JP5780721B2 - Protein crystal growth apparatus and method - Google Patents

Description

  The present invention relates to a protein crystal growth apparatus and a method thereof.

  Proteins play a fundamental role in supporting complex biological phenomena. For example, it is known that many diseases and diseases are caused by abnormalities (functional abnormalities) in various functions of proteins. Therefore, for example, by analyzing the structure and function of a protein that is related to or involved in a specific disease, it is possible to foresee compounds that can regulate and control the function of the protein. It is considered possible to significantly reduce costs.

  In order to determine the three-dimensional structure of such a protein, the most frequently used technique at present is an X-ray structure analysis by an X-ray diffraction method using a protein single crystal as a sample. Until now, the three-dimensional structure of many proteins has been determined at the atomic level, and has contributed greatly to the understanding of life science.

  On the other hand, some or all of proteins cause various reactions in the living body as enzymes, but most of them are involved in the enzyme activity of proteins, such as hydrogen atoms (protons), hydrogen Observation of these hydrogen chemical species, which are hydrogen atoms in ions and water molecules, is essential for elucidating the mechanism of action of proteins as enzymes.

  However, the above-described X-ray diffraction method uses X-rays and scattering by electrons in the atom, and since the contribution to the diffraction intensity increases as the atomic number increases, X by the electrons of the hydrogen atom. Line scattering is usually very difficult to detect, or below the detection limit and cannot be observed. In particular, observation of protons lacking electrons is impossible in principle. On the other hand, neutron structure analysis using neutron (line) diffractometry among diffraction techniques is equivalent to protons and easily produces elastic scattering. Since the diffraction intensity is relatively large, there is an advantage that position information (atomic coordinates) of hydrogen atoms and protons in proteins can be observed with high accuracy.

In such a neutron diffraction method, neutrons emitted from a neutron source are collimated and irradiated to nuclei existing in the protein, and the scattering angle and intensity of the neutron are measured, so that hydrogen atoms and protons in the protein are measured. Position information is acquired selectively and the three-dimensional structure of the protein containing them is analyzed. At this time, since the neutron intensity (flux: the number of neutrons passing through the unit area per second) is almost 10 orders of magnitude smaller than the X-ray intensity, in order to compensate for the small neutron intensity and increase the detection sensitivity, the inventor's knowledge According to the above, it is necessary to grow a high-quality crystal having a protein crystal volume of, for example, 1 mm ³ or more and use it for a neutron diffraction experiment.

However, so far, it has been difficult to form large, high-quality protein crystals of 1 mm ³ or more with good reproducibility. Here, generally, protein molecules in a crystal are regularly arranged (arranged) three-dimensionally according to the symmetry of the crystal, and each protein molecule is surrounded by, for example, water molecules without gaps. The proportion of water molecules in the crystal is about 30 to 70%. Then, for example, the amount of protein in a 1 mm ³ protein crystal is about 0.3 to 0.7 mg. On the other hand, in the vapor diffusion method used in protein crystallization, for example, if 20 μl of a protein aqueous solution having a concentration of 30 mg / ml is used, 0.6 mg of protein molecules are dissolved therein. Quantitatively enough to make a 1 mm ³ protein crystal.

However, it is extremely difficult to produce a large, high-quality protein crystal of 1 mm ³ or more even by such a vapor diffusion method. As a result of diligent research and investigation by the present inventors, in normal crystallization, many small crystals tend to be formed individually in an aqueous solution, and the mass of protein contained in one small crystal is small, and one large crystal It cannot be. Moreover, even if the amount of the protein aqueous solution is increased, an enormous number of small crystals are formed. In addition, recently, a macroscopic seeding method using a seed crystal has been sought to increase the size of protein crystals, but crystals that are not suitable for structural analysis (for example, flat or needle-like) have grown. In some cases, a large protein crystal can be formed accidentally after trial and error. However, a method for growing a large, high-quality protein crystal with good reproducibility has not been established yet, and there is an urgent need to develop a technology capable of improving the yield.

  In view of this, the present invention proposes a protein crystal growth apparatus capable of growing (forming) high-quality protein crystals large enough to be used in neutron diffraction with good reproducibility, and a method therefor.

  In order to solve such problems, a protein crystal growth apparatus according to the present invention includes a chamber (container) portion to which a protein solution and a crystallization agent solution are supplied, a protein solution supply portion to supply a protein solution in the chamber portion, and The relationship between the concentration of the crystallization agent and the concentration of the protein in the mixed solution of the protein solution and the crystallization agent solution contained in the chamber, And a control unit that adjusts between an unsaturated region, a metastable region, and an automatic nucleation region in the dissolution state diagram of the protein and the crystallization agent. The protein solution and the crystallization agent solution may be mixed in advance to form a “mixed solution”, and the protein solution may be a so-called “stock solution” of protein, an aqueous solution prepared at an appropriate concentration, etc. Or other suitable reagents for crystallization may be included.

  That is, the present invention utilizes a protein and crystallizing agent in a mixed solution of protein and crystallizing agent accommodated in a chamber part by using a “protein and crystallizing agent dissolution state diagram” described later for large and high-quality crystals of protein. It is intended to prevent the formation of many small crystals by appropriately adjusting the concentration of the agent.

  As a more specific device configuration, for example, an inert gas supply unit that is connected to the control unit and supplies an inert gas into the chamber unit, and a decompression unit that is connected to the control unit and depressurizes the inside of the chamber unit. And a depressurization stop unit that is connected to the control unit and stops depressurization in the container, and a pressurization unit that is connected to the control unit and pressurizes the inside of the container. The protein solution supply unit and the crystallization agent solution supply unit may also be connected to the control unit.

