Method for screening protein allosteric modulators by observing size change of micron-sized vesicles through light microscope
The size changes of micron-scale protein vesicles were observed by optical microscope, combined with the microfluidic chip method and magnetic stirring method, the problem of high cost and complexity of screening protein allosteric regulators in the prior art was solved, and efficient and simple The screening process broadened the application direction and promoted the development of targeted drugs.
Patent Information
- Application Number
- CN202411407563.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2024-12-31
AI Technical Summary
The existing screening methods for protein allosteric regulators are expensive in detection instruments and complex in fluorescent labeling methods, which limits large-scale screening and drug development, especially in cancer treatment, with limited number of protein allosteric regulators used.
The size changes of micron-scale protein vesicles were observed by optical microscope to screen protein allosteric regulators, protein self-assembly formed vesicles, and uniform vesicles were prepared by microfluidic chip method and magnetic stirring method, and stable combined with crosslinking agent. Vesicle structure, real-time visualization of in-situ dimensional tracer.
The screening process is simplified, the cost is reduced, the efficiency is improved, and the research and application direction of protein allosteric regulators is broadened, which is helpful for targeted drug development and achieves efficient screening of protein allosteric regulators.
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Figure CN119223963A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for screening protein allosteric regulators, in particular to a method for screening protein allosteric regulators by means of a microscope and vesicle size changes, and belongs to the technical field of biomedicine. Background Art
[0002] Proteins are the direct executors of various life activities of organisms. The realization of various specific functions is closely related to the specific structure of specific proteins, especially the three-dimensional conformation of proteins. However, the conformation of proteins is not static. When certain substances bind to proteins, the spatial structure of proteins will change, thereby changing the activity and biological function of proteins. These substances are protein allosteric regulators. This characteristic of protein allosteric regulators has been applied to drug development: according to the binding site, drugs based on protein allosteric regulators include orthosteric drugs and allosteric drugs; according to the function, protein allosteric regulators include protein inhibitors and protein activators.
[0003] Taking cancer treatment as an example, allosteric proteins that have important biological functions for tumor cells are a large class of anticancer drug targets, such as calmodulin (CaM), physiological activity regulating enzymes, etc. Good progress has been made in the search for allosteric regulators of these proteins and the screening of targeted drugs. For example, the calmodulin inhibitor trifluoperazine has a positive therapeutic effect on lung cancer. However, the number of protein allosteric regulators that have been developed for cancer treatment is very limited, and the existing protein allosteric regulator screening methods have disadvantages such as expensive detection instruments and complex fluorescent labeling methods, which seriously limit the large-scale screening of protein allosteric regulators and drug development.
[0004] Vesicles are a type of molecular ordered assembly with a closed double-layer structure that is spontaneously formed when amphiphilic molecules are dispersed in water. They are used in the fields of cell membrane simulation, drug transportation, targeted release, etc. For example, Chinese patent CN110563829A provides a method for constructing liposome vesicles based on the biochemical characteristics of RA1, promoting membrane fusion, and spatial tubular morphology, which can form giant vesicle monomers with a scale of microns, and have the potential to be used in the fields of biopharmaceuticals, drug target positioning, tumor treatment technology, etc. However, the current direction of vesicle research and application is relatively fixed, and most of the research objects are liposome vesicles or surfactant vesicles. Summary of the invention
[0005] Purpose of the invention: The purpose of the present invention is to provide a method for screening protein allosteric regulators, wherein the screening criterion is the size change of micron-scale protein vesicles, and this change can be detected using an optical microscope.
[0006] Technical solution: The present invention provides a method for screening protein allosteric regulators by observing the size changes of micron-scale vesicles through light microscopy, comprising the following steps: In the first step, the proteins are assembled into micrometer-sized protein vesicles to obtain a solution containing protein vesicles; The second step is to observe the protein vesicles in the solution under an optical microscope and record the original size of the protein vesicles; The third step is to add the substance to be screened into the solution and observe the protein vesicles under an optical microscope. The substance to be screened that changes the size of the protein vesicles compared with the original size is the protein allosteric regulator.
