Device for protein crystallization in microgravity environment and protein crystallization method using same

Abstract

According to an embodiment of the present invention, a device for crystallizing proteins in a microgravity environment may include: a gas storage unit that stores at least one gas and inserts the stored at least one gas into a bubble generation unit; a solution storage unit that stores a solution for protein crystallization and inserts the stored solution into the bubble generation unit; the bubble generation unit that generates a solution containing bubbles on the basis of the at least one gas received from the gas storage unit and the stored solution received from the solution storage unit, and inserts the solution containing the generated bubbles into a protein crystallization chamber; the protein crystallization chamber in which protein crystallization of the bubble-containing solution received from the bubble generation unit is performed in the microgravity environment; a pressure control unit for adjusting the pressure in the protein crystallization chamber; and a temperature control unit for adjusting the temperature in the protein crystallization chamber.

Description

Protein crystallization device in a microgravity environment and method for crystallizing proteins using the same

[0001] The present invention relates to a method for crystallizing a protein using a solution containing gas bubbles in a microgravity environment and a device supporting the same.

[0002] Structural studies of biopolymers, particularly proteins, are crucial for studying biological functions and developing structure-based medicines. Protein crystals are necessary for studying protein conformations. The production of protein crystals typically takes more than six months, and sometimes, manufacturing them is impossible. One widely known protein crystallization method involves mixing a protein solution on a glass slide surface, placing the slide with the protein solution attached as droplets in a crystallization vessel, sealing the glass plate and vessel, and slowly evaporating the solvent to produce protein crystals. Another example of a protein crystallization method involves controlling solubility in a protein solution batch through temperature control, antisolvent addition, evaporation, and pH adjustment, thereby producing protein crystals.

[0003] Prior art worth referring to includes Korean Patent Publication No. 10-1025228 (published on April 1, 2011) and Korean Patent Publication No. 10-1762963 (published on July 28, 2017).

[0004] The present invention relates to a method for crystallizing a protein using a solution containing gas bubbles in a microgravity environment such as space, and a device supporting the method.

[0005] In one embodiment of the present invention, a device for crystallizing a protein in a microgravity environment may include a gas storage unit that stores at least one gas and inserts the stored at least one gas into a bubble generation unit, a solution storage unit that stores a solution for protein crystallization and inserts the stored solution into the bubble generation unit, the bubble generation unit that generates a solution containing bubbles based on the at least one gas received from the gas storage unit and the stored solution received from the solution storage unit, and inserts the solution containing the generated bubbles into a protein crystallization chamber, the protein crystallization chamber that performs protein crystallization in the microgravity environment with the solution containing bubbles received from the bubble generation unit, and a pressure control unit that regulates a pressure in the protein crystallization chamber.

[0006] In one embodiment of the present invention, the device for crystallizing a protein in a microgravity environment may further include a monitoring unit for checking a solution or protein crystals containing the bubbles stored in the protein crystallization chamber.

[0007] In one embodiment of the present invention, the gas storage unit may include polymer capsules containing at least one chemical substance. In addition, the gas storage unit may include microparticles, nanoparticles, etc. that can serve as templates for heterogeneous nucleation. In one embodiment of the present invention, the bubble generation unit (130) may generate a solution containing bubbles based on the at least one gas and the stored solution through at least one technique selected from the group consisting of microfluidic, electrically induced bubble generation, sonication, baffle, saline shaking, and freeze drying.

[0008] In one embodiment of the present invention, the solution storage unit comprises a plurality of solution storage units configured in parallel, the protein crystallization chamber comprises a plurality of protein crystallization chambers configured in parallel, the pressure control unit comprises a plurality of pressure control units configured in parallel, and each of the plurality of pressure control units can control the pressure of each of the protein crystallization chambers configured in parallel.

[0009] In one embodiment of the present invention, the bubble generating unit can insert a solution containing bubbles generated based on the at least one gas and a solution received from each of the plurality of solution storage units configured in parallel into each of the plurality of protein crystallization chambers.

[0010] In one embodiment of the present invention, the temperature control unit adjusts the temperature of each of the plurality of protein crystallization chambers configured in parallel, and the pressure control unit adjusts the pressure of each of the plurality of pressure control units configured in parallel.

[0011] In one embodiment of the present invention, a method for crystallizing a protein in a microgravity environment may include the steps of inserting a solution containing bubbles generated based on at least one gas and solution into a protein crystallization chamber, controlling the temperature and pressure of the solution containing the bubbles, and culturing the solution containing the bubbles to perform protein crystallization in the microgravity environment.

[0012] According to one embodiment of the present invention, by crystallizing a protein in a microgravity environment other than standard gravity on Earth, crystallized protein crystals can be produced while minimizing gravitational convection and sedimentation.

[0013] According to one embodiment of the present invention, by performing a protein crystallization process in a microgravity environment rather than the standard gravity on Earth, high-quality, more precise and uniform crystallized protein crystals can be produced with less mosaicity.

[0014] FIG. 1 is a drawing illustrating an example of a protein crystallization device according to one embodiment of the present invention.

[0015] FIG. 2A is a perspective view illustrating an example of a protein crystallization device according to one embodiment of the present invention.

[0016] FIG. 2b is a diagram illustrating another example of a protein crystallization device according to one embodiment of the present invention.

