Method for improving protein crystallization efficiency by using hydrogel
By preparing a precipitant using a hydrogel precursor solution and a buffer solution, the supersaturation of the protein solution was controlled, solving the problem of low protein crystallization efficiency and achieving efficient and uniform protein crystal production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- EAST CHINA UNIV OF SCI & TECH
- Filing Date
- 2022-09-14
- Publication Date
- 2026-06-19
AI Technical Summary
Protein crystallization is inefficient and produces poor crystal quality. Current technologies lack proactive methods for controlling solution supersaturation, resulting in long crystallization times and low efficiency.
A hydrogel precursor solution was prepared, and a photoinitiator was added. The solution was then irradiated with ultraviolet light to form a hydrogel. A precipitant was prepared using a buffer solution to soak the hydrogel. The supersaturation of the protein solution was adjusted, and the protein solution was dropped onto the surface of the hydrogel to crystallize.
This method enables crystallization to be completed within 60 minutes, improving crystallization efficiency and yielding high-quality protein crystals with uniform particle size distribution, making it suitable for large-scale production.
Smart Images
Figure CN115260281B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of protein crystallization methods, and in particular to a method for improving protein crystallization efficiency by utilizing hydrogel regulation. Background Technology
[0002] Proteins are the material basis of life activities, and elucidating their three-dimensional structure is essential for understanding the fundamental mechanisms of these activities, serving as a prerequisite and foundation for functional research. Single-crystal X-ray diffraction is the most mature and effective technique for determining the three-dimensional structure of proteins, but this technique relies on the availability of high-quality protein crystals. Furthermore, protein crystals have wide applications in protein purification, biosensing, and drug delivery. However, low protein crystallization efficiency and poor crystal quality limit the resolution of protein structures and related applications, representing a critical problem that urgently needs to be solved.
[0003] Proteins are difficult to crystallize due to their large molecular weight, complex spatial structure, and surface charge. Therefore, it is necessary to regulate the supersaturation of the protein solution to a specific range by adjusting the concentration of the precipitant and effectively induce the nucleation process. The protein crystallization process mainly depends on the regulation of solution supersaturation and the induction of nucleation. This is because, at low supersaturation, protein molecules cannot form metastable clusters before nucleation; at high supersaturation, protein molecules aggregate too quickly and excessively, forming amorphous precipitates. Patent application CN105254706 discloses a method for crystallizing proteins using a superhydrophobic surface. This method uses a superhydrophobic interface as the protein crystallization interface and slowly evaporates the solution in a closed container to create supersaturation, thus promoting protein crystallization. However, in this method, the solution supersaturation is controlled by a random evaporation process, resulting in a slow and uncontrollable protein crystallization process; furthermore, the crystallization time is long, and the crystallization efficiency is low.
[0004] Hydrogels, due to their good biocompatibility and unique surface and spatial structures, have been increasingly applied in the field of protein crystallization in recent years. Currently, research on the regulation of protein crystallization using hydrogel systems is still in its early stages both domestically and internationally. Profio et al. (Profio GD, Polino M, Nicoletta FP, et al. Tailored hydrogel membranes for efficient protein crystallization[J]. Advanced Functional Materials, 2014, 24(11):1582-1590.) utilized the surface and internal structures of hydrogels to regulate protein crystallization. They added a lysozyme solution to the hydrogel surface and then added an equal volume of precipitant to form a mixed droplet. This droplet was then placed in a pendant plate for crystallization to obtain lysozyme crystals. This method can provide nucleation sites for proteins and induce nucleation, but it lacks proactive methods for controlling solution supersaturation. It requires extensive screening of precipitant concentrations to determine the supersaturation range. Furthermore, using this method, the crystallization time for lysozyme is as long as 48 hours, resulting in low efficiency. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art by providing a method for improving protein crystallization efficiency using hydrogel regulation, which can effectively solve the problem of low efficiency in protein crystallization while ensuring the quality of protein crystals.