[0007] By utilizing protein self-assembly, single-molecule proteins can be used as building blocks and arranged in order to form protein vesicles. Thus, the conformational changes of single-molecule proteins at the nanoscale can be magnified into changes in the size of protein vesicles at the micron level. Therefore, by observing protein vesicles with an optical microscope, it is possible to infer whether the protein has undergone conformational changes, thereby realizing the screening of protein allosteric regulators. The single-molecule protein can be purchased or made by oneself, the latter of which includes conventional means such as constructing a target gene expression vector and an engineered bacterium, culturing the engineered bacterium, expressing protein monomers, and purifying them.
[0008] In the first step, the solution containing the protein vesicles can also be dialyzed to transfer the protein vesicles to the aqueous phase, thereby removing the surfactant and oily substances in the system to prevent them from affecting the interaction between the protein allosteric regulator and the protein.
[0009] Preferably, in the first step, the protein is an allosteric protein.
[0010] Preferably, the allosteric protein comprises calmodulin (CaM) or Src homology 2 domain-containing protein tyrosine phosphatase (SHP2).
[0011] Preferably, a protein allosteric regulator in an amount not less than 1 / 40 of the allosteric protein equivalent can change the size of the protein vesicles.
[0012] Preferably, in the first step, a cross-linking agent is used to stabilize the structure of the protein vesicle. In order to ensure the integrity of the protein vesicle and avoid rupture of the vesicle due to protein conformational changes, the protein on the surface of the vesicle can be cross-linked. A suitable cross-linking agent can maintain the structural stability of micron-sized protein vesicles and does not affect the change in vesicle size caused by protein conformational changes. For example, glutaraldehyde can be used as a cross-linking agent to react with the exposed amino molecules on the surface of the protein to produce a Schiff base reaction, thereby stabilizing the protein vesicle structure.
[0013] Preferably, in the first step, the method for assembling the micron-sized protein vesicles comprises: firstly mixing the protein with a surfactant to form an amphiphilic protein-surfactant complex; and then assembling the complex at an oil-water interface to form micron-sized protein vesicles.
[0014] A surfactant with an opposite charge to that of the protein in a neutral buffer is selected, and the two are coupled to form a complex by electrostatic interaction. For example, in a neutral buffer, there are a large number of negatively charged amino acid residues on the surface of calmodulin, and a positively charged surfactant can be used to construct a complex with it, such as the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). The success of the complex construction can be determined by methods such as protein surface Zeta potential analysis.
[0015] The hydrophilic protein part of the amphiphilic complex tends to aggregate in the water phase, and the lipophilic surfactant part tends to aggregate in the oil phase, thereby assembling to form protein vesicles at the oil-water interface. The oily substance is such as 2-ethylhexanol. The size of the vesicle can be regulated by conditions such as the protein concentration, protein / surfactant ratio or oil-water ratio in the system. The size uniformity of the vesicle can be achieved by high-speed stirring or ultrasound and optimizing assembly conditions.
[0016] Preferably, the method of assembling the complex at the oil-water interface to form micron-sized protein vesicles includes a magnetic stirring method or a microfluidic chip method. Vesicle self-assembly can be achieved by controlling conditions such as stirring speed or microfluid flow rate.
[0017] Preferably, the structure of the chip used in the microfluidic chip method includes an oil phase inlet 1, an aqueous phase inlet 2, a collection port 3 and a flow channel, the complex is added to the aqueous phase inlet 2, the oily substance is added to the oil phase inlet 1, the flow rate is controlled, the complex and the oily substance are assembled into micron-sized protein vesicles at the intersection of the flow channel, and the protein vesicles are collected at the collection port 3. The size uniformity of the protein vesicles obtained by the microfluidic chip method is better. A cross-linking agent can be placed in advance in the container for collecting vesicles, or a cross-linking agent inlet and a corresponding flow channel can be added on the chip, and the flow channel length can be set according to the needs of the cross-linking reaction.