  In the protein crystal growth apparatus configured as described above, a control unit is connected to each unit to control each unit. For example, first, the pressure in the chamber unit is controlled as desired by the control unit. In other words, the control unit adjusts the evaporation of water in the mixed solution stored in the chamber, and protein crystals are formed and grown from the mixed solution. Specifically, the controller controls the decompression unit to decompress the interior of the chamber, thereby evaporating water in the mixed solution and increasing the protein concentration. Thus, when the mixed solution enters the automatic nucleation region, protein crystals are precipitated from the mixed solution. As a result, the protein concentration in the mixed solution drops, and the control unit controls the depressurization stop unit to stop depressurization in the chamber unit, so that the mixed solution enters the metastable region. Since no new nucleation occurs there, the protein in the mixed solution is consumed for the crystal growth, so that one protein crystal grows significantly without forming many seed crystals. At the same time, the protein concentration in the mixture drops and crystal growth stops when the solubility curve is reached. Therefore, the control unit controls the protein solution supply unit to additionally inject the protein solution into the mixed solution. The mixed solution enters the unsaturated region, and the crystal surface is slightly dissolved. This also leads to cleaning the crystal surface. Then, protein crystal growth can be resumed by reducing the pressure of the controller again to reach the metastable region in the chamber.

  Further, the chamber part includes a substrate for holding a crystal of the growing protein, the substrate is supplied with an inert gas, and a hole part into which the protein solution and the crystallizing agent solution or mixed solution are injected, and the substrate part is inert. The first path through which the gas flows and the second path through which the protein solution flows are formed. The first path connects the upper portion of the hole portion and the end portion of the substrate and is formed along the upper surface of the substrate. The second path may be configured to connect the lower part of the hole part and the end part of the substrate and be formed along the bottom surface of the substrate.

  In this way, protein crystal growth is performed in the hole portion formed in the substrate of the chamber portion. In addition, the inert gas flows into the hole part from the upper part of the hole part and the protein stock solution is injected into the hole part from the lower part of the hole part by each path provided in the substrate. As a result, protein crystal growth can be controlled to a higher degree.

  In addition, the control unit may be configured to operate the decompression unit and the decompression stop unit alternately and / or repeatedly.

  Furthermore, the detection unit may be connected to the control unit and detect the state of the crystal of the protein. In this case, the detection unit may be a growth state of the crystal in the protein and / or a crystal surface of the protein. Examples include those that detect the dissolved state. Thus, by detecting the state of the protein to be crystal-grown, there is an advantage that a protein crystal having a desired property can be more reliably grown.

  Further, in the protein crystal growth apparatus according to the present invention, if attention is paid to the operation of the control unit, the control unit is in a state where the protein seed crystal is previously accommodated in the chamber unit, or the protein seed crystal is formed in the chamber unit. In the dissolved state diagram of the protein and crystallization agent, the state of the mixture is automatically changed from the unsaturated region by operating the decompression unit to decompress the interior of the chamber and evaporating the water in the mixture. After transitioning to the formation region and starting the growth of the seed crystal, the decompression stop section is operated to stop the decompression in the chamber section, and the protein in the protein solution is used for the growth of the protein crystal to By reducing the concentration, the state of the mixed solution is shifted from the automatic nucleation region to the unsaturated region through the metastable region. In addition, the control unit may be configured to increase the protein concentration in the mixture by supplying (adding) the protein solution to the mixed solution that has reached the unsaturated region by operating the protein solution supply unit. Useful.

  In addition, the protein crystal growth method according to the present invention is particularly a method that can be effectively performed using the protein crystal growth apparatus according to the present invention, and includes a step of supplying a protein solution into the chamber portion, The step of supplying the crystallizing agent solution and the relationship between the protein concentration and the crystallizing agent concentration in the mixed solution of the protein solution and the crystallizing agent solution contained in the chamber part, the dissolved state of the protein and the crystallizing agent Adjusting between the unsaturated region, the metastable region, and the autonucleation region in the figure.

  Further, from the step of replacing the inside of the chamber containing the mixed solution with an inert gas, the state in which the protein seed crystal is previously stored in the chamber part, or the state in which the protein seed crystal is formed in the chamber, Reducing the pressure in the chamber, stopping the pressure reduction in the chamber when the protein crystals start growing, and supplying the protein solution to the mixture when the protein crystal surface is dissolved. Further, it may be included.

  In this case, it is also preferable that the step of reducing the pressure and the step of stopping the pressure reduction are executed alternately and / or repeatedly.

  Furthermore, a step of detecting a crystal state of the protein may be included. At this time, in the step of detecting the crystal state of the protein, the growth state of the crystal in the protein and / or the dissolution state of the crystal surface in the protein are determined. You may make it detect.

  In other words, the protein crystal growth method according to the present invention reduces the pressure in the chamber part from the state in which the protein seed crystal is previously stored in the chamber part or the state in which the protein seed crystal is formed in the chamber part, By evaporating the water in the mixed solution, in the dissolution phase diagram of the protein and the crystallization agent, the state of the mixed solution is changed from the unsaturated region to the automatic nucleation region, and the growth of the seed crystal is started. By stopping the decompression in the part and using the protein in the protein solution for protein crystal growth to reduce the protein concentration in the mixture, the state of the mixture is changed from the automatic nucleation region to the metastable region. In this case, the protein solution is additionally supplied to the mixed solution that has reached the unsaturated region, and the tank in the mixed solution is supplied. It may be increased click quality levels.

  According to the present invention, the crystal growth method by the vapor diffusion method is applied, that is, the protein is controlled by controlling the vapor pressure in the protein solution (droplet) without using the vapor pressure difference with the crystallization agent (reservoir) solution. Can be grown to a desired size and high quality crystal with good reproducibility, and the crystal growth is carried out in an inert gas atmosphere, preventing oxidation of the protein crystal surface. can do.

