Method for producing acrylamide gel embedding of protein crystal

Abstract

To provide a very versatile method for producing a hydrogel-embedded material of a protein crystal, which can be applied to any protein as long as the protein can be crystallized.SOLUTION: Acrylamide is subjected to a radical polymerization reaction in an extrudable shape containing an acrylamide solution in which a crystallized protein is present to form a gel, and then the content of the shape is extruded to obtain an acrylamide gel in which a protein crystal is embedded.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a hydrogel-embedded protein crystal. [Background technology]

[0002] Protein crystal structure analysis is an essential means for elucidating the function of proteins, but in order to perform accurate analysis, it is important to obtain high-quality crystals suitable for analysis, as well as to handle the obtained crystals. This is because protein crystals are weak and easily broken by physical impact, so they must be handled with great care. Therefore, research into methods for handling protein crystals without damaging them has been actively conducted, and the present inventors' research group has also proposed a method in Patent Document 1 in which a solution containing crystallized protein in a shape that can be extruded with a liquid agent is extruded with either an alginic acid solution or a polyvalent metal ion solution, and then one solution is extruded into the other solution to produce an alginic acid gel-embedded product of protein crystals. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent Publication No. 2021-84907 Summary of the Invention [Problem to be solved by the invention]

[0004] The method proposed by the present inventors' research group in Patent Document 1 has been evaluated as a method of embedding a crystallized protein in a hydrogel, rather than crystallizing the protein in the hydrogel. However, this method has room for improvement in that, depending on the type of protein to be crystallized, polyvalent metal ions may change the structure of the protein crystal, causing the protein crystal to dissolve, and depending on the type of precipitant used for crystallization, a poorly soluble salt may be formed with the polyvalent metal ions, hindering structural analysis of the protein crystal.

[0005] Therefore, an object of the present invention is to provide a versatile method for producing hydrogel-embedded protein crystals that can be applied to any protein that can be crystallized. [Means for solving the problem]

[0006] In consideration of the above, the method of the present invention for producing a hydrogel-embedded protein crystal involves, as described in claim 1, gelling acrylamide by radical polymerization inside an extrudable shaped object containing an acrylamide solution containing crystallized protein, and then extruding the contents of the shaped object to obtain an acrylamide gel in which protein crystals are embedded. In addition, the production method described in claim 2 is the production method described in claim 1, in which acrylamide is gelled by reacting ammonium peroxodisulfate as a polymerization initiator and tetramethylethylenediamine as a polymerization accelerator with acrylamide. In addition, the production method described in claim 3 is the production method described in claim 2, in which the acrylamide solution containing the crystallized protein inside the extrudable shaped object is brought into contact with an ammonium peroxodisulfate solution or a tetramethylethylenediamine solution outside the shaped object, thereby gelling the acrylamide inside the shaped object (however, when an ammonium peroxodisulfate solution is used, at least one of the acrylamide solution containing the crystallized protein and the ammonium peroxodisulfate solution contains tetramethylethylenediamine, and when a tetramethylethylenediamine solution is used, at least one of the acrylamide solution containing the crystallized protein and the tetramethylethylenediamine solution contains ammonium peroxodisulfate). In addition, the production method described in claim 4 is the production method described in claim 2, in which an acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine, prepared outside the extrudable shaped object and containing crystallized protein, is placed in the shaped object to gel the acrylamide inside the shaped object. In addition, the production method described in claim 5 is the production method described in claim 2, in which the acrylamide solution containing the crystallized protein being delivered and the ammonium peroxodisulfate solution or tetramethylethylenediamine solution being delivered are joined and delivered into an extrudable shaped object, thereby gelling the acrylamide inside the shaped object (however, when ammonium peroxodisulfate solution is used, at least one of the acrylamide solution containing the crystallized protein and the ammonium peroxodisulfate solution contains tetramethylethylenediamine, and when tetramethylethylenediamine solution is used, at least one of the acrylamide solution containing the crystallized protein and the tetramethylethylenediamine solution contains ammonium peroxodisulfate). Effect of the Invention

[0007] The method of the present invention for producing hydrogel-embedded protein crystals does not use polyvalent metal ions, which can dissolve protein crystals or form poorly soluble salts with the precipitant used for crystallization, and is therefore highly versatile and can be applied to any protein that can be crystallized. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic explanatory diagram (front view) of one example of Method 1 in the method of the present invention for producing a hydrogel-embedded protein crystal. [Diagram 2] FIG. 11 is a schematic explanatory view (front view) of an example of Method 2 of the first embodiment. [Diagram 3] FIG. 11 is a schematic explanatory view (front view) of an example of Method 3 of the first embodiment. [Figure 4] 1 is a stereomicroscope photograph of hen egg white lysozyme crystals embedded in a continuous acrylamide gel, obtained in Example 2 (scale bar: 1 mm). [Diagram 5] 1 is a stereomicroscope photograph of hen egg white lysozyme crystals embedded in a continuous acrylamide gel, obtained in Example 3 (scale bar: 1 mm). [Figure 6] 1 is a stereomicroscope photograph of hen egg white lysozyme crystals embedded in a continuous acrylamide gel, obtained in Example 5 (scale bar: 500 μm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The method of the present invention for producing a hydrogel-embedded protein crystal involves gelling acrylamide through a radical polymerization reaction inside an extrudable shaped object containing an acrylamide solution containing crystallized protein, and then extruding the contents of the shaped object to obtain an acrylamide gel in which protein crystals are embedded.

