JP2007230841A - Protein crystal production method, protein crystallization promotion apparatus, and protein crystallization promotion system - Google Patents

Abstract

It is possible to crystallize a protein using a protein solution having a low protein concentration and a low concentration condition, so that the crystallization condition can be searched and screened.
A protein crystal production method for producing protein crystals from protein solution droplets includes the step (A) of forming crystal nuclei by increasing the vapor diffusion rate of the protein solution droplets. In this method, the vapor diffusion rate is increased by using a solvent adsorbent contained in the droplet of the protein solution.
[Selection] Figure 2

Description

  The present invention relates to a protein crystal generation method, a protein crystallization promotion apparatus, and a protein crystallization promotion system. More specifically, the present invention relates to a protein crystal generation method, a protein crystallization promotion apparatus, and a protein crystallization promotion system suitable for screening. .

  In recent years, the DNA base sequences of many biological species including humans have been rapidly deciphered, and the three-dimensional structure of the proteins encoded by individual genes has been revealed for a huge number of genes extracted from a large amount of genome sequence information. Thus, elucidating the relationship with function has become an important issue.

  The number of proteins subject to structural analysis in structural genomics to clarify the three-dimensional structure of such proteins is about 100,000 when it comes to human genes.

  Here, the three-dimensional structure of a protein is formed from a combination of thousands to thousands of basic structural units, and it is considered that a diversity of functions is realized by the combination. For this reason, research on systematic and comprehensive structural analysis aimed at clarifying the relationship between the structure and function of proteins by elucidating all these basic structural units has been advanced.

In general, in order to efficiently advance systematic and comprehensive structural analysis research, it is required to speed up and automate each process in the structural analysis.

  In particular, since proteins are inactivated or lost due to denaturation, degradation, adsorption, etc., when preparing a target protein sample, a series of operations can be performed in as short a time as possible while avoiding these losses. It is desirable to do this, and speeding up and automation of each process in the structural analysis is an urgent issue.

By the way, the X-ray method is known as one of the protein structure analysis techniques. In this X-ray method, the target protein is purified and concentrated, then crystallized, and the crystallized protein is used for measurement. In order to use it as a sample, protein crystallization has been required to be performed in a short time with high efficiency.

  Furthermore, since the protein to be subjected to the structural analysis by the X-ray method is valuable and only a small amount can be obtained, it has been desired to develop a method for crystallizing the protein from a solution containing the protein only at a lower concentration.

  Conventionally, the hanging drop method, which is a kind of vapor equilibrium diffusion method, has been widely used as a protein crystallization method for preparing a protein crystal that can be used as a measurement sample for structural analysis by the X-ray method. ing.

  In carrying out this hanging drop method for protein crystallization, a commercially available crystallization plate container is generally used.

  The operation procedure of the hanging drop method using such a commercially available crystallization plate container will be described. First, a reservoir solution (crystallization solution) composed of a buffer solution, a salt and a precipitant is placed in the container. Next, after dropping a droplet obtained by equally mixing the protein solution and the reservoir solution onto the cover glass surface, or after dropping the protein solution and the reservoir solution equally onto the cover glass, the cover glass The cover glass surface on which the liquid droplets are attached is repeatedly opposed to the container, and the cover glass is sealed and fixed to the container with grease or the like.

  According to such a hanging drop method, the droplets attached to the cover glass sealed from the outside spontaneously diffuse by vapor equilibrium diffusion and take a crystal growth process. A protein crystal that can be used as a measurement sample for structural analysis by the method can be obtained.

  In addition, when obtaining a protein crystal that can be used as a measurement sample for structural analysis by the X-ray method, as a technique often used other than the above-described hanging drop method, for example, a sitting drop method is known. Yes.

However, according to the conventional vapor equilibrium diffusion methods such as the hanging drop method and the sitting drop method, it takes a long time to grow a protein to a crystal suitable for the structural analysis by the X-ray method. There is a problem that the efficiency is low and the yield is very low.

  In other words, in the hanging drop method and sitting drop method, crystals suitable for structural analysis are deposited only under conditions (crystallization conditions) in which the concentration and molecular weight of the reservoir solution and protein characteristics are optimized. In addition, it takes several tens of hours to several weeks to precipitate (it usually takes about 1 to 4 weeks to crystallize) and takes time.

  Furthermore, when a new protein crystal is obtained by the hanging drop method or sitting drop method, as shown in the example of the process flow shown in FIG. For example, since a condition for combining about 100 or more kinds of reservoir solutions or the like is combined with a protein by trial and error, that is, screening is performed, more work time is required, and a large amount of protein sample and reservoir solution are added. It was a lot of waste.

  Further, even when crystallization is reached, it is currently impossible to determine whether it is a true protein crystal or a precipitated crystal such as a salt by external observation with a microscope.

  In other words, according to the conventional protein crystallization technique, in order to crystallize one new protein molecule, the conditions of the reservoir solution consisting of buffer solution, salt, and precipitant are changed in various ways and tested by trial and error. Therefore, a large amount of samples are required for screening for crystallization conditions, and further, it usually takes about 1 to 4 weeks to determine the success or failure of crystallization, There was a problem that time efficiency was not good.

