Production of protein crystal materials

Abstract

To provide a method for producing protein crystals capable of analyzing three-dimensional structures in a short time with little effort.SOLUTION: A method for producing protein crystals, which comprises a step of incubating a crystallization solution containing a cell-free protein synthesis system, a crystallization control agent, and a nucleic acid encoding the protein, is employed.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the production of protein crystal materials. [Background technology]

[0002] In order to analyze the three-dimensional structure of a protein, it is necessary to prepare a large amount of the target protein and form crystals from the obtained protein. Conventionally, a method has been used in which a gene encoding the target protein is introduced into E. coli or cultured cells to express the target protein.

[0003] However, there is a limit to the type and expression amount of a target protein in living cells, so a method of expressing a large amount of any target protein using a cell-free protein synthesis system has been adopted (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2018 / 024428 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in order to obtain crystals of a target protein using the method described in Patent Document 1, it is necessary to purify the target protein expressed in a cell-free protein synthesis system and then maintain the target protein solution under appropriate conditions to crystallize it.

[0006] Such a crystallization process requires a lot of time and effort. In addition, the crystals obtained by the method described in Patent Document 1 may not be large enough for three-dimensional structure analysis or may not have sufficient periodicity. Therefore, it may not be possible to analyze the three-dimensional structure of the obtained crystals.

[0007] Therefore, an object of the present invention is to provide a method for producing protein crystals that enable analysis of their three-dimensional structure in a short time and with little effort. [Means for solving the problem]

[0008] The present invention includes the following aspects. A first aspect of the present invention is a method for producing protein crystals, which comprises a step of incubating a crystallization solution containing a cell-free protein synthesis system, a crystallization controlling agent, and a nucleic acid encoding the protein.

[0009] In the first aspect, the crystallization control agent preferably contains a compound that suppresses the formation of twin crystals of the protein.

[0010] In the first aspect, the crystallization control agent preferably contains a compound that promotes the formation of the crystals.

[0011] In the first aspect, the crystallization control agent preferably contains a compound that improves the resolution of the crystal in three-dimensional structural analysis.

[0012] In the first aspect, the crystal is preferably a crystal amenable to X-ray crystal structure analysis.

[0013] In the first aspect, in the step, the crystallization solution is contained in one storage space of a container divided by a dialysis membrane, and a dialysis external solution containing an amino acid is contained in the other storage space, and it is preferable that the crystallization solution contained in the one storage space and the dialysis external solution contained in the other storage space come into contact with the dialysis membrane, and the amino acid is capable of moving from the dialysis external solution to the crystallization solution through pores in the dialysis membrane.

[0014] In the first aspect, it is preferable that a plurality of the crystallization solutions are contained in the one of the containing spaces.

[0015] Alternatively, in the first aspect, in the step, it is preferable to form a bilayer by contacting the crystallization solution with a substrate solution containing an amino acid.

[0016] A second aspect of the present invention is a protein crystal manufacturing apparatus comprising a container having a first storage space and a second storage space, and a dialysis membrane facing both the first storage space and the second storage space, wherein the first storage space contains a crystallization solution containing a cell-free protein synthesis system and a nucleic acid encoding a protein, and the second storage space contains a dialysis external solution containing amino acids, the dialysis membrane divides the internal space of the container into the first storage space and the second storage space, and is in contact with both the crystallization solution contained in the first storage space and the dialysis external solution contained in the second storage space, and the amino acids are capable of moving from the dialysis external solution to the crystallization solution via pores in the dialysis membrane.

[0017] In the second aspect, it is preferable that a plurality of the crystallization solutions are accommodated in the first accommodation space.

[0018] In the second aspect, it is preferable that the first housing space is housed in the container by being supported by a support member.

[0019] In the second aspect, it is preferable that the first accommodating space is positioned with respect to the support member by a positioning portion.

[0020] A third aspect of the present invention is a kit for cell-free crystal production, comprising a nucleic acid encoding a protein, a crystallization controlling agent, and a reagent for a cell-free protein synthesis system.