[0017] FIG. 3 is a diagram illustrating a process for performing protein crystallization according to one embodiment of the present invention.

[0018] FIG. 4A and FIG. 4B are drawings illustrating an example of a crystallized protein according to one embodiment of the present invention.

[0019] FIG. 5 is a drawing illustrating an example of a crystallized protein according to one embodiment of the present invention.

[0020] FIG. 6 is a drawing illustrating an example in which at least one component of a protein crystallization device is configured in multiple pieces and connected in parallel, according to one embodiment of the present invention.

[0021] FIG. 7 is a diagram illustrating another example in which at least one component of a protein crystallization device is configured in multiple pieces and connected in parallel, according to one embodiment of the present invention.

[0022] FIGS. 8A to 8C are diagrams illustrating another example of a protein crystallization device according to one embodiment of the present invention.

[0023] FIG. 9 is a diagram illustrating a flowchart for performing protein crystallization according to one embodiment of the present invention.

[0024] Hereinafter, embodiments disclosed in this specification will be described in detail with reference to the attached drawings. Regardless of the drawing reference numerals, identical or similar components will be assigned the same reference numerals, and redundant descriptions thereof will be omitted. Furthermore, when describing embodiments disclosed in this specification, if a detailed description of a related known technology is judged to obscure the gist of the embodiments disclosed in this specification, the detailed description thereof will be omitted.

[0025] Terms that include ordinal numbers, such as first, second, etc., may be used to describe various components, but the components are not limited by these terms. These terms are used solely to distinguish one component from another.

[0026] Singular expressions include plural expressions unless the context clearly indicates otherwise.

[0027] In this application, each step described may be performed regardless of the listed order, except in cases where a special causal relationship requires that the steps be performed in the listed order.

[0028] In this application, terms such as “include” or “have” are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but should be understood not to exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

[0029] According to various embodiments of the present invention, a technique for crystallizing a protein using a protein crystallization device (100) under a microgravity environment is described. Here, a microgravity environment may refer to a state in which the influence of gravity is very small. For example, a microgravity environment may occur within the International Space Station (ISS) or within a spacecraft. As another example, a microgravity environment may occur for a short period of time, such as when an aircraft draws a large parabolic trajectory. As a further example, a microgravity environment may occur for a short period of time, such as when experimental equipment is dropped from a drop tower. As another further example, a microgravity environment may occur for a certain period of time, such as when a rocket ascends to an altitude near the boundary of space and then falls back to Earth. As yet another example, a microgravity environment may persist while the protein crystallization device (100) is located in space.

[0030] According to various embodiments of the present invention, when crystallizing a protein using a protein crystallization device (100) under a microgravity environment, various conditions may differ from those under the Earth's gravity environment. For example, in the Earth's gravity environment, convection, in which air or liquid rises and falls due to temperature differences, generally occurs, which may affect temperature distribution and heat transfer. In addition, the produced protein crystals may sediment due to gravity, which may affect crystal-molecule interactions. In a microgravity environment, convection and sedimentation may hardly occur due to weak gravity. Since the microgravity environment does not cause distortion due to gravity, more uniform and perfect crystals can grow.

[0031] FIG. 1 is a drawing illustrating an example of a protein crystallization device (100) according to one embodiment of the present invention.

[0032] According to various embodiments, the protein crystallization device (100) may include a gas storage unit (110), a solution storage unit (120), a bubble generation unit (130), a protein crystallization chamber (140), a pressure control unit (150), and a temperature control unit (160). The protein crystallization device (100) may omit at least one of the components or may additionally include other components.

[0033] According to various embodiments, the gas storage unit (110) can store at least one gas. According to one embodiment, the at least one gas may be nitrogen, argon, helium, oxygen, carbon dioxide, etc. In addition, various gases may be selected and applied in consideration of solubility, polarizability, zeta potential, and interaction with a target substance. Here, solubility may be a measure of how well a specific substance dissolves in a solvent. Polarizability indicates the degree to which an electron cloud within a molecule can be modified by an external electric field, and may be an important factor in determining the dielectric reactivity of a molecule. Zeta potential may be a physical property indicating an electrical potential difference between the surface of dispersed particles (e.g., colloidal particles) and the surrounding solvent.

[0034] In another embodiment, the at least one gas may be a mixture of at least two of nitrogen, argon, helium, oxygen, and carbon dioxide. Alternatively, the at least one gas may be a mixture of nitrogen, argon, helium, oxygen, and carbon dioxide, and, depending on the desired substance, air.

[0035] According to various embodiments, the gas storage unit (110) can insert at least one gas into the bubble generation unit (130). The protein crystallization device (100) or the gas storage unit (110) can include a pump, an actuator, a valve, etc. that provide related functions to enable the insertion of at least one gas into the bubble generation unit (130).

[0036] The protein crystallization device (100) or the gas storage unit (110) may utilize various technical methods to insert at least one gas into the bubble generation unit (130), and may include additional devices supporting each technical method. For example, pneumatic pressure utilizing the pressure difference of air or gas may be utilized to move at least one gas from the gas storage unit (110) to the bubble generation unit (130). As another example, a syringe pump that precisely moves a gas or liquid using a piston like a syringe may be utilized. As a further example, a diaphragm pump that compresses and moves a gas using a diaphragm may be utilized. As yet another additional example, a piezoelectric pump that moves a gas using the piezoelectric effect may be utilized. As another example, a peristaltic pump that pushes a gas or the like by compressing a flexible hose in a certain direction may be utilized. However, the protein crystallization device (100) or gas storage unit (110) is not limited to the above example in terms of technology for inserting at least one gas into the bubble generation unit (130), and may include various widely known pumps or actuators.

