Marking and purifying method of exosome membrane protein

Abstract

The invention discloses a marking and purifying method of exosome membrane protein, and belongs to the technical field of membrane protein. Comprising the following steps: labeling exosome membrane protein by using N-hydroxy sulfosuccinimide ester biotin, removing free biotin by using an ultracentrifugation method, then cracking an exosome structure, and finally purifying the labeled exosome membrane protein by using streptavidin agarose gel. The invention provides an innovative experimental scheme for separation and purification of exosome membrane protein markers. The method comprises the following steps: firstly, efficiently reacting N-hydroxy sulfosuccinimide ester biotin with primary amino (-NH2) of exosome membrane surface membrane protein to form stable amido bond marked exosome membrane surface membrane protein, and then removing free biotin by utilizing an ultracentrifugation method; and purifying the labeled membrane protein by utilizing efficient combination of biotin and affinity streptavidin, wherein the purified sample can be used for protein mass spectrometry analysis or other molecular biology function researches.

Description

Technical Field

[0001] The present invention relates to the technical field of membrane proteins, and in particular to a method for labeling and purifying exosome membrane proteins. Background Art

[0002] Exosomes, as a type of extracellular vesicles that mediate important functions such as cell-to-cell interactions and communication, have received extensive attention. Exosomes are widely present and distributed in various body fluids. They carry and transmit important signal molecules, forming a new cell-to-cell information transmission system. The exosome membrane structure can carry biologically active substances such as proteins, lipids, and nucleic acids, and transmit them to receptor cells, thereby regulating the physiological or pathological functions of the receptor cells, playing an important role in intercellular information exchange.

[0003] As a natural information carrier, exosomes have the advantages of low immunogenicity, high biocompatibility, and stable existence in the blood. Therefore, exosomes can be used as drug carriers and have great application potential in the medical field. In addition, exosomes usually carry specific receptors of parent cells, have certain targeting, and are helpful for targeted drug delivery. There are more and more related studies on exosomes as drug delivery carriers. However, although exosomes have brought hope in the field of new medicines, exosomes injected in vivo will be enriched in large quantities in immune clearance organs such as the liver, spleen, and lungs, and very few exosomes will eventually play a therapeutic role in the lesion site. In order to enhance the therapeutic effect of exosomes and reduce toxicity to normal cells, it is necessary to improve the ability of exosomes to deliver drugs in a targeted manner. Among them, exosome membrane proteins play a very important role in exosome targeting and recognition with receptor cells. Therefore, it is of great significance to deeply understand the molecular composition of exosome membrane proteins and the targeting function and molecular mechanism of exosomes.

[0004] Membrane proteins refer to a class of proteins contained in biological membranes. As the main embodiment of membrane functions, membrane proteins play a vital role in biological membrane recognition, transport and other functions. According to the different ways in which proteins bind to biological membranes, membrane proteins can be divided into three categories: extrinsic membrane proteins, intrinsic membrane proteins and lipid-anchored membrane proteins. Extrinsic membrane proteins are distributed on the inner and outer surfaces of the membrane, accounting for about 20% to 30% of membrane proteins, mainly on the inner surface, and are mostly water-soluble proteins. They bind to the polar heads of membrane lipid molecules through ionic bonds and hydrogen bonds, or indirectly bind to the membrane through interactions with intrinsic membrane proteins. Lipid-anchored membrane proteins are covalently linked to lipid molecules, which are inserted into the lipid bilayer of the membrane, thereby anchoring the protein to the cytoplasmic membrane. Intrinsic membrane proteins account for about 70% to 80% of membrane proteins, are amphiphilic molecules, and can be embedded in lipid bilayer molecules to varying degrees. Some run through the entire lipid bilayer, with both ends exposed to the inner and outer surfaces of the membrane. This type of membrane protein is also called a transmembrane protein. The part of the intrinsic membrane protein exposed outside the membrane contains more polar amino acids, which are hydrophilic and adjacent to the hydrophilic head of the phospholipid molecule; the membrane protein embedded in the lipid bilayer is composed of some non-polar amino acids, which bind to the hydrophobic tail of the lipid molecule. Intrinsic membrane proteins are usually very tightly bound to the membrane and can only be separated after the membrane structure is destroyed by using detergents. As transmembrane proteins, intrinsic membrane proteins can be divided into three major domains: the extracytoplasmic domain, the transmembrane domain and the intracytoplasmic domain. At present, the field of exosome research mainly focuses on the abundance and types of exosome proteins. There is a lack of research methods for exosome membrane proteins, which to a certain extent limits scientists' understanding of the identification and targeting functions involved in exosome membrane proteins. Summary of the invention

