Protein liquid-liquid phase separation condition screening kit based on biomacromolecule crowding agent and use method

Abstract

The present invention relates to a protein liquid-liquid phase separation condition screening kit based on a biomacromolecule crowding agent and a method of use, with a biomacromolecule crowding agent as a key component, and other additives to form a reagent mixture, forming a protein liquid-liquid phase separation condition screening kit of 96 reagent conditions. When used, the target protein is dissolved in ultrapure water to prepare a protein solution, and is mixed with each screening reagent in the kit in a certain volume ratio to obtain a protein phase separation condition screening array, which is placed in a temperature-controlled box and incubated for a certain period of time and then taken out for observation. When the phase separation phenomenon occurs, the corresponding screening reagent is the chemical condition that can promote the target protein to achieve phase separation. The present invention can conveniently and quickly obtain protein liquid-liquid phase separation conditions. At the same time, it is also possible to obtain conditions that inhibit phase separation, which has important reference value for the research and development of new drugs based on phase separation regulation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a protein liquid-liquid phase separation condition screening kit based on a biomacromolecule crowding agent and a use method. Background Art

[0002] Liquid-liquid phase separation of biomacromolecules has been one of the frontier hotspots in the field of life sciences in recent years, and is crucial in the fields of biology, medicine, biophysics, physical chemistry, etc. Liquid-liquid phase separation is the main driving force for the dynamic assembly of membraneless organelles (nucleoli, nuclear speckles, processed bodies, stress granules, etc.) in organisms, transport channels in nuclear pore complexes, membrane receptor clusters related to signal transduction, and other subcellular structures, as well as biomolecular aggregates. In addition, more studies have shown that the liquid-liquid phase separation of proteins is closely related to the occurrence of diseases (such as TAU, β-amyloid protein and Alzheimer's disease, α-synuclein and Parkinson's disease, hIAPP and type 2 diabetes, FUS protein and amyotrophic lateral sclerosis, etc.). When multivalent proteins interact with each other, they undergo liquid-liquid phase separation to form micron-sized droplets, which can fuse and coalesce. This property is determined by their surface tension, and then further assembled to form aggregates through liquid-solid phase transformation. Liquid-liquid phase separation is a reversible molecular dynamic self-assembly process. Two macromolecular solutions can be reversibly separated, with one part concentrating into a dense phase that usually resembles a droplet and the other forming a dilute liquid phase.

[0003] Phase separation is highly dependent on the concentration of biomacromolecules (such as proteins, RNA, etc.) in the solution, their own physicochemical properties, and environmental conditions (such as temperature, pH, salt ion concentration, salt ion type, etc.). Macromolecular crowding reagents, such as PEG, Dextran, etc., are often used in in vitro phase separation experiments. Macromolecular crowding reagents are used to simulate the environment of high molecular concentration in cells. The crowded environment is more favorable for protein folding, oligomerization, complexation and aggregation. The study of the conditions for the formation of protein liquid-liquid phase separation has important academic value, and obtaining highly repeatable phase separation conditions has important guiding significance for disease treatment strategies and drug development. In order to obtain the conditions for the occurrence of protein liquid-liquid phase separation, a Chinese patent with publication number CN111175514A discloses a method for screening and characterizing proteins with phase separation ability, which provides a high-throughput screening protein phase separation kit, which includes a buffer solution, salt, polymer, positively charged reagent, negatively charged reagent, biological reagent, surfactant, and protein binder. However, the types of polymers in the kit are relatively small. There is currently no phase separation screening kit based on macromolecular crowding reagents.

[0004] Therefore, there is an urgent need in the art to develop more types of kits for screening protein liquid-liquid phase separation conditions. Summary of the invention

[0005] Technical issues to be solved

[0006] In order to avoid the shortcomings of the prior art, the present invention proposes a protein liquid-liquid phase separation condition screening kit based on a biomacromolecule crowding agent and a method of use, which overcomes the shortcomings of the prior art in the protein liquid-liquid phase separation condition screening of a small number of screening reagents.