[0006] The objective of this invention can be achieved through the following technical solutions:
[0007] The purpose of this invention is to provide a method for improving protein crystallization efficiency using hydrogel regulation, comprising the following steps:
[0008] S1. Polyethylene glycol diacrylate and N,N-dimethylacrylamide are mixed to obtain a prepolymer solution. Water and photoinitiator are added to the obtained prepolymer solution and stirred to obtain a hydrogel precursor solution. After stabilizing the obtained hydrogel precursor solution, it is irradiated with ultraviolet light to obtain a rough hydrogel. The rough hydrogel is soaked in water and then dried to obtain a dried hydrogel.
[0009] S2. Prepare a buffer solution and use the resulting buffer solution to prepare a precipitant;
[0010] S3. Soak the dried hydrogel obtained in step S1 in the precipitant obtained in step S2 to make the dried hydrogel obtained in S1 swell, wash and dry to obtain the target hydrogel.
[0011] S4. Dissolve the protein in the buffer solution obtained in step S2 to obtain a protein solution;
[0012] S5. The protein solution obtained in step S4 is dropped onto the surface of the target hydrogel obtained in step S3 to crystallize and obtain protein crystals.
[0013] Furthermore, the molecular weight of the polyethylene glycol diacrylate mentioned in step S1 is 200-1000.
[0014] The reasons for choosing polyethylene glycol diacrylate as the monomer are as follows: polyethylene glycol diacrylate is biocompatible and non-toxic, as certified by the FDA.
[0015] Further, the photoinitiator in step S1 is one or a combination of 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP), 2-methyl-1-(4-methylthiophenyl)-2-morpholino-1-propanone, and tetramethylethylenediamine (TEMED).
[0016] More preferably, the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP).
[0017] More preferably, the photoinitiator in step S1 is 2-hydroxy-2-methyl-1-phenyl-1-propanone.
[0018] More preferably, the water in step S1 is ultrapure water.
[0019] Furthermore, in step S1, the mass ratio of polyethylene glycol diacrylate to N,N-dimethylacrylamide is 5:1 to 10:1. Within this range, a hydrogel with a special structure can be formed to regulate the supersaturation of the protein solution; the mass ratio of the prepolymer solution to water is 1:10 to 1:20; and the mass fraction of the photoinitiator in the hydrogel precursor solution is 0.5% to 1.5%.
[0020] Furthermore, the stirring speed used in step S1 is 100-500 rpm, and the stirring time is 0.5-2 h.
[0021] More preferably, the stirring speed in step S1 is 200-400 rpm, and the stirring time is 1 hour.
[0022] Furthermore, the stabilization condition described in step S1 is stabilization under a nitrogen atmosphere for 0.5 to 1 hour.
[0023] Furthermore, the ultraviolet light wavelength used in the ultraviolet irradiation in step S1 is 365nm, and the irradiation time is 10-20min.
[0024] Further, the buffer solution in step S2 is a sodium acetate solution; the concentration of the sodium acetate solution is 0.1-0.2M, and the pH is 4-5. The buffer solution can reduce pH fluctuations in the system and maintain the acidity or alkalinity of the solution system.
[0025] Further, the precipitant in step S2 includes the buffer solution and sodium chloride; the concentration of sodium chloride in the precipitant is 1-2M.
[0026] Furthermore, the soaking time in step S3 is 6 to 12 hours.
[0027] More preferably, the protein in step S4 is lysozyme.
[0028] Furthermore, the concentration of protein in the protein solution described in step S4 is 10–50 mg / mL.
[0029] Furthermore, the crystallization temperature in step S5 is 20–30°C.
[0030] Furthermore, the crystallization described in step S5 is carried out in a sealed crystallization container.
[0031] More preferably, the crystallization container is a petri dish.
[0032] Compared with the prior art, the present invention has the following beneficial effects:
[0033] 1) The method for improving protein crystallization efficiency by using hydrogel regulation provided by the present invention controls the supersaturation of the protein solution by hydrogel regulation, and the complete crystal growth time is controlled within 60 minutes, which greatly improves the crystallization efficiency.
