A method for preparing a polypeptide or protein single crystal or amorphous material

Abstract

The present application relates to the technical field of single crystal or amorphous preparation, and particularly relates to a method for preparing a polypeptide or protein single crystal or amorphous substance, which is suitable for the preparation of single crystals or amorphous substances of any polypeptide or protein molecules that can be dissolved in a solvent. The method comprises the following steps: (a1) preparing a polypeptide or protein solution, wherein the solvent used for preparing the solution is a freezable solvent; (a2) freezing the polypeptide or protein solution of step (a1), and optionally maturing, to obtain a mixed system containing polypeptide or protein single crystals or amorphous substances and a frozen solvent; and optionally (a3) separating the polypeptide or protein single crystals or amorphous substances from the mixed system containing polypeptide or protein single crystals or amorphous substances and the frozen solvent of step (a2). The method of the present application is universal and can be applied to all polypeptide or protein molecules that have single crystals or amorphous substances.

Description

[0001] This application claims priority to an earlier application filed on October 30, 2018, with patent application number 201811279200X entitled "A method for preparing single crystals of polypeptides or proteins", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of single-crystal or amorphous material preparation technology, specifically to a method for preparing single-crystal or amorphous polypeptides or proteins. This method is applicable to the preparation of single-crystal or amorphous polypeptide or protein molecules that can be dissolved in a solvent. Background Technology

[0003] Polypeptides or proteins are the most basic substances in living organisms, essential for growth and life maintenance, and also play catalytic and immune functions. Understanding protein structure is crucial for structural genomics, peptide or protein research and development, and protein design and modification. Common methods for peptide or protein crystallization include batch crystallization, liquid-liquid diffusion, hanging drop, precipitation, and dialysis. However, these methods all suffer from difficulties in nucleation, leading to polycrystalline or amorphous formation. For some peptide or protein molecules, obtaining crystals using these methods is even difficult. Therefore, efficiently preparing perfect single crystals of peptides or proteins remains a significant challenge, but it is of paramount importance for both basic research and industrial production. Furthermore, amorphous peptides or proteins are also of great significance for pharmaceutical industrial production and basic research. Summary of the Invention

[0004] To address the shortcomings of existing technologies in the preparation of single crystals or amorphous peptides or proteins, this invention aims to provide a method for preparing and cultivating single crystals or amorphous peptides or proteins by controlling the supply and aggregation rate of crystalline materials through solution freezing and optional ripening. This invention is the first to achieve controllable preparation of single crystals or amorphous peptides or proteins using a frozen solvent. Specifically, by controlling the solution freezing and optional ripening process, the supply and aggregation rates of solute molecules (i.e., peptides or proteins) are regulated, thereby controlling the nucleation and crystallization of solute molecules and their crystal growth, achieving efficient preparation of single crystals or amorphous peptides or proteins.

[0005] This invention is achieved through the following technical solution:

[0006] A method for preparing single crystals or amorphous forms of polypeptides or proteins, the method comprising the following steps:

[0007] (a1) Prepare a solution of a polypeptide or protein, wherein the solvent used to prepare the solution is a freezeable solvent;

[0008] (a2) Freeze the solution of the polypeptide or protein from step (a1), optionally ripening it, to prepare a mixture of a single crystal or amorphous material containing the polypeptide or protein and a frozen solvent; optionally,

[0009] (a3) The single crystal or amorphous material containing polypeptide or protein is separated from the mixture of single crystal or amorphous material containing polypeptide or protein and frozen solvent in step (a2).

[0010] In this invention, the freezeable solvent refers to a solvent that can form a solid state under certain temperature and pressure.

[0011] In this invention, the polypeptide includes homopeptides or heteropeptides. The homopeptides include linear peptides or cyclic peptides; the heteropeptides include pigment peptides, glycopeptides, lipopeptides, or ketal peptides.

[0012] In this invention, the proteins include simple proteins and bound proteins.

[0013] In this invention, the simple proteins include albumins, globulins, histones, protamines, glutenins, and sclerosins.

[0014] In this invention, the binding proteins include glycoproteins, nucleoproteins, lipoproteins, phosphoproteins, metalloproteins, and pigment proteins.

[0015] In this invention, the solubility of the polypeptide or protein in the solvent is readily soluble, soluble, slightly soluble, or sparingly soluble.

[0016] In this invention, step (a2) specifically includes the following steps:

[0017] The solution of the polypeptide or protein from step (a1) is cooled and frozen into a solid mixture, and optionally subjected to aging treatment to prepare a mixture system containing a single crystal or amorphous material of the polypeptide or protein and a frozen solvent.

[0018] In step (a2) of the present invention, freezing is the process of converting the solution of polypeptide or protein from step (a1) from a liquid state to a solid state.

[0019] In this invention, the freezing method includes, but is not limited to, one or a combination of several of the following freezing methods: natural cooling freezing, cooling freezing using a compression refrigeration device, cooling freezing using a semiconductor refrigeration device, cooling freezing using liquid nitrogen, cooling freezing using liquid helium, cooling freezing using liquid carbon dioxide, cooling freezing using liquid oxygen, cooling freezing using liquid ethane, cooling freezing using dry ice, and cooling freezing using ice.

[0020] In this invention, the freezing process includes, but is not limited to, one or a combination of several freezing processes such as rapid cooling, slow cooling, step-by-step cooling, and heating followed by cooling.

[0021] In this invention, the freezing includes, but is not limited to, complete freezing and incomplete freezing.

[0022] In this invention, the ripening process refers to keeping a solution of polypeptides or proteins in a frozen state for a period of time.

[0023] In this invention, the curing time refers to the time required to raise or lower the temperature to the curing temperature after the freezing process is completed, and the time to maintain the temperature at the curing temperature.

[0024] In one embodiment, step (a2) involves freezing the solution of the pseudo-crystallizing substance from step (a1) to prepare a mixed system containing a single crystal of the pseudo-crystallizing substance and a frozen solvent.

[0025] In one embodiment, step (a2) includes a aging process, in which the solution of the crystalline substance from step (a1) is frozen and aged to prepare a mixed system containing a single crystal or amorphous material of the crystalline substance and a frozen solvent.

[0026] In one embodiment, step (a2) involves raising or lowering the temperature at a rate greater than or equal to 10°C / min during the aging process, and the aging time is less than 25 min, thereby preparing a mixed system of an amorphous material containing a pseudo-crystallizing substance and a frozen solvent.

[0027] In another embodiment, the greater the difference between the reached temperature and the freezing temperature, the larger the particle size of the resulting amorphous material. Therefore, the particle size of the obtained amorphous material can be controlled by adjusting the magnitude of this temperature difference.