[0007] Technical Solution

[0008] A protein liquid-liquid phase separation condition screening kit based on a biomacromolecule crowding agent, characterized in that: the screening kit is composed of a collection of a biomacromolecule crowding agent, a pH buffer, a salt and an additive, wherein the characteristic parameters of the biomacromolecule crowding agent are molecular weight and concentration, and the concentration is 5-50% w / v; the concentration of the pH buffer is 50-100 mM, and the characteristic parameter is the pH value, i.e., 3.5-10.5; the characteristic parameter of the salt is concentration, which is distributed in the range of 100 mM to 500 mM; and the characteristic parameter of the additive is concentration.

[0009] The biomacromolecule crowding agents include Dextran T-500, PEG 200, PEG 600, PEG 1000, PEG 2000, PEG 3350, PEG 5000, PEG 6000, PEG 8000, PEG 10000, and PEG 20000.

[0010] The buffer solution includes Tris-HCl buffer, HEPES buffer, citric acid-sodium citrate buffer, Bis-Tris buffer, acetic acid-sodium acetate buffer or phosphate buffer.

[0011] The salt includes sodium chloride, potassium chloride, magnesium acetate, calcium acetate monohydrate, calcium chloride dihydrate, zinc chloride, barium chloride, sodium acetate, ammonium sulfate, anhydrous potassium carbonate, magnesium chloride hexahydrate or a combination thereof.

[0012] The additives include Proclin 300 liquid biological preservative, glycerol, dimethyl sulfoxide, dithiothreitol, L-glutathione and reduced form, D-anhydrous glucose, D-(+)-mannose, D-galactose or a combination thereof.

[0013] The reagent set is a set of 96 reagent conditions:

[0014]

[0015]

[0016]

[0017]

[0018]

[0019] A method for using the protein liquid-liquid phase separation condition screening kit based on a biomacromolecule crowding agent, characterized in that: a target protein is dissolved in ultrapure water to prepare a protein solution, and the volume ratio of different screening reagents to the protein solution is 0.1 to 10:1, so as to obtain a protein phase separation condition screening array; the screening array is placed in a temperature-controlled box for incubation, and samples are taken at fixed points and at regular intervals for observation using an inverted microscope. When a phase separation phenomenon occurs, the corresponding screening reagent is the chemical condition that can promote the target protein to achieve phase separation.

[0020] The volume ratio of the different screening reagents to the protein solution is 1:1.

[0021] The screening array was placed in a temperature-controlled box at 37°C.

[0022] Steps for using the kit:

[0023] Step 1: Prepare the protein liquid-liquid phase separation screening reagent according to the above-designed kit formula table, and add Proclin 300 liquid biological preservative with a final concentration of 0.05%, filter the prepared reagent solution with a 0.22μm filter membrane, and the kit is prepared.

[0024] Step 2: Select and obtain the target protein for liquid-liquid phase separation.

[0025] Step 3: Prepare the selected target protein into protein solutions of different concentrations using ultrapure water, and filter out impurities using a 0.22 μm filter membrane.

[0026] Step 4: premix the protein solution prepared in step 3 with the screening reagent in step 1.

[0027] Use a dispenser to drop a certain volume of protein solution (2 μL is recommended) in the center of the 96-well plate, mix the 96 screening reagents in step 1 with the protein solution (recommended volume ratio 1:1), and quickly seal with transparent sealing tape to ensure a tight seal.

[0028] Alternatively, use an automatic pipetting workstation (Screenmaker 96+8) to add samples, the volume ratio of different screening reagents to protein solution is 0.1 to 10:1, the sample volume is 0.1 μL to 2.0 μL, add the solution to the crystallization plate, and seal it with transparent sealing tape.