[0037] According to various embodiments, the protein crystallization device (100) or gas storage unit (110) may include various types of valves to control the direction of the flow of at least one gas. Here, the valve may provide a function to control the start, stop, or change of direction of the gas or flow.

[0038] According to various embodiments, the gas storage unit (110) may store polymer capsules containing at least one chemical substance. Here, the polymer capsule may be a microscopic capsule composed of a polymer material outer shell and containing liquid, gas, and solid substances inside. The polymer capsule may respond to specific conditions (e.g., pH changes, temperature, presence of enzymes) to release the drug at a precise time at the target site. In addition, the gas storage unit (110) may include microparticles, nanoparticles, etc. that may serve as templates for heterogeneous nucleation.

[0039] According to various embodiments, the solution storage unit (120) can store a solution for protein crystallization. Here, the solution for protein crystallization can be any protein molecule that is the target molecule for protein crystallization. For example, the solution for protein crystallization can be enzymes, structural proteins, transport proteins, signaling proteins, immunoproteins, motor proteins, storage proteins, etc.

[0040] According to one embodiment, the solution for protein crystallization may include, in addition to proteins, biomacromolecules that constitute living organisms, such as nucleic acids (DNA, RNA), polysaccharides, and lipids, and low-molecular-weight pharmaceuticals. According to a further embodiment, the solvent stored in the solution storage unit (120) may be water, an organic solvent (e.g., methanol, ethanol, acetone, chloroform, dichloromethane, ethyl acetate, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), etc.). According to another further embodiment, the solution for protein crystallization may include salts, surfactants, etc. that affect the crystallization process of the protein.

[0041] According to various embodiments, the solution storage unit (120) can insert the stored solution into the bubble generation unit (130). The protein crystallization device (100) or the solution storage unit (120) can include a pump (e.g., a syringe pump, a piezoelectric pump, etc.), an actuator, a valve, etc. that provide related functions so that the stored solution can be inserted into the bubble generation unit (130). Here, inserting the stored solution into the bubble generation unit (130) can include injecting, putting, administering, injecting, or inserting the stored solution into the bubble generation unit (130).

[0042] According to various embodiments, the bubble generating unit (130) can generate a solution containing bubbles based on at least one gas received from the gas storage unit (110) and a stored solution received from the solution storage unit (120).

[0043] According to one embodiment, the bubble generation unit (130) can generate a bubble-containing solution based on at least one gas and a stored solution through at least one of microfluidic, electrically induced bubble generation, sonication, baffle, saline shaking, and freeze drying techniques.

[0044] According to one embodiment, the bubble generating unit (130) can generate a solution containing bubbles based on at least one gas and a stored solution through microfluidic technology. Microfluidic technology may be a technology for precisely manipulating liquids through microfluidic channels. For example, a microfluidic T-junction may be a technology in which two fluid channels meet in a 'T' shape, and bubbles are formed as fluid flows from one channel to the other. When the bubble generating unit (130) uses the microfluidic T-junction technology, the bubble generating unit (130) may be formed as a microchannel having a 'T'-shaped intersection, with a main channel being vertically connected to another channel. The main material of the microfluidic T-junction may be a material such as polydimethylsiloxane (PDMS), glass, or a polymer. The microfluidic T-junction may be manufactured through photolithography and soft lithography processes.

[0045] For additional examples, microfluidic flow-focusing can be a technology where a fluid flows through two opposing channels, concentrating it into a narrow region, and forming bubbles as it passes through that region. A microfluidic coflowing device can be a technology where particles or bubbles are formed as two fluids flow in parallel at different speeds or through changes in their relative positions. A microfluidic terrace-based microchannel (MC) can be a technology where bubbles are formed as a fluid flows over the steps in a microchannel with a stepped structure. A microfluidic partitioned-edge droplet formation unit utilizes edge (EDGE) technology to partition the fluid within the channel, which can produce precisely sized droplets. Microfluidic flow-focusing, microfluidic coflowing device, microfluidic terrace-based microchannel (MC), and microfluidic partitioned-edge droplet formation unit can be made of materials such as polydimethylsiloxane (PDMS), glass, or polymers.

[0046] In one embodiment, the bubble generation unit (130) can generate a solution containing bubbles through electrically induced bubble generation. When the bubble generation unit utilizes an electrically induced bubble generation method, a container, a power supply, a metallic probe capable of applying voltage, and the like can be configured.

[0047] In one embodiment, the bubble generating unit (130) may generate a solution containing bubbles based on at least one gas and a stored solution through sonication technology. The sonication technology may be a technology that finely pulverizes particles in a liquid or uniformly mixes a mixture using ultrasonic energy. When the bubble generating unit (130) uses sonication technology, the bubble generating unit (130) may be composed of a container, an ultrasonic generator, an interface for controlling temperature, time, and ultrasonic intensity, an ultrasonic probe, a piezoelectric element, a cooling system, a power supply, etc. In one embodiment, the bubble generating unit (130) may induce, impede, or mix a flow within a fluid through a baffle. When the bubble generating unit (130) uses a baffle, the baffle may be configured in the shape of a flat plate or a rod and may be configured vertically or inclinedly with respect to the inner wall of the reactor or container. Here, the baffle may be made of various materials such as stainless steel, titanium, plastic, or ceramic.