Therefore, it is possible to crystallize the protein even when using a protein solution under a low concentration condition with a low protein concentration, and to search and screen for the crystallization conditions, It is recognized that the development of methods that enable rapid determination of the state of crystallization is an extremely urgent and important technical issue in protein structural analysis research, and the development of these methods is extremely strong. There was a request.

As a technique that makes it possible to easily produce a polymer compound crystal and to easily screen the crystallization conditions of the polymer, for example, JP-A-2004-307335 presented as Patent Document 1 The polymer crystal growth vessel disclosed in the Japanese Patent No. 2 is proposed.

However, according to the polymer crystal growth vessel disclosed in Japanese Patent Application Laid-Open No. 2004-307335, since the vapor diffusion rate cannot be varied according to the concentration of the protein solution, the protein solution may be dried up. There is a problem that precipitates such as the above may occur.
JP 2004-307335 A

    The present invention has been made in view of the above-described various demands and problems of the prior art, and the object of the present invention is to use a protein solution in a low concentration condition with a low protein concentration. A protein crystal generation method and a protein crystallization promoting device that can be crystallized, and can search and screen for crystallization conditions even using a low concentration protein solution having a low protein concentration. It is something to be offered.

  Another object of the present invention is to provide a protein crystal production method and a protein crystallization promoting device capable of arbitrarily controlling the vapor diffusion rate of a protein solution.

  Furthermore, an object of the present invention is to provide a protein crystallization promoting system capable of quickly determining the state of protein crystallization.

  In order to achieve the above object, the protein crystal production method and the protein crystallization promotion apparatus according to the present invention actively control the vapor diffusion rate of the protein solution instead of the conventional passive vapor equilibrium diffusion method, The protein solution is concentrated.

  That is, the protein crystal production method and the protein crystallization promoting apparatus according to the present invention provide a solvent adsorbing substance (for example, a water-absorbing agent such as silica gel) that adsorbs the solvent in the protein solution in order to increase the protein concentration in the protein solution. Is used to evaporate solvent molecules such as water molecules to reduce the volume of the protein solution and concentrate the protein solution. During this concentration, simple evaporation of the solvent molecules causes the protein solution to evaporate. However, the vapor diffusion rate of the protein solution is arbitrarily controlled because various solids other than protein crystals may be precipitated during the evaporation process.

  In addition, the protein crystallization promoting system according to the present invention can observe the concentration of protein droplets in real time by Raman spectroscopy in order to arbitrarily control the vapor diffusion rate while detecting the state during crystallization. It is a thing. In protein crystallization, pseudo-crystals may be generated. With this Raman spectroscopy, it is possible to determine in real time whether a crystal-like substance in a droplet is a protein crystal.

That is, the invention according to claim 1 of the present invention is a protein crystal production method for producing protein crystals from protein solution droplets, whereby crystal nuclei are formed by increasing the vapor diffusion rate of the protein solution droplets. A protein crystal production method comprising the step (A) of: Here, “accelerate” refers to increasing the speed compared to the vapor diffusion speed when a general vapor diffusion equilibrium method, which is a protein crystal production method, is performed. In addition, when the initial concentration of the protein solution is 2 mg / ml to 10 mg / ml, etc., in the case of a low concentration, it is slower than the step (A) until the concentration of the protein solution is increased to some extent. Vapor diffusion is preferred.
In the protein crystal production method according to claim 1, the invention according to claim 2 of the present invention uses the solvent adsorbent contained in the droplet of the protein solution to increase the vapor diffusion rate. This is a method for producing protein crystals.

  Moreover, invention of Claim 3 among this invention is compared with the said process (A) after the said process (A) in the protein crystal | crystallization production | generation method of any one of Claim 1 or 2. The method for producing a protein crystal further comprises a step (B) of growing a protein crystal by slowing the vapor diffusion rate.

  Moreover, the invention according to claim 4 of the present invention is the protein crystal production method according to any one of claims 1, 2, or 3, wherein the control of the vapor diffusion rate of the droplets of the protein solution is as follows: The protein crystal production method is performed according to the concentration of the protein solution.

  The invention according to claim 5 of the present invention is the protein crystal production method according to any one of claims 1, 2, 3 or 4, wherein the initial concentration of the protein solution is 2 mg / ml to 10 mg. / Ml of protein crystal production method.

  According to a sixth aspect of the present invention, in the protein crystallization promoting device for promoting the production of protein crystals from the protein solution droplets, the first partition for arranging the protein solution droplets is provided. A chamber, a second compartment for disposing a solvent-adsorbing substance that adsorbs a solvent in the protein solution, a first passage communicating the first compartment and the second compartment, A first valve that opens and closes the first passage to maintain the first compartment and the second compartment in a communication state and a non-communication state; and It is a protein crystallization promotion apparatus characterized by controlling and opening / closing of said 1st channel | path.

  Moreover, invention of Claim 7 among this invention is a control method of the protein crystallization promotion apparatus of Claim 6, Comprising: Opening the said 1st valve | bulb, the said 1st compartment and the said 1st The crystal nuclei are generated by the step (A) of maintaining the two compartments in communication with each other and forming the crystal nuclei by increasing the vapor diffusion rate of the droplets in the first compartment, A step of growing a protein crystal by closing the first valve to limit the ventilation between the first compartment and the second compartment and by reducing the vapor diffusion rate compared to the step (A) (B) is a method for controlling a protein crystallization promoting apparatus, characterized in that a protein crystal is generated by performing crystal growth around the nucleus. Here, the above-mentioned “restricting ventilation” includes a non-communication state, and the “reducing the vapor diffusion rate” includes stopping vapor diffusion.