[0029] Step 5: Place the 96-well plate or crystallization plate completed in step 4 into a temperature-controlled box for incubation.

[0030] Step 6: Samples are taken at fixed points and at fixed times, and an inverted microscope is used to observe whether liquid-liquid phase separation of proteins occurs under different conditions, and photographs are taken for record and statistical analysis.

[0031] Beneficial Effects

[0032] The present invention proposes a protein liquid-liquid phase separation condition screening kit based on a biomacromolecule crowding agent and a method of use, which lays a foundation for the study of protein phase separation conditions. The present invention uses a biomacromolecule crowding agent as a key component, and is combined with other additives to form a reagent mixture to form a protein liquid-liquid phase separation condition screening kit with 96 reagent conditions. When used, the target protein is dissolved in ultrapure water to prepare a protein solution, and is mixed with each screening reagent in the kit in a certain volume ratio to obtain a protein phase separation condition screening array, which is placed in a temperature-controlled box and incubated for a certain period of time and then taken out for observation. When the phase separation phenomenon occurs, the corresponding screening reagent is the chemical condition that can promote the target protein to achieve phase separation. The present invention can conveniently and quickly obtain protein liquid-liquid phase separation conditions. At the same time, conditions for inhibiting phase separation can also be obtained, which has important reference value for the development of new drugs based on phase separation regulation.

[0033] The present invention increases the types of protein liquid-liquid phase separation condition screening kits, provides more types of crowded environments, and obtains more conditions that inhibit or promote phase separation. The method of using the kit is: first prepare a protein liquid-liquid phase separation kit, then select a target protein to achieve phase separation, and prepare a protein solution with ultrapure water. The prepared protein solution is mixed with the screening reagent in the kit and added to a 96-well plate or a crystallization plate, the 96-well plate or the crystallization plate is placed in a temperature-controlled box and incubated for a certain period of time, and the occurrence of protein liquid-liquid phase separation is observed regularly using an inverted fluorescence microscope. Using the kit of the present invention, respectively mixed with the target protein, a variety of conditions for protein liquid-liquid phase separation are found, which solves the problem of few types of protein liquid-liquid phase separation condition screening kits.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) Compared with the prior art, the present invention provides more options for crowded environments;

[0036] (2) The present invention can provide new conditions for promoting or inhibiting phase separation;

[0037] (3) New conditions that promote or inhibit phase separation have important reference value for the screening of new drugs based on phase separation regulation;

[0038] (4) The kit designed by the present invention for efficiently screening protein liquid-liquid phase separation conditions based on biomacromolecule crowding agents has low cost, convenient use and high repeatability;

[0039] (5) The 96-condition screening kit designed by the present invention contains a variety of screening reagents, and the biomacromolecule crowding agent includes Dextran T-500 and PEG of different molecular weights, with a concentration distribution ranging from 5% w / v to 50% w / v, covering a wide range. Different combinations provide more possibilities for the occurrence of phase separation;

[0040] (6) The acid-base environment designed by the present invention selects 6 buffer solutions with strong buffering capacity, and the pH distribution ranges from 3.5 to 10.5, thereby improving the success rate of screening and increasing the probability of phase separation.

[0041] (7) The salts used in the present invention are rich in variety, covering most of the salts required for phase separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It shows that the 96 conditions of the kit of the present invention are divided into 6 categories according to 6 buffer solutions and distinguished by different colors.

[0043] Figure 2 The classification principle is shown, which is divided into 3 categories and distinguished by different colors. (a) is phase separation; (b) is precipitation; (c) is clear and transparent.

[0044] Figure 3 This is the screening statistical result of the FUS protein liquid-liquid phase separation screening kit obtained in Example 1 of the method of the present invention.

[0045] Figure 4 It is the screening statistical result of the α-synuclein liquid-liquid phase separation screening kit obtained in Example 2 of the method of the present invention.