[0018] Preferably, the protein is calmodulin, the injection speed of the complex is 10-22 μL / min, and the injection speed of the oily substance is 1-10 μL / min. By regulating the injection speed of the microfluidic system, protein vesicles of different sizes and uniformity can be constructed.
[0019] Preferably, the protein allosteric regulators include protein inhibitors and protein activators.
[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The use of an optical microscope to perform real-time visual in situ size tracing of vesicles can realize the screening of protein allosteric regulators without the need for precision instruments or fluorescent labels. The method is simple and efficient, which broadens the direction of vesicle research and application and contributes to the development of targeted drugs with protein allosteric regulators as the core; 2. The use of a microfluidic chip method can accurately prepare micron-sized protein vesicles of uniform size, which is conducive to the screening of protein allosteric regulators based on changes in vesicle size. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 are the size exclusion chromatograms of the purified proteins (a is CaM; b is SHP2); Figure 2 Schematic diagram of a microfluidic chip for constructing micron-sized protein vesicles; Figure 3 This is the morphology of calmodulin vesicles under light microscopy; Figure 4 The morphological changes of vesicles before and after treatment with four different CaM allosteric regulators under light microscopy (a / c / e / g are before treatment; b / d / f / h are after treatment); Figure 5 The morphology of vesicles before and after treatment with warfarin, a non-CaM allosteric modulator, under a light microscope (a is before treatment; b is after treatment); Figure 6 The morphological changes of vesicles before and after treatment with three different SHP2 allosteric regulators under light microscopy (a / b / d are before treatment; e / f / g are after treatment); Figure 7 The morphological changes of vesicles before and after treatment with non-SHP2 allosteric regulators under light microscopy (a is before treatment; b is after treatment); Figure 8 The surface pressure and area curves of the vesicles before and after trifluoperazine treatment; Fig. 9 The surface pressure and area curves corresponding to the vesicles before and after treatment with different concentrations of trifluoperazine. DETAILED DESCRIPTION
[0022] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.
[0023] The embodiment uses two methods, magnetic stirring method and microfluidic chip method, and two proteins, CaM and SHP2, to construct micron-scale protein vesicles. Both proteins are from recombinant expression and purified and collected. The protein purification results are detected by size exclusion chromatography (SEC), see Figure 1 The microfluidic chips used were purchased from Anhui Chixin Biotechnology Co., Ltd.
[0024] The corresponding micron-sized protein vesicles were treated with known CaM allosteric modulators, CaM non-allosteric modulators and SHP2 allosteric modulators, and the changes in vesicle size were observed with an optical microscope to verify the feasibility of the method of screening protein allosteric modulators by observing the changes in micron-sized vesicle size under an optical microscope. On this basis, the screening method provided by the present invention was further characterized by analyzing the relationship between the surface pressure and the surface area of the vesicles.
[0025] Example 1 Assembling micron-sized protein vesicles using a microfluidic chip method This example uses a microfluidic chip method to assemble micron-sized CaM vesicles, and the specific steps are as follows: 1. Preparation of complex: The concentration of CaM collected by the purifier was adjusted to 10 μM and filtered with a 0.22 μM water filter. Prepare 4 mM CTAB and filter with a filter. Place the filtered CaM and CTAB on a magnetic stirrer and stir thoroughly, set the speed to 700 rpm, and stir for 1 h. The buffer solution is a 10 mM HEPES solution containing 0.5 mM CaCl2, pH 7.5. The isoelectric point of the CaM is about 4.0. In a neutral HEPES buffer solution, there are a large number of negatively charged amino acid residues on the surface of CaM. Through electrostatic interaction forces, the positively charged surfactant and the negatively charged CaM combine to form a complex of protein and surfactant. The Zeta potential of the protein surface before and after binding was analyzed using a Zeta potential meter to verify the construction results of the protein-surfactant complex.