  Moreover, invention of Claim 8 among this invention is a protein crystallization promotion apparatus of Claim 6, Furthermore, the 3rd compartment for storing water, The said 1st compartment, The second passage communicating with the third compartment and the second passage are opened and closed to maintain the communication state and the non-communication state between the first compartment and the third compartment. And a second valve for controlling the opening and closing of the second passage by controlling the second valve.

  The invention according to claim 9 of the present invention is the method for controlling a protein crystallization promoting apparatus according to claim 8, wherein the first valve and the first compartment are opened by opening the first valve. The crystal nuclei are generated by the step (A) of maintaining the two compartments in communication with each other and forming the crystal nuclei by increasing the vapor diffusion rate of the droplets in the first compartment, The first valve is closed to restrict ventilation between the first compartment and the second compartment, and the second valve is opened to connect the first compartment and the third compartment. Maintaining the state of communication and producing a protein crystal by growing the crystal around the nucleus by the step (B) of growing the protein crystal by slowing the vapor diffusion rate compared to the step (A). Is a method for controlling a protein crystallization promoting device.

  The invention according to claim 10 of the present invention is the protein crystallization promoting device according to any one of claims 6 or 8, wherein a plurality of the first compartments are arranged. This is a protein crystallization promoting device.

  The invention according to claim 11 of the present invention is the protein crystallization promoting device according to any one of claims 6 or 8, wherein the first compartment has a large number of liquids on a slide glass. A protein crystallization accelerating device comprising a sealed container capable of dropping drops.

  The invention according to claim 12 of the present invention is the protein crystallization promoting device according to claim 6 and the first valve based on the observation result of the protein concentration in the droplet by Raman spectroscopy. A protein crystallization promotion system comprising: a valve opening / closing control device that performs opening / closing control.

  The invention according to claim 13 of the present invention is the protein crystallization promoting device according to claim 8, and the first valve based on the observation result of the protein concentration in the droplet by the Raman spectroscopy. And a protein crystallization promotion system comprising: a valve opening / closing control device that performs opening / closing control of the second valve.

  Since the present invention is configured as described above, it is possible to crystallize a protein even when a protein solution having a low protein concentration and a low concentration condition is used. There is an excellent effect of being able to.

  In addition, since the reservoir solution in the container is not necessary in the present invention, only a reservoir solution to be mixed with the protein solution is required, and an excellent effect is achieved that the amount of the reservoir solution can be remarkably suppressed.

  Further, since the present invention is configured as described above, it has an excellent effect that the vapor diffusion rate of the protein solution can be arbitrarily controlled.

  Hereinafter, an example of an embodiment of a protein crystal production method, a protein crystallization promotion apparatus, and a protein crystallization promotion system according to the present invention will be described in detail with reference to the accompanying drawings.

FIG. 2 is a conceptual diagram of a partial end face conceptual configuration of an embodiment of a protein crystallization promoting apparatus according to the present invention.

  This protein crystallization promoting apparatus 10 is formed by a first container 12 configured to be able to hold a droplet 14 of a protein solution in a first compartment 12a formed by an internal space, and the internal space. The second container 16 configured to accommodate the solvent adsorbing substance 18 that adsorbs the solvent in the protein solution in the second compartment 16a, and the first compartment 12a and the second compartment 16a communicate with each other. A tube 20 forming the passage 20a and a valve 22 capable of opening and closing the passage 20a formed by the tube 20 and maintaining the communication state and the non-communication state of the passage 20a are configured.

  Further, the first compartment 12a, the passage 20a, and the second compartment 16a are maintained in a sealed state with respect to the outside.

  Here, the 1st container 12 has the main-body part 12b provided with the concave shape which the upper part opened, and the cover part 12c sealed by the opening part of the main-body part 12b, and is comprised. In addition, as such 1st container 12, the commercially available crystallization plate container used for the hanging drop method etc. which are prevailing widely for screening the protein crystallization conditions can be utilized, for example.

  Similarly to the first container 12, the second container 16 includes a main body part 16b having a concave shape with an open top, and a lid part 16c sealed in the opening part of the main body part 16b. Yes. In addition, as such 2nd container 16, the various containers excellent in airtightness can be used suitably, for example.

In the above configuration, in order to generate a protein crystal by the protein crystallization promoting device 10, the droplet 14 hangs after dropping the droplet 14 of the protein solution on one surface of the lid portion 12c of the first container 12. The lid 12c is repeatedly pulled in the state, and the lid 12c to which the droplets 14 are attached is sealed and fixed to the opening of the main body 12b with grease or the like (see FIG. 2).

  On the other hand, with respect to the second container 16, the lid portion 16c is removed and a solvent adsorbing substance 18 (for example, a hygroscopic solid such as a water absorbent such as silica gel) is disposed in the main body portion 16b. 16c is sealed and fixed to the opening of the main body 16b with grease or the like (see FIG. 2).