[0010] As a method for gelling acrylamide by radical polymerization, for example, a method of reacting acrylamide (bisacrylamide may be contained at a ratio of 1 part by weight per 10 to 50 parts by weight of acrylamide) with ammonium peroxodisulfate (APS: also called ammonium persulfate) as a polymerization initiator (radical generator) and tetramethylethylenediamine (TEMED) as a polymerization promoter can be adopted. It is well known that when ammonium peroxodisulfate and tetramethylethylenediamine are reacted with acrylamide, a radical polymerization reaction of acrylamide occurs, and an acrylamide gel is obtained as a hydrogel, and this reaction can be used to embed a crystallized protein inside an extrudable shaped object in the acrylamide gel.

[0011] The method of reacting ammonium peroxodisulfate and tetramethylethylenediamine with acrylamide is not particularly limited as long as it is a method that can gel acrylamide inside an extrudable shaped object containing an acrylamide solution containing crystallized protein, and for example, the following three methods can be adopted.

[0012] Method 1: A method of gelling the acrylamide inside the extrudable shape by contacting an acrylamide solution containing crystallized protein inside the shape with an ammonium peroxodisulfate solution or a tetramethylethylenediamine solution outside the shape (however, when an ammonium peroxodisulfate solution is used, at least one of the acrylamide solution containing the crystallized protein and the ammonium peroxodisulfate solution contains tetramethylethylenediamine, and when a tetramethylethylenediamine solution is used, at least one of the acrylamide solution containing the crystallized protein and the tetramethylethylenediamine solution contains ammonium peroxodisulfate)

[0013] FIG. 1 is a schematic explanatory diagram (front view) of one example of Method 1, showing a method in which an acrylamide solution containing ammonium peroxodisulfate, in which crystallized protein is present inside a capillary tube as an extrudable object, is brought into contact with a tetramethylethylenediamine solution filled in a petri dish, thereby gelling the acrylamide inside the capillary tube.

[0014] In the method shown in FIG. 1, first, a capillary tube (with an inner diameter of, for example, 0.1 to 3 mm (the upper limit of the inner diameter is preferably 2 mm, more preferably 1 mm) containing an acrylamide solution containing ammonium peroxodisulfate in which a crystallized protein exists is prepared. The capillary tube containing the acrylamide solution containing ammonium peroxodisulfate in which a crystallized protein exists can be prepared by adding an acrylamide solution containing ammonium peroxodisulfate to a solution in which a protein crystallized outside the capillary tube exists using a method such as a vapor diffusion method or a free interface diffusion method, and then placing the acrylamide solution containing ammonium peroxodisulfate in which a crystallized protein exists into the capillary tube. The crystallization of a protein outside the capillary tube may be performed according to known conditions (protein concentration, type and concentration of precipitant, pH, temperature, etc.) according to the protein to be crystallized. The operation of introducing the acrylamide solution containing ammonium peroxodisulfate in which the crystallized protein is present into the capillary tube can be performed, for example, by sucking the acrylamide solution containing ammonium peroxodisulfate in which the crystallized protein is present into a syringe connected to one end of the capillary tube via a synthetic resin tube, or by using a micropipette.

[0015] The acrylamide concentration in the acrylamide solution containing ammonium peroxodisulfate in which the crystallized protein is present and placed in the capillary tube is preferably 5 to 15%. The concentration of ammonium peroxodisulfate is preferably 0.1 to 5%. If the concentration of acrylamide or ammonium peroxodisulfate is too low, the radical polymerization reaction of acrylamide to be performed next may not be sufficiently initiated, while if the concentration is too high, the protein crystals may be dissolved. In the acrylamide solution containing ammonium peroxodisulfate in which the crystallized protein is present, the precipitant used in the crystallization of the protein is preferably contained at a concentration similar to that used in the crystallization of the protein, and the pH of the solution is preferably set to the same as that used in the crystallization of the protein, in order to prevent the protein crystals from dissolving (here, "same" means within a range of ±10%. The same applies below).

[0016] Next, the lower end of the capillary tube containing the acrylamide solution containing ammonium peroxodisulfate in which the crystallized protein exists is immersed in a tetramethylethylenediamine solution filled in a petri dish, and the acrylamide solution containing ammonium peroxodisulfate in which the crystallized protein exists inside the capillary tube is brought into contact with the tetramethylethylenediamine solution, thereby causing a radical polymerization reaction of acrylamide inside the capillary tube. In this way, the acrylamide inside the capillary tube is gelled, and the contents of the capillary tube are converted into an acrylamide gel in which protein crystals are embedded. In order to complete the gelation of acrylamide, it is desirable to set the reaction time to 15 minutes or more. It is desirable to put the acrylamide solution containing ammonium peroxodisulfate in which the crystallized protein exists into the capillary tube by, for example, 10 to 50 mm of the length of the capillary tube. If an acrylamide solution containing ammonium peroxodisulfate in which crystallized protein of a length greater than this exists is placed in a capillary tube, it may take a long time to cause a radical polymerization reaction throughout the acrylamide placed in the capillary tube, or it may not occur even if a long time is spent on it. The concentration of tetramethylethylenediamine in the tetramethylethylenediamine solution is preferably 10% or more. If the concentration of tetramethylethylenediamine is too low, the radical polymerization reaction of acrylamide may not occur sufficiently. The upper limit of the concentration of tetramethylethylenediamine may be, for example, 30%. It is desirable to contain the precipitant used in protein crystallization in the tetramethylethylenediamine solution at a concentration similar to that used in protein crystallization, and to set the pH of the solution to the same as that used in protein crystallization, in order to prevent the protein crystals from dissolving after contact with an acrylamide solution containing ammonium peroxodisulfate in which crystallized protein exists.