[0026] 2. Protein vesicle assembly: Microfluidic chips such as Figure 2 , including oil phase inlet 1, water phase inlet 2, collection port 3 and flow channel. Turn on the microfluidic instrument and install the microfluidic chip. Add 1 mL of 6% 2-ethylhexanol solution to the syringe corresponding to the oil phase inlet 1, and add 1 mL of the complex to the syringe corresponding to the water phase inlet 2. After the chip is hydrophilic, the injection speed is 10 μL / min for the complex and 10 μL / min for the oil phase, which can stably generate water-in-oil droplets, that is, micron-sized protein vesicles. Place a 96-well plate at the collection port 3 of the chip to collect the generated protein vesicles. Glutaraldehyde is pre-added to each well to cross-link the proteins on the surface of the collected vesicles. Collect 100 μL of the solution containing vesicles in each well.
[0027] The collected micron-sized CaM vesicles were characterized using an optical microscope. Figure 3 .
[0028] Based on the above method, the injection speed of the complex was changed to 22 μL / min and the oil phase was changed to 1 μL / min. The experiment showed that this method can produce micron-sized protein vesicles.
[0029] Based on the above method, the injection speed of the complex was changed to 22 μL / min and the oil phase was changed to 10 μL / min. The experiment showed that this method can produce micron-sized protein vesicles.
[0030] Based on the above method, the injection speed of the complex was changed to 44 μL / min and the oil phase was changed to 1 μL / min. The experiment showed that this method could not produce micron-sized protein vesicles.
[0031] On the basis of the above method, the method of adding the crosslinking agent is changed, specifically, a crosslinking agent inlet and a corresponding flow channel are added on the microfluidic chip, and the protein vesicles are collected after completing the crosslinking on the chip. According to the test, the method can produce micron-sized protein vesicles.
[0032] Example 2 Assembly of micron-sized protein vesicles using magnetic stirring In this example, the magnetic stirring method was used to construct micron-sized CaM vesicles and SHP2 vesicles, respectively. The specific steps were as follows: 1. Stir 10 μM protein and 4 mM CTAB with a magnetic stirrer at 700 r for 1 h; 2. After step 1, add 6% 2-ethylhexanol and stir for 4 h; 3. After step 2, add glutaraldehyde; 4. Transfer the vesicles to the aqueous phase by multiple dialysis to remove CTAB and 2-ethylhexanol.
[0033] Using an optical microscope, the micron-sized SHP2 vesicles were observed. Figure 6 As shown in a, b, d.
[0034] Example 3 Verification of the method of screening protein allosteric modulators by observing micron-sized vesicles through light microscopy Micron-sized CaM vesicles were treated with known CaM allosteric modulators and non-allosteric modulators. The CaM allosteric modulators used were trifluoperazine, chlorpromazine, chlorprothixene and thioridazine, and the non-allosteric modulator was warfarin. The specific steps were: observe the vesicle morphology in the 96-well plate with a light microscope and record the size, add the above substances equivalent to CaM to different wells for co-incubation, and observe and record the vesicle size. The results are shown in Figure 4 and Figure 5 The results showed that the inhibition of CaM molecules by allosteric modulators caused the size of micron-sized protein vesicles to shrink, while non-allosteric modulators did not change the size of micron-sized protein vesicles.
[0035] The micron-sized SHP2 vesicles were treated with known SHP2 allosteric modulators. The SHP2 allosteric modulators used were cefsulodin sodium, cryptotanshinone and SHP099, and the non-allosteric modulator was thioridazine. The specific steps were: add a solution containing vesicles to a well of a 96-well plate, observe the vesicle morphology and record the size under a light microscope, then add the above substances equivalent to SHP2 and incubate together, and observe and record the vesicle size. The results are shown in Figure 6 and Figure 7 The results showed that the inhibition of SHP2 molecules by allosteric modulators cefsulodin sodium, cryptotanshinone and SHP099 caused the size of micron-sized protein vesicles to shrink, while non-allosteric modulators did not change the size of micron-sized protein vesicles.