[0048] In one embodiment, the bubble generating unit (130) can generate a solution containing bubbles based on at least one gas and a stored solution through a salt water shaking technique. In one embodiment, when the bubble generating unit (130) utilizes the salt water shaking technique, the bubble generating unit (130) can be configured with a mixing vessel containing a sample and salt water, a device for shaking the mixing vessel using an electric motor, and the like.

[0049] In one embodiment, the bubble generating unit (130) can generate a solution containing bubbles based on at least one gas and a stored solution through a freeze drying technique. In one embodiment, when the bubble generating unit (130) uses a freeze drying technique, the bubble generating unit (130) can be configured with a cooling device for cooling a sample, a vacuum chamber for providing a low-pressure environment, a vacuum pump for removing air and moisture within the vacuum chamber, a heat source for providing temperature to the sample, a condenser for removing moisture that has become a gaseous state by converting it back to a solid state, etc.

[0050] According to various embodiments, the bubble generation unit (130) can insert a solution containing the generated bubbles into a protein crystallization chamber (140). The protein crystallization device (100) or the bubble generation unit (130) can include a pump (e.g., a syringe pump, a piezoelectric pump, etc.), an actuator, a valve, etc. that provide related functions so that the stored solution can be inserted into the bubble generation unit (130). Here, inserting the solution containing the generated bubbles into the protein crystallization chamber (140) can include injecting, placing, administering, injecting, or inserting the solution containing the generated bubbles into the protein crystallization chamber (140).

[0051] According to various embodiments, the protein crystallization chamber (140) can perform protein crystallization in a microgravity environment using a solution containing bubbles received from the bubble generation unit (130). Here, the microgravity environment is a state in which the influence of gravity is very small, and may be an environment in space or a specific state (e.g., a drop tower, etc.).

[0052] According to various embodiments, the protein crystallization chamber (140) may be a device including a space for protein crystallization. The protein crystallization chamber (140) may be manufactured in various sizes and shapes, and the materials used may be determined according to the purpose of the experiment or the characteristics of the reacting substance. According to one embodiment, the protein crystallization chamber (140) may be composed of an optically transparent material. For example, the protein crystallization chamber (140) may be manufactured from a material such as polycarbonate, polymethyl methacrylate (PMMA), or a silicone-based polymer that allows light of a specific wavelength. The protein crystallization chamber (140) may be manufactured by taking into consideration the specific gravity, flow rate, length, viscosity, etc. of the fluid to secure a laminar flow, which is a uniform and orderly flow of the fluid.

[0053] According to various embodiments, the protein crystallization chamber (140) may include one or more inlets for inserting a solution containing bubbles received from the bubble generating unit (130) into the top, bottom, or bottom. Here, the inlet is for injecting various fluids, such as a solution containing bubbles, a sample, etc., into the protein crystallization chamber (140), and may be mounted or connected to a pipeline, a syringe, a pump, a valve, or a control device.

[0054] According to various embodiments, the protein crystallization chamber (140) may include a channel connected to a pressure control unit (150) at the top, bottom, or bottom of the protein crystallization chamber (140). Here, the channel may be a passage connecting the protein crystallization chamber (140) and the pressure control unit (150). The pressure within the chamber may be controlled through the channel.

[0055] According to various embodiments, the pressure control unit (150) can control the pressure of the protein crystallization chamber (140). In one embodiment, the pressure control unit (150) can be designed to control the pressure of the protein crystallization chamber (140) when a solution containing bubbles is introduced into the protein crystallization chamber (140). For example, the pressure control unit (150) can be configured in the form of an expandable reservoir, and can be designed to effectively control pressure changes due to the inflow of fluid. As another example, the pressure control unit (150) can be configured in the form of a cylinder having a plunger, and the plunger can be configured in a form that moves inside the cylinder.

[0056] According to various embodiments, the pressure control unit (150) can store or move a solution or fluid containing bubbles included in the protein crystallization chamber (140). According to one embodiment, the pressure control unit (150) can be connected to a temperature control unit (160). For example, when the pressure control unit (150) is connected to the temperature control unit (160), simultaneous control of temperature and pressure is possible, enabling precise environmental control and enabling changes in the state of the fluid.

[0057] According to various embodiments, the temperature control unit (160) can adjust the temperature of the protein crystallization chamber (140). In one embodiment, the temperature control unit (160) may be a device that adjusts the temperature of the protein crystallization chamber (140). For example, the temperature control unit (160) may be a device that utilizes a Peltier element. Here, the Peltier element may be an electronic cooler that uses electricity to cool one side and heat the opposite side.

[0058] According to various embodiments, the protein crystallization device (100) may further include a monitoring unit that checks the solution containing the bubbles or protein crystals stored in the protein crystallization chamber (140). According to one embodiment, the monitoring unit is a device that can observe the bubbles or crystallized proteins in the protein crystallization chamber (140), and may be a microscope (e.g., an optical microscope, an electron microscope, an in situ emission microscope, etc.), a spectrometer, an electrochemical device, etc.