  As described above, in the state where the droplet 14 is arranged in the first container 12 and the solvent adsorbing substance 18 is arranged in the second container 16, the valve 22 is operated to open and close the passage 20a formed by the tube 20, When the passage 20a is appropriately maintained in the communication state and the non-communication state, a humidity gradient is formed between the melters, and the adsorption of the solvent of the droplet 14 to the solvent adsorbing material 18 is appropriately controlled. It can be concentrated while controlling the evaporation of the solvent.

  That is, since the first compartment 12a of the first container 12 and the second compartment 16a of the second container 16 are partitioned by the valve 22, the evaporation rate, that is, the vapor diffusion rate is adjusted by adjusting the opening and closing of the valve 22. Can be adjusted.

  Here, when the passage 20a is opened by operating the valve 22 (the passage 20a is in a communicating state) and the vapor diffusion speed is increased, the solvent of the droplet 14 is rapidly evaporated. When called “evaporation shock” as appropriate, crystal nuclei are generated as a trigger for crystallization in the droplet 14, and crystal growth is performed around the nuclei to obtain protein crystals. Presumed.

  If crystal nuclei are generated in the droplet 14 by the evaporation shock, it is preferable to close the passage 20a by operating the valve 22 (the passage 20a is in a non-communication state) to slow the vapor diffusion rate.

Next, in FIG. 3, crystals are produced by crystal growth by the hanging drop method as a conventional natural vapor diffusion method and evaporation shock of the protein crystal production method according to the present invention (hereinafter referred to as “the present crystallization method” as appropriate). The graph of the experimental result of the crystal growth performed using the lysozyme (Lysozyme) solution as a protein solution about the crystal growth which produced | generated this nucleus is shown. In the graph shown in FIG. 3, the vertical axis represents the spectrum intensity of lysozyme at a Raman shift of 1557 cm ⁻¹ (this indicates lysozyme concentration (mg / ml)), and the horizontal axis represents time.

  This experiment by the inventor of the present application includes Experiment 1, Experiment 2, and Experiment 3, and details thereof are as follows.

  Experiment 1: By using a hanging drop method as a conventional natural vapor diffusion method, a lysozyme concentration was adjusted using a reservoir solution composed of a buffer solution (HEPES 0.1 M, pH 7.5), a salt (0.8 M), and a precipitant. Lysozyme crystals were produced from a 50 mg / ml lysozyme solution.

  Experiment 2: According to the crystallization method of the present invention, lysozyme crystals were generated from a lysozyme solution having a lysozyme concentration of 50 mg / ml by evaporation shock using silica gel as the solvent adsorbing material 18 without using a reservoir solution.

  Experiment 3: According to the crystallization method of the present invention, lysozyme crystals were generated from a lysozyme solution having a lysozyme concentration of 20 mg / ml by evaporation shock using silica gel as the solvent adsorbing material 18 without using a reservoir solution.

  From this experiment 1, according to the hanging drop method, which is a conventional natural vapor diffusion method, the lysozyme concentration in the lysozyme solution was supersaturated (the supersaturated concentration of lysozyme was 310 mg / ml (Albert M. Schwartz, and Kris A. Berglund: J. Cryst. Growth. 210 (2000) 753.)) until it reaches a supersaturated concentration, and the presence of lysozyme crystals is confirmed, and thereafter the concentration decreases slowly. It is considered that the lysozyme crystal grows.

  On the other hand, from Experiment 2 and Experiment 3, according to the crystallization method of the present invention, the concentration of lysozyme in the lysozyme solution rapidly increased due to evaporation shock, and the presence of lysozyme crystals was confirmed before reaching the supersaturated concentration. Thereafter, the lysozyme concentration increased rapidly, but it was found that the lysozyme crystal grew rapidly as the lysozyme concentration decreased rapidly before reaching the supersaturated concentration.

  From the experimental results shown in FIG. 3, it can be seen that the crystallization method of the present invention is concentrated at a speed 6 times faster than the hanging drop method.

4 to 6, the lysozyme concentration of the lysozyme solution is 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg. The experimental results of the Raman spectrum when changing to / ml and 100 mg / ml are shown. 5 is an enlarged view of region A in FIG. 4, and FIG. 6 is an enlarged view of region B in FIG. From these FIG. 4 to FIG. 6, the accuracy of the data shown in FIG. 3 is clear.

FIG. 7 shows the experiment by the inventor of the present invention described above regarding crystal growth by the hanging drop method as a conventional natural vapor diffusion method and crystal growth in which crystal nuclei are generated by evaporation shock, which is the crystallization method of the present invention. It is the graph showing the characteristic curve which shows the relationship between the protein concentration in the droplet obtained from the result, and processing time. In the graph shown in FIG. 7, the vertical axis represents the protein concentration of the protein solution, and the horizontal axis represents time.

  As shown in the graph of FIG. 7, according to the hanging drop method, which is a conventional natural vapor diffusion method, the protein concentration in the protein solution gradually increases until reaching the supersaturated concentration, and when the supersaturated concentration is reached, the crystal After that, the concentration gradually decreases and the crystal grows.

  On the other hand, according to the crystallization method of the present invention, the protein concentration in the protein solution rapidly increases due to evaporation shock, and the nucleus of the crystal is generated, and then the protein concentration further increases rapidly, but reaches the supersaturated concentration. The concentration rapidly decreases before, and the crystal grows around the nucleus.