[0028] In one embodiment, step (a2) involves raising or lowering the temperature to a certain temperature at a rate of less than 10°C / min during the aging process, and / or the aging time is at least 25 min, to prepare a mixed system containing a single crystal of a pseudo-crystallizable substance and a frozen solvent.

[0029] For example, during the aging process, the temperature is raised or lowered at a rate of less than 10°C / min to a certain temperature and held for a period of time to prepare a mixed system containing a single crystal of a pseudo-crystallizable substance and a frozen solvent.

[0030] For example, during the aging process, the temperature is raised or lowered at any rate to a certain temperature, and the aging process is carried out for at least 25 minutes to prepare a mixed system containing a single crystal of a pseudo-crystallizable substance and a frozen solvent.

[0031] For example, during the aging process, the temperature is raised or lowered at a rate of less than 10°C / min to a certain temperature, and the aging process is carried out for at least 25 minutes to prepare a mixed system containing a single crystal of a pseudo-crystallizable substance and a frozen solvent.

[0032] In this invention, in step (a3), the separation is achieved by using physical and / or chemical methods to separate the solvent frozen into a solid from the mixed system.

[0033] In this invention, the physical methods include, but are not limited to, one or a combination of several of the following: rapid cooling separation, sublimation (such as vacuum sublimation), and dissolution.

[0034] In this invention, the chemical methods include, but are not limited to, one or a combination of chemical reactions and electrolysis.

[0035] In this invention, the method further includes the following steps:

[0036] (a4) Collect the single crystals or amorphous materials prepared in step (a3).

[0037] In this invention, in step (a4), the collection includes, but is not limited to, one or a combination of optical microscope collection, scanning electron microscope collection, dual-beam electron microscope collection, and transmission electron microscope collection.

[0038] The present invention also provides a method for culturing single crystals of polypeptides or proteins, the method comprising the above-described method for preparing single crystals.

[0039] In this invention, the method for culturing polypeptide or protein single crystals further includes the following steps:

[0040] (b1) Transfer the single crystals of the prepared polypeptides or proteins to the mother liquor of the polypeptides or proteins for culturing.

[0041] (b2) Collect the single crystals from step (b1).

[0042] In this invention, in step (b1), the transfer can be to transfer the mixed system of single crystal and solvent containing the crystallizing substance from step (a2) to the mother liquor of the crystallizing substance for single crystal culture; or the transfer can be to directly transfer the single crystal after solvent removal from step (a3) ​​to the mother liquor of the polypeptide or protein for single crystal culture; or the single crystal collected in step (a4) can be transferred to the mother liquor of the polypeptide or protein for single crystal culture.

[0043] In this invention, the transfer includes, but is not limited to, one or a combination of several of the following: optical microscope transfer, scanning electron microscope transfer, dual-beam electron microscope transfer, and transmission electron microscope transfer.

[0044] In this invention, in step (b1), the method for cultivating the single crystal includes, but is not limited to, one or a combination of several of the following: evaporation, cooling, and diffusion.

[0045] In this invention, in step (b2), the collection includes, but is not limited to, one or a combination of optical microscope collection, scanning electron microscope collection, dual-beam electron microscope collection, and transmission electron microscope collection.

[0046] Beneficial effects

[0047] 1. Addressing the shortcomings of traditional methods in preparing single crystals or amorphous peptides or proteins, such as difficulty in controlling molecular supply, aggregation, and nucleation rates, this invention proposes for the first time a method for inducing solute molecule nucleation and crystallization through solution freezing. By controlling the ripening process of the frozen peptide or protein solution, and optionally the ripening process, single crystals or amorphous peptides or proteins can be prepared rapidly and effectively. Simultaneously, this method solves the problem of single crystal preparation of molecules that are difficult to crystallize in traditional single crystal culture, and also addresses the difficulty in forming amorphous substances, especially high-purity amorphous substances.

[0048] 2. Compared to the hanging drop method and precipitation method, the freezing treatment method used in this invention allows for a wider range of concentration control for peptides or proteins, enabling the preparation of single crystals or amorphous peptides or proteins from very low concentrations to supersaturated concentrations. This invention achieves, for the first time, the acquisition of single crystals or amorphous peptides or proteins at extremely low solution concentrations; it also solves the problems of uncontrollable formation of single crystals or amorphous substances and the tendency to form polycrystalline or twinned crystals due to the rapid aggregation of solute molecules at high concentrations; furthermore, this invention has the advantage of obtaining single crystals or amorphous peptides or proteins in a very short time (minutes to hours).

[0049] 3. Solution freezing is a key technical aspect of this invention. The freezing process refers to freezing the solution in any manner; the freezing time, freezing temperature, freezing temperature gradient, freezing method, and freezing process are not particularly limited. Experiments have confirmed that the essence of preparing solute single crystals or amorphous substances through solution freezing lies in the fact that during the freezing process, while the solvent freezes into a solid state (e.g., water molecules form ice crystals), solute molecules are released and aggregate at the interface between the solid solvent and the solid state (e.g., at the ice crystal interface). By controlling the solution freezing process and the recrystallization process of the solidified solvent (e.g., controlling the water crystallization process and the ice crystal recrystallization process), the release and aggregation rate of solute molecules can be further controlled, effectively achieving the control of solute molecule nucleation and growth, thereby obtaining single crystals or amorphous substances of the target molecules.

[0050] 4. The ripening process described in this invention refers to maintaining the frozen solution in a solid state or a solid-liquid mixture for a certain period of time, with no temperature limitation, but the heating or cooling rate needs to be controlled. Experiments have confirmed that the ripening process described in this invention, optionally used as a supplement to the freezing process, can optimize the control of the recrystallization process of the crystallized solvent, thereby controlling the release rate of solute molecules and the aggregation rate of solute molecules at the interface of the crystallizing solvent. This is beneficial for further optimizing the growth of amorphous materials and / or the nucleation and growth of single crystals after the solution is frozen. Moreover, since the ripening process does not have excessive temperature limitations, the frozen system does not need to be frozen again; instead, it can obtain single crystals or amorphous materials with particle sizes in the nanometer to micrometer range through the ripening process. This allows for the selection of more economical temperatures and higher efficiency in the optimized preparation of single crystals or amorphous materials, which is beneficial for reducing energy consumption and thus greatly saving costs. Compared with traditional methods, this invention optimizes the recrystallization of frozen solvent by controlling the heating or cooling rate of the ripening process. It can further control the aggregation rate of solute molecules in the crystallizing solvent to the interface of the crystallizing solvent, thereby effectively obtaining single crystals or amorphous products of solute molecules. It has advantages such as energy saving and is more conducive to the large-scale industrial production of single crystals or amorphous products of target molecules.