[0017] Finally, the lower end of the capillary tube is raised from the tetramethylethylenediamine solution, and the acrylamide gel in which the protein crystals are embedded, which is the content of the capillary tube, is pushed out, and the acrylamide gel in which the protein crystals are embedded, is taken out of the capillary tube. This operation may be performed, for example, by connecting an air-filled syringe to one end of the capillary tube via a synthetic resin tube, and pushing the air out of the syringe. The content of the capillary tube may be pushed out by the air filled in the syringe manually, or mechanically using a syringe pump or the like. The pushing speed is preferably 0.001 to 100 mL / min, and more preferably 0.01 to 10 mL / min. The acrylamide gel in which the protein crystals are embedded may be pushed out from a place on a glass plate or in a precipitant solution, where the extruded acrylamide gel in which the protein crystals are embedded is easy to handle. When the acrylamide gel in which the protein crystals are embedded is pushed out onto a glass plate, it is desirable to occasionally drip a precipitant solution onto the acrylamide gel in which the protein crystals are embedded in order to prevent the gel from drying.

[0018] The capillary tube containing the acrylamide solution containing ammonium peroxodisulfate in which the crystallized protein exists may be prepared by replacing the solution in which the crystallized protein exists inside the capillary tube, obtained by crystallizing the protein inside the capillary tube using a method such as counter diffusion (liquid-liquid diffusion method), with an acrylamide solution containing ammonium peroxodisulfate. The replacement of the solution in which the crystallized protein exists inside the capillary tube, obtained by crystallizing the protein inside the capillary tube, with an acrylamide solution containing ammonium peroxodisulfate may be performed, for example, by connecting a syringe filled with an acrylamide solution containing ammonium peroxodisulfate to one end of the capillary tube via a synthetic resin tube and pushing out the acrylamide solution containing ammonium peroxodisulfate from the syringe. The crystallized protein inside the capillary tube is not pushed out together with the solution inside the capillary tube, but remains inside the capillary tube. This is because the protein crystallized inside the capillary tube is not present in a state of being suspended in the solution, but is present in a state of being adsorbed or adhered to the inner wall of the capillary tube (because the crystallization of the protein starts from the inner wall of the capillary tube). However, it should be noted that some protein crystals may be pushed out together with the solution inside the capillary tube depending on the location of the protein crystals inside the capillary tube and the degree of adsorption or adhesion to the inner wall of the capillary tube, so that not all protein crystals inside the capillary tube necessarily remain inside the capillary tube. The acrylamide solution containing ammonium peroxodisulfate filled in the syringe may be pushed out manually or mechanically using a syringe pump or the like. The upper limit of the extrusion speed is preferably 100 mL / min, more preferably 10 mL / min. If the extrusion speed is too fast, there is a risk that the protein crystals adsorbed or adhered to the inner wall of the capillary tube may be pushed out together with the solution inside the capillary tube.The concentrations of acrylamide and ammonium peroxodisulfate in the acrylamide solution containing ammonium peroxodisulfate and containing the crystallized protein thus placed in the capillary tube may be the same as those described above. Also, as described above, it is desirable to contain the precipitant used in protein crystallization in the acrylamide solution containing ammonium peroxodisulfate and containing the crystallized protein at a concentration similar to that used in protein crystallization, and to adjust the pH of the solution to the same level as that used in protein crystallization, in order to prevent dissolution of the protein crystals.

[0019] In addition, it is effective to add ammonium peroxodisulfate to the acrylamide solution as in the method shown in Fig. 1 in that it facilitates the radical polymerization reaction of acrylamide, but it may be added to the tetramethylethylenediamine solution rather than to the acrylamide solution, or it may be added to both the acrylamide solution and the tetramethylethylenediamine solution.

[0020] Furthermore, the shape of the object that can be extruded is not limited to a capillary tube, so long as it is capable of extruding the acrylamide gel in which the protein crystals are embedded as the content.

[0021] The tetramethylethylenediamine solution may be filled into a microtube or the like (a smaller amount of tetramethylethylenediamine solution may be used than when filling a petri dish).

[0022] Method 2: A method of gelling the acrylamide inside the extrudable shape by pouring into the shape an acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in the presence of crystallized protein, which was prepared outside the shape.

[0023] FIG. 2 is a schematic explanatory diagram (front view) of an example of method 2, showing a method in which an acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in the presence of crystallized protein, which has been prepared outside the capillary tube as an extrudable shape, is placed in the capillary tube to gel the acrylamide inside the capillary tube.