[0005] The purpose of the present invention is to provide a method for labeling and purifying exosome membrane proteins to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a method for labeling and purifying exosome membrane proteins, comprising the following steps:

[0008] Exosome membrane proteins were labeled with N-hydroxysulfosuccinimidyl ester biotin, and free biotin was removed by ultracentrifugation. The exosomes were then lysed and finally purified using streptavidin agarose gel.

[0009] Based on the above technical solution, the present invention has the following technical effects:

[0010] (1) The study of exosome membrane surface proteins is of great significance for understanding their biological functions and developing new diagnostic and therapeutic strategies. However, there is currently a lack of effective technical solutions for the separation and purification of exosome membrane proteins. Therefore, the present invention proposes a technical method for purifying exosome membrane proteins by biotin labeling of exosome membrane proteins. The effect of biotin labeling concentration on experimental results is particularly significant: low concentrations of biotin labeling cannot completely label exosome membrane surface proteins, resulting in insufficient labeling; while excessively high concentrations of biotin labeling may lead to false positive membrane protein results. Therefore, formulating appropriate labeling and purification schemes is crucial for subsequent membrane protein research and can ensure the accuracy of experimental results.

[0011] (2) Sulfo-NHS-Biotin reacts specifically with primary amine groups (-NH2) in proteins through its sulfonic acid NHS active ester to achieve biotinylation labeling of proteins. This process is carried out in physiological to weakly alkaline buffers (pH 7-9) to form stable amide bonds. Due to its water solubility and membrane impermeability, Sulfo-NHS-Biotin is particularly suitable for labeling surface proteins. The labeled proteins can be effectively separated by a biotin-affinity streptavidin separation column. The separation column uses the high affinity between streptavidin and biotin to capture the biotinylated proteins from the mixture. The purified proteins can be used for subsequent analysis and functional studies. The efficient labeling of Sulfo-NHS-Biotin and the purification ability of the biotin affinity separation column make the labeling and purification process of exosome membrane surface proteins more accurate and efficient. This provides a reliable foundation for subsequent membrane surface protein research and helps to deeply understand the role of exosomes in intercellular communication. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 In the figure, A is the immunoblotting result of exosome membrane surface proteins at different biotin labeling concentrations. B is the result of gray value analysis of the protein bands in image A using Image J software.

[0014] Figure 2 The results of purification and verification of biotinylated exosome proteins. A is the verification of silver staining results. B is the verification of Dot Blot results. C is the quantitative analysis of DotBlot verification of biotin-labeled samples in unpurified and residual samples after the first purification. DETAILED DESCRIPTION

[0015] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0016] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0017] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0018] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0019] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0020] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0021] The present invention provides a method for labeling and purifying exosome membrane proteins, comprising the following steps:

[0022] Exosome membrane proteins were labeled with N-hydroxysulfosuccinimidyl ester biotin, and free biotin was removed by ultracentrifugation. The exosomes were then lysed and finally purified using streptavidin agarose gel.

[0023] In some specific embodiments, the method of labeling exosome membrane proteins with N-hydroxysulfosuccinimide ester biotin is: adding NHS-Biotin to the exosome solution for labeling.

[0024] In some specific embodiments, the reaction temperature for labeling exosome membrane proteins with N-hydroxysulfosuccinimide ester biotin is room temperature, the reaction time is 10 min, and the concentration of the exosome solution is 1×10 11 The dosage of NHS-Biotin is 1.5 mg / mL.

[0025] In some specific embodiments, the step of adding a termination reaction solution to terminate the reaction is also included.