It is a block diagram which shows schematic structure of one Embodiment of the protein crystal growth apparatus by this invention. It is a top view which shows typically the principal part of the protein crystal growth apparatus 1 shown in FIG. It is a side view which shows typically the principal part of the protein crystal growth apparatus. It is sectional drawing which shows the chamber part 3 in FIG. 2A along a III-III line. It is a dissolution state figure which shows an example of the phase diagram of protein crystal growth. It is a melt | dissolution state figure which shows an example of the conventional hanging drop method. It is the schematic which shows the state which is performing the crystal growth of protein using an example of the conventional hanging drop method shown in FIG. It is a melt | dissolution state figure which shows an example of the phase diagram of the crystal growth of protein based on one Embodiment of this invention. It is a dissolution state figure which shows an example of the density | concentration of the liquid mixture of the protein used by one Embodiment of this invention, and a crystallization agent (reservoir) solution. It is a side view of the chamber part 3 in FIG. 2A.

  Embodiments of the present invention will be described below with reference to the drawings. In the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the positional relationship such as up, down, left and right is based on the positional relationship shown in the drawings unless otherwise specified. Furthermore, the dimensional ratios in the drawings are not limited to the illustrated ratios. Further, the following embodiments are exemplifications for explaining the present invention, and are not intended to limit the present invention only to the embodiments. Furthermore, the present invention can be variously modified without departing from the gist thereof.

(1) Configuration of Protein Crystal Growth Device FIG. 1 is a block diagram showing a schematic configuration of an embodiment of a protein crystal growth device according to the present invention, and FIG. 2A is a main part of the protein crystal growth device 1 shown in FIG. FIG. 2B is a side view schematically showing the main part of the protein crystal growth apparatus 1. As shown in the figure, the protein crystal growth apparatus 1 includes a control unit 2 and a chamber unit 3, and further includes an atmospheric pressure adjustment unit 4, a gas adjustment unit 5, an injection unit 6, a sensor unit 7, connected thereto. And the detection part 8 is comprised.

  The control unit 2 controls each unit of the atmospheric pressure adjustment unit 4, the gas adjustment unit 5, the injection unit 6, the sensor unit 7, and the detection unit 8, and may be an electronic circuit such as a CPU or a processor. Alternatively, it may be a normal computer device, a dedicated system, or a general-purpose information processing device. Furthermore, for example, in an information processing apparatus having a general configuration, by executing software (programmed) including an algorithm for each process in the protein crystal growth method described later, the information processing apparatus is operated as the control unit 2 of the present embodiment. You can also.

The chamber part 3 defines a sealed space for growing protein crystals, and uses a crystal growth substrate 31 provided in the chamber part 3 to produce large and high-quality protein crystals. is there. The chamber part 3 of the present embodiment is a colorless and transparent sealed container having a volume of, for example, 10 ⁶ mm ³ , and a container that can be kept airtight for at least one month or more can be used. The detailed configuration of the chamber unit 3 will be described later.

  The atmospheric pressure adjusting unit 4 adjusts the inside of the chamber unit 3 to a desired pressure (vapor pressure) based on an instruction from the control unit 2 and stops the adjustment. More specifically, the atmospheric pressure adjustment unit 4 increases or decreases the pressure in the chamber unit 3. In this respect, the atmospheric pressure adjustment unit 4 also serves as the “pressurization unit”, “decompression unit”, and “decompression stop unit” in the present invention. For example, the atmospheric pressure adjustment unit 4 includes a syringe 41 (see FIG. 2A), and one end of the syringe 41 is connected to the connection unit 35 of the chamber unit 3. An appropriate opening / closing valve such as an electromagnetic valve 42 is provided between one end of the syringe 41 and the connection portion 35 of the chamber portion 3, and the electromagnetic valve 42 is opened / closed based on an instruction from the control unit 2. The

  On the other hand, the gas adjustment unit 5 (inert gas supply unit) has a function of adjusting the inside of the chamber unit 3 to a desired gas component based on an instruction from the control unit 2. Before starting the growth of protein crystals, the gas adjustment unit 5 releases the atmosphere (air) in the chamber unit 3 to the outside of the chamber unit 3, and the inert gas (for example, rare gas or nitrogen) Gas) and the supplied inert gas is adjusted to a desired pressure.

  As shown in FIG. 2A, the gas adjusting unit 5 includes, for example, a gas regulator 51, a gas flow rate adjuster 52, an inert gas supply valve 53, and an atmosphere release valve 54. The gas regulator 5 is connected to a gas regulator 51 that keeps the gas pressure constant and a gas flow rate regulator 52, and is connected to the regulator 52, an inert gas supply valve 53, and an atmosphere release valve 54. Further, the valves 53 and 54 and the chamber portion 3 are connected. In this embodiment, a mass flow controller (MFC: Mass Flow Controller) is used as a gas flow controller to control the flow rate of the inert gas. However, the MFC function is provided in the control unit 2 itself, You may omit it. Further, the inert gas supply valve 53 and the atmosphere release valve 54 can be opened and closed based on instructions from the gas flow rate regulator 52 or the control unit 2.

  Furthermore, the injection unit 6 injects appropriate amounts of various reagents for growing protein crystals into the chamber unit 3 at a desired timing based on instructions from the control unit 2. The injection unit 6 includes, for example, a protein injection unit 61 (protein solution supply unit) holding a protein stock solution (protein solution) and a crystallization agent injection unit 62 holding a crystallization agent (precipitating agent) solution. (Crystallizing agent solution supply unit).

  The type of crystallization agent is not particularly limited, and examples thereof include inorganic salts (ammonium sulfate, sodium sulfate, lithium sulfate, sodium chloride, potassium chloride, ammonium chloride, lithium chloride, ammonium acetate, sodium acetate, ammonium phosphate, phosphoric acid. Sodium or potassium phosphate), polyethylene glycol (PEG) (eg, PEG400, PEG1000, PEG4000, PEG6000, PEG10000), or an organic compound (2-methyl-2,4-pentanediol (MDP), isopropropanol, Ethanol, methanol, dioxane, butanol, propanol) and the like.