[0024] In the method shown in FIG. 2, a capillary tube (with an inner diameter of, for example, 0.1 to 3 mm (the upper limit of the inner diameter is preferably 2 mm, and more preferably 1 mm). It may be made of glass, metal, or synthetic resin) containing an acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which a crystallized protein is present is prepared. The capillary tube containing the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which a crystallized protein is present can be prepared by adding an acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which a crystallized protein is present to a solution containing a protein crystallized outside the capillary tube using, for example, a vapor diffusion method or a free interface diffusion method, to the capillary tube. The operation of introducing the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine, in which the crystallized protein is present, into the capillary tube can be performed, for example, by sucking the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine, in which the crystallized protein is present, into a syringe connected to one end of the capillary tube via a synthetic resin tube, or by using a micropipette.

[0025] It is essential that the operation of preparing the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which the crystallized protein is present and the operation of putting the prepared acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which the crystallized protein is present into the capillary tube are performed quickly. It is desirable that the time from the completion of preparation of the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine to the completion of putting the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which the crystallized protein is present into the capillary tube is within 1 minute. If it takes more than this time, the radical polymerization reaction of acrylamide may start before the completion of putting the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which the crystallized protein is present into the capillary tube, or the radicals generated from ammonium peroxodisulfate may react with oxygen in the air, making it difficult or impossible to gel acrylamide inside the capillary tube.

[0026] The acrylamide concentration in the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which the crystallized protein is present and placed in the capillary tube is preferably 1 to 10%. The ammonium peroxodisulfate concentration is preferably 0.1 to 1%. The tetramethylethylenediamine concentration is preferably 0.1 to 5%. If the concentrations of acrylamide, ammonium peroxodisulfate, or tetramethylethylenediamine are too low, the radical polymerization reaction of acrylamide may not be sufficiently initiated, while if the concentrations are too high, the protein crystals may be dissolved, or the heat generated by the rapid radical polymerization reaction may damage the protein crystals. In the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which the crystallized protein is present, the precipitant used in the crystallization of the protein is preferably contained at a concentration similar to that used in the crystallization of the protein, and the pH of the solution is preferably set to the same as that used in the crystallization of the protein, in order to prevent the protein crystals from dissolving. In order to complete the gelation of acrylamide, the reaction time is preferably 15 minutes or more.

[0027] Finally, the acrylamide gel containing the embedded protein crystals is extruded from the capillary tube in the same manner as described in Method 1.

[0028] Method 3: A method in which the acrylamide solution containing the crystallized protein being delivered and the ammonium peroxodisulfate or tetramethylethylenediamine solution being delivered are joined and delivered to an extrudable shaped object, thereby gelling the acrylamide inside the shaped object (however, when an ammonium peroxodisulfate solution is used, at least one of the acrylamide solution containing the crystallized protein and the ammonium peroxodisulfate solution contains tetramethylethylenediamine, and when a tetramethylethylenediamine solution is used, at least one of the acrylamide solution containing the crystallized protein and the tetramethylethylenediamine solution contains ammonium peroxodisulfate).

[0029] FIG. 3 is a schematic explanatory diagram (front view) of an example of Method 3, showing a method in which an acrylamide solution containing tetramethylethylenediamine in which crystallized protein is present and an ammonium peroxodisulfate solution are each sent to a T-shaped connector, joined together, and sent into a synthetic resin tube having an extrudable shape, thereby gelling the acrylamide inside the tube.

[0030] In the method shown in FIG. 3, a synthetic resin tube for feeding an acrylamide solution containing tetramethylethylenediamine in which a crystallized protein exists and a synthetic resin tube for feeding an ammonium peroxodisulfate solution are connected to the opposing connection ports of the T-shaped connector, and a synthetic resin tube as an extrudable shape is connected to the remaining connection port of the T-shaped connector. As the synthetic resin tubes to be connected to the three connection ports of the T-shaped connector, a silicon tube with an inner diameter of, for example, 0.1 to 3 mm (the upper limit of the inner diameter is preferably 2 mm, more preferably 1 mm) can be used. It is desirable that the synthetic resin tube for feeding an acrylamide solution containing tetramethylethylenediamine in which a crystallized protein exists and the synthetic resin tube for feeding an ammonium peroxodisulfate solution have the same inner diameter (to facilitate equal division and mixing).

[0031] The delivery of the acrylamide solution containing tetramethylethylenediamine in which the crystallized protein exists to the T-shaped connector can be performed, for example, by connecting a capillary tube (with an inner diameter of, for example, 0.1 to 3 mm (the upper limit of the inner diameter is preferably 2 mm, and more preferably 1 mm). It may be made of glass, metal, or synthetic resin) containing the acrylamide solution containing tetramethylethylenediamine in which the crystallized protein exists upstream of a synthetic resin tube connected to the connection port of the T-shaped connector, and further connecting a syringe filled with the acrylamide solution containing tetramethylethylenediamine via a synthetic resin tube upstream of the capillary tube, and pushing out the acrylamide solution containing tetramethylethylenediamine from the syringe. The capillary tube containing the acrylamide solution containing tetramethylethylenediamine in which the crystallized protein exists can be prepared by adding the acrylamide solution containing tetramethylethylenediamine to a solution containing the protein crystallized outside the capillary tube using, for example, a vapor diffusion method or a free interface diffusion method, and feeding the acrylamide solution containing tetramethylethylenediamine in which the crystallized protein exists into the capillary tube. The operation of introducing the tetramethylethylenediamine-containing acrylamide solution containing the crystallized protein into the capillary tube can be performed, for example, by sucking the tetramethylethylenediamine-containing acrylamide solution containing the crystallized protein into a syringe connected to one end of the capillary tube via a synthetic resin tube, or by using a micropipette.