[0026] In some specific embodiments, the method for lysing exosomes is: adding 100X protease inhibitor to the exosomes, diluting to 1X, and then adding 10X RIPA solution for lysis.

[0027] In some specific embodiments, the method for purifying the labeled exosome membrane protein using streptavidin agarose gel is as follows: 450 μL of biotin affinity agarose resin is filled into a chromatography column, and after centrifuging the chromatography column at 1000 g, 500 μL of PBS is added to the gel, and centrifuged at 1000×g for 1 minute, and this step is repeated twice;

[0028] Then, the bottom cap was placed on the lower end of the chromatography column, 200 μL of exosome lysate was added to the agarose resin, and the top cap was placed on the column; incubated at room temperature for 360 minutes, during which a rotator was used for rotation mixing;

[0029] After the incubation and binding is completed, remove the upper and lower covers of the chromatography column and centrifuge at 1000g for 1 minute;

[0030] Add 500 μL PBS to the column and mix well, then centrifuge at 1000 g for 1 min;

[0031] Close the lower cover of the chromatography column, collect all the resin into a new centrifuge tube with 300 μL PBS, and add 100 μL protein buffer to the resin;

[0032] To elute the labeled exosomal membrane proteins from the agarose resin, the resin was boiled in protein buffer at 100 °C for 10 min;

[0033] The centrifuge tube was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected, which was the labeled exosome membrane protein.

[0034] The present invention proposes an innovative experimental scheme for the separation and purification of exosome membrane markers. First, N-hydroxysulfosuccinimidyl ester biotin (Sulfo-NHS-Biotin) is used to efficiently react with the primary amine group (-NH2) of the exosome surface membrane protein to form a stable amide bond to label the exosome membrane surface protein, and then ultracentrifugation is used to remove free biotin, and then the labeled membrane protein is purified by efficient binding of biotin affinity streptavidin. The purified sample can be used for protein mass spectrometry analysis or other molecular biological function studies. In summary, the present invention provides an efficient and reliable experimental method for the study of exosome membrane proteins, which helps to promote the functional and structural research of exosome membrane proteins.

[0035] Example 1

[0036] 1. Labeling and purifying exosome membrane proteins using NHS-Biotin

[0037] The applicant team of the present invention used the lentiviral vector plasmid phage-CD63-Nluc-T2A-puroR to construct a cell line stably expressing CD63-Nluc, selected the exosomes secreted by the cell line as the research object, and labeled them with NHS-Biotin, in order to provide technical support for subsequent exosome-related research. 11 NHS-Biotin was added to 2mL of exosome solution with a concentration of 1.5mg / mL for labeling, and the final concentration of NHS-Biotin reached 1.5mg / mL. After the exosomes reacted with EZ-Link Sulfo-NHS biotin for 10 minutes, the reaction was terminated by adding the termination solution. Subsequently, pre-cooled PBS was added to dilute the exosome solution to about 34mL, and then ultracentrifuged at 160,000g for 2 hours to collect the exosomes. 100X protease inhibitors were added to the collected exosomes, diluted to 1X, and then 10X RIPA solution was added to lyse the exosomes. The protein samples after lysis were quantified using the BCA method, and the quantified samples were uniformly diluted to a protein concentration of 100μg / mL.

[0038] Next, add 450 μL of biotin affinity agarose resin (NeutrAvidin Agaroseslurry) to the column. After centrifuging the column at 1000g, add 500 μL of PBS to the gel and centrifuge at 1000×g for 1 minute, repeating this step twice. Then, cover the lower end of the column with a bottom cap, add 200 μL of exosome lysate to the agarose resin, and cover the column with a top cap. Incubate at room temperature for 360 minutes, using a rotator to rotate and mix during the period. After the incubation and binding are completed, remove the upper and lower caps of the column and centrifuge at 1000g for 1 minute. Next, add 500 μL of PBS to the column and mix, and centrifuge at 1000g for 1 minute. Close the lower cap of the column, collect all the resin in a new centrifuge tube with 300 μL PBS, and add 100 μL of protein buffer to the resin. To elute the biotinylated protein from NeutrAvidin agarose resin, the resin was boiled in protein sample buffer at 100°C for 10 minutes. Finally, the centrifuge tube was centrifuged at 5000 rpm for 10 minutes, and the supernatant (purified protein) was collected and stored in a -80°C refrigerator.