  In this embodiment, in addition to the protein injection part 61 and the crystallization agent injection part 62, a water injection part 63 is provided, and the water injection part 63 holds water. Moreover, the various injection | pouring parts 61, 62, and 63 are comprised by the syringe, for example (refer FIG. 2A), and the end of the syringe is connected to the chamber part 3. FIG. Appropriate on-off valves such as electromagnetic valves 64, 65, 66, for example, are provided between one end of each syringe and the chamber portion 3. Based on instructions from the control unit 2, the electromagnetic valves 64, 65, 66 is opened and closed. For example, according to an instruction from the control unit 2, a liquid amount of 1 μl (microliter) / time from the protein injection unit 61, 5 μl / time from the crystallization agent injection unit 62, and 1 μl / time from the water injection unit 63 is stored in the chamber 3. Injected (supplied).

  In addition, the form of the various injection | pouring parts 61, 62, and 63 is not restricted to the syringe of this embodiment, What is necessary is just to inject | pour appropriate amounts of various reagents into the chamber part 3 at a desired timing.

  The sensor unit 7 detects the temperature or atmospheric pressure (pressure) in the chamber unit 3. The sensor unit 7 in this embodiment includes a temperature sensor 71 and an atmospheric pressure sensor 72, and each sensor 71, 72 detects the temperature or atmospheric pressure of the chamber unit 3. The temperature sensor 71 is provided on the crystal growth substrate 31 so that the temperature in the chamber portion 3 can be measured, and the atmospheric pressure sensor 72 is the atmospheric pressure in the hole portion 32 provided on the crystal growth substrate 31 of the chamber portion 3. It is provided in the hole 32 so that can be measured. Furthermore, the sensor unit 7 is preferably provided with a monitor 73, and in this case, the detected values detected by the sensors 71 and 72 are displayed on the monitor 73. Depending on the detection value detected by the sensor unit 7, the control unit 2 can perform control so as not to exceed preset threshold values (reference temperature and reference atmospheric pressure).

  The detection unit 8 is for detecting a protein crystal growth process (degree of crystal growth). In the present embodiment, the detection unit 8 includes an imaging device 81 such as a CCD camera and a monitor 82 on the back surface of the chamber unit 3, and a crystal image detected by the imaging device 81 is displayed on the monitor 82. In accordance with the crystal growth state displayed on the monitor 81, the control unit 2 gives a predetermined instruction to each unit to control the atmospheric pressure (vapor pressure) in the chamber unit 3. In the present embodiment, the imaging device 81 and the monitor 82 may be provided separately, or of course, a device that can integrally perform imaging and display of crystals may be used.

(2) Configuration of Chamber Unit As shown in FIG. 2A, the chamber unit 3 has a crystal growth substrate 31 disposed substantially at the center thereof. One side surface of the chamber portion 3 is provided with connection portions 33 to which the various injection portions 61, 62, and 63 are connected, and the various injection portions 61, 62, and 63 are connected through the connection portions 33. Is connected to the crystal growth substrate 31. Further, each connection part 34 to which the gas adjustment part 5 is connected is provided on the other side surface of the chamber part 3, and the gas adjustment part 5 is connected to the crystal growth substrate 31 through each connection part 34. Has been.

  Here, FIG. 3 is a sectional view showing the chamber portion 3 (particularly, the crystal growth substrate 31) in FIG. 2A along the line III-III. The crystal growth substrate 31 mainly includes a hole portion 32, a gas connection portion 36, a gas path portion 37, an aqueous solution connection portion 38, and an aqueous solution path portion 39 as a constant defined space.

  The hole portion 32 is a container that is formed substantially at the center of the crystal growth substrate 31, has a bottom surface, and has a cylindrical hole formed from the bottom surface toward the top surface. As shown in FIG. 9, a mixed solution 15 of protein and crystallization agent is injected into the hole portion 32 from the injection portion 6, and, for example, a protein seed crystal 16 is mixed in the mixed solution. The hole portion 32 is covered with a cover glass 9, and the cover glass 9 has a through hole h for inserting the atmospheric pressure sensor 72 into the hole portion 32.

  The gas connection portion 36 is formed on the upper surface of the crystal growth substrate 31 and connects the gas adjustment portion 5 and the crystal growth substrate 31. As shown in FIG. 2A, in the present embodiment, three gas connection portions 36 are formed on the left side of the upper surface of the crystal growth substrate 31 as shown in the figure, but it is sufficient that gas can be injected into the hole portion 32. Is not limited to this form.

  The gas path part 37 is a path that connects the gas connection part 36 and the hole part 32 (see FIG. 3), and is formed radially inside the substrate 31 from the hole part 32 (see FIG. 2A). In the gas path part 37, the inert gas delivered from the gas adjustment part 5 flows into the hole part 32 through the gas path part 37. As shown in FIG. 3, the gas path portion 37 is formed along the vicinity of the upper surface 311 of the crystal growth substrate 31 so as to reach the upper side surface 321 of the hole portion 32 from the gas connection portion 36 of the crystal growth substrate 31. .

  The aqueous solution connection portion 38 is formed on the upper surface of the crystal growth substrate 31 and connects the injection portion 6 and the crystal growth substrate 31. As shown in FIG. 2A, the aqueous solution connection portions 38 are formed at three positions on the right side of the upper surface 311 of the crystal growth substrate 31. Each connection portion 38 includes the protein aqueous solution injection portion 61, the crystallization agent from the front of the page. The aqueous solution injection part 62 and the water injection part 63 are connected. The aqueous solution connecting portion 38 is not limited to this form as long as it can produce a mixed solution of an aqueous protein solution and an aqueous crystallization agent solution in the hole portion 32.

  The aqueous solution path portion 39 is a path connecting the aqueous solution connection portion 38 and the hole portion 32, and is formed in the substrate 31 radially from the hole portion 32 as shown in FIG. 2A. Various aqueous solutions sent from the injection parts 61 to 63 flow into the hole part 32 through the aqueous solution path part 39. As shown in FIG. 3, the aqueous solution path portion 39 extends along the vicinity of the bottom surface 312 of the crystal growth substrate 31 so as to reach the bottom surface 323 along the bottom side surface 322 of the hole portion 32 from the aqueous solution connection portion 38 of the crystal growth substrate 31. Is formed.