  According to the experiments of the present inventor, protein can be crystallized from a protein solution at high speed by the crystallization method of the present invention, and the protein concentration is low (for example, about 2 mg / ml. In the hanging drop method, crystallization with a protein solution generally having a concentration higher than about 14 mg / ml could be realized.

  For example, according to the crystallization method of the present invention, lysozyme crystals can be produced using a lysozyme solution having a lysozyme concentration of 2 mg / ml, and when a lysozyme solution having a lysozyme concentration of 50 mg / ml is used, In the conventional hanging drop method, the crystallization time required for one day or more can be drastically reduced to about 5 minutes.

Furthermore, according to the experiment of the present inventor, xylanase crystals could be precipitated from a xylanase solution having a xylanase concentration of 36 mg / ml.

  In addition, according to the crystallization method of the present invention, crystals could be precipitated even at a salt concentration of 0.1 M, where crystals were not grown by the conventional natural vapor diffusion method.

  In addition, since it is possible to arrange a plurality of droplets 14 of the same kind or a plurality of droplets 14 for crystallization on the lid portion 12c of the protein crystallization promoting device 10, conventional natural vapor diffusion is possible. Compared with the case where one droplet is placed in one crystallization plate container as in the method, protein crystals can be generated extremely efficiently.

As described above, according to the protein crystallization promoting apparatus 10 and the protein crystal generation method according to the present invention, a protein crystal is generated from a protein solution having a low protein concentration, for example, a protein solution having a protein concentration of 1 mg / ml. can do. That is, it is now possible to precipitate crystals using a protein solution in a protein concentration region, which could not be crystallized by the conventional natural vapor diffusion method.

  In addition, as a protein solution used for the protein crystallization promotion apparatus 10 and the protein crystal production method according to the present invention, it is preferable to use a protein solution having a protein concentration of 2 mg / ml or more.

  Further, according to the protein crystallization promoting apparatus 10 and the protein crystal production method according to the present invention, the conventional natural vapor diffusion method requires a long time (for example, about 1 to 4 weeks) for crystal production. Crystals can be precipitated in an extremely short time (for example, about 5 minutes).

  For this reason, when the protein crystallization promoting apparatus 10 and the protein crystal production method according to the present invention are used, high-throughput screening can be performed with a small amount of sample.

  Furthermore, in the protein crystallization promoting apparatus 10 and the protein crystal production method according to the present invention, the reservoir solution in the container is unnecessary, and the apparatus configuration is simple, so that the cost for producing crystals can be reduced. .

Next, a protein crystallization promotion system according to the present invention will be described with reference to FIG.

  The protein crystallization determination system 100 includes the above-described protein crystallization promoting apparatus 10 according to the present invention and an image observation microscope 102a for observing a region centered on the droplet 14, and a crystal is a true crystal by Raman spectroscopy. A Raman spectral crystallization determination device 102 that determines whether or not there is, and a valve opening / closing control device 104 that performs opening / closing control of the valve 22 based on the observation result of the image observation microscope 102a of the Raman spectral crystallization determination device 102. Configured.

  Here, the protein crystallization promoting apparatus 10 can use, as the first container 12, for example, a crystallization plate container used for a conventional hanging drop method made of a transparent material.

  Next, the Raman spectroscopic crystallization determination apparatus 102 is provided with an image observation microscope 102a capable of observing the crystallization process in the droplet 14 over time, and a crystal generated from the droplet 14 by Raman spectroscopy. Is a device that determines whether or not is a true crystal, and has the configuration and the action disclosed in, for example, “PR Carey and J. Dong: J. Biochemistry, 43 (2004) 8885”. Is.

Further, the valve opening / closing control device 104 includes control means for controlling the opening / closing of the valve 22, and controls the opening / closing of the valve 22 by the control means based on the observation result by the image observation microscope 102 a of the Raman spectral crystallization determination device 102.

In the above configuration, according to the protein crystallization determination system 100, the Raman spectroscopic crystallization determination apparatus 102 performs the crystallization process while observing the crystallization process in the droplet 14 with the image observation microscope 102a over time. In addition, a signal for controlling the valve opening / closing control device 104 is output. For example, the valve opening / closing control device 104 adjusts the opening degree of the valve 22 to 100% first, then 50%, 25%, 0%, etc. based on the signal output from the Raman spectral crystallization determination device 102. Adjust the crystallization process.

  Then, after crystal precipitation, the Raman spectral crystallization determination device 102 is used to determine whether the crystal is a true crystal or a pseudo crystal based on the Raman spectrum.

  Therefore, according to the protein crystallization determination system 100, the crystallization process of the droplet 14 of the protein solution can be monitored in situ, and it can be immediately discriminated whether it is a true protein crystal or a pseudo crystal. In other words, crystallization can be immediately determined on-site.

  In addition, each component of the protein crystallization determination system 100 can be specifically configured as follows.

  That is, for the protein crystallization promoting apparatus 10, for example, a crystallization plate container can be used as the first container 12, and a centrifuge tube can be used as the second container 16, for example. As the tube 20, for example, a rubber tube can be used, and as the valve 22, a needle valve or a PC-controlled electromagnetic solenoid valve can be used for visualizing the degree of opening.