[0034] 2) The method for improving protein crystallization efficiency using hydrogel regulation provided by this invention produces high-quality protein crystals with less amorphous precipitation, improved crystal morphology, and uniform crystal size distribution.
[0035] 3) The method for improving protein crystallization efficiency by utilizing hydrogel regulation provided by this invention can realize the large-scale production and preparation of protein crystals. Attached Figure Description
[0036] Figure 1 This is a SEM image of protein crystallization controlled by hydrogel as provided in Example 1.
[0037] Figure 2 This is an SEM image of protein crystallization controlled by hydrogel, provided in Example 2. Detailed Implementation
[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.
[0039] Any preparation methods, materials, structures, or composition ratios not explicitly described in this technical solution are considered common technical features disclosed in the prior art.
[0040] During the conceptualization process, this technical solution fully recognized the lack of proactive methods for controlling solution supersaturation during protein crystallization in existing technologies. This necessitates extensive screening of precipitant concentrations to determine the supersaturation range, leading to low protein crystallization efficiency. Therefore, this solution innovatively incorporates a method where the precipitant, which is typically added after the protein solution is dropped onto the hydrogel surface, is added during the hydrogel preparation process. This proactively controls solution supersaturation, avoids extensive screening of precipitant concentrations, and improves protein crystallization efficiency.
[0041] Lysozyme: Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥20000U / mg.
[0042] Polyethylene glycol diacrylate: Shanghai Aladdin Biochemical Technology Co., Ltd., average molecular weight ≈ 400.
[0043] Example 1
[0044] This embodiment provides a method for improving protein crystallization efficiency using hydrogel regulation. The protein in this embodiment is lysozyme, and the crystallization of lysozyme is regulated using a hydrogel with a prepolymer ratio of 5:1. The method includes the following steps:
[0045] S1. Under a nitrogen atmosphere, polyethylene glycol diacrylate (PEGDA) and N,N-dimethylacrylamide (DMAA) were mixed in a mass ratio of 5:1 to obtain a prepolymer solution. Ultrapure water was added to the obtained prepolymer solution, with a mass ratio of 1:10 between the prepolymer solution and ultrapure water. Then, the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP) was added. The mixture was stirred electromagnetically at a stirring rate of 300 rpm for 1 h to obtain a hydrogel precursor solution. The mass fraction of HMPP in the hydrogel precursor solution was 1%. 10 mL of the obtained hydrogel precursor solution was stabilized under a nitrogen atmosphere for 30 min and then irradiated with ultraviolet light at a wavelength of 365 nm for 10 min to obtain a rough hydrogel. The rough hydrogel was soaked in ultrapure water and then dried in a vacuum drying oven for 6 h to obtain a dried hydrogel.
[0046] S2. Prepare a buffer solution with a sodium acetate concentration of 0.1M and a pH value of 4.6, and use the obtained buffer solution to prepare a precipitant with a sodium chloride concentration of 1M.
[0047] S3. Soak the dried hydrogel obtained in step S1 in the precipitant obtained in step S2 for 6 hours to make the dried hydrogel obtained in S1 swell, wash and dry to obtain the target hydrogel.
[0048] S4. Dissolve the lysozyme in the buffer solution obtained in step S2 to obtain a lysozyme solution with a concentration of 30 mg / mL.
[0049] S5. Add 0.01 mL of the lysozyme solution obtained in step S4 to the surface of the target hydrogel obtained in step S3 to crystallize until the number and size of crystals no longer change, and obtain lysozyme crystals. The crystallization is carried out in a sealed petri dish at a crystallization temperature of 25°C.
[0050] The crystallization time in this embodiment was 55.1 min. The SEM image of the lysozyme crystals obtained in this embodiment is shown below. Figure 1 As shown, the lysozyme crystals are hexagonal crystals with a uniform crystal size distribution.