[0059] FIG. 2A is a perspective view illustrating an example of a protein crystallization device (100) according to one embodiment of the present invention. According to various embodiments, the protein crystallization device (100) may include a bubble generation unit (130), a protein crystallization chamber (140), and a pressure control unit (150). The protein crystallization device (100) may omit at least one of the components or may additionally include other components.

[0060] According to various embodiments, the bubble generating unit (130) can generate a solution containing bubbles based on at least one gas received from the gas storage unit (110) and a stored solution received from the solution storage unit (120).

[0061] In one embodiment, the bubble generating unit (130) can generate a solution containing bubbles based on at least one gas and a stored solution through microfluidic technology. When the bubble generating unit (130) utilizes microfluidic T-junction technology, the bubble generating unit (130) can be formed as a microchannel having a 'T'-shaped intersection, with a main channel being vertically connected to another channel.

[0062] According to various embodiments, the protein crystallization chamber (140) may include at least one lower inlet (141a) and at least one upper inlet (143a). Here, the lower portion of the protein crystallization chamber (140) including the at least one lower inlet (141a) of the protein crystallization chamber (140) and the upper portion of the protein crystallization chamber (140) including the at least one upper inlet (143a) of the protein crystallization chamber (140) may be made of a metal having high conductivity.

[0063] The protein crystallization chamber (140) can inject a solution containing bubbles received from the bubble generating unit (130) through at least one lower inlet (141a). Here, the at least one lower inlet (141a) is for injecting various fluids, such as a solution containing bubbles, a sample, etc., into the protein crystallization chamber (140), and can be mounted or connected to a pipeline, syringe, pump, valve, or control device.

[0064] At least one upper inlet (143a) of the protein crystallization chamber (140) may be connected to a pressure control unit (150) that controls the pressure of the protein crystallization chamber (140). For example, the pressure control unit (150) may be configured in a cylinder shape with a plunger so that the pressure of the protein crystallization chamber (140) can be controlled when a solution containing bubbles is inserted into the protein crystallization chamber (140), and the plunger may be configured in a shape that moves inside the cylinder.

[0065] FIG. 2b is a drawing illustrating another example of a protein crystallization device (100) according to one embodiment of the present invention.

[0066] According to various embodiments, the protein crystallization device (100) may include a bubble generation unit (130), a protein crystallization chamber (140), and a pressure control unit (150). The protein crystallization device (100) may omit at least one of the components or additionally include other components.

[0067] According to various embodiments, the bubble generating unit (130) can generate a solution containing bubbles based on at least one gas received from the gas storage unit (110) and a stored solution received from the solution storage unit (120).

[0068] According to various embodiments, the protein crystallization chamber (140) may include at least one lower tubing (141b), at least one upper tubing (143b), a peltier (145), and a metal block (metal block 147) having a hole (hole 147a). Here, a monitoring device (e.g., an optical microscope, an electron microscope, an in situ microscope, etc.), a spectrometer, an electrochemical device, etc., can check the solution containing bubbles or protein crystals stored in the protein crystallization chamber through the hole (hole 147a).

[0069] The protein crystallization chamber (140) may be configured in a form in which a peltier (145) is mounted in a form in which a metal block (metal block 147) is in contact with at least one side of the metal block (metal block 147). For example, the protein crystallization chamber (140) may be configured in a form in which a square frame structure (e.g., a rectangular parallelepiped structure, etc.) composed of a peltier (145) and a metal block (metal block 147) is positioned in parallel. Here, the peltier (145) may be a device that generates a peltier effect by allowing current to pass through it, in which one side absorbs heat and the other side releases heat. Here, the metal block (metal block 147) may be manufactured in various shapes and sizes as a metal piece.

[0070] According to one embodiment, the protein crystallization chamber (140) can insert a solution containing bubbles received from the bubble generating unit (130) through at least one lower end tubing (141b) of the protein crystallization chamber (140). Here, the at least one lower end tubing (141b) is for injecting various fluids such as a solution containing bubbles, a sample, etc. into the protein crystallization chamber (140), and can be mounted or connected to a pipeline, a syringe, a pump, a valve, or a control device. At least the lower end tubing (141b) can penetrate at least one surface of the lower end lid when the lower end of the protein crystallization chamber (140) has a lower end lid.

[0071] According to one embodiment, at least one upper tubing (143b) of the protein crystallization chamber (140) may be connected to a pressure control unit (150) that controls the pressure of the protein crystallization chamber (140). For example, the pressure control unit (150) may be configured as an inflatable bag, which may be a bag whose volume can be controlled by injecting air or other gases. In addition, at least one upper tubing (143b) may penetrate at least one surface of the upper lid, if the upper lid of the protein crystallization chamber (140) is provided. FIG. 3 is a diagram illustrating a process of performing protein crystallization according to one embodiment of the present invention.

[0072] Referring to step (301), the protein crystallization chamber (140) can insert a solution containing bubbles received from the bubble generating unit (130) through at least one lower inlet (141a).