[0021] The third aspect preferably further includes the manufacturing apparatus according to the second aspect. Effect of the Invention

[0022] According to the present invention, a method for producing protein crystals capable of analyzing their three-dimensional structure can be provided in a short time and with little labor. [Brief description of the drawings]

[0023] [Figure 1] 1A to 1C are diagrams illustrating a manufacturing method according to the present embodiment. [Diagram 2] 1A to 1C are diagrams illustrating a manufacturing method according to the present embodiment. [Diagram 3] FIG. 1 is a diagram showing a schematic diagram of a manufacturing apparatus 1A according to the present embodiment. [Figure 4] FIG. 2 is a diagram showing a schematic diagram of a manufacturing apparatus 1B according to the present embodiment. [Diagram 5] FIG. 13 is a diagram illustrating a modified example of the manufacturing apparatus according to the present embodiment. [Figure 6] FIG. 2 is a schematic diagram of a production apparatus used in Experimental Example 1. [Figure 7] 1 shows an electrophoretic image of CipA protein subjected to SDS-PAGE and stained with CBB in Experimental Example 1. [Figure 8] 1 is a photograph of CipA crystals obtained using a crystallization inhibitor in Experimental Example 1. [Figure 9A] 1 is an SEM image of CipB crystals obtained in Experimental Example 2 without adding a crystallization inhibitor. [Figure 9B] 1 is an SEM image of CipB crystals obtained using a crystallization inhibitor in Experimental Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] [Manufacturing method] In one aspect, the present invention provides a method for producing protein crystals, comprising the step of incubating a crystallization solution containing a cell-free protein synthesis system, a crystallization control agent, and a nucleic acid encoding the protein. In the production method according to the present embodiment, the protein to be crystallized is expressed from nucleic acid in a crystallization solution in the presence of a crystallization controlling agent by a cell-free protein synthesis system, and protein crystals are formed.

[0025] (Protein crystals) In the production method according to this embodiment, the protein to be crystallized is not particularly limited as long as the effects of the present invention are achieved. The protein to be crystallized may be, for example, a crystalline protein or a protein other than a crystalline protein. The protein other than a crystalline protein is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include soluble proteins, membrane proteins, etc.

[0026] As used herein, a crystalline protein refers to a protein that can spontaneously self-assemble to form a crystal under physiological conditions, such as in the cytoplasm, and a protein crystal refers to a crystal formed from the above-mentioned crystalline protein.

[0027] The crystalline protein is not particularly limited. Even if a protein does not normally form crystals, if it can form crystals in a crystallization solution by techniques such as chemical modification of the protein, creation of a mutant, or fusion protein, the protein is also included in the crystalline protein of the present specification.

[0028] Examples of the crystalline protein include any of the proteins described in (A) to (D) below. (A) Cytoplasmic polyhedrin protein, nuclear polyhedrin protein, insecticidal protein, cathepsin B, luciferase, reovirus nonstructural protein (μNS), Crystalline Inclusion Protein A (CipA), Crystalline Inclusion Protein B (CipB), or Fusolin protein (B) A protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the protein (A) and having crystal-forming ability. (C) A fusion protein of the protein (A) or (B) with a target peptide. (D) A fusion protein of the protein (A) or (B) and a target protein.

[0029] The cytoplasmic polyhedrin protein is a polyhedrin protein derived from a cytoplasmic polyhedrosis virus (Cypovirus, CPV), which is a pathogenic virus that causes cytoplasmic polyhedrosis. For example, the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 is a wild-type polyhedrin protein expressed by a cytoplasmic polyhedrosis virus that infects silkworm moths. Furthermore, the nuclear polyhedrin protein is a polyhedrin protein derived from a nuclear polyhedrosis virus (Nucleopolyhedrovirus, NPV), which is a pathogenic virus of nuclear polyhedrosis. For example, the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 is a wild-type polyhedrin protein expressed by a nuclear polyhedrosis virus that infects Autographa californica.

[0030] The insecticidal protein is an insecticidal protein produced by Bacillus thuringiensis. SEQ ID NO: 3 shows the amino acid sequence of the Cry3A protein produced by Bacillus thuringiensis.