Next, FIG. 9 shows a partial end face conceptual configuration explanatory diagram of another embodiment of the protein crystallization promoting apparatus according to the present invention. Note that the same or corresponding components as those of the protein crystallization promoting device 10 shown in FIG. 2 are denoted by the same reference numerals as those of the protein crystallization promoting device 10, so that the detailed configuration and description of the operation are omitted. .

  The protein crystallization promoting apparatus 200 shown in FIG. 9 can be used instead of the protein crystallization promoting apparatus 10 of the protein crystallization determination system 100 described above, but is formed by an internal space. A third container 202 configured to be able to contain water 204 in the third compartment 202a, a tube 206 forming a passage 206a communicating the first compartment 12a and the third compartment 202a, It differs from the protein crystallization promoting device 10 in that it has a valve 208 capable of opening and closing the passage 206a formed by the tube 206 and maintaining the passage 206a in a communication state and a non-communication state.

  The first compartment 12a, the passage 206a, and the third compartment 202a are maintained in a sealed state with respect to the outside.

  Here, the third container 202 is configured to include a main body 202b having a concave shape with an open top, and a lid 202c sealed in the opening of the main body 202b. In addition, as such 3rd container 202, the various container excellent in airtightness can be used suitably, for example.

In the above configuration, according to the protein crystallization promoting device 200, when water is put into the third compartment 202a of the third container 202, the third compartment 12a is filled with water via the valve 208. Since the container 202 is connected, the humidity in the first compartment 12a can be increased by opening the valve 208 and opening the passage 206a of the tube 206 to bring the passage 206a into communication.

  Therefore, by appropriately opening and closing the valve 22 and the valve 208, the vapor diffusion rate in the first compartment 12a can be controlled more precisely, and a higher quality crystal can be generated in a shorter time. It becomes possible.

  When the protein crystallization promoting device 200 is used in the protein crystallization determination system 100, the valve opening / closing control device 104 may control the opening / closing of the valve 22 similarly to the valve 206.

  In addition, a plurality of third compartments 202a having different amounts of stored water and valves 208 corresponding to the plurality of third compartments 202a are provided, and the first compartment 12a is controlled by opening / closing control of the valve 208. You may make it electrically connect with either of the some 3rd compartment 202a.

Here, an experiment result of the present inventor using the protein crystallization promoting apparatus 200 will be described.

  In this experiment, in order to adjust the vapor diffusion rate, the third compartment 202a not containing water, the third compartment 202a containing 100 μl of water, and the third compartment 202a containing 200 μl of water, The third compartment 202a containing 500 μl of water is connected to the first compartment 12a via the corresponding valve 208, and the four third compartments 202a are selectively used as the first compartment 12a. It was configured to be conductive. When any one of the third compartments 202a and the first compartment 12a is electrically connected, the valve 208 corresponding to the third compartment 202a electrically connected to the first compartment 12a is maintained in a fully opened state. The valves 208 corresponding to the other three third compartments 202a remained fully closed.

  In this experiment, a lysozyme solution having a lysozyme concentration of 2 mg / ml, a buffer solution (HEPES 0.1M), and a salt (0.8M) were used as protein solutions.

  In this experiment, an experiment was conducted in which the third compartment 202a was replaced to produce lysozyme crystals by the crystallization method of the present invention. In either case, the valve 22 was initially fully opened, When the nucleus was generated, the valve 22 was fully closed. FIG. 12 shows a photograph of the lysozyme crystal produced by the above-described experiment. The photograph shown in FIG. 12 is obtained when the first compartment 12a is electrically connected to the third compartment 202a containing 500 μl of water.

  In addition, when producing a crystal with a low concentration protein solution using the crystallization method of the present invention, an appropriate evaporation rate is required. However, it is difficult to obtain an appropriate evaporation rate from the beginning. Therefore, water was put into the third compartment 202a to suppress the evaporation rate from the protein solution droplets.

  In the above experiment, the crystallization experiment was performed by adding only 0 μl, 100 μl, 200 μl, and 500 μl of water into each of the four third compartments 202a. If the water absorption rate by the solvent adsorbing material 18 is constant, As the amount of water put into the third compartment 202a increases, the amount of water evaporated from the third compartment 202a increases, while the evaporation rate from the droplets decreases. In the experiment described above, the valve 208 was fully opened, and the evaporation rate was controlled by the amount of water placed in the third compartment 202a.

The above-described embodiment can be modified as shown in the following (1) to (6).

  (1) In the above-described embodiment, the case where one first compartment 12a is provided via the valve 22 to the second compartment 16a has been described. However, the present invention is not limited to this. In this case, a plurality of first compartments 12a may be provided via the valve 22 with respect to the second compartment 16a. When a plurality of first compartments 12a are provided, a valve 22 is provided corresponding to each of the plurality of first compartments 12a, and the vapor diffusion rate is controlled according to each first compartment 12a. You may make it do.

  (2) In the above-described embodiment, the case where one first compartment 12a is provided via the valve 208 for the third compartment 202a has been described. However, the present invention is not limited to this. Thus, a plurality of first compartments 12a may be provided via the valve 208 for the third compartment 202a. Further, when a plurality of first compartments 12a are provided, a valve 208 is provided corresponding to each of the plurality of first compartments 12a, and the vapor diffusion rate is controlled according to each first compartment 12a. You may make it do.