[0073] Referring to steps (303) and (305), in a microgravity environment, a laminar flow phenomenon may occur in which a solution containing bubbles flows smoothly in parallel layers within a protein crystallization chamber (140). Here, laminar flow may refer to a phenomenon in which each layer of a solution containing bubbles does not mix with each other and each layer moves at different speeds. In a microgravity environment, laminar flow can be maintained more stably because there is no change in flow due to gravity. In addition, in a microgravity environment, since the rising or settling of bubbles due to gravity is minimal, bubbles inside the fluid can remain for a long time without floating or settling within the solution and without being adjacent to the wall.

[0074] Referring to step (307), a crystallized protein can be produced by controlling the pressure of the protein crystallization chamber (140), adjusting the temperature of the protein crystallization chamber (140), and incubating. According to various embodiments, protein crystallization may mean that the protein is aligned to form a regular lattice structure to form a crystal. A crystallized protein can be produced by controlling various environmental factors such as the type of protein, solvent, pH, and concentration.

[0075] FIG. 4A and FIG. 4B are diagrams illustrating an example of a protein crystallization process according to one embodiment of the present invention.

[0076] Referring to Fig. 4a, there are two graphs for the protein crystallization process, one of which is a phase diagram showing the state of the protein solution according to the protein concentration and salt concentration, and the other graph may be a graph showing the change in Gibbs free energy.

[0077] First, by adjusting the concentrations of protein and salt, the solution can be made supersaturated, and when the solution is supersaturated, conditions for the formation of protein molecules can be created. The phase diagram can include a stable state where the protein molecules do not crystallize in a region where the solution is not saturated, a metastable state where moderate supersaturation is formed and crystal growth proceeds, a labile state where nucleation and crystal growth proceed, and a precipitation state where the solution is highly supersaturated and an amorphous precipitate is formed.

[0078] Nucleation can be the process by which protein molecules, under supersaturation, assemble to form small crystal cores. It can be represented by the Gibbs free energy graph, which represents the largest crystal core that overcomes the energy barrier required for nucleation. For example, nucleation can occur through homogeneous nucleation, in which nuclei form due to random fluxation within the solution, or heterogeneous nucleation, in which nuclei form on existing solid surfaces or around impurities.

[0079] The growth phase may be the stage after nucleation, when more protein molecules bind to the crystal nucleus, causing the crystal to grow. This process occurs as the Gibbs free energy decreases, allowing the crystal to transition to a more stable state. In the Gibbs free energy graph, represents the Gibbs free energy change, can represent the critical energy required for nucleation, C can represent the concentration, and Cs can represent the saturation concentration. Sizer on the graph represents controlling the crystal size, and can be a factor that determines the size of the crystal that grows after nucleation.

[0080] Through the above process, experimental conditions for protein crystallization can be optimized. For example, protein and salt concentrations for protein crystallization can be adjusted, or temperature and pH conditions can be optimized.

[0081] Referring to Fig. 4b, the protein crystallization process involving heterogeneous nucleation with gas bubbles can be described.

[0082] Figure 4b compares the protein crystallization process under conditions with and without gas bubbles, and the Gibbs free energy change ( ) is a diagram showing the energy change from the solution state of a protein to the formation of a crystal nucleus.

[0083] First, examining the change in Gibbs free energy within a protein solution, protein adsorption can occur, where protein molecules are adsorbed onto the surface of a gas bubble. This adsorption process occurs with a decrease in Gibbs free energy, resulting in the protein being stably positioned on the surface in contact with the gas. Protein molecules can then begin to form nuclei on the surface. These nuclei serve as the starting point for crystal growth, and the Gibbs free energy initially increases and then decreases once a critical nucleus is formed. Once the critical nucleus is formed, additional protein molecules can bind to the nucleus, allowing crystal growth. Throughout this process, the Gibbs free energy can continuously decrease.

[0084] When gas bubbles are present, their surfaces can provide a favorable substrate for protein nucleation. The presence of gas bubbles facilitates crystal nucleation, making protein crystallization more efficient when gas bubbles are present. In the absence of gas bubbles, protein crystallization may be slower or less rapid, as a higher Gibbs free energy barrier must be overcome without the substrate provided by the gas bubbles.

[0085] Regarding the gas bubbles in the crystal growth process, section (b) at the bottom of the graph shows the boundary between the gas bubbles and the protein clusters, and the contact angle ( ) and surface energy ( ) can be expressed. Also, is the boundary surface energy between the protein cluster and the solution, is the boundary surface energy between the gas bubble and the solution, This could be the surface energy of the boundary between the protein cluster and the gas bubble. Such information could be an important factor in determining how energetically advantageous it is for the protein cluster to form on the surface of the gas bubble.

[0086] More specifically, the protein crystallization process involving heterogeneous nucleation in the presence of gas bubbles can be described as follows.

[0087] As a first step, we can confirm the phenomenon of protein adsorption on the gas bubble surface. The change in Gibbs free energy ( ) can be the sum of the energy change values ​​according to the adsorption and nucleation processes. The adsorption energy change ( ) is negative, it is energetically favorable, which may mean that adsorption occurs more easily than homogeneous nucleation. In other words, the adsorption process is thermodynamically favorable, which allows heterogeneous nucleation to occur more easily on the bubble surface.