[0046] Figure 5 This is the screening statistical result of the bovine serum albumin liquid-liquid phase separation screening kit obtained in Example 3 of the method of the present invention.

[0047] Figure 6 The results of screening of the FUS protein liquid-liquid phase separation screening kit in Example 1 of the present invention are shown. (a) is the screening result of the condition of kit A11 after incubation of 100 μM FUS protein at 25°C for 1 day. (b) is the screening result of the condition of kit A12 after incubation of 100 μM FUS protein at 25°C for 1 day. (c) is the screening result of the condition of kit C7 after incubation of 100 μM FUS protein at 25°C for 1 day. (d) is the screening result of the condition of kit D11 after incubation of 100 μM FUS protein at 25°C for 1 day.

[0048] Figure 7The results of screening of the α-synuclein liquid-liquid phase separation screening kit in Example 2 of the method of the present invention are shown. (a) is the screening result of the conditions of the kit A3 after incubation of 100 μM α-synuclein at 37°C for 4 hours. (b) is the screening result of the conditions of the kit A12 after incubation of 100 μM α-synuclein at 37°C for 4 hours. (c) is the screening result of the conditions of the kit E6 after incubation of 100 μM α-synuclein at 37°C for 4 hours. (d) is the screening result of the conditions of the kit F4 after incubation of 100 μM α-synuclein at 37°C for 4 hours.

[0049] Figure 8 The results of screening of the bovine serum albumin liquid-liquid phase separation screening kit in Example 3 of the method of the present invention are shown. (a) is the screening result of the condition of kit B9 after incubation at 25°C for 1 day with 100 μM bovine serum albumin. (b) is the screening result of the condition of kit B10 after incubation at 25°C for 1 day with 100 μM bovine serum albumin. (c) is the screening result of the condition of kit D6 after incubation at 25°C for 1 day with 100 μM bovine serum albumin. (d) is the screening result of the condition of kit E6 after incubation at 25°C for 1 day with 100 μM bovine serum albumin. DETAILED DESCRIPTION

[0050] The present invention will now be further described with reference to the embodiments and the accompanying drawings:

[0051] Example 1

[0052] Taking 100 μM FUS protein as an example, different phase separation conditions were screened using the protein liquid-liquid phase separation screening kit. The screening statistical results are shown in Figure 2. Figure 4 shown.

[0053] Step 1: According to the reagent kit formula table 1 designed in the present invention, a protein liquid-liquid phase separation screening reagent is prepared, and a final concentration of 0.05% of Proclin 300 liquid biopreservative is added, and the prepared reagent solution is filtered with a 0.22 μm filter membrane, and the preparation of the reagent kit is completed.

[0054] Step 2: Select FUS protein as the target protein for liquid-liquid phase separation.

[0055] Step 3: Prepare the selected target protein into a 100 μM FUS protein solution with ultrapure water, and filter out impurities with a 0.22 μm filter membrane.

[0056] Step 4: premix the 100 μM FUS protein solution prepared in step 3 with the screening reagent in step 1.

[0057] Use a dispenser to pipette 2 μL of 100 μM FUS and drop it in the center of a 96-well plate. Mix the 96 screening reagents in step 1 with the protein solution in a 1:1 volume ratio and quickly seal the plate with transparent sealing tape to ensure a tight seal.

[0058] Step 5: Place the 96-well plate completed in step 4 in a temperature-controlled box and incubate at 25°C for 1 day.

[0059] Step 6: Use an inverted microscope to observe whether FUS protein undergoes liquid-liquid phase separation under different conditions, take photos and record them, and make statistics. Figure 2 The classification principles are divided into three categories, namely phase separation, precipitation, and clarity, and they are distinguished by different colors. Figure 3 The statistical results are shown. Figure 6 Some of the screening results are shown.

[0060] In this example, FUS protein was mixed with 96 screening agents, and the results obtained by imaging at a final concentration of 50 uM were classified according to the above standards. Figure 3 From the results shown, it can be seen that FUS protein undergoes obvious phase separation in Tris-HCl buffer.