[0039] For non-denaturing elution conditions, the protein was biotinylated using a thiol-cleavable biotinylation reagent, NHS-SS-biotin, 10 mM DTT was prepared in 100 mM tris-bis-carbonate (TEAB), and reacted with the resin at 25°C for 45 minutes. The eluted target protein was collected by centrifugation.

[0040] 2. Verification of exosome membrane protein labeling efficiency

[0041] Exosomes were placed in N-hydroxysulfosuccinimidyl ester biotin (NHS-Biotin) solutions of different concentrations (0-2 mg / mL) for labeling. The particle concentration of exosomes was kept at 1×10 11 / mL. At room temperature, the exosomes were reacted with EZ-Link Sulfo-NHS-biotin for 10 minutes to achieve efficient biotinylation of exosome membrane surface proteins. After labeling, the exosomes were collected by ultracentrifugation to remove unbound biotin labeling reagents. The collected exosomes were lysed in RIPA lysis buffer, and the protein was quantified by BCA method. The quantified exosome protein samples were subjected to immunoblotting experiments. The exosome samples were separated by SDS-PAGE electrophoresis, and then the proteins were transferred to PVDF membranes. First, the PVDF membrane was incubated with biotin-labeled antibodies, and then incubated with horseradish peroxidase (HRP)-labeled secondary antibodies. After the HRP-labeled secondary antibodies were bound to the biotin antibodies, the exosome membrane protein labeling levels were detected by chemiluminescent substrates. Then, the grayscale value of the selected area of ​​the image was analyzed using Image J software, and the result graph was drawn using GraphPad Prism software.

[0042] Depend on Figure 1 It can be seen that different biotin labeling concentrations affect the results of labeling surface protein bands. As the biotin labeling concentration gradually increases, the color depth of the protein band shows a clear upward trend. This phenomenon indicates that the abundance of the labeled surface protein gradually increases with the increase of the biotin labeling concentration. When the biotin labeling concentration reaches 1.5 mg / mL, the color depth of the protein band is no longer significantly enhanced, indicating that the labeling saturation concentration has been reached at this time. This shows that at a labeling concentration of 1.5 mg / mL, the exosome membrane protein has been completely labeled with biotin, and further increasing the labeling concentration will not lead to a significant change in protein abundance.

[0043] 3. Verification of the separation and purification efficiency of exosome-labeled membrane proteins

[0044] Biotin-labeled exosomes were added to RIPA lysis buffer for lysis, and the protein samples after lysis were quantified by BCA method. The quantified samples were uniformly diluted to a protein concentration of 100 μg / mL to ensure the standardized operation and comparability of the results of subsequent affinity purification experiments. The chromatography column was inserted into the collection tube, 450 μL of NeutrAvidin agarose resin was added and the chromatography column was covered, and equilibrated with PBS three times to build a stable affinity chromatography system. 200 μL of clarified exosome protein solution was added to the gel, incubated at room temperature for 360 minutes, and mixed using a rotator. The long incubation and mixing ensured that the biotin-labeled protein was fully bound to the NeutrAvidin agarose resin, improving the specificity and efficiency of purification. After the incubation was completed, the top and bottom caps were removed from the column, the chromatography column was centrifuged at 1000g for 1 minute, the effluent was collected, and it was marked. Place the column back into the collection tube, add 500 μL of wash buffer, centrifuge at 1000 g for 1 min, collect the flow-through and rinse the top cover, repeat this step 3 times to thoroughly wash the resin.