  (3) In the above-described embodiment, the crystallization plate container used is one in which droplets of the protein solution are set by the hanging drop method, which is a vapor diffusion method, but is not limited to this. Of course, a crystallization plate container in which droplets of the protein solution are set by a sitting drop method or a sandwich drop method may be used.

  (4) Further, as another embodiment of the crystallization plate container, a plurality of droplets set in a commercially available slide glass and put in a container containing the glass to have a sealed structure, etc. Can do.

  For example, in the protein crystallization promoting apparatus shown in FIG. 9, instead of the first container 12, a sealed container 303 configured to set a plurality of droplets on the crystallization multiwell plate 302 or the slide glass may be used. Alternatively, as in the protein crystal determination system 300 shown in FIG. 10, a sealed container 303 in which a plurality of droplets are set on the crystallization multiwell plate 302 or the slide glass is used as the first container 12. Also good.

  Here, FIG. 9 and FIG. 11 (FIG. 11 shows the protein crystallization promotion system 400), a schematic configuration cross-sectional explanatory diagram of the sealed container 303 is shown. Is a container with an O-ring with an open top (this container is made of plastic or glass, etc. In FIGS. 9 and 11, examples using plastic containers made of plastic are shown. ), A slide glass disposed in the container, and a plate for sealing the upper surface of the container. The container can communicate with the second container 16 and the third container 202 via a tube and a valve.

  In such an airtight container 303, after many droplets 14 are spotted on the slide glass, the slide glass is put into the container with an O-ring, and the upper part of the container is sealed with a plate.

  By using such a sealed container 303, the efficiency of crystal generation can be improved.

  In the protein crystallization determination system 300, the centrifuge tube 304 is used as the second container 16, the silica gel 306 is used as the solvent adsorbing substance 18, the silicon rubber tube 308 is used as the tube 20, and the valve opening / closing control device is used as the valve 22. A controlled electromagnetic solenoid valve 310 is used. Further, as the valve 22, a needle valve may be used instead of the electromagnetic solenoid valve 310 for visualization of the degree of opening.

  Here, as an example of the experimental procedure, 15 ml of silica gel 308 is put into a centrifuge tube 306. Note that an excessive amount of silica gel 308 is placed in the centrifuge tube 306.

  Next, the centrifugal tube 306 and the crystallization multiwell plate 302 are connected by a silicon rubber tube 308 provided with an electromagnetic solenoid valve 310. The electromagnetic solenoid valve 310 is completely closed.

  Next, a lysozyme solution (5 mg / ml, 10 mg / ml, 15 mg / ml) is prepared, and a buffer solution (salt 0.8 M, HEPES 0.1 M, pH 7.5) is prepared.

  Then, 2 μl of the buffer solution is taken into a pipetman, a droplet is dropped on the cover glass to form a spot of the droplet, and an equal amount of lysozyme solution is added to the droplet (drop), and then the cover is covered. Place the glass on the multiwell plate and seal. Here, in order to form a large number of spots of droplets, a sealed container 303 using a slide glass shown in FIGS. 9 and 11 may be used in place of the crystallization multiwell plate 302.

  The crystallization multiwell plate 302 is set in the Raman spectroscopic crystallization determination apparatus 102, and the crystallization process is observed with the image observation microscope 102a of the Raman spectroscopic crystallization determination apparatus 102 as time passes. At this time, the degree of opening of the electromagnetic solenoid valve 310 is first adjusted to 100%, then 50%, 25%, and 0% to adjust the crystallization process.

  Then, after crystal precipitation, it is discriminated whether it is a protein crystal or a pseudo crystal based on the Raman spectrum obtained by the Raman spectral crystallization determination device 102.

  (5) The specific numerical values shown in the above-described embodiments are merely examples for facilitating the understanding of the present invention, and the present invention is limited to the numerical values shown in the above-described embodiments. Is not to be done.

  (6) The above-described embodiment and the modifications shown in (1) to (5) described above are, for example, a combination of the embodiment illustrated in FIG. 8 and the embodiment illustrated in FIG. As shown in FIG. 11, they may be combined as appropriate.

  The present invention can be used for screening.