[0088] In a second process, nucleation can begin at the surface of a gas bubble. The bubble itself is the primary catalyst for nucleation, and nucleation can be promoted by gas bubble shrinkage, which increases local concentration as the bubble size decreases, and gas dissolution, which increases local concentration as the gas dissolves. In a third process, protein crystal growth can occur around the gas bubble. After a nucleus forms, more protein molecules can bind to the nucleus, promoting crystal growth.

[0089] FIG. 5 is a drawing illustrating an example of a crystallized protein according to one embodiment of the present invention.

[0090] According to one embodiment of the present invention, FIG. 5 is a diagram visually illustrating gas bubbles, proteins, and crystallized proteins through the process of FIGS. 3 to 4B through the protein crystallization device (100) included in FIGS. 1, 2A, and 2B. Proteins may refer to protein molecules dissolved in a solution. Gas bubbles may be used as a substrate for heterogeneous nucleation. Protein crystals may refer to proteins crystallized into cubes.

[0091] FIG. 6 is a drawing illustrating an example in which at least one component of a protein crystallization device (100) is configured in multiple pieces and connected in parallel according to one embodiment of the present invention.

[0092] According to various embodiments, at least one component among the gas storage unit (110), the solution storage unit (120), the bubble generation unit (130), the protein crystallization chamber (140), the pressure control unit (150), and the temperature control unit (160) included in the protein crystallization device (100) may be configured in multiple units and connected in parallel.

[0093] According to one embodiment, the solution storage unit (120) may be configured with a plurality of solution storage units (120a, 120b, 120c) arranged in parallel. The protein crystallization chamber (140) may be configured with a plurality of protein crystallization chambers (140a, 140b, 140c) arranged in parallel. The pressure control unit (150) may be configured with a plurality of pressure control units (150a, 150b, 150c) arranged in parallel, and each of the plurality of pressure control units (150a, 150b, 150c) may control the pressure of each of the protein crystallization chambers (140a, 140b, 140c) arranged in parallel. The bubble generation unit (130) can insert a solution containing bubbles generated based on a solution received from each of a plurality of solution storage units (120a, 120b, 120c) configured in parallel and at least one gas into each of a plurality of protein crystallization chambers (140a, 140b, 140c).

[0094] According to one embodiment, the temperature control unit (160) can adjust the temperature of each of a plurality of protein crystallization chambers (140a, 140b, 140c) configured in parallel. The pressure control unit (150) is configured such that a plurality of pressure control units (150a, 150b, 150c) are configured in parallel, and each pressure control unit (150a, 150b, 150c) adjusts the pressure of each of the protein crystallization chambers (140a, 140b, 140c) configured in parallel.

[0095] FIG. 7 is a drawing illustrating another example in which at least one component of a protein crystallization device (100) is configured in multiple pieces and connected in parallel, according to one embodiment of the present invention.

[0096] According to various embodiments, at least one component among the gas storage unit (110), the solution storage unit (120), the bubble generation unit (130), the protein crystallization chamber (140), the pressure control unit (150), and the temperature control unit (160) included in the protein crystallization device (100) may be configured in multiple units and connected in parallel.

[0097] According to one embodiment, the solution storage unit (120) may be configured with a plurality of solution storage units (120a, 120b, 120c) arranged in parallel. The pressure control unit (150) may be configured with a plurality of pressure control units (150a, 150b, 150c) arranged in parallel, and each of the plurality of pressure control units (150a, 150b, 150c) may control the pressure of each of the protein crystallization chambers (140a, 140b, 140c) arranged in parallel. The pressure control unit (150) may control the pressure of the protein crystallization chamber (140) through a valve (151).

[0098] FIGS. 8A to 8C are drawings illustrating various examples of a protein crystallization device (100) according to one embodiment of the present invention.

[0099] Referring to FIG. 8a, the protein crystallization device (100) may include a gas storage unit (110), a solution storage unit (120a, 120b, 120c), a bubble generation unit (130), a protein crystallization chamber (140), a pressure control unit (150), a temperature control unit (160), and a mixer (170). Detailed descriptions of each component of the protein crystallization device (100) corresponding to at least the components of FIGS. 1 to 3 are omitted.

[0100] According to one embodiment, the mixer (170) of the protein crystallization device (100) can mix solutions received from each of the plurality of solution storage units (120a, 120b, 120c). For example, the mixer (170) is a mixing device that mixes a plurality of different solutions to control the solubility of a protein, and the mixer (170) can be a magnetic stirrer, an overhead stirrer, a microfluidic mixer, or the like. The mixer (170) can transfer the mixed solution to the protein crystallization chamber (140).

[0101] Referring to FIG. 8b, according to various embodiments, at least one solution storage unit (120c) included in the protein crystallization device (100) can insert a solution (e.g., a solution for protein crystallization, a pure solvent, etc.) into the protein crystallization chamber (140) without going through the mixer (170) and / or the bubble generation unit (130). The protein crystallization chamber (140) can receive a solution containing bubbles from the bubble generation unit (130) and receive a solution from at least one solution storage unit (120c).

[0102] Referring to FIG. 8c, according to various embodiments, the mixer (170) included in the protein crystallization device (100) can insert a mixed solution or a single solution (e.g., when the solution storage unit (120) is single, etc.) into the bubble generation unit (130), while inserting the mixed solution or a single solution (e.g., when the solution storage unit (120) is single, etc.) into the protein crystallization chamber (140). The protein crystallization chamber (140) can receive a solution containing bubbles from the bubble generation unit (130) and a solution for protein crystallization from the mixer (170).