[0045] Collect all the resin into a new centrifuge tube with 300 μL PBS, and add 100 μL protein buffer to the resin in the centrifuge tube. In order to elute the biotinylated protein from NeutrAvidin agarose resin, boil the resin and protein buffer mixture in a 100°C water bath for 10 minutes. The high temperature treatment effectively destroys the strong binding between biotin and NeutrAvidin, and promotes the smooth dissociation of biotinylated protein from the resin. After that, centrifuge the centrifuge tube containing the resin at 5000 rpm for 10 minutes, and collect the supernatant after centrifugation. The purified protein samples with and without biotin labeling were verified by silver staining experiments. The purification effect and protein integrity were evaluated by observing the color clarity and distribution of the protein bands. At the same time, the samples were subjected to dot blot experiments with the help of Dot Blot technology. By detecting the signal intensity of HRP labeling, the binding efficiency of biotinylated protein and NeutrAvidin agarose resin and the specificity and accuracy of the purification process were accurately verified. Image J software was used to perform grayscale value analysis on the selected area of ​​the image, and the results were plotted using GraphPad Prism software.

[0046] Figure 2The verification results of the protein purification process are shown. Biotin-labeled exosome membrane proteins are bound to avidin agarose resin to obtain purified exosome proteins. The purified samples were stained by silver staining, and the results showed that only the bands in the biotinylated protein samples were displayed. The purification scheme of the present invention successfully and efficiently purified the biotinylated protein, and no bands of non-biotinylated protein were detected, which confirmed that the purification process had high accuracy and specificity. The Dot Blot experiment further verified the efficient adsorption and purification rate of the scheme. The signal intensity generated after the biotinylated protein combined with HRP was significant, and there was no obvious nonspecific binding. Through the visualization of gray value analysis, the adsorption purification efficiency reached 98%, indicating that the purification adsorption efficiency was high and the membrane surface protein could be effectively enriched. The efficient adsorption performance of this experimental scheme laid a good foundation for subsequent mass spectrometry analysis, ensured the accuracy and reliability of mass spectrometry detection, and contributed to the in-depth study of the composition and function of surface proteins.

[0047] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for labeling and purifying exosome membrane proteins, characterized in that: The following steps are involved: Exosome membrane proteins were labeled with N-hydroxysulfosuccinimidyl ester biotin, and free biotin was removed by ultracentrifugation. The exosomes were then lysed and finally purified using streptavidin agarose gel.

2. The labeling and purification method according to claim 1, characterized in that: The method of labeling exosome membrane proteins using N-hydroxysulfosuccinimide ester biotin is: adding NHS-Biotin to the exosome solution for labeling.

3. The labeling and purification method according to claim 2, characterized in that: The reaction temperature for labeling exosome membrane proteins with N-hydroxysulfosuccinimide ester biotin was room temperature and the reaction time was 10 min. The concentration of the exosome solution was 1×10 11 The dosage of NHS-Biotin is 1.5 mg / mL.

4. The labeling and purification method according to claim 2, characterized in that: The step of adding a terminating reaction solution to terminate the reaction is also included.

5. The labeling and purification method according to claim 1, characterized in that: The method for lysing the exosomes is: adding 100X protease inhibitor to the exosomes, diluting to 1X, and then adding 10X RIPA solution for lysing.

6. The labeling and purification method according to claim 1, characterized in that: The method for purifying the labeled exosome membrane protein using streptavidin agarose gel is as follows: 450 μL of biotin affinity agarose resin is filled into a chromatography column, after centrifuging the chromatography column at 1000 g, 500 μL of PBS is added to the gel, and centrifuged at 1000×g for 1 minute, and this step is repeated twice; Then, the bottom cap was placed on the lower end of the chromatography column, 200 μL of exosome lysate was added to the agarose resin, and the top cap was placed on the column; incubated at room temperature for 360 minutes, during which a rotator was used for rotation mixing; After the incubation and binding is completed, remove the upper and lower covers of the chromatography column and centrifuge at 1000g for 1 minute; Add 500 μL PBS to the column and mix well, then centrifuge at 1000 g for 1 min; Close the lower cover of the chromatography column, collect all the resin into a new centrifuge tube with 300 μL PBS, and add 100 μL protein buffer to the resin; To elute the labeled exosomal membrane proteins from the agarose resin, the resin was boiled in protein buffer at 100 °C for 10 min; The centrifuge tube was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected, which was the labeled exosome membrane protein.

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