(3) Conventional General Protein Crystal Growth Method Before explaining the procedure (process) in the protein crystal growth method according to the present invention using the protein crystal growth apparatus 1 configured as described above, the conventional general crystal growth method is used. The protein crystallization method that has been described will be described with reference to FIG.

  FIG. 4 is a dissolution state diagram showing an example of a phase diagram of protein crystal growth. The solubilization state diagram shown in this figure shows the solubilization state of protein, the vertical axis indicates the protein concentration (Cp), and the horizontal axis indicates the crystallization agent concentration (Cc). In the dissolution phase diagram showing this phase diagram, the solubility curve C1 is used as a boundary, and the region is divided into an unsaturated region A1 and supersaturated regions A2 and A3. And automatic nucleation region A3.

  The unsaturated region A1 is a region below the solubility curve C1, and is a region in which proteins are dissolved and dispersed without forming protein crystal nuclei. The metastable region A2 is a region located between the solubility curve C1 and the supersaturation curve C2, and no crystal nucleus is formed (generated). For example, when a seed crystal or a crystal nucleus already exists, This is the region where the crystal grows. The automatic nucleation region A3 is a region that exceeds the supersaturation curve C2, and is a region in which crystal nuclei are formed by the interaction between protein molecules.

  Based on this phase diagram, it will be understood that the following points are necessary to form an ideal protein crystal. First, prior to crystallization, it is necessary that the protein be in an unsaturated region where it is completely dissolved. Secondly, if the crystallizing agent concentration is too high, the probability of forming crystal nuclei is too high and many crystals are formed. Therefore, in order to form a good quality crystal nuclei, the metastable region A2 is as close as possible. It is necessary to grow crystal nuclei in the automatic nucleation region A3. Third, the growth of crystal nuclei is also performed in the automatic nucleation region A3. However, in order to grow the crystal largely, it is necessary to perform it in the metastable region A2 without new nucleation. Fourth, crystal growth stops when it reaches a solubility curve where the incorporation and dissolution of protein molecules into the crystal achieve an equilibrium state.

  However, in the past, it was widely performed using the vapor diffusion method, which is one of the methods for crystallizing proteins, which is a method according to the phase diagram as a phenomenon of protein crystallization. It was not done with a strong awareness of the figure.

  Such a vapor diffusion method is a technique for obtaining crystals by precipitating protein from a protein solution by evaporating water in the protein solution and gradually increasing the protein concentration. As a typical method of such a vapor diffusion method, for example, a method called a hanging drop method or a sitting drop method is used. Here, the hanging drop method will be described with reference to FIGS. 5 and 6, and the sitting drop method is substantially the same in principle, and thus the description thereof is omitted here.

  In order to realize protein crystallization by the hanging drop method, as shown in FIG. 5, first, a protein solution (a solution containing water molecules, crystallization agent molecules, and protein molecules) and a reservoir (crystallization agent) aqueous solution 11 are used. And prepare. Next, a sufficient amount of the reservoir aqueous solution is placed in the reservoir container 12. Then, the protein solution is dropped on the cover glass 13 (hereinafter referred to as protein solution droplet 10), and the cover glass 13 is suspended from the reservoir container 12.

  In such a state, since the crystallization agent concentration (Cci) in the protein solution droplet 10 is lower than the concentration in the aqueous reservoir solution, the vapor pressure of the protein solution droplet 10 is in the aqueous reservoir (crystallization agent) solution 11. Therefore, the water evaporates from the protein solution droplet 10. As a result, when the protein concentration (Cpi) in the protein solution droplet 10 increases with the crystallization agent concentration (Cci) and eventually reaches the automatic nucleation region A3, crystal nuclei are automatically formed and crystallization starts. Is done. When the crystallization agent concentration (Cc) in the protein solution droplet 10 reaches the same concentration as the crystallization agent concentration (Ccr) in the reservoir aqueous solution 11, the vapor pressures of both solutions become equal. Water evaporation inside stops. Thus, the protein crystal growth process based on the hanging drop method can be represented by a straight line (start point (Cci, Cpi,), end point (Ccr, Cpf)) passing through the origin as shown in FIG. it can.

  Here, the reservoir aqueous solution 11 in the vapor diffusion method evaporates moisture from the protein solution droplet 10 as described above, and the crystallization agent concentration (Cc) in the protein solution droplet 10 is the crystallization agent concentration in the reservoir aqueous solution 11. It has a function (role) for controlling the vapor pressure in the protein solution droplet 10 such that the evaporation of moisture from the protein solution droplet 10 is stopped when the same concentration as (Ccr) is reached. However, as described above, in the conventional crystal growth method, a large-sized and high-quality protein crystal cannot be produced with good reproducibility.

(4) Protein Crystal Growth Method According to the Present Invention Therefore, as a result of detailed studies by the present inventors, the difference in vapor pressure from the reservoir aqueous solution, which has been one of the constituent elements in growing protein crystals, is used. The present inventors have found a method for controlling the vapor pressure in protein solution droplets without completing the present invention. Before describing this control method, the feasibility of the control method of this embodiment using the protein crystal growth apparatus 1 will be described first.

(4-1) Examination of vapor pressure control First, when a non-volatile substance is dissolved in a liquid, the vapor pressure of the aqueous solution decreases in proportion to the solute concentration (Raoul's law). When the vapor pressure of a pure solvent (for example, pure water) is Po, the vapor pressure drop ΔP of the aqueous solution is expressed by the following formula (1).
ΔP = (n ₂ / n ₁ ) Po (1)
In the formula, n ₁ represents the number of moles of the pure solvent, and n ₂ represents the number of moles of the solute.