[0103] FIG. 9 is a diagram illustrating a flowchart for performing protein crystallization according to one embodiment of the present invention.

[0104] Referring to step (901), the protein crystallization device (100) can insert a solution containing bubbles into the protein crystallization chamber (140) based on at least one gas (e.g., nitrogen, argon, carbon dioxide, etc.) and a solution. The description of the protein crystallization device (100) inserting into the solution containing bubbles corresponds to at least FIGS. 1 to 3, and thus a detailed description thereof will be omitted.

[0105] Referring to step (903), the protein crystallization device (100) can control the temperature and pressure of the solution containing bubbles. The description of how the protein crystallization device (100) controls the pressure and temperature within the protein crystallization chamber (140) through the temperature control unit (160) and the pressure control unit (150) corresponds to at least FIGS. 1 to 3, and thus a detailed description thereof will be omitted.

[0106] Referring to step (905), the protein crystallization device (100) can perform protein crystallization in a microgravity environment by culturing a solution containing bubbles. The description of the protein crystallization device (100) performing protein crystallization in a microgravity environment by culturing a solution containing bubbles corresponds to at least FIGS. 1 to 3, and thus a detailed description thereof will be omitted.

[0107] The technical features disclosed in each embodiment of the present invention are not limited to that embodiment, and, unless they are mutually incompatible, the technical features disclosed in each embodiment may be combined and applied to different embodiments. Those skilled in the art will further understand that any disjunctive words and / or phrases indicating two or more alternative terms, whether in the detailed description, claims, or drawings of the present invention, are to be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" is to be understood to encompass the possibility of "A" or "B," or "A and B." Furthermore, as used herein, the term "each," in addition to its ordinary meaning, may mean any subset of the set of elements to which the term "each" applies. Combinatory language such as the phrase "at least one of X, Y, and Z," unless specifically stated otherwise, is to be understood in the context in which it is generally used to convey that an item, term, etc., can be X, Y, or Z. Thus, such binding language is not generally intended to imply that a particular embodiment requires the presence of at least one of X, at least one of Y, or at least one of Z.

[0108] Therefore, although each embodiment focuses on its own technical features, each technical feature can be applied in combination with each other as long as they are not mutually incompatible.

[0109] The present invention is not limited to the above-described embodiments and the attached drawings, and various modifications and variations are possible within the scope of those skilled in the art. Therefore, the scope of the present invention should be defined not only by the claims of this specification but also by equivalents thereof.

Claims

1. In a device for crystallizing proteins in a microgravity environment, A gas storage unit storing at least one gas and inserting the stored at least one gas into a bubble generating unit; A solution storage unit for storing a solution for protein crystallization and inserting the stored solution into the bubble generating unit; The bubble generating unit generating a solution containing bubbles based on the at least one gas received from the gas storage unit and the stored solution received from the solution storage unit, and inserting the solution containing the generated bubbles into a protein crystallization chamber; The protein crystallization chamber that performs protein crystallization in the microgravity environment using the solution containing the bubbles received from the bubble generation unit; A pressure control unit for controlling the pressure of the protein crystallization chamber; and A temperature control unit for adjusting the temperature of the protein crystallization chamber, A device for crystallizing proteins in a microgravity environment.

2. In paragraph 1, Further comprising a monitoring unit for checking the solution or protein crystals containing the bubbles stored in the protein crystallization chamber. A device for crystallizing proteins in a microgravity environment.

3. In paragraph 1, The above gas storage unit, Comprising at least one of polymer capsules, microparticles, and nanoparticles containing at least one chemical substance; A device for crystallizing proteins in a microgravity environment.

4. In paragraph 1, The above bubble generating unit, Generating a bubble-containing solution based on said at least one gas and said stored solution through at least one of microfluidic, electrically induced bubble generation, sonication, baffle, saline shaking, and freeze drying techniques. A device for crystallizing proteins in a microgravity environment.

5. In paragraph 1, The above solution storage unit is, A plurality of the above solution storage units are configured in parallel, The above protein crystallization chamber, A plurality of the above protein crystallization chambers are configured in parallel, The above pressure control unit, A plurality of the pressure control units are configured in parallel, and each of the plurality of pressure control units controls the pressure of each of the protein crystallization chambers configured in parallel. Protein crystallization device.

6. In paragraph 5, The above bubble generating unit, Inserting a solution containing a bubble generated based on the at least one gas and a solution received from each of the plurality of solution storage units configured in parallel into each of the plurality of protein crystallization chambers, Protein crystallization device.

7. In paragraph 6, The above temperature control unit, Adjusting the temperature of each of the plurality of protein crystallization chambers configured in parallel, The above pressure control unit, A plurality of the pressure control units are configured in parallel, and each of the pressure control units controls the pressure of each of the protein crystallization chambers configured in parallel. Protein crystallization device.

8. In a method for crystallizing a protein in a microgravity environment, A step of inserting a solution containing bubbles generated based on at least one gas and solution into a protein crystallization chamber; A step of controlling the temperature and pressure of the solution containing the above bubbles; and A step of culturing a solution containing the bubbles to perform protein crystallization in the microgravity environment, A method for crystallizing proteins in a microgravity environment.

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