[0051] 5. The method for preparing single crystals or amorphous substances and the further method for cultivating single crystals provided by this invention have a wide range of applications, applicable to existing peptides or proteins. Furthermore, this method can be used to obtain single crystals of substances that are difficult to crystallize using traditional methods, and to obtain amorphous forms of substances that are difficult to obtain. The experimental methods are simple and highly operable. The methods described in this invention are applicable not only to basic laboratory research but also to the needs of industrial production.

[0052] 6. The solvent of this invention is easy to select; it can be either polar or non-polar, as long as it can be frozen. This provides different options for dissolving different molecules, especially for water-soluble pseudo-crystallizable substances, eliminating the need for large amounts of organic solvents, which not only reduces costs but also has advantages such as being green and environmentally friendly. Attached Figure Description

[0053] Figure 1 This is a scanning electron microscope image of a single crystal of L-glutathione.

[0054] Figure 2 A scanning electron microscope image of a single crystal of L-carnosine.

[0055] Figure 3 This is a scanning electron microscope image of a single crystal of diglycinate.

[0056] Figure 4 This is a scanning electron microscope image and molecular formula of a single crystal of aminopeptidase.

[0057] Figure 5 This is a scanning electron microscope image of a single crystal of lysozyme, which is derived from eggs.

[0058] Figure 6 This is a scanning electron microscope image of a single crystal of a protease, which is derived from Bacillus subtilis.

[0059] Figure 7 This is a scanning electron microscope image of a single crystal of albumin, which is derived from egg white.

[0060] Figure 8 This is a scanning electron microscope image of a single crystal of corn protein, which is derived from corn.

[0061] Figure 9 This is a scanning electron microscope image of a single crystal of proteinase K.

[0062] Figure 10 These are optical photographs and the molecular formula of a single crystal of phalloidin.

[0063] Figure 11 This is a schematic diagram illustrating the principle of single crystal formation of polypeptides or proteins according to the present invention.

[0064] Figure 12 This is a diagram illustrating the process of AIE35 forming a single crystal.

[0065] Figure 13 This is a diagram illustrating the process of p-toluenesulfonic acid forming a single crystal. Detailed Implementation

[0066] [Methods for preparing single crystals]

[0067] The polypeptides or proteins used in this invention refer to those polypeptides or proteins that exist as single crystals or amorphous forms.

[0068] In this invention, "optionally" means to perform or not perform subsequent steps.

[0069] In this invention, the amorphous form of the polypeptide or protein is an amorphous polypeptide or protein.

[0070] [Methods for preparing single crystals or amorphous materials]

[0071] As mentioned above, the present invention provides a method for preparing single crystals or amorphous forms of polypeptides or proteins, the method comprising the following steps:

[0072] (a1) Prepare a solution of a polypeptide or protein, wherein the solvent used to prepare the solution is a freezeable solvent;

[0073] (a2) Freeze the solution of the polypeptide or protein from step (a1), optionally ripening it, to prepare a mixture of a single crystal or amorphous material containing the polypeptide or protein and a frozen solvent; optionally,

[0074] (a3) The single crystal or amorphous material containing polypeptide or protein is separated from the mixture of single crystal or amorphous material containing polypeptide or protein and frozen solvent in step (a2).

[0075] [Methods for preparing single crystals]

[0076] As mentioned above, the present invention provides a method for preparing single crystals or amorphous forms of polypeptides or proteins, the method comprising the following steps:

[0077] (a1) Prepare a solution of a polypeptide or protein, wherein the solvent used to prepare the solution is a freezeable solvent;

[0078] (a2) Freeze the solution of the polypeptide or protein from step (a1), optionally ripening it, to prepare a mixed system containing a single crystal of the polypeptide or protein and a frozen solvent; optionally,

[0079] (a3) The single crystal of the polypeptide or protein is separated from the mixture of single crystal of polypeptide or protein and frozen solvent in step (a2).

[0080] Wherein, the heating or cooling rate during the ripening process is less than 10℃ / min, and / or the ripening time during the ripening process is at least 25min.

[0081] For example, during the aging process, the temperature is raised or lowered at a rate of less than 10°C / min to a certain temperature and held for a period of time to obtain a mixed system containing a single crystal of a pseudo-crystallizable substance and a frozen solvent.

[0082] For example, during the aging process, the temperature is raised or lowered at any rate to a certain temperature, and the aging process lasts for at least 25 minutes, thereby obtaining a mixed system containing a single crystal of a pseudo-crystallizable substance and a frozen solvent.

[0083] For example, during the aging process, the temperature is raised or lowered at a rate of less than 10°C / min to a certain temperature, and the aging is carried out for at least 25 minutes to obtain a mixed system containing a single crystal of a pseudo-crystallizable substance and a frozen solvent.

[0084] For example, the temperature reached may be less than or equal to 0°C, or less than or equal to -5°C; specifically, it may be -10°C, -15°C, -18°C, -20°C, -24°C, -25°C, -30°C, -72°C, -80°C, -90°C, -100°C, or liquid nitrogen temperature, etc.

[0085] As mentioned above, the heating or cooling rate is less than 10°C / min, for example, less than 9°C / min, and further, for example, less than or equal to 5°C / min; depending on the different substances intended for crystallization. It is easy to understand that if the rate is 0°C / min, it means maintaining a temperature for aging at the same temperature as the freezing temperature.

[0086] As described above, the ripening time is at least 25 minutes, but can be, for example, 30 minutes, 40 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, 100 minutes, 120 minutes, 150 minutes, 200 minutes, 300 minutes, 500 minutes or longer, depending on the different crystallizing substances.

[0087] [Methods for preparing amorphous materials]

[0088] As mentioned above, the present invention provides a method for preparing amorphous polypeptides or proteins, the method comprising the following steps:

[0089] (a1) Prepare a solution of a polypeptide or protein, wherein the solvent used to prepare the solution is a freezeable solvent;

[0090] (a2) Freeze and mature the solution of the polypeptide or protein from step (a1) to prepare a mixed system containing the polypeptide or protein in amorphous and frozen solvent; optionally,

[0091] (a3) The amorphous form of the polypeptide or protein is separated from the mixture of the amorphous and frozen solvent containing the polypeptide or protein in step (a2).

[0092] During the ripening process, the heating or cooling rate is greater than or equal to 10℃ / min, and the ripening time is less than 25min.

[0093] For example, in the aging process of step (a2), the temperature is raised or lowered at a rate of 10°C / min or higher to a certain temperature and aged for less than 25 minutes to obtain a mixed system of an amorphous material containing a crystalline substance and a frozen solvent.

