0068] The method for extracting and purifying small rubber particle proteins according to the present invention will be described in detail below with reference to specific operating steps.
[0069] A method for extracting and purifying small rubber particle proteins from Brazilian rubber tree latex includes the following steps:
[0070] 1. Tap rubber trees in the rubber plantation and collect latex. When collecting latex, place the centrifuge tubes receiving the latex on ice to prevent protein degradation. Take 15-20 mL of latex into a 50 mL centrifuge tube and add Tris-HCl buffer (pH>7) to make the latex alkaline to maintain its stability.
[0071] 2. Add a strong anionic detergent, such as sodium dodecyl sulfate or sodium dodecyl sulfonate solution or dry powder, to the centrifuge tube to a final concentration between 0.05% and 2%, and place the centrifuge tube on ice and shake for 10-60 minutes.
[0072] 3. Add an organic solvent (such as acetonitrile) to the centrifuge tube. The volume ratio of latex to organic solvent should be 1:(0.3-2). Place the centrifuge tube on a shaker and shake at maximum speed for 10-60 minutes to precipitate the natural rubber, other solid substances, and most of the protein in the latex, while retaining a small amount of protein, mainly small rubber particles. Centrifuge at 5000 rpm for 10 minutes to clarify the mixture in the centrifuge tube. Retain the clear liquid at the top of the centrifuge tube (referred to as crude protein extract).
[0073] 4. Transfer the clarified crude protein extract to a new centrifuge tube. Add ammonium sulfate solution (4 M) at 10-100% of the crude protein extract volume. Place the centrifuge tube on a shaker and shake for about 10 minutes to allow the protein to precipitate. Centrifuge at 5000 rpm for 10 minutes. The protein precipitate will appear at the aqueous-organic phase interface. Transfer the protein precipitate at the interface to a smaller centrifuge tube using a pipette and centrifuge again to collect the protein precipitate. Add 4-20 times the volume of ultrapure water to disperse and wash the protein precipitate. Then centrifuge to collect the protein precipitate to remove salts (alternatively, the precipitate can be dissolved in urea or a solution containing detergent first, and then the protein can be precipitated with an organic solvent to remove salts and other impurities). The desalted protein can be dissolved in a suitable solution.
[0074] 5. Anion exchange chromatography separation of crude protein extract: Separation is performed using a strong anion exchange column (Q, quaternary ammonium resin) or a weak anion exchange column. The mobile phases used are as follows: Mobile phase A (e.g., 3-9 M urea, 10-50 mM Tris-HCl, pH 6-9), Mobile phase B (e.g., 3-9 M urea, 10-50 mM Tris-HCl, pH 6-9, 1 M NaCl). After loading the sample, wash with 80 mM NaCl for two column volumes to remove some non-target proteins. Use a linear gradient elution; the elution solution for the target protein should have a concentration of 80-200 mM NaCl.
[0075] 6. Precipitate the protein contained in the main elution peak of anion exchange using the three-phase method: Add protein solution, 4 M ammonium sulfate and tert-butanol (or n-butanol or pentanol) at a volume ratio of 1:(0.1-0.5):(0.2-2), mix on a shaker for 5-60 min and then centrifuge (400-15000 rpm, 5-10 min). The resulting intermediate layer is the protein precipitate. Transfer the precipitate at the interface to a smaller centrifuge tube using a pipette and centrifuge again to remove the liquid carried out during the transfer and obtain the protein precipitate. Add 4-20 times the volume of ultrapure water to the precipitate, disperse the precipitate by pipetting, wash away the salt, and then centrifuge to collect the protein precipitate.
[0076] 7. Separate the protein from the main anion exchange elution peak using gel filtration chromatography: Dissolve the protein precipitate obtained in step 6 in the mobile phase (containing 3-9M urea or 0.2-2% Brij 35, 50-250 mM NaCl, pH 7-10). Collect the elution fraction; the main elution peak is small rubber particle protein. Precipitate it using the aforementioned three-phase method, and after desalting, obtain purified small rubber particle protein.
[0077] like Figure 2 The image shows SDS gel electrophoresis of protein samples during the extraction of latex protein and purification of small rubber particle protein. Lane M represents the protein molecular weight standard.
[0078] Lane 1 contains a protein sample (crude protein extract) extracted from latex using the method of this invention. The thickest band is composed of small rubber particle protein (confirmed by mass spectrometry analysis).
[0079] Lane 2: The main elution peak protein obtained after anion exchange separation of crude protein extract, mainly containing the target protein (approximately 24 kDa) and a small molecular weight protein of approximately 10 kDa.
[0080] In lane 3, the anion exchange main elution peak protein was further purified by gel filtration chromatography to obtain single small rubber-like protein particles.