[0032] The acrylamide concentration in the tetramethylethylenediamine-containing acrylamide solution in which the crystallized protein is present to be delivered is preferably 5 to 15%. The tetramethylethylenediamine concentration is preferably 0.1 to 5%. If the acrylamide or tetramethylethylenediamine concentration is too low, there is a risk that the radical polymerization reaction of acrylamide cannot be sufficiently caused inside the synthetic resin tube as an extrudable shape, while if the concentration is too high, there is a risk that the protein crystals will dissolve, or that the heat generated by the rapid radical polymerization reaction after merging with the ammonium peroxodisulfate solution will damage the protein crystals. In order to prevent the protein crystals from dissolving, it is preferable to include the precipitant used in the crystallization of the protein in the tetramethylethylenediamine-containing acrylamide solution in which the crystallized protein is present at a concentration similar to that used in the crystallization of the protein, and to set the pH of the solution to the same as that used in the crystallization of the protein.

[0033] The ammonium peroxodisulfate solution can be fed to the T-shaped connector by, for example, connecting a syringe filled with the ammonium peroxodisulfate solution to the upstream of a synthetic resin tube connected to the connection port of the T-shaped connector and pushing the ammonium peroxodisulfate solution out of the syringe. The concentration of ammonium peroxodisulfate in the ammonium peroxodisulfate solution to be fed is desirably 5 to 15%. If the concentration of ammonium peroxodisulfate is too low, there is a risk that the radical polymerization reaction of acrylamide cannot be sufficiently caused inside the synthetic resin tube as an extrudable shape, while if the concentration is too high, there is a risk that the protein crystals will dissolve, or that the heat generated by the sudden radical polymerization reaction after merging with the acrylamide solution containing tetramethylethylenediamine in which the crystallized protein is present will damage the protein crystals. It is desirable to contain the ammonium peroxodisulfate solution with the precipitant used in protein crystallization at a concentration similar to that used in protein crystallization, and to adjust the pH of the solution to be similar to that used in protein crystallization, in order to prevent the protein crystals from dissolving.

[0034] The extrusion of the acrylamide solution containing tetramethylethylenediamine from the syringe filled with the acrylamide solution containing tetramethylethylenediamine and the extrusion of the ammonium peroxodisulfate solution from the syringe filled with the ammonium peroxodisulfate solution may be performed manually or mechanically using a syringe pump or the like. The extrusion speeds of the respective solutions are the same, and are preferably 0.001 to 1 mL / min, more preferably 0.01 to 0.1 mL / min. If the extrusion is continued at a too high extrusion speed, there is a risk that the mixture of the acrylamide solution containing tetramethylethylenediamine containing the crystallized protein and the ammonium peroxodisulfate solution, which is fed into the synthetic resin tube as an extrudable shape, will be discharged from the tube before the acrylamide gels inside the tube even if the length of the tube is increased. If the extrusion is continued at the above extrusion speed, the gelation of the acrylamide can be completed inside the tube by setting the length of the tube to 10 to 50 cm, and the acrylamide gel in which the protein crystals are embedded can be discharged from the tube. If extrusion is continued at too slow an extrusion speed, the radical polymerization reaction of acrylamide may begin before the mixture of the acrylamide solution containing tetramethylethylenediamine in which the crystallized protein is present and the ammonium peroxodisulfate solution is sent into the synthetic resin tube in an extrudable shape, making it difficult or impossible to gel the acrylamide inside the tube.

[0035] The location where the acrylamide gel with embedded protein crystals is discharged from the synthetic resin tube as an extrudable object may be on a glass plate, in a precipitant solution, etc., where the discharged acrylamide gel with embedded protein crystals is easy to handle. When the acrylamide gel with embedded protein crystals is discharged onto a glass plate, it is desirable to occasionally drip the precipitant solution onto the acrylamide gel to prevent it from drying.

[0036] Although it is effective to add tetramethylethylenediamine to the acrylamide solution as in the method shown in Fig. 3 in that it facilitates the radical polymerization reaction of acrylamide, it may be added to the ammonium peroxodisulfate solution rather than to the acrylamide solution, or may be added to both the acrylamide solution and the ammonium peroxodisulfate solution.

[0037] In addition, instead of connecting a capillary tube containing an acrylamide solution containing tetramethylethylenediamine in which a crystallized protein is present, upstream of a synthetic resin tube connected to a connection port of a T-shaped connector, a capillary tube containing a solution containing a protein crystallized outside the capillary tube using a method such as vapor diffusion or free interface diffusion, or a capillary tube containing a solution containing a crystallized protein obtained by crystallizing a protein inside the capillary tube using a method such as counter diffusion may be connected in order to feed the acrylamide solution containing tetramethylethylenediamine in which a crystallized protein is present to a T-shaped connector. Regardless of which capillary tube is connected, the amount of solution inside the capillary tube is small, so it can be said that there is no hindrance to feeding the acrylamide solution containing tetramethylethylenediamine in which a crystallized protein is present.

[0038] Furthermore, the extrudable shape is not limited to a synthetic resin tube (it may be a glass or metal tube, etc.) as long as it is capable of ejecting the acrylamide gel containing the protein crystals embedded therein as its contents.