[0094] In one embodiment, the greater the difference between the reached temperature and the freezing temperature, the larger the particle size of the resulting amorphous material. Therefore, the particle size of the obtained amorphous material can be controlled by adjusting this temperature. For example, the reached temperature may be less than or equal to 0°C, or less than or equal to -5°C; specifically, it could be -5°C, -7°C, -8°C, -10°C, -12°C, -20°C, -45°C, etc. Preferably, the temperature is increased from the liquid nitrogen temperature at a heating rate of greater than or equal to 10°C / min.

[0095] As described above, the heating or cooling rate is greater than or equal to 10℃ / min, for example, greater than or equal to 15℃ / min, and can be 15℃ / min, 16℃ / min, 17℃ / min, 18℃ / min, 19℃ / min, 20℃ / min, 21℃ / min, 22℃ / min, 23℃ / min, 24℃ / min, 25℃ / min, 26℃ / min, 27℃ / min, 28℃ / min, 29℃ / min, 30℃ / min or higher; the ripening time is less than 25 min, for example, it can be less than 25 min, less than or equal to 23 min, less than or equal to 22 min, less than or equal to 21 min, less than or equal to 20 min, less than or equal to 19 min, less than or equal to 18 min, less than or equal to 17 min or less than or equal to 16 min, etc., depending on the different crystallizing substances.

[0096] [Specific solutions within the above methods]

[0097] According to an embodiment of the present invention, in step (a1), the preparation of the polypeptide or protein solution can be carried out in accordance with the operation methods known to those skilled in the art, such as the method for preparing a standard solution.

[0098] According to an embodiment of the present invention, in step (a1), the freezeable solvent includes, but is not limited to, water and / or organic solvents.

[0099] The water includes, but is not limited to, recycled water, distilled water, and ultrapure water.

[0100] The so-called freezeable organic solvent refers to an organic solvent that can form a solid state under certain temperature and pressure.

[0101] The organic solvents include, but are not limited to, hydrocarbon organic solvents, halogenated hydrocarbon organic solvents, alcohol organic solvents, phenolic organic solvents, ether and acetal organic solvents, ketone organic solvents, acid and anhydride organic solvents, ester organic solvents, nitrogen-containing organic solvents, sulfur-containing organic solvents, and multifunctional organic solvents.

[0102] The hydrocarbon organic solvents include aliphatic hydrocarbons (straight-chain aliphatic hydrocarbons, branched aliphatic hydrocarbons, alicyclic hydrocarbons) and aromatic hydrocarbons; for example: methane, ethane, propane, butane, pentane, 2-methylbutane, hexane, petroleum ether, butene, cyclopentane, cyclohexane, benzene, styrene, toluene, xylene, ethylbenzene, diethylbenzene, biphenyl, naphthalene, etc.; the halogenated hydrocarbon organic solvents are halogen-substituted hydrocarbon organic solvents, such as dichloromethane, chloroform, carbon tetrachloride, chloroethane, dichloroethane, trichloroethane, dibromomethane, bromoethane, dibromoethane, dibromopropane, chlorobenzene, dichlorobenzene, dichlorotoluene, dibromobenzene, etc.; the alcohol solvents include, for example: methanol, ethanol. The solvents include: propanol, isopropanol, butanol, isobutanol, pentanol, 2-methyl-1-butanol, cycloethanol, phenethyl alcohol, ethylene glycol, propylene glycol, glycerol, butanediol, pentanediol, ethylene glycol, etc.; the phenolic solvents include, for example: phenol, hydroquinone, cresol, xylenol, etc.; the ether and acetal solvents include, for example: methyl ethyl ether, propyl ether, butyl ether, pentanol ether, ethyl butyl ether, anisole, diphenyl ether, ethylene oxide, propylene oxide, butane oxide, dioxane, furan, tetrahydrofuran, ethylene glycol methyl ether, ethylene glycol butyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol methyl ether, glycerol ether, crown ether, benzaldehyde, cinnamaldehyde, etc.; the ketone solvents include, for example: Acetone, methyl ethyl ketone, methyl acetone, pentanone, cyclohexanone, acetophenone, etc.; the acid and anhydride solvents are, for example: formic acid, acetic acid, oxalic acid, propionic acid, butyric acid, acetic anhydride, propionic anhydride, etc.; the ester solvents are, for example: methyl formate, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl butyrate, methyl benzoate, ethyl benzoate, ethyl cinnamate, dimethyl phthalate, butyrolactone, etc.; the nitrogen-containing compound solvents include nitro solvents, nitrile solvents, amine solvents, amide solvents, lactam solvents, etc., for example: nitrobenzene, acetonitrile, propionitrile, methylamine, dimethylamine, ethylamine, diethylamine, triethylamine, etc. Aniline, pyrrole, tetrahydropyrrole, piperidine, pyridine, tetrahydropyridine, ethylenediamine, propylenediamine, formamide, acetamide, N,N-dimethylformamide, N,N-dimethylacetamide, pyrrolidone, N-methylpyrrolidone, caprolactam, etc.; the sulfur-containing compound solvents include, for example: carbon disulfide, methyl sulfide, thiophene, tetrahydrothiophene, dimethyl sulfoxide, dimethyl sulfone, etc.; the multifunctional solvents include, for example: ethylene glycol monomethyl ether, diethylene glycol, polyethylene glycol, polypropylene glycol, 2-chloroethanol, allyl alcohol, acrylonitrile, diethanolamine, p-methoxybenzyl alcohol, morpholine, N-methylmorpholine, lactic acid, methyl acetoacetate, ethyl acetoacetate, etc.

[0103] According to an embodiment of the present invention, the organic solvent further includes a combination of the above-mentioned organic solvents.

[0104] According to an embodiment of the present invention, the polypeptide includes a homopeptide or a heteropeptide.

[0105] In this invention, the homopeptide includes linear peptides or cyclic peptides; the heteropeptide includes pigment peptides, glycopeptides, lipopeptides or ketal peptides.

[0106] For example, the polypeptide is selected from, but is not limited to, L-glutathione, L-carnosine, glutathione, or phalloidin.

[0107] In this invention, the proteins include simple proteins and bound proteins.

[0108] In this invention, the simple proteins include albumins, globulins, histones, protamines, glutenins, and sclerosins.

[0109] In this invention, the binding proteins include glycoproteins, nucleoproteins, lipoproteins, phosphoproteins, metalloproteins, and pigment proteins.

[0110] For example, the protein is selected from aminopeptidase, lysozyme (derived from eggs), protease (derived from Bacillus subtilis), albumin (derived from egg white), corn protein (derived from corn), proteinase K, etc.

[0111] According to an embodiment of the present invention, the polypeptide or protein may be a hydrophilic substance or a hydrophobic substance.