[0081] Figure 3The column chromatography purification process of small rubber particle protein is shown in Figure A. The crude protein extract is separated by anion exchange, and the chromatographic curve represents the separation process of anion exchange chromatography (the gray area indicates the main elution peak). The broken line represents the conductivity (indicating the change in NaCl concentration). Figure B shows the separation process of gel filtration chromatography. The main elution peak of ion exchange is further subjected to gel filtration chromatography to obtain purified small rubber particle protein (marked by the gray area).
[0082] according to Figure 2 and Figure 3 As a result, the present invention has the following advantages:
[0083] (1) By using strong anionic detergent (low concentration) and organic solvent to precipitate and remove most of the protein from rubber tree latex, small rubber particle protein is selectively retained, simplifying the complexity of the sample and thus improving the efficiency of subsequent extraction and purification processes.
[0084] (2) The separation effect of target protein (small rubber particle protein) and non-target protein is good, and the process is easy to operate and takes little time.
[0085] It should be understood that the various forms of processes shown above can be used to rearrange, add, or delete steps. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this disclosure can be achieved, and this is not limited herein.
[0086] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means two or more, unless otherwise explicitly specified.
[0087] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.
Claims
1. A method for extracting and purifying small rubber particle proteins from Brazilian rubber tree latex, characterized in that, The method includes: An alkaline buffer solution is added to the rubber latex to stabilize it; the alkaline buffer solution is a Tris-HCl buffer solution with pH > 7. A strong anionic detergent and an organic solvent are added sequentially to precipitate most of the proteins (excluding small rubber particle proteins) and solid substances mainly composed of rubber hydrocarbons in the latex. The solids are collected by centrifugation, and the clarified liquid is transferred to obtain a crude protein extract. The strong anionic detergent is selected from sodium dodecyl sulfate or sodium dodecyl sulfonate, and the concentration of the strong anionic detergent is 0.05%-2%. The organic solvent is acetonitrile. The volume ratio of rubber latex to organic solvent is 1:0.3 to 1:
2. Ammonium sulfate was added to the crude protein extract and shaken to precipitate the protein. The protein precipitate was collected by centrifugation to obtain the crude protein extract. The crude protein extract was separated by ion exchange chromatography, and the main elution peak was collected. Ion exchange chromatography was performed using either anion exchange or cation exchange. For anion exchange chromatography, the mobile phase was: A: 3-9 M urea, 10-50 mM Tris-HCl, pH 6-9; B: 3-9 M urea, 10-50 mM Tris-HCl, 1 M sodium chloride, pH 6-9. For cation exchange chromatography, the mobile phase included: A: 3-9 M urea, 10-50 mM acetic acid or succinic acid, citric acid, lactic acid, pH 2-4.2; B: 3-9 M urea, 10-50 mM acetic acid or succinic acid, citric acid, lactic acid, 1 M sodium chloride, pH 2-4.
2. The proteins contained in the main elution peak of ion exchange are mainly composed of small rubber particle proteins. The precipitation of these proteins using a three-phase method includes: mixing the ion exchange main elution peak components, 4 M ammonium sulfate, and an alcohol reagent; centrifuging and transferring the protein precipitate from the intermediate layer; dispersing the protein precipitate in ultrapure water, washing away salts, and centrifuging again to collect the precipitate; the volume ratio of the ion exchange main elution peak components, 4 M ammonium sulfate, and the alcohol reagent is 1:(0.1-0.5):(0.2-2); the alcohol reagent is selected from tert-butanol, n-butanol, or pentanol. The proteins contained in the main elution peak of ion exchange were further separated by gel filtration chromatography to obtain purified small rubber particle proteins. Specifically, the protein precipitate was dissolved in the mobile phase, gel filtration chromatography was performed, and the elution fraction was collected. The mobile phase included: 1) 50-250 mM sodium chloride, 2) 3-9M urea or 0.2%-2% of Brij35 detergent which has no UV absorption at 280 nm wavelength; the proteins in the main elution fraction were precipitated and desalted to obtain purified small rubber particle proteins.
2. The method according to claim 1, characterized in that, After adding strong anionic detergents and organic solvents, most of the proteins (excluding small rubber particle proteins) and solid substances mainly composed of rubber hydrocarbons in the latex are precipitated, including: Add a strong anionic detergent to the rubber latex to a concentration of 0.5%-2%, and incubate on ice with shaking for 10-60 minutes; Add an organic solvent and shake for 10-60 minutes to precipitate most of the proteins in the latex except for small rubber particle proteins, and at the same time precipitate solid substances in the latex mainly composed of rubber hydrocarbons.
3. The method according to claim 1, characterized in that, Protein components are precipitated from the crude protein extract to obtain a crude protein extract, including: Add ammonium sulfate to the crude protein extract and shake. Centrifuge to collect and transfer the protein precipitate between the upper organic phase and the lower aqueous phase. Add ultrapure water to disperse and wash the protein precipitate to remove salts; The desalted protein precipitate is then dissolved to obtain crude protein extract.