[0039] The acrylamide gel with embedded protein crystals obtained by the method of the present invention can be cut to an appropriate length to contain the protein crystals as necessary, and can be used in a variety of situations in various fields, including protein crystal structure analysis, including the life science field, where protein crystals are used. EXAMPLES

[0040] The present invention will be described in detail below with reference to examples, but the present invention should not be construed as being limited to the following description.

[0041] Example 1: Preparation of egg white lysozyme crystals embedded in acrylamide gel by method 1 (part 1) (1) In accordance with a known method, hen egg white lysozyme was crystallized on a cover glass by the vapor diffusion method (hanging drop method). Specifically, a cover glass on which a 2 μL drop of mother liquid prepared by mixing 1 μL of 10 mg / mL hen egg white lysozyme aqueous solution and 1 μL of precipitant solution was dropped was placed upside down on a general-purpose crystallization plate containing 1 mL of precipitant solution (1.5 M sodium chloride + 0.1 M sodium acetate, pH 4.5; the same applies below), and the cover glass was sealed with liquid paraffin and left to stand at room temperature to allow the precipitant solution and mother liquid in the general-purpose crystallization plate to diffuse between each other. After 24 hours, multiple hen egg white lysozyme crystals were visible to the naked eye on the cover glass. A 2 μL acrylamide solution containing ammonium peroxodisulfate (24% acrylamide-bisacrylamide (29:1) + 5.4% ammonium peroxodisulfate + 1.5 M sodium chloride + 0.1 M sodium acetate, pH 4.5) was dropped onto 2 μL of the mother liquor containing multiple hen egg white lysozyme crystals on the cover glass. The 4 μL acrylamide solution containing ammonium peroxodisulfate (hen egg white lysozyme crystals + 12% acrylamide-bisacrylamide (29:1) + 2.7% ammonium peroxodisulfate + 1.5 M sodium chloride + 0.1 M sodium acetate, pH 4.5) containing hen egg white lysozyme crystals was then introduced into a glass capillary tube (outer diameter 1 mm, inner diameter 0.6 mm, length 60 mm) by aspirating the solution with a syringe connected to one end of the capillary tube via a silicone tube (approximately 20 mm from the end of the capillary tube).

[0042] (2) The end of the capillary tube containing the acrylamide solution containing ammonium peroxodisulfate in which hen egg white lysozyme crystals exist was immersed in a petri dish filled with a tetramethylethylenediamine solution (20% tetramethylethylenediamine + 1.5M sodium chloride + 0.1M sodium acetate, pH 4.5) to cause a radical polymerization reaction of acrylamide inside the capillary tube. After 30 minutes, the end of the capillary tube was pulled out of the tetramethylethylenediamine solution, and a syringe filled with air was connected via a silicon tube to the end of the capillary tube opposite to the end containing the acrylamide solution containing ammonium peroxodisulfate in which hen egg white lysozyme crystals exist (the end immersed in the tetramethylethylenediamine solution). The air was manually pushed out of the syringe (pushing speed: about 1mL / min) to push the contents of the capillary tube onto a cover glass, and a continuous acrylamide gel in which hen egg white lysozyme crystals were dispersed and embedded was obtained on the cover glass.

[0043] Example 2: Preparation of egg white lysozyme crystals embedded in alginate hydrogel using method 1 (part 2) (1) According to a known method, hen egg white lysozyme was crystallized inside a capillary tube by the counter diffusion method. Specifically, a 15 mg / mL hen egg white lysozyme aqueous solution was first introduced into a glass capillary tube with an outer diameter of 1 mm, an inner diameter of 0.6 mm, and a length of 60 mm by sucking it with a syringe connected to one end of the capillary tube via a silicon tube (total content of the capillary tube). In addition, 1 mL of agarose gel (2% agarose + 1.5 M sodium chloride + 0.1 M sodium acetate, pH 4.5) was placed at the bottom of a 50 mL test tube with a lid, and 8 mL of precipitant solution was added on top of it. The lower end of the capillary tube containing the hen egg white lysozyme solution was pierced into the agarose gel at the bottom of the lidded test tube, the upper end of the capillary tube was sealed with aluminum foil, the test tube was closed, and the tube was left to stand at room temperature, allowing the hen egg white lysozyme solution in the capillary tube and the precipitant solution outside to diffuse between each other through the agarose gel. After 24 hours, multiple hen egg white lysozyme crystals adsorbed or attached to the inner wall of the capillary tube were visible to the naked eye. The capillary tube in which the hen egg white lysozyme had crystallized was removed from the lidded test tube, and a portion of the capillary tube with a length of about 30 mm was cut out, including the area where the hen egg white lysozyme crystals were present. A syringe filled with an appropriate amount (5-10 mL) of an acrylamide solution containing ammonium peroxodisulfate (12% acrylamide-bisacrylamide (29:1) + 1.8% ammonium peroxodisulfate + 1.5 M sodium chloride + 0.1 M sodium acetate, pH 4.5) was connected to one end of the excised capillary tube via a silicone tube, and the acrylamide solution containing ammonium peroxodisulfate was manually pushed out of the syringe (extrusion rate: approximately 1 mL / min) to replace the solution inside the capillary tube with the acrylamide solution containing ammonium peroxodisulfate. However, the hen egg white lysozyme crystals remained inside the capillary tube.