[0112] According to an embodiment of the present invention, the polypeptide or protein has a certain solubility in the solvent; those skilled in the art will understand that the amount of the polypeptide or protein dissolved in the solvent can be arbitrary, that is, the polypeptide or protein can be dissolved in the solvent, and there is no particular limitation on the amount dissolved in the solvent; it can be understood that the solubility of the polypeptide or protein in the solvent can be sparingly soluble, slightly soluble, soluble, and readily soluble.

[0113] According to an embodiment of the present invention, preferably, the amount of the polypeptide or protein dissolved in the solvent is greater than or equal to 1 × 10⁻⁶. -7 g / 100g (solvent used), for example, greater than or equal to 0.001g / 100g (solvent used), such as greater than or equal to 0.01g / 100g (solvent used), such as greater than or equal to 0.1g / 100g (solvent used), such as greater than or equal to 1g / 100g (solvent used), such as greater than or equal to 10g / 100g (solvent used).

[0114] According to embodiments of the present invention, the concentration of the polypeptide or protein solution is not particularly limited, i.e., the polypeptide or protein only needs to be soluble in the solvent. As those skilled in the art know, the polypeptide or protein in the solvent can be an unsaturated solution, a saturated solution, or a supersaturated solution. Of course, the concentration of the polypeptide or protein solution has a significant impact on the aggregation rate of the polypeptide or protein. At lower concentrations, the polypeptide or protein aggregates more slowly, and the time required to obtain single crystals or amorphous products increases accordingly. At higher concentrations, the polypeptide or protein aggregates more quickly, and the time required to obtain single crystals or amorphous products decreases accordingly. Therefore, by rationally selecting the concentration, the preparation time of single crystals or amorphous products can be controlled by the solution concentration. Of course, the preparation time of single crystals or amorphous products depends not only on the solution concentration but also closely on the aging process.

[0115] According to an embodiment of the present invention, the concentration of the polypeptide or protein solution is greater than or equal to 1 × 10⁻⁶. -7 The concentration of the organic solvent solution of the polypeptide or protein is not specifically limited, and it can be a supersaturated or saturated solution of the polypeptide or protein in the solvent. For example, it can be greater than or equal to 0.001 g / 100 g (solvent used), greater than or equal to 0.01 g / 100 g (solvent used), greater than or equal to 0.1 g / 100 g (solvent used), greater than or equal to 1 g / 100 g (solvent used), or greater than or equal to 10 g / 100 g (solvent used).

[0116] Preferably, the concentration of the polypeptide or protein solution is 1×10⁻⁶. -7 From g / 100g (solvent used) to 1g / 100g (solvent used).

[0117] According to the present invention, step (a2) specifically includes the following steps:

[0118] The solution of the polypeptide or protein from step (a1) is cooled and frozen into a solid, and optionally subjected to aging treatment to prepare a mixed system containing a single crystal or amorphous material of polypeptide or protein and a frozen solvent.

[0119] According to embodiments of the present invention, the inventors unexpectedly discovered that during the freezing process of the solution, the solvent freezes into a solid, while the pseudo-crystallization substance dissolved in the solution accumulates at the solvent interface, providing the possibility of forming single crystals or amorphous substances. Furthermore, in the solution of the frozen pseudo-crystallization substance, during the freezing process and optionally further aging process, the grain size of a certain amount of the frozen solid solvent gradually increases, and the pseudo-crystallization substance is gradually released from the disappearing solid solvent. Thus, the pseudo-crystallization substance continuously accumulates at each interface of the frozen solid solvent, forming single crystals or amorphous substances that continue to grow, or existing single crystals or amorphous substances continue to grow, ultimately yielding polypeptide or protein nanoparticles with particle sizes ranging from tens to hundreds of nanometers, such as... Figure 11 As shown.

[0120] Taking an aqueous system as an example, to demonstrate that ice crystals aggregate solute molecules at their interfaces during freezing or, optionally, further ripening, we selected the aggregated luminescent material AIE35 to verify this process (aggregated luminescent materials, in their free molecular state, cannot be excited to emit light by any wavelength, but when the molecules exist in an aggregated state, they will be excited to emit fluorescence). During the experiment, the AIE35 aqueous solution was frozen into a solid by any method, and the ice formed independently existing polycrystalline systems, such as... Figure 12 As shown, at the interface between any two contacting ice crystals, AIE35 forms aggregates and then crystallizes. Figure 12 As shown in section a, the enhanced fluorescence at the interface indicates that AIE35 molecules can aggregate at the interface and gradually transition from an amorphous state to form AIE35 nanocrystals. Furthermore, from... Figure 12 As shown in b, the aggregates formed at the interface underwent a transformation from an amorphous state to a single crystal, and the volume of the single crystal gradually increased. Figure 12 The results are from transmission electron microscopy and electron diffraction characterization.

[0121] The molecular structure of AIE35 is as follows:

[0122]

[0123] To further demonstrate the principle of single crystal formation, we used p-toluenesulfonic acid molecules and employed in-situ low-temperature attenuated total internal reflection infrared spectroscopy (TEM) to observe the aggregation and formation of single crystals of p-toluenesulfonic acid during the freezing and ripening processes in water, as well as the continuous growth of these single crystals. The results showed that p-toluenesulfonic acid single crystals formed during the freezing process, and these single crystals gradually grew during ripening. Simultaneously, the characteristic peak of p-toluenesulfonic acid was observed at -1035 cm⁻¹. -1 The generation and blue shift of (the stretching vibration of the sulfonate group) further strongly demonstrates that with ripening, the continuous aggregation of p-methylbenzenesulfonic acid molecules leads to the continuous growth of the formed single crystal (see...). Figure 13 ).

[0124] According to embodiments of the present invention, the freezing includes, but is not limited to, complete freezing and incomplete freezing. Those skilled in the art will understand that complete freezing refers to the complete freezing of a polypeptide or protein solution into a solid; incomplete freezing refers to the partial freezing of a polypeptide or protein solution into a solid state, while a portion remains in a liquid state.

[0125] According to embodiments of the present invention, those skilled in the art will understand that the freezing can be any cooling method used to freeze a solution of polypeptides or proteins of arbitrary volume and shape into a solid or a solid-liquid mixture. That is, the freezing is the freezing of a polypeptide or protein solution into a solid or a solid-liquid mixture. Compared with traditional evaporation and cooling crystallization methods, the freeze-crystallization method allows for a wider range of concentration control for the polypeptide or protein solution and significantly reduces the time required to obtain polypeptide or protein crystals.