[0036] Example 4 Investigation of the mechanism of protein vesicle size change In order to further explore the mechanism of vesicle size change, the Langmuir-Blodgett (LB) membrane multifunctional film drawing machine was used to analyze the surface tension before and after vesicle deformation. 100 μL of CaM vesicle solution without trifluoperazine treatment and 100 μL of CaM vesicle solution treated with trifluoperazine were evenly dropped into the Langmuir-Blodgett (LB) membrane multifunctional film drawing machine, and the relationship between the vesicle surface pressure and surface area was analyzed. The vesicle surface pressure and area curve was drawn, as shown in Figure 2. Figure 8 This verifies that the change in vesicle size is caused by the change in vesicle surface tension.
[0037] 1 / 40 equivalent, 1 / 20 equivalent, 1 / 10 equivalent, and 1 / 5 equivalent of trifluoperazine were added to the solution containing CaM vesicles collected in the 96-well plate and incubated together. 100 μL of each solution was evenly dropped into the LB film multifunctional film drawing machine to analyze the relationship between the surface pressure and surface area of the vesicles and draw the curve of the surface pressure and area of the vesicles, as shown in Figure 2. Fig. 9 The results showed that the lower the concentration of the allosteric modulator, the smaller the effect on the surface tension of the vesicles, but even 1 / 40 CaM equivalent of trifluoperazine could change the surface tension of the vesicles, indicating the sensitivity of the system.
Claims
1. A method for screening protein allosteric regulators by observing the size changes of micron-sized vesicles through light microscopy, characterized in that: The following steps are involved: In the first step, the proteins are assembled into micrometer-sized protein vesicles to obtain a solution containing protein vesicles; The second step is to observe the protein vesicles in the solution under an optical microscope and record the original size of the protein vesicles; The third step is to add the substance to be screened into the solution, and observe the protein vesicles under an optical microscope. The substance to be screened that changes the size of the protein vesicles compared with the original size is the protein allosteric regulator.
2. The method according to claim 1, characterized in that In the first step, the protein is an allosteric protein.
3. The method according to claim 2, characterized in that The allosteric protein includes calmodulin or a Src homology 2 domain-containing protein tyrosine phosphatase.
4. The method according to claim 3, characterized in that A protein allosteric regulator at least 1 / 40 equivalent of the allosteric protein can change the size of the protein vesicles.
5. The method according to claim 1, characterized in that In the first step, a cross-linking agent is used to stabilize the structure of the protein vesicles.
6. The method according to claim 1, characterized in that In the first step, the method for assembling the micron-sized protein vesicles includes: firstly mixing the protein with the surfactant to form an amphiphilic protein-surfactant complex; and then assembling the complex at the oil-water interface to form the micron-sized protein vesicles.
7. The method according to claim 6, characterized in that The method of assembling the complex at the oil-water interface to form micron-sized protein vesicles includes a magnetic stirring method or a microfluidic chip method.
8. The method according to claim 7, wherein the structure of the chip used in the microfluidic chip method comprises an oil phase inlet (1), a water phase inlet (2), a collection port (3) and a flow channel, characterized in that: The complex is added to the water phase inlet (2), and the oily substance is added to the oil phase inlet (1). The flow rate is controlled so that the complex and the oily substance are assembled into micrometer-sized protein vesicles at the intersection of the flow channel, and the protein vesicles are collected at the collection port (3).
9. The method according to claim 8, characterized in that The protein is calmodulin, the injection speed of the complex is 10-22 μL / min, and the injection speed of the oily substance is 1-10 μL / min.
10. The method according to claim 1, characterized in that The protein allosteric regulators include protein inhibitors and protein activators.