[0044] (2) One end of a capillary tube, the inside of which was filled with an acrylamide solution containing ammonium peroxodisulfate in which hen egg white lysozyme crystals exist, was immersed in a petri dish filled with a tetramethylethylenediamine solution (20% tetramethylethylenediamine + 1.5M sodium chloride + 0.1M sodium acetate, pH 4.5), to cause a radical polymerization reaction of acrylamide inside the capillary tube. After 30 minutes, the end of the capillary tube was pulled out of the tetramethylethylenediamine solution, and an air-filled syringe was connected to one end of the capillary tube via a silicon tube. The air was manually pushed out of the syringe (pushing speed: about 1mL / min) to push the contents of the capillary tube onto a cover glass, resulting in a continuous acrylamide gel on the cover glass in which hen egg white lysozyme crystals were dispersed and embedded. A stereomicroscope photograph of hen egg white lysozyme crystals embedded in the continuous acrylamide gel is shown in Figure 4 (scale bar: 1mm). When the obtained hen egg white lysozyme crystals embedded in acrylamide gel (cut into appropriate lengths from the continuum) were immersed in a methylene blue solution containing a precipitant, the hen egg white lysozyme crystals were stained blue in about 30 minutes, indicating that this embedment can be used for soaking hen egg white lysozyme crystals. When this embedment was immersed in pure water, the hen egg white lysozyme crystals dissolved and disappeared in about 30 minutes, and a cavity was formed in the acrylamide gel. Furthermore, when X-ray structural analysis of the hen egg white lysozyme crystals was performed using this embedment, the results matched the known structure, indicating that the structure of the hen egg white lysozyme crystals was not affected by embedding in acrylamide gel.

[0045] Example 3: Preparation of egg white lysozyme crystals embedded in acrylamide gel by method 2 (part 1) According to a known method, hen egg white lysozyme was crystallized on a cover glass by the vapor diffusion method (hanging drop method). Specifically, a cover glass on which a droplet of 4 μL of mother liquid prepared by mixing 2 μL of 10 mg / mL hen egg white lysozyme aqueous solution and 2 μL of precipitant solution was dropped was placed upside down on a general-purpose crystallization plate containing 1 mL of precipitant solution, and the cover glass was sealed with liquid paraffin and left to stand at room temperature to allow the precipitant solution and mother liquid in the general-purpose crystallization plate to diffuse between each other. After 24 hours, multiple hen egg white lysozyme crystals were visible to the naked eye on the cover glass. A mixture of 6 μL of acrylamide solution (12% acrylamide-bisacrylamide (29:1) + 0.91% ammonium peroxodisulfate + 1.5 M sodium chloride + 0.1 M sodium acetate, pH 4.5) and 2 μL of tetramethylethylenediamine solution (10% tetramethylethylenediamine + 1.5 M sodium chloride + 0.1 M sodium acetate, pH 4.5) containing ammonium peroxodisulfate and tetramethylethylenediamine was added dropwise to 4 μL of mother liquor containing multiple hen egg white lysozyme crystals on a cover glass. A 12μL solution of acrylamide containing diamine (hen egg white lysozyme crystals + 6% acrylamide-bisacrylamide (29:1) + 0.46% ammonium peroxodisulfate + 1.7% tetramethylethylenediamine + 1.5M sodium chloride + 0.1M sodium acetate, pH 4.5) was introduced into a glass capillary tube with an outer diameter of 1mm, inner diameter of 0.6mm, and length of 60mm by sucking it with a syringe connected to one end of the capillary tube through a silicone tube (integration of the entire contents of the capillary tube), and a radical polymerization reaction of acrylamide was caused inside the capillary tube. The operation of introducing the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine, in which hen egg white lysozyme crystals were present, into the capillary tube was performed within 10 seconds after the completion of the preparation of the acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine.After 30 minutes, an air-filled syringe was connected to one end of the capillary tube via a silicon tube, and the contents of the capillary tube were pushed out onto the cover glass by manually pushing the air out of the syringe (extrusion rate: approximately 1 mL / min), yielding a continuous acrylamide gel on the cover glass with hen egg white lysozyme crystals dispersed and embedded therein. A stereomicroscope photograph of hen egg white lysozyme crystals embedded in the acrylamide gel is shown in Figure 5 (scale bar: 1 mm).

[0046] Example 4: Preparation of egg white lysozyme crystals embedded in acrylamide gel by method 2 (part 2) A continuous acrylamide gel in which hen egg white lysozyme crystals were dispersed and embedded was obtained on a cover glass in the same manner as in Example 3, except that an acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in which hen egg white lysozyme crystals were present was placed in a glass capillary tube having an outer diameter of 1.5 mm, an inner diameter of 0.9 mm, and a length of 90 mm.