[0126] According to embodiments of the present invention, the freezing time, freezing temperature, freezing temperature gradient, freezing method, and freezing process are not particularly limited; any solution of polypeptides or proteins of any volume and shape can be frozen into a solid. Of course, the concentration of the polypeptide or protein solution can be considered during the freezing process for appropriate selection, with the aim of controlling the diffusion rate of the polypeptide or protein and thus affecting its crystallization process. For example, if the concentration of the polypeptide or protein solution is high, the freezing time can be appropriately shortened and the freezing temperature can be appropriately lowered; this is to prevent the polypeptide or protein in the high-concentration solution from uncontrollably forming polycrystalline structures. If the concentration of the polypeptide or protein solution is low, the freezing time can be appropriately extended and the freezing temperature can be appropriately increased; this is to achieve effective aggregation of the polypeptide or protein, thereby controllably forming amorphous substances or single crystals.

[0127] According to an embodiment of the present invention, the freezing method is an operation known to those skilled in the art, such as using any refrigeration device for cooling and freezing or using any low-temperature substance for cooling and freezing; for example, the freezing method includes, but is not limited to, one or a combination of several cooling and freezing methods such as compression refrigeration equipment cooling and freezing, semiconductor refrigeration equipment cooling and freezing, liquid nitrogen cooling and freezing, liquid helium cooling and freezing, liquid carbon dioxide cooling and freezing, liquid oxygen cooling and freezing, liquid ethane cooling and freezing, dry ice cooling and freezing, ice cooling and freezing.

[0128] According to an embodiment of the present invention, the freezing operation pressure is not limited, and it can be freezing under normal pressure, or freezing under high pressure or low pressure.

[0129] According to an embodiment of the present invention, the freezing process is an operation known to those skilled in the art, such as freezing a solution of polypeptides or proteins from a liquid state to a solid state or a solid-liquid mixture through any process. Exemplarily, the freezing process includes, but is not limited to, one or a combination of several freezing processes such as rapid cooling, slow cooling, stepwise cooling, and heating followed by cooling.

[0130] According to embodiments of the present invention, the volume and shape of the polypeptide or protein solution are not particularly limited; similarly, the volume and shape of the solid formed by freezing the polypeptide or protein solution are not particularly limited, as long as it can be frozen into a solid; those skilled in the art will understand that the freezing can be the overall freezing of a polypeptide or protein solution of any volume, the freezing of a membrane formed by a polypeptide or protein solution of any volume, or the freezing of droplets formed by a polypeptide or protein solution of any volume.

[0131] According to an embodiment of the present invention, the solution of a polypeptide or protein frozen into a solid may optionally undergo a ripening treatment. The ripening temperature, ripening time, and ripening process are not particularly limited, but it is necessary to ensure that the frozen polypeptide or protein solution remains at least partially or completely in a solid state during the ripening treatment; that is, the polypeptide or protein solution remains frozen during the ripening process. For example, the solid may be ripened using the same method as the freezing treatment, or other methods may be used. The purpose of the ripening treatment is to regulate the aggregation of polypeptides or proteins and the growth rate of nanoparticles, thereby obtaining single crystals or amorphous products of polypeptides or proteins. Those skilled in the art will understand that the ripening temperature should be lower than the temperature at which the frozen polypeptide or protein solution remelts (i.e., T). 融化 Preferably, the ripening temperature is lower than T. 融化 Above 5°C, preferably below T 融化 Above 10℃.

[0132] According to an embodiment of the present invention, the ripening process involves keeping the solution of the polypeptide or protein in a frozen state for a period of time. This frozen state can be completely frozen or partially frozen, and can be selected according to procedures known to those skilled in the art.

[0133] According to an embodiment of the present invention, the ripening process, for example, employs rapid heating (or cooling) or slow heating (or cooling), and exemplaryly, the heating or cooling rate of the ripening process is greater than or equal to 10°C / min. This range of heating or cooling rates causes solute molecules to be rapidly released from the solid mixture and to generate disordered aggregation. By limiting the ripening time, the preparation of amorphous materials is guaranteed.

[0134] For example, the heating or cooling rate of the ripening process is less than 10°C / min. This range of heating or cooling rates will cause solute molecules to be slowly released from the solid mixture and thus generate ordered aggregation, which can prepare single crystals.

[0135] According to an embodiment of the present invention, the aging temperature (i.e., the reached temperature) controls the size of the crystallites in the freezing solvent, thereby controlling the aggregation rate of the pseudo-crystalline material. Specifically, the greater the temperature difference between the aging temperature and the freezing temperature, the larger the crystallites in the freezing solvent, the faster the aggregation rate of the pseudo-crystalline material, and the shorter the time required to form single crystals or amorphous materials. Consequently, the particle size of the prepared single crystals or amorphous materials is also larger. Conversely, the smaller the temperature difference between the aging temperature and the freezing temperature, the smaller the crystallites in the freezing solvent, the slower the aggregation rate of the pseudo-crystalline material, and the longer the time required to form single crystals or amorphous materials. In other words, the greater the temperature difference between the aging temperature and the freezing temperature, the larger the particle size of the prepared single crystals or amorphous materials.

[0136] According to embodiments of the present invention, the ripening time is not particularly limited and can be a process known to those skilled in the art. As can be seen from the above description of the mechanism of the method of this application, the ripening process can be understood as the nucleation and growth of amorphous materials or the formation and growth of crystals. Appropriately extending the ripening time can yield crystals with complete particle size and morphology. However, it should be noted that since adjusting the ripening time essentially regulates the aggregation rate and concentration of polypeptides or proteins, excessively long ripening may lead to excessively high aggregation rates and concentrations, which is detrimental to the formation of single crystals or amorphous materials. Exemplarily, the ripening time is greater than 1 picosecond; preferably, the ripening time is 1 to 1000 minutes; more preferably, the ripening time is 10 to 300 minutes.

[0137] For example, the ripening time is less than 25 minutes. By controlling the heating or cooling rate of the ripening process, amorphous materials can be prepared. When the ripening time is at least 25 minutes, the aggregation concentration of the crystalline material can be further controlled, for example, a single crystal can be prepared. However, the ripening time cannot be too long, as an excessively long ripening time may cause the known single crystal to further transform into a polycrystalline structure.

[0138] According to an embodiment of the present invention, the maturation process can employ any refrigeration device or any low temperature, as long as the solution of the polypeptide or protein remains frozen; for example, natural cooling, compression refrigeration equipment, semiconductor refrigeration equipment, or a combination of one or more of the following methods: liquid nitrogen, liquid helium, liquid carbon dioxide, liquid oxygen, liquid ethane, dry ice, ice, etc.