[0051] Example 2
[0052] This embodiment provides a method for improving protein crystallization efficiency using hydrogel regulation. The protein in this embodiment is lysozyme, and lysozyme crystallization is regulated using a hydrogel with a prepolymer ratio of 8:1. The method includes the following steps:
[0053] S1. Under a nitrogen atmosphere, polyethylene glycol diacrylate (PEGDA) and N,N-dimethylacrylamide (DMAA) were mixed in a mass ratio of 8:1 to obtain a prepolymer solution. Ultrapure water was added to the obtained prepolymer solution, with a mass ratio of prepolymer solution to ultrapure water of 1:15. Then, the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP) was added. The mixture was stirred electromagnetically at a stirring rate of 300 rpm for 1 h to obtain a hydrogel precursor solution. The mass fraction of HMPP in the hydrogel precursor solution was 1%. 10 mL of the obtained hydrogel precursor solution was stabilized under a nitrogen atmosphere for 30 min and then irradiated with ultraviolet light at a wavelength of 365 nm for 10 min to obtain a rough hydrogel. The rough hydrogel was soaked in ultrapure water and then dried in a vacuum drying oven for 6 h to obtain a dried hydrogel.
[0054] S2. Prepare a buffer solution with a sodium acetate concentration of 0.1M and a pH of 4.8, and use the obtained buffer solution to prepare a precipitant with a sodium chloride concentration of 1.8M.
[0055] S3. Soak the dried hydrogel obtained in step S1 in the precipitant obtained in step S2 for 6 hours to make the dried hydrogel obtained in S1 swell, wash and dry to obtain the target hydrogel.
[0056] S4. Dissolve the lysozyme in the buffer solution obtained in step S2 to obtain a lysozyme solution with a concentration of 40 mg / mL.
[0057] S5. Add 0.01 mL of the lysozyme solution obtained in step S4 to the surface of the target hydrogel obtained in step S3 to crystallize until the number and size of crystals no longer change, and obtain lysozyme crystals. The crystallization is carried out in a sealed petri dish at a crystallization temperature of 25°C.
[0058] The crystallization time in this embodiment was 55.1 min. The SEM image of the lysozyme crystals obtained in this embodiment is shown below. Figure 2 As shown, the lysozyme crystals are hexagonal crystals with a uniform crystal size distribution.
[0059] Comparative Example 1
[0060] This comparative example provides a method for regulating protein crystallization using hydrogels. The protein in this comparative example is lysozyme, and the crystallization of lysozyme is regulated using a hydrogel with a prepolymer ratio of 10:1. The method includes the following steps:
[0061] S1. Under a nitrogen atmosphere, polyethylene glycol diacrylate (PEGDA) and N,N-dimethylacrylamide (DMAA) were mixed in a mass ratio of 10:1 to obtain a prepolymer solution. Ultrapure water was added to the obtained prepolymer solution at a mass ratio of 1:15. Then, photoinitiators 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP) and tetramethylethylenediamine (TEMED) were added. The mixture was stirred electromagnetically at a stirring speed of 300 rpm for 1 hour to obtain a hydrogel precursor solution. The mass fraction of HMPP in the hydrogel precursor solution was 0.6%, and the mass fraction of TEMED in the hydrogel precursor solution was 0.0015%. 10 mL of the obtained hydrogel precursor solution was used to prepare a liquid film with a thickness of 450 μm on a polypropylene support film using a microcoating device. The obtained liquid film was irradiated with a high-pressure mercury lamp at a wavelength of 253 nm for 5 min to obtain a rough hydrogel. The obtained rough hydrogel was washed with ultrapure water and dried in a vacuum drying oven for 6 h to obtain a dried hydrogel.
[0062] S2. Prepare a buffer solution with a sodium acetate concentration of 0.1M and a pH value of 4.6, and use the obtained buffer solution to prepare a precipitant with a sodium chloride concentration of 7wt%.