Here, when Po = 760 mmHg (1 atm), for example, when 5 wt% of sodium chloride is used as a crystallization agent, the molar mass (molecular weight) of sodium chloride is about 58.5 gmol ⁻¹ , and the molar mass of water. Is 18 gmol ⁻¹ , the molar fraction (n ₂ / n ₁ ) is about 0.0142 from equation (1), and the vapor pressure drop ΔP = 10.8 mmHg of the aqueous solution.

Further, when as another crystalline agent eg PEG4000 was used 50 wt%, the molar mass of PEG4000 is about 4000Gmol ^-1, molar mass of water so 18Gmol ^-1, the equation (1), the molar fraction (n ₂ / N ₁ ) becomes 0.0025, and the vapor pressure drop ΔP = 1.9 mmHg of the aqueous solution.

  Thus, if the vapor pressure in the protein solution droplet can be controlled, it is possible to reliably grow protein crystals without using the vapor pressure difference with the aqueous reservoir solution. Therefore, in the protein crystal growth apparatus 1, the vapor pressure in the protein solution droplet is controlled by controlling the atmospheric pressure in the chamber unit 3 using a mixed aqueous solution composed of protein and a crystallization agent.

(4-2) Examination of Vapor Pressure Change in Chamber Part 3 Next, a vapor pressure change in the chamber part 3 of the protein crystal growth apparatus 1 will be examined. When the temperature in the chamber part 3 is constant, the product of the volume in the chamber part 3 and the atmospheric pressure is constant (Boyle's law), so the relationship between the atmospheric pressure change ΔP and the volume change ΔV is expressed by the following equation (2) It is represented by
(ΔP / P) = − (ΔV / V) (2)

Here, the maximum volume change in the chamber 3 of the protein crystal growth apparatus 1 is (ΔV) max, the minimum volume change is (ΔV) min, the maximum pressure change in the chamber 3 is (ΔP) max, and the minimum pressure change. Is (ΔP) min, the maximum atmospheric pressure change (ΔP) max and the minimum atmospheric pressure change (ΔP) min can be obtained by the following equations (3) and (4), respectively.
(ΔP) max = −P (ΔV) max / V (3)
(ΔP) min = −P (ΔV) min / V (4)

As an example, (ΔV) max of the chamber portion 3 of the protein crystal growth apparatus 1 is set to 30 × 10 ³ mm ³ and (ΔV) min is set to 0.3 × 10 ³ mm ³ , and the respective values are expressed by the above formula (3). When substituting into the equation (4), (ΔP) max is 22 mmHg, and (ΔP) min is 0.2 mmHg.

  In the above example, the chamber part 3 of the protein crystal growth apparatus 1 is capable of changing the vapor pressure between 0.2 and 22 mmHg in order to grow protein crystals.

(4-3) Examination of Vapor Pressure Drop in Chamber Part 3 Examining the drop value ΔP of the vapor pressure required for evaporating water M μl, first, the volume V occupied when 1 μl of water becomes water vapor is 1.24. Since it is × 10 ³ mm ³ , if it is substituted into the equation (2) as described above, the drop value ΔP of the vapor pressure required to evaporate M μl of water is expressed by the following equation (5).
ΔP = −PM (1.24 / 10 ³ ) = − 1.24 × 10 ⁻³ PM (5)
Therefore, in order to evaporate 1 μl of water (M = 1) under 1 atm (P = 760 mmHg), the protein crystal growth apparatus 1 has only to reduce the pressure to −0.94 mmHg.

(4-4) Protein Crystal Growth Method According to the Present Invention Next, a protein crystal growth process in the present embodiment will be described based on a crystal growth phase diagram based on a dissolution state diagram. FIG. 7 is a dissolution state diagram of an example of a phase diagram of protein crystal growth according to an embodiment of the present invention, and FIG. 8 is a protein and crystallization agent (reservoir) solution used in an embodiment of the present invention. It is a melt | dissolution state figure which shows an example of the density | concentration of the liquid mixture. FIG. 9 is a side view of the chamber portion 3 in FIG. 2A.

  First, as shown in FIG. 9, the protein injected from the protein injection part 61, the crystallization agent injected from the crystallization agent injection part 62, and the water injection part 63 are injected into the hole part 32 in the chamber part 3. A mixed aqueous solution 15 (mixed solution) made of pure water is injected, and a protein seed crystal 16 is put into the mixed aqueous solution 15. At this time, on the dissolution diagram, the protein state is located in the initial state (CdropA: Va) in which the protein concentration (Cpa) and the crystallization agent concentration (Cca) are (m1 + m2): m3 (FIG. 7). Process P0).

  The control unit 2 controls the pressure adjusting unit 4 so that the chamber unit 3 is depressurized, and when the water in the mixed aqueous solution 15 is evaporated, the protein concentration and the crystallization agent concentration are changed as shown in FIG. Ascend and move on a straight line rising right through the origin (process P1 shown in FIG. 7).

  Then, when the state of the mixed aqueous solution 15 (protein concentration and crystallization agent concentration) exceeds the supersaturation curve C2 shown in FIG. 7, that is, the control unit 2 determines one crystal based on the detection result in the detection unit 8. If it is determined that nuclei have started to form, the evaporation of moisture in the mixed aqueous solution 15 is stopped (process P2 shown in FIG. 7). Thereby, it is possible to avoid the protein from being dispersed in many crystal nuclei and to grow the crystal greatly.

The protein molecules in the mixed aqueous solution 15 are consumed for the growth of crystal nuclei, so that the protein concentration in the mixed aqueous solution 15 decreases and the protein state reaches the metastable region A2 (process shown in FIG. 7). P3). In the metastable region A2, no new crystal nucleus is formed, so that only one crystal nucleus formed by the process P1 continues to grow. When the state of the mixed aqueous solution 15 (protein concentration and crystallization agent concentration) reaches the solubility curve C1 (process P4, CdropB: Vb shown in FIG. 7), the growth of crystal nuclei stops. If the crystal nucleus has grown to a desired size (preferably 1 mm ³ or more) at this stage, the operation of the protein crystal growth apparatus 1 is terminated.