[0139] According to an embodiment of the present invention, in step (a3), the separation may be achieved by physical and / or chemical methods to separate the solvent frozen into a solid from the system. After the curing process, a single crystal or amorphous material has been prepared. At this point, the single crystal or amorphous material exists at the solvent crystal interface and needs to be separated by an appropriate method; or the solvent needs to be removed.

[0140] According to embodiments of the present invention, the physical methods include, but are not limited to, one or a combination of several of the following: rapid cooling separation, sublimation (such as vacuum sublimation), and dissolution. Sublimation may be performed, for example, by freeze-drying; vacuum sublimation may be performed, for example, by freeze-drying under vacuum conditions; and dissolution may be performed, for example, by dissolving a frozen solvent with another liquid solvent.

[0141] According to embodiments of the present invention, the chemical method includes, but is not limited to, one or a combination of chemical reactions and electrolysis.

[0142] According to the present invention, the method further includes the following steps:

[0143] (a4) Collect the single crystals or amorphous materials prepared in step (a3).

[0144] According to an embodiment of the present invention, in step (a4), the collection includes, but is not limited to, one or a combination of optical microscope collection, scanning electron microscope collection, dual-beam electron microscope collection, and transmission electron microscope collection.

[0145] [Methods for growing single crystals]

[0146] As mentioned above, the present invention also provides a method for cultivating single crystals, the method comprising the above-described method for preparing single crystals.

[0147] According to an embodiment of the present invention, the method for cultivating single crystals further includes the following steps:

[0148] (b1) Transfer the single crystals of the prepared polypeptides or proteins to the mother liquor of the polypeptides or proteins for culturing.

[0149] (b2) Collect the single crystals from step (b1).

[0150] According to embodiments of the present invention, the transfer is any method known to those skilled in the art capable of transferring a single crystal, including but not limited to one or a combination of optical microscopy transfer, scanning electron microscopy transfer, dual-beam electron microscopy transfer, and transmission electron microscopy transfer.

[0151] According to an embodiment of the present invention, the mother liquor is a mother liquor system known to those skilled in the art that is compatible with the single crystal phase to be cultured. For example, it can be a saturated solution system, a supersaturated solution system, or an unsaturated solution system; for example, when the substance to be crystallized is L-glutathione, an aqueous solution of L-glutathione is selected as the mother liquor.

[0152] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0153] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0154] The curing time mentioned in the following embodiments refers to the time required to raise or lower the temperature to the curing temperature after the freezing process is completed, and the time to maintain the temperature at the curing temperature; the maintenance time refers to the time to maintain the temperature at the curing temperature.

[0155] Example 1

[0156] A 0.5 mg / mL L-glutathione solution was prepared using water. 1 mL of the solution was taken with a syringe and spread onto a silicon wafer. The wafer was then slowly cooled to -24°C until completely frozen. Finally, it was aged at -10°C for 20 minutes, followed by freeze-drying to completely sublimate the solid water (ice), yielding L-glutathione single crystals. A high-quality single crystal was selected from the beaker (the selection method is conventional for those skilled in the art, such as judging by morphology and structure) and transferred to a saturated L-glutathione aqueous solution. The crystals were then placed in a constant temperature and humidity environment of 25°C and 40% for a period of time to grow larger L-glutathione crystals. (See Appendix) Figure 1 .

[0157] Example 2

[0158] A 1 mg / mL L-glutathione solution was prepared using water. 100 mL of this solution was transferred to a beaker using a graduated cylinder and slowly cooled to -24°C until completely frozen. Finally, it was placed in a -15°C freezer for 60 minutes to mature, followed by rapid cooling to remove the frozen ice, yielding L-glutathione single crystals. High-quality single crystals were then selected from silicon wafers and transferred to a saturated L-glutathione aqueous solution. These crystals were then placed in a constant temperature and humidity environment (25°C, 40% relative humidity) for a period of time to grow larger L-glutathione crystals.

[0159] Example 3

[0160] A 0.2 mg / mL L-glutathione solution was prepared using water. 20 μL of this solution was pipetted onto a silicon wafer at -90°C. The wafer temperature was controlled using a hot-cold stage. The temperature was then increased to -10°C at a rate of 15°C / min and maintained at this temperature for 30 min. The frozen ice was then removed by rapid cooling to obtain L-glutathione single crystals. High-quality single crystals were selected from the silicon wafer and transferred to a saturated L-glutathione aqueous solution. These crystals were then placed in a constant temperature and humidity environment (25°C, 40% relative humidity) for a period of time, resulting in the growth of larger L-glutathione crystals.

[0161] Example 4

[0162] A 0.1 mg / mL L-carnosine solution was prepared using dimethyl sulfoxide (DMSO). 100 mL of this solution was transferred to a beaker using a graduated cylinder and slowly cooled to -24°C until completely frozen. Finally, it was aged at -20°C for 90 minutes. The sample was then freeze-dried to completely sublimate the frozen DMSO, yielding L-carnosine single crystals. The best-quality single crystals were then transferred to a saturated L-carnosine DMSO solution and placed in a constant temperature and humidity environment (25°C, 40% relative humidity) for a period of time. This process resulted in the growth of larger L-carnosine crystals. (See attached image.) Figure 2 .

[0163] Example 5

[0164] A 0.1 mg / mL L-carnosine solution was prepared using dimethyl sulfoxide. 15 μL of this solution was pipetted onto a silicon wafer at -90°C. The wafer temperature was controlled using a hot-cold stage. The temperature was then increased to -18°C at a rate of 5°C / min and maintained at this temperature for 60 min. The sample was then freeze-dried to completely sublimate the solid ice. High-quality single crystals were then selected from the silicon wafer and transferred to a saturated L-carnosine solution. These crystals were placed in a constant temperature and humidity environment at 25°C and 40% relative humidity for a period of time, resulting in the growth of larger L-carnosine crystals.

[0165] Example 6

[0166] A 0.2 mg / mL diglycinate solution was prepared using water. 15 μL of this solution was pipetted onto a silicon wafer at -90°C. The wafer temperature was controlled using a hot-cold stage. The temperature was then increased to -10°C at a rate of 20°C / min and maintained at this temperature for 120 min. The sample was then freeze-dried to completely sublimate the ice. High-quality single crystals were then selected from the silicon wafer and transferred to a saturated diglycinate solution. These crystals were placed in a constant temperature and humidity environment (25°C, 40% relative humidity) for a period of time, resulting in the growth of larger diglycinate crystals.