[0063] S3. Dissolve the lysozyme in the buffer solution obtained in step S2 to obtain a lysozyme solution, wherein the concentration of lysozyme is 50 mg / mL;
[0064] S4. Add 0.01 mL of the lysozyme solution obtained in step S3 to the surface of the dried hydrogel obtained in step S1, and then add an equal volume of precipitant solution to form a mixed droplet. Place the mixed droplet in a pendant plate for crystallization until the number and size of crystals no longer change, and obtain lysozyme crystals at a crystallization temperature of 20°C.
[0065] The crystallization time in this comparative example was 48 hours.
[0066] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.
[0067] The crystallization times of Examples 1-2 and Comparative Example 1 are shown in Table 1. By using the method of improving protein crystallization efficiency by regulating hydrogel provided by the present invention, the crystallization time is shortened from 48 hours to less than 1 hour, which can effectively solve the problem of low efficiency in protein crystallization.
[0068] Table 1 Crystallization Time
[0069] Example 1 Example 2 Comparative Example 1 Crystallization time 55.1min 47.0min 48h
Claims
1. A method for improving the efficiency of protein crystallization by using a hydrogel, characterized in that, Includes the following steps: S1. Polyethylene glycol diacrylate and N,N-dimethylacrylamide are mixed to obtain a prepolymer solution. Water and photoinitiator are added to the obtained prepolymer solution and stirred to obtain a hydrogel precursor solution. After stabilizing the obtained hydrogel precursor solution, it is irradiated with ultraviolet light to obtain a rough hydrogel. The rough hydrogel is soaked in water and then dried to obtain a dried hydrogel. S2. Prepare a buffer solution and use the resulting buffer solution to prepare a precipitant; S3. Soak the dried hydrogel obtained in step S1 in the precipitant obtained in step S2 to make the dried hydrogel obtained in S1 swell, wash and dry to obtain the target hydrogel. S4. Dissolve the protein in the buffer solution obtained in step S2 to obtain a protein solution; S5. The protein solution obtained in step S4 is dropped onto the surface of the target hydrogel obtained in step S3 to crystallize and obtain protein crystals. The method uses hydrogel to regulate the supersaturation of the protein solution, and the complete crystal growth time is controlled within 60 min. The method yields high-quality protein crystals with minimal amorphous precipitates and uniform crystal size distribution. The mass ratio of polyethylene glycol diacrylate and N,N-dimethylacrylamide in step S1 is 5:1 to 10:1; The mass ratio of the prepolymer liquid to water is 1:10 to 1:20; The photoinitiator has a mass fraction of 0.5% to 1.5% in the hydrogel precursor solution; The stirring speed used in step S1 is 100~500 rpm, and the stirring time is 0.5~2 h; The stabilization condition described in step S1 is stabilization under a nitrogen atmosphere for 0.5 to 1 hour; The ultraviolet light wavelength used in the ultraviolet irradiation is 365 nm, and the irradiation time is 10~20 min; The protein mentioned in step S4 is lysozyme; The protein concentration in the protein solution described in step S4 is 10~50 mg / mL.
2. The method for improving protein crystallization efficiency using hydrogel regulation according to claim 1, characterized in that, The molecular weight of the polyethylene glycol diacrylate mentioned in step S1 is 200-1000; The photoinitiator is one or a combination of 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-methyl-1-(4-methylthiophenyl)-2-morpholino-1-propanone, and tetramethylethylenediamine.
3. The method for improving the efficiency of protein crystallization by using hydrogel according to claim 1, wherein, The buffer solution mentioned in step S2 is a sodium acetate solution; the concentration of the sodium acetate solution is 0.1~0.2 M, and the pH is 4~5; The precipitant comprises the buffer solution and sodium chloride; the concentration of sodium chloride in the precipitant is 1~2 M.
4. The method for improving the efficiency of protein crystallization by using hydrogel according to claim 1, wherein, The soaking time described in step S3 is 6~12 hours.
5. The method for improving the efficiency of protein crystallization by using hydrogel according to claim 1, wherein, The crystallization temperature in step S5 is 20~30℃.