  Subsequently, the control unit 2 opens the electromagnetic valve 64 connected to the protein injection unit 61, and injects a predetermined amount of the protein stock solution into the mixed aqueous solution 15 (process P5 shown in FIG. 7). In the unsaturated region A1 in the process P5, the surface of the crystal nucleus oxidized in the processes P2 to P4 is dissolved. At this time, since the crystallization agent is not injected, the concentration of the protein in the mixed aqueous solution 15 is increased (increased), while the concentration of the crystallization agent in the mixed aqueous solution 15 is decreased (decreased). The state (state of protein concentration and crystallization agent concentration) becomes the initial state (CdropA: Va) (process P6 shown in FIG. 7).

  Here, a method of injecting a protein stock solution to bring the mixed aqueous solution 15 into a state (protein concentration and crystallization agent concentration) (CdropA: Va) will be further described with reference to a dissolution state diagram shown in FIG.

In this embodiment, the concentration and volume of the protein stock solution are (Cp0) and (Vp0), respectively, and the concentration and volume of the crystallization agent are (Cc0) and (Vc0). In addition, the protein concentration, crystallization concentration, and volume in the mixed solution in the state of the mixed aqueous solution 15 (protein concentration and crystallization agent concentration) (CdropB) are (Cpb), (Ccb), and (Vb), respectively. To do. Furthermore, the protein concentration, the crystallization concentration, and the volume in the mixed solution at the time of the mixed aqueous solution 15 (protein concentration and crystallization agent concentration) (CdropA) are (Cpa), (Cca), and (Va), respectively. To do. The protein stock solution and the crystallization agent have a concentration ratio represented by the following formulas (6) to (8).
| 0- (Cca) | = | (Cpa)-(Cp0) | = m1 (6)
| (Cca) − (Ccb) | = | (Cpb) − (Cpa) | = m2 (7)
| (Ccb) − (Cc0) | = | 0− (Cpb) | = m3 (8)

  Therefore, in the state of the mixed aqueous solution 15 (protein concentration and crystallization agent concentration) (CdropB), the control unit 2 prepares a mixed solution so as to satisfy (Vc0) :( Vp0) = (m1 + m2): m3, and In the state (CdropA), the protein stock solution may be injected so as to satisfy (Vb) :( Vp0) = m1: m2.

  The control unit 2 again controls the atmospheric pressure adjustment unit 4 to depressurize the inside of the chamber unit 3 to evaporate water in the mixed aqueous solution 15 (process P7 shown in FIG. 7). Further, when the control unit 2 determines that one crystal nucleus starts to be formed based on the detection result of the detection unit 8, the control unit 2 stops the evaporation of moisture in the mixed aqueous solution 15 (process P8 and process shown in FIG. 7). P9). Since the concentration of the crystallization agent in the process P8 is thinner than that in the process P2, the state of the mixed aqueous solution 15 (protein concentration and crystallization agent concentration) is lowered before the position of the process P2 in FIG. Will be.

  When the state of the mixed aqueous solution 15 (protein concentration and crystallization agent concentration) reaches the solubility curve C1 (process P10 shown in FIG. 7), the control unit 2 opens the electromagnetic valve connected to the protein injection unit 6, By injecting a predetermined amount of the protein stock solution into the mixed aqueous solution 15, the state of the mixed aqueous solution 15 (protein concentration and crystallization agent concentration) is set to the initial state (Cdrop) (process P11 shown in FIG. 7).

  As described above, the control unit 2 performs vapor diffusion (process P0, P1, P2, and process P6, P7, P8), crystal growth (process P2, P3, P4, and process P8, P9, P10), crystal nucleus The steps of surface dissolution (process P4, P5, P0, and process P10, P11, P6) are repeated, and when the crystal nucleus grown to a desired size reaches the automatic formation region, the crystal nucleus is taken out. The growth process is completed.

  According to the protein crystal growth apparatus and protein crystal growth method of the present invention having such a configuration, the crystal growth method by the vapor diffusion method is applied, and the vapor pressure difference with the aqueous solution of the crystallization agent (reservoir, precipitation agent) is not used. Instead, by controlling the vapor pressure in the protein solution droplet based on the crystal growth phase diagram based on the dissolution phase diagram, the protein can be grown to a desired size and to a good quality crystal with good reproducibility. it can. Therefore, when structural analysis of such a large protein crystal is performed by neutron diffraction, neutrons are reliably irradiated to the water-derived nuclei (protons) present in the protein, and the scattering analysis analyzes the hydrogen atoms and protons in the protein. It is possible to selectively acquire the positional information of the proteins and to analyze the three-dimensional structure of the protein containing them in more detail.

  In particular, by growing protein crystal nuclei in the metastable region A2, it has been difficult to quantitatively control the evaporation of water conventionally caused by the difference in vapor pressure, the difficulty of adding an aqueous protein solution to a protein solution droplet, etc. It is possible to solve such troubles in work and complexity of control. In addition, since crystal growth is performed in an inert gas atmosphere, protein crystal surface oxidation can be prevented.

  As described above, according to the protein crystal growth method and apparatus of the present invention, a large-sized and high-quality protein crystal can be obtained with good reproducibility, and by structural analysis by neutron diffraction method, Proton position information can be acquired selectively and the three-dimensional structure of the protein containing them can be analyzed in more detail, so it is widely and effectively used for protein structure analysis that has been difficult until now. be able to.