[0061] Example 2

[0062] Taking 100 μM α-synuclein as an example, the kit was used to screen different phase separation conditions. The screening statistical results are as follows Figure 4 shown.

[0063] Step 1: Prepare a protein liquid-liquid phase separation screening kit according to the kit formula table designed in the present invention, see Table 1.

[0064] Step 2: Select α-synuclein as the target protein for liquid-liquid phase separation.

[0065] Step 3: Prepare the selected target protein into a 100 μM α-synuclein solution with ultrapure water, and filter out impurities with a 0.22 μm filter membrane.

[0066] Step 4: premix the 100 μM α-synuclein solution prepared in step 3 with the screening reagent in step 1.

[0067] Use a dispenser to pipette 2 μL of 100 μM α-synuclein and drop it in the center of a 96-well plate. Mix the 96 screening reagents in step 1 with the protein solution in a 1:1 volume ratio and quickly seal with transparent sealing tape to ensure a tight seal.

[0068] Step 5: Place the 96-well plate completed in step 4 in a temperature-controlled box and incubate at 37°C for 4 hours.

[0069] Step 6: Use an inverted microscope to observe whether α-synuclein undergoes liquid-liquid phase separation under different conditions, take photos and record, and make statistics. Figure 4 Shows the Figure 2 Classification principles The statistical results after classification, Figure 7 Some of the screening results are shown.

[0070] Example 3

[0071] Taking 20 mg / mL bovine serum albumin as an example, different phase separation conditions were screened using the protein liquid-liquid phase separation screening kit. The screening statistical results are as follows Figure 5 shown.

[0072] Step 1: Prepare a protein liquid-liquid phase separation screening kit according to the kit formula table designed by the present invention.

[0073] Step 2: Select bovine serum albumin as the target protein for liquid-liquid phase separation.

[0074] Step 3: Prepare the selected target protein into a 20 mg / mL bovine serum albumin solution with ultrapure water, and filter out impurities with a 0.22 μm filter membrane.

[0075] Step 4: premix the 20 mg / mL bovine serum albumin solution prepared in step 3 with the screening reagent in step 1.

[0076] Use a dispenser to pipette 2 μL of 20 mg / mL bovine serum albumin solution and drop it in the center of the 96-well plate. Mix the 96 screening reagents in step 1 with the protein solution in a 1:1 volume ratio and quickly seal with transparent sealing tape to ensure a tight seal.

[0077] Step 5: Place the 96-well plate completed in step 4 in a temperature-controlled box and incubate at 25°C for 1 day.

[0078] Step 6: Use an inverted microscope to observe whether liquid-liquid phase separation of bovine serum albumin occurs under different conditions, take photos and record, and make statistics. Figure 5 Shows the Figure 2 The statistical results after classification are as follows: Figure 8 Some of the screening results are shown.

Claims

1. A protein liquid-liquid phase separation condition screening kit based on biomacromolecule crowding agent, characterized in that: The reagent set is a set of 96 reagent conditions: 。 2. A method for using the protein liquid-liquid phase separation condition screening kit based on biomacromolecule crowding agent according to claim 1, characterized in that: The target protein is dissolved in ultrapure water to prepare a protein solution, and the volume ratio of different screening reagents to the protein solution is 0.1~10:1 to obtain a protein phase separation condition screening array; the screening array is placed in a temperature-controlled box for incubation, and samples are taken at fixed points and timed for observation using an inverted microscope. When phase separation occurs, the corresponding screening reagent is the chemical condition that can promote the phase separation of the target protein.

3. The method of use according to claim 2, characterized in that: The volume ratio of the different screening reagents to the protein solution is 1:

1.

4. The method of use according to claim 2, characterized in that: The screening array was placed in a temperature-controlled box at 37°C.

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