FIG. 1 is an explanatory diagram showing an example of a process flow of a conventional natural vapor diffusion method. FIG. 2 is a partial end face conceptual configuration explanatory diagram of an embodiment of a protein crystallization promoting device according to the present invention. FIG. 3 shows a crystal growth by a hanging drop method as a conventional natural vapor diffusion method and a crystal growth in which crystal nuclei are generated by evaporation shock, which is the crystallization method of the present invention, using a lysozyme solution as a protein solution. It is a graph which shows the experimental result of the crystal growth performed. FIG. 4 shows that the lysozyme concentration of the lysozyme solution is 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml. It is a graph which shows the experimental result of a Raman spectrum when making it change. FIG. 5 is an enlarged view of region A in FIG. FIG. 6 is an enlarged view of region B in FIG. FIG. 7 shows the protein concentration in the droplet and the crystal growth by the hanging drop method as a conventional natural vapor diffusion method and the crystal growth in which the crystal nuclei are generated by the evaporation shock according to the present invention. It is a graph showing the characteristic curve which shows the relationship with processing time. FIG. 8 is a partial end face conceptual configuration explanatory diagram of an embodiment of a protein crystallization promotion system according to the present invention. FIG. 9 is a partial end face conceptual configuration explanatory view of another embodiment of the protein crystallization promoting apparatus according to the present invention. FIG. 10 is a partial end face conceptual configuration explanatory view of another embodiment of the protein crystallization promotion system according to the present invention. FIG. 11 is a partial end face conceptual configuration explanatory diagram of another embodiment of the protein crystallization promotion system according to the present invention. FIG. 12 is a photograph of a lysozyme crystal produced by an experiment by the present inventor.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Protein crystallization promotion apparatus 12 1st container 12a 1st compartment 12b Main body part 12c Lid part 14 Droplet 16 2nd container 16a 2nd compartment 16b Main body part 16c Lid part 18 Solvent adsorption substance 20 Tube 20a Passage 22 Valve 100 Protein crystallization determination system 102 Protein crystallization determination device 102a Image observation microscope 104 Valve opening / closing control device 200 Protein crystallization promotion device 202 Third container 202a Third compartment 202b Main body portion 202c Lid portion 204 Water 206 Tube 206a Passage 208 Valve 300 Protein crystallization determination system 302 Crystallization multiwell plate 303 Sealed container 304 Centrifugal tube 306 Silica gel 308 Silicon rubber tube 310 Electromagnetic solenoid valve 400 Protein crystallization promotion system

Claims (13)

In a protein crystal production method for producing protein crystals from droplets of a protein solution,
A method for producing a protein crystal comprising the step (A) of forming a crystal nucleus by increasing the vapor diffusion rate of the droplet of the protein solution. The protein crystal production method according to claim 1,
A method for producing a protein crystal, wherein the vapor diffusion rate is increased by using a solvent adsorbent contained in a droplet of a protein solution. In the protein crystal production | generation method of any one of Claim 1 or 2,
Following the step (A), the method further comprises the step (B) of growing a protein crystal by slowing the vapor diffusion rate as compared with the step (A). In the protein crystal production method according to any one of claims 1, 2, and 3,
Control of vapor diffusion rate of the droplet of the protein solution is performed according to the concentration of the protein solution. The method for producing a protein crystal according to any one of claims 1, 2, 3 or 4.
An initial concentration of the protein solution is 2 mg / ml to 10 mg / ml. In a protein crystallization promoting apparatus that promotes the production of protein crystals from droplets of a protein solution,
A first compartment for placing droplets of the protein solution;
A second compartment for disposing a solvent adsorbing material that adsorbs the solvent in the protein solution;
A first passage communicating the first compartment and the second compartment;
A first valve that opens and closes the first passage to maintain the first compartment and the second compartment in a communication state and a non-communication state;
The protein crystallization promoting device, wherein the first valve is controlled to control opening and closing of the first passage. It is a control method of the protein crystallization promotion apparatus of Claim 6, Comprising:
The first valve is opened to maintain the first compartment and the second compartment in communication with each other, and the crystal nucleus is formed by increasing the vapor diffusion speed of the droplet in the first compartment. In the step (A) of forming, crystal nuclei are generated, and then the first valve is closed to limit the ventilation between the first compartment and the second compartment, and the step (A) A method for controlling a protein crystallization accelerating device, characterized in that a protein crystal is generated by performing crystal growth around the nucleus in the step (B) of growing a protein crystal by slowing down a vapor diffusion rate. . The protein crystallization promoting apparatus according to claim 6, further comprising:
A third compartment for storing water;
A second passage communicating the first compartment and the third compartment;
A second valve that opens and closes the second passage and maintains the communication state and the non-communication state between the first compartment and the third compartment;
A protein crystallization promoting device, wherein the second valve is controlled to control opening and closing of the second passage. It is a control method of the protein crystallization promotion apparatus of Claim 8, Comprising:
The first valve is opened to maintain the first compartment and the second compartment in communication with each other, and the crystal nucleus is formed by increasing the vapor diffusion speed of the droplet in the first compartment. In the forming step (A), crystal nuclei are generated, and then the first valve is closed to limit the ventilation between the first compartment and the second compartment, and the second valve is A step (B) of opening and maintaining the first compartment and the third compartment in communication and growing a protein crystal by slowing the vapor diffusion rate compared to step (A); A method for controlling a protein crystallization promoting device, characterized in that crystal growth is performed around the nucleus to produce a protein crystal. In the protein crystallization promotion apparatus of any one of Claim 6 or 8,
A protein crystallization promoting device, wherein a plurality of the first compartments are arranged. In the protein crystallization promotion apparatus of any one of Claim 6 or 8,
The first compartment is composed of an airtight container capable of dropping a large number of droplets on a slide glass. A protein crystallization promoting device according to claim 6;
A protein crystallization promotion system comprising: a valve opening / closing control device that controls opening / closing of the first valve based on a result of observing a protein concentration in the droplet by Raman spectroscopy. The protein crystallization promoting device according to claim 8,
A protein crystallization promotion system comprising: a valve opening / closing control device that performs opening / closing control of the first valve and the second valve based on the observation result of the protein concentration in the droplet by the Raman spectroscopy. .