[0047] Example 5: Preparation of egg white lysozyme crystals embedded in acrylamide gel by method 3 A silicon tube with an outer diameter of 2 mm and an inner diameter of 1 mm was connected to each of three ports of a polypropylene T-shaped connector with an outer diameter of 1.5 mm at the tip. An acrylamide solution containing tetramethylethylenediamine in which hen egg white lysozyme crystals existed (hen egg white lysozyme crystals + 12% acrylamide-bisacrylamide (29:1) + 1% tetramethylethylenediamine + 1.5 M sodium chloride + 0.05 M sodium acetate, pH 4.5) was delivered to one of the opposing ports. An ammonium peroxodisulfate solution (10% ammonium peroxodisulfate + 1.5 M sodium chloride + 0.05 M sodium acetate, pH 4.5) was delivered to the other port. The two solutions were then joined and sent to a 20 cm long silicon tube connected to the remaining port. The delivery of the acrylamide solution containing tetramethylethylenediamine in which hen egg white lysozyme crystals were present was performed by connecting a capillary tube containing the acrylamide solution containing tetramethylethylenediamine in which hen egg white lysozyme crystals were present upstream of a silicone tube connected to the connection port of the T-connector, and connecting a syringe filled with an acrylamide solution containing tetramethylethylenediamine (12% acrylamide-bisacrylamide (29:1) + 1% tetramethylethylenediamine + 1.5 M sodium chloride + 0.05 M sodium acetate, pH 4.5) upstream of that via a silicone tube, and pushing the acrylamide solution containing tetramethylethylenediamine out of the syringe.The capillary tube containing the acrylamide solution containing tetramethylethylenediamine in which hen egg white lysozyme crystals exist was prepared by dropping 2 μL of an acrylamide solution containing tetramethylethylenediamine (24% acrylamide-bisacrylamide (29:1) + 2% tetramethylethylenediamine + 1.5 M sodium chloride + 0.05 M sodium acetate, pH 4.5) into 2 μL of the mother liquor containing hen egg white lysozyme crystals in accordance with the method described in Example 1 (1). A 4 μL solution of tetramethylethylenediamine-containing acrylamide (hen egg white lysozyme crystals + 12% acrylamide-bisacrylamide (29:1) + 1% tetramethylethylenediamine + 1.5 M sodium chloride + 0.05 M sodium acetate, pH 4.5) containing lysozyme crystals was prepared by aspirating 4 μL of the solution into a glass capillary tube (1 mm outer diameter x 0.6 mm inner diameter x 20 mm length) with a syringe connected to one end of the capillary tube via a silicon tube. Ammonium peroxodisulfate solution was delivered by connecting a syringe filled with ammonium peroxodisulfate solution (10% ammonium peroxodisulfate + 1.5 M sodium chloride + 0.05 M sodium acetate, pH 4.5) to the upstream of the silicon tube connected to the connection port of the T-shaped connector, and pushing the ammonium peroxodisulfate solution out of the syringe. The delivery of the acrylamide solution containing tetramethylethylenediamine in which hen egg white lysozyme crystals existed and the delivery of the ammonium peroxodisulfate solution were performed by continuously extruding the acrylamide solution containing tetramethylethylenediamine from a syringe filled with the acrylamide solution containing tetramethylethylenediamine and the ammonium peroxodisulfate solution from a syringe filled with the ammonium peroxodisulfate solution at an extrusion rate of 0.03 mL / min using a syringe pump. As a result, the acrylamide gelled inside the 20 cm long tube, and a continuous acrylamide gel in which hen egg white lysozyme crystals were dispersed and embedded was discharged from the tube (the discharged location was on the cover glass). A stereomicroscope photograph of the hen egg white lysozyme crystals embedded in the continuous acrylamide gel is shown in Figure 6 (scale bar: 500 μm). [Industrial Applicability]

[0048] The present invention has industrial applicability in that it provides a versatile method for producing hydrogel-embedded protein crystals that can be applied to any protein that can be crystallized.

Claims

1. A method for producing a hydrogel-embedded protein crystal, comprising: gelling acrylamide through a radical polymerization reaction inside an extrudable shaped object containing an acrylamide solution containing crystallized protein; and then extruding the contents of the shaped object to obtain an acrylamide gel in which protein crystals are embedded.

2. 2. The method according to claim 1, wherein acrylamide is gelled by reacting it with ammonium peroxodisulfate as a polymerization initiator and tetramethylethylenediamine as a polymerization promoter.

3. The method according to claim 2, wherein the acrylamide solution containing the crystallized protein inside the extrudable shape is contacted with an ammonium peroxodisulfate solution or a tetramethylethylenediamine solution outside the shape, thereby gelling the acrylamide inside the shape (provided that when an ammonium peroxodisulfate solution is used, at least one of the acrylamide solution containing the crystallized protein and the ammonium peroxodisulfate solution contains tetramethylethylenediamine, and when a tetramethylethylenediamine solution is used, at least one of the acrylamide solution containing the crystallized protein and the tetramethylethylenediamine solution contains ammonium peroxodisulfate).

4. 3. The method of claim 2, wherein an acrylamide solution containing ammonium peroxodisulfate and tetramethylethylenediamine in the presence of the crystallized protein, prepared outside the extrudable shape, is poured into the shape to gel the acrylamide inside the shape.

5. The method according to claim 2, in which the acrylamide solution containing the crystallized protein being delivered and the ammonium peroxodisulfate solution or tetramethylethylenediamine solution being delivered are joined and delivered into an extrudable shaped object, thereby gelling the acrylamide inside the shaped object (however, when an ammonium peroxodisulfate solution is used, at least one of the acrylamide solution containing the crystallized protein and the ammonium peroxodisulfate solution contains tetramethylethylenediamine, and when a tetramethylethylenediamine solution is used, at least one of the acrylamide solution containing the crystallized protein and the tetramethylethylenediamine solution contains ammonium peroxodisulfate).

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