  DESCRIPTION OF SYMBOLS 1 ... Protein crystal growth apparatus, 15 ... Mixed aqueous solution, 16 ... Seed crystal, 2 ... Control part, 3 ... Chamber part, 31 ... Crystal growth substrate, 32 ... Hole part, 33, 34, 35 ... Connection part, 36 ... Gas Connection part (end part), 37 ... Gas path part (first path), 38 ... Aqueous solution connection part (end part), 39 ... Aqueous solution path part (second path), 4 ... Pressure adjustment part, 42 ... Solenoid valve, DESCRIPTION OF SYMBOLS 5 ... Gas adjustment part, 53 ... Nitrogen gas supply valve, 54 ... Atmospheric release valve, 6 ... Injection | pouring part, 61 ... Protein injection | pouring part, 62 ... Crystallizer injection | pouring part, 63 ... Water injection | pouring part, 64, 65, 66 ... Solenoid valve, 7 ... sensor part, 8 ... detection part.

Claims (16)

A chamber portion to which a protein solution and a crystallization agent solution are supplied;
In the chamber part, a protein solution supply part for supplying the protein solution;
In the chamber part, a crystallization agent solution supply part for supplying the crystallization agent solution;
The relationship between the concentration of the protein and the concentration of the crystallization agent in the mixed solution of the protein solution and the crystallization agent solution accommodated in the chamber portion is not saturated in the dissolution state diagram of the protein and the crystallization agent. A controller that adjusts between the region, the metastable region, and the automatic nucleation region;
Equipped with a,
The control unit performs control to repeat a process cycle of transition from the unsaturated region to the metastable region through the automatic nucleation region,
Protein crystal growth device. An inert gas supply unit connected to the control unit and supplying an inert gas into the chamber unit;
A decompression unit connected to the control unit and decompressing the inside of the chamber unit;
A depressurization stop unit that is connected to the control unit and stops depressurization in the chamber unit;
A pressurizing unit connected to the control unit and pressurizing the inside of the chamber unit;
A protein crystal growth apparatus according to claim 1. The protein solution supply unit is connected to the control unit,
The crystallization agent solution supply unit is connected to the control unit,
The protein crystal growth apparatus according to claim 1 or 2. The chamber portion includes a substrate that holds crystals of the protein to be grown,
The substrate is supplied with the inert gas, and a hole portion into which the protein solution and the crystallization agent solution or the mixed solution are injected, a first path through which the inert gas flows, and the protein solution And a second route through which
The first path connects an upper portion of the hole portion and an end portion of the substrate, and is formed along the upper surface of the substrate.
The second path connects the lower part of the hole and the end of the substrate, and is formed along the bottom surface of the substrate.
The protein crystal growth apparatus of any one of Claims 1-3. The control unit operates the decompression unit and the decompression stop unit alternately and / or repeatedly,
The protein crystal growth apparatus of any one of Claims 1-4. A detection unit connected to the control unit and detecting a state of the protein crystal;
The protein crystal growth apparatus of any one of Claims 1-5. The detection unit detects a crystal growth state in the protein and / or a crystal surface dissolution state in the protein;
The protein crystal growth apparatus according to claim 6. The control unit operates the decompression unit in a state in which the protein seed crystal is accommodated in the chamber part in advance or in a state in which the protein seed crystal is formed in the chamber part. By reducing the pressure and evaporating the water in the mixed solution, the state of the mixed solution is transitioned from the unsaturated region to the automatic nucleation region in the dissolution state diagram of the protein and the crystallization agent. After starting the growth of the seed crystal, the decompression stop portion is operated to stop the decompression in the chamber portion, and the protein in the protein solution is used for the growth of the protein crystals to use the protein in the protein solution. By reducing the concentration of the mixture, the state of the mixed solution is changed from the automatic nucleation region to the unsaturated region through the metastable region. That,
The protein crystal growth apparatus of any one of Claims 1-7. The protein according to claim 8, wherein the control unit operates the protein solution supply unit to supply the protein solution to the mixed solution that has reached the unsaturated region to increase the protein concentration in the mixed solution. Crystal growth equipment. Supplying a protein solution into the chamber;
Supplying a crystallization agent solution into the chamber part;
The relationship between the concentration of the protein and the concentration of the crystallization agent in the mixed solution of the protein solution and the crystallization agent solution accommodated in the chamber portion is not saturated in the dissolution state diagram of the protein and the crystallization agent. Adjusting between the region, the metastable region, and the autonucleation region;
Only including,
Repeating the process cycle of transitioning from the unsaturated region to the metastable region through the automatic nucleation region ,
Protein crystal growth method. Replacing the inside of the chamber containing the mixed solution with an inert gas;
Depressurizing the inside of the chamber part from a state in which the protein seed crystal is preliminarily accommodated in the chamber part or a state in which the protein seed crystal is formed in the chamber;
Stopping the decompression in the chamber when the protein crystal begins to grow;
Supplying the protein solution to the mixed solution when the crystal surface of the protein is dissolved;
The method for growing protein crystals according to claim 10. Alternately and / or repeatedly performing the depressurizing step and the depressurizing step.
The protein crystal growth method according to claim 11. Detecting the crystalline state of the protein,
The protein crystal growth method according to any one of claims 10 to 12. In the step of detecting the crystal state of the protein, the crystal growth state in the protein and / or the dissolution state of the crystal surface in the protein are detected.
The protein crystal growth method according to claim 13. From the state in which the protein seed crystal is preliminarily accommodated in the chamber part or the state in which the protein seed crystal is formed in the chamber part, the inside of the chamber part is decompressed to evaporate water in the mixed solution. Thus, in the dissolution state diagram of the protein and the crystallization agent, after the transition of the state of the mixed solution from the unsaturated region to the automatic nucleation region to start the growth of the seed crystal, By stopping the decompression and using the protein in the protein solution for the growth of the protein crystals to reduce the concentration of the protein in the mixed solution, the state of the mixed solution is changed from the automatic nucleation region. Transition to the unsaturated region via the metastable region,
The protein crystal growth method according to any one of claims 10 to 14. Supplying the protein solution to the mixed solution that has reached the unsaturated region to increase the protein concentration in the mixed solution;
The protein crystal growth method according to claim 15.

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