[0167] See attached Figure 3

[0168] The operating steps are the same as in Example 1. This application also prepared single crystals of the following polypeptides or proteins, and the preparation conditions differ from those in Example 1 as shown in the table below:

[0169]

[0170]

[0171] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing single crystals of polypeptides or proteins, characterized in that, This method differs from traditional evaporation and cooling crystallization methods; it utilizes solution freezing to induce peptide or protein crystallization. The method for preparing single crystals of polypeptides or proteins includes the following steps: (a1) Preparing a solution of a polypeptide or protein, said solution of a polypeptide or protein being composed of a polypeptide or protein and a freezeable solvent; (a2) Freeze and mature the solution of the polypeptide or protein from step (a1) to prepare a mixed system containing a single crystal of polypeptide or protein and a frozen solvent. During the maturation process, the heating rate is less than 10°C / min; the maturation temperature is lower than T. 融化 Above 5℃, the T 融化 The temperature at which a frozen solution of polypeptides or proteins can be rethawed; optionally, (a3) The single crystal of the polypeptide or protein is separated from the mixture of single crystal of polypeptide or protein and frozen solvent in step (a2). The freezing process involves transforming the solution of peptides or proteins from a liquid state to a solid state in step (a1). The ripening process involves keeping the solution of peptides or proteins in a frozen state for a period of time. During the freezing process, the solvent freezes into a solid state, while the peptides or proteins dissolved in the solution accumulate at the interface between the solid and solvent states. That is, during the freezing process, as the solvent freezes into a solid state, the peptides or proteins are released and accumulate at the interface between the solid and solvent states. By regulating the freezing process of the solution and the recrystallization process of the solidified solvent, the release and aggregation rate of peptides or proteins can be further regulated to form single crystals of peptides or proteins.

2. The method according to claim 1, characterized in that, In step (a2), during the aging process, the temperature is increased at a rate of less than 10℃ / min to a certain temperature and maintained for a period of time to obtain a mixed system containing single crystals of polypeptides or proteins and frozen solvent.

3. The method according to claim 1, characterized in that, The freezeable solvents include water and / or organic solvents.

4. The method according to any one of claims 1-3, characterized in that, The polypeptide includes homopeptides or heteropeptides; the homopeptides include linear peptides or cyclic peptides; the heteropeptides include pigment peptides, glycopeptides, lipopeptides or ketal peptides.

5. The method according to any one of claims 1-3, characterized in that, The proteins include simple proteins and conjugated proteins; the simple proteins include albumins, globulins, histones, protamines, glutenins, and sclerosins; the conjugated proteins include glycoproteins, nucleoproteins, lipoproteins, phosphoproteins, metalloproteins, and pigment proteins.

6. The method according to any one of claims 1-3, characterized in that, The solubility of the polypeptide or protein in the solvent is readily soluble, soluble, slightly soluble, or sparingly soluble.

7. The method according to claim 1, characterized in that, The amount of the polypeptide or protein dissolved in the solvent is greater than or equal to 1 × 10⁻⁶. -7 Solvent used per 100g.

8. The method according to claim 7, characterized in that, The amount of the polypeptide or protein dissolved in the solvent is greater than or equal to 0.001 g / 100 g of the solvent used.

9. The method according to claim 8, characterized in that, The amount of the polypeptide or protein dissolved in the solvent is greater than or equal to 0.01g / 100g of the solvent used.

10. The method according to claim 9, characterized in that, The amount of the polypeptide or protein dissolved in the solvent is greater than or equal to 0.1g / 100g of the solvent used.

11. The method according to claim 10, characterized in that, The amount of the polypeptide or protein dissolved in the solvent is greater than or equal to 1g / 100g of the solvent used.

12. The method according to claim 11, characterized in that, The amount of the polypeptide or protein dissolved in the solvent is greater than or equal to 10g / 100g of the solvent used.

13. The method according to any one of claims 1-3, characterized in that, Step (a2) specifically includes the following steps: The solution of the polypeptide or protein from step (a1) is cooled and frozen into a solid mixture, and then subjected to aging treatment to prepare a mixed system containing polypeptides or proteins in single crystal-frozen solvent.

14. The method according to any one of claims 1-3, characterized in that, The freezing method includes one or a combination of several of the following methods: natural cooling freezing, cooling freezing using a compression refrigeration device, cooling freezing using a semiconductor refrigeration device, cooling freezing using liquid nitrogen, cooling freezing using liquid helium, cooling freezing using liquid carbon dioxide, cooling freezing using liquid oxygen, cooling freezing using liquid ethane, cooling freezing using dry ice, and cooling freezing using ice.

15. The method according to any one of claims 1-3, characterized in that, The freezing process includes one or a combination of several of the following freezing processes: rapid cooling, slow cooling, step-by-step cooling, and heating followed by cooling.

16. The method according to any one of claims 1-3, characterized in that, The freeze refers to a complete freeze.

17. The method according to any one of claims 1-3, characterized in that, In step (a3), the separation is achieved by using physical and / or chemical methods to separate the solvent frozen into a solid from the mixed system; The physical methods mentioned include one or a combination of several of the following: rapid cooling separation, sublimation, and dissolution; The chemical method mentioned includes one or a combination of chemical reactions and electrolysis.

18. The method according to any one of claims 1-3, characterized in that, The method further includes the following step: (a4) collecting the single crystals prepared in step (a3); In step (a4), the collection includes one or a combination of optical microscope collection, scanning electron microscope collection, dual-beam electron microscope collection, and transmission electron microscope collection.

19. A method for culturing single crystals of polypeptides or proteins, characterized in that, The method includes the method for preparing single crystals of polypeptides or proteins as described in any one of claims 1-18; the method for culturing single crystals of polypeptides or proteins further includes the following steps: (b1) Transfer the prepared single crystal of the polypeptide or protein to the mother liquor of the polypeptide or protein for culture. (b2) Collect the single crystals obtained in step (b1).

20. The method according to claim 19, characterized in that, In step (b1), the transfer is to transfer the mixture of single crystal containing polypeptide or protein and frozen solvent from step (a2) to the mother liquor of polypeptide or protein for single crystal culture; or the transfer is to directly transfer the single crystal after solvent removal from step (a3) ​​to the mother liquor of polypeptide or protein for single crystal culture. Alternatively, the single crystals collected in step (a4) can be transferred to the mother liquor of the polypeptide or protein for single crystal culture.

21. The method according to claim 19, characterized in that, The transfer includes one or a combination of optical microscope transfer, scanning electron microscope transfer, dual-beam electron microscope transfer, and transmission electron microscope transfer.

22. The method according to claim 19, characterized in that, In step (b1), the method for cultivating the single crystal includes one or a combination of several of the following: evaporation, cooling, and diffusion.

23. The method according to claim 19, characterized in that, In step (b2), the collection includes one or a combination of optical microscope collection, scanning electron microscope collection, dual-beam electron microscope collection, and transmission electron microscope collection.

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