[0031] Cathepsin B is a protease that has endopeptidase and exopeptidase activities. Cathepsin B is a protein that forms crystals in cultured insect cells. SEQ ID NO: 4 shows the amino acid sequence of cathepsin B derived from Trypanosoma brucei.

[0032] Luciferase is a general term for enzymes that catalyze the chemical reactions that cause luminescent substances to emit light in bioluminescence, such as in luminous bacteria and fireflies. Luciferase is a protein that forms crystals in insect cells. SEQ ID NO:5 shows the amino acid sequence of firefly luciferase, and SEQ ID NO:6 shows the amino acid sequence of Renilla luciferase.

[0033] μNS is a crystalline nonstructural protein of reovirus. SEQ ID NO:7 shows the amino acid sequence of μNS from reovirus.

[0034] Fusolin is a constituent protein of crystalline protein inclusion bodies formed by entomopoxviruses in host cells. SEQ ID NO: 8 shows the amino acid sequence of entomopoxvirus-derived Fusolin.

[0035] Crystalline inclusion protein A (CipA) is a constituent protein of the crystal protein inclusion body of the genus Photorhabdus. SEQ ID NO: 9 shows the amino acid sequence of CipA derived from Photorhabdus laumondii.

[0036] Crystalline inclusion protein B (CipB) is a constituent protein of the crystal protein inclusion body of the genus Photorhabdus. SEQ ID NO: 10 shows the amino acid sequence of CipB derived from Photorhabdus laumondii.

[0037] In this specification, one or more means, for example, 1 to 50, for example, 1 to 35, for example, 1 to 20, for example, 1 to 15, for example, 1 to 10, for example, 1 to 5.

[0038] As long as the crystalline protein has the ability to form crystals, it may be a mutant crystalline protein having a mutation in the above-mentioned cytoplasmic polyhedrin protein, nuclear polyhedrin protein, insecticidal protein, cathepsin B, luciferase, μNS, CipA, Fusolin, etc. The mutant crystalline protein may be, for example, a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence set forth in the above-mentioned SEQ ID NOs: 1 to 10.

[0039] The crystalline protein may be a fusion of the above-mentioned wild-type crystalline protein or mutant crystalline protein with a target peptide.

[0040] Here, the target peptide may be, for example, a peptide whose three-dimensional structure is to be analyzed. As described later, the crystalline protein, which is a fusion protein of the above-mentioned crystalline protein and the target peptide, is expressed in a crystallization solution to form a crystal, and the crystal of the crystalline protein is subjected to X-ray crystal structure analysis, whereby the three-dimensional structure of the target peptide can be easily analyzed.

[0041] In this case, the target peptide may be any peptide for which there is a need to analyze the three-dimensional structure. The length of the amino acids in the target peptide is preferably, for example, about 5 to 50 amino acids, from the viewpoint of enabling the crystalline protein to maintain its crystal-forming ability.

[0042] The crystalline protein may be a fusion of the above-mentioned wild-type crystalline protein or mutant crystalline protein with a target protein.

[0043] Here, the target protein may be, for example, a crystalline protein or a protein other than a crystalline protein. The protein other than a crystalline protein is not particularly limited as long as the effect of the present invention is exhibited, and examples thereof include soluble proteins, membrane proteins, etc. The target protein may be a wild-type target protein, or a mutant target protein having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of the wild-type target protein.

[0044] The crystalline protein, which is a fusion protein between the above-mentioned crystalline protein and a target protein, is expressed in a crystallization solution to form crystals, and the crystals of the crystalline protein are subjected to X-ray crystal structure analysis, thereby making it possible to easily analyze the three-dimensional structure of the target protein.

[0045] In the production method of this embodiment, a crystallization solution containing a nucleic acid encoding a protein is incubated.

[0046] (nucleic acid) In the production method according to this embodiment, the nucleic acid encoding the protein may be DNA or RNA. The base sequence of the nucleic acid encoding the protein may be one that has synonymous substitution so that the expression of the protein is optimized in a crystallization solution described below.

[0047] In the production method according to this embodiment, the number of proteins expressed in the same crystallization solution may be one or more than one. In the production method according to this embodiment, the number of nucleic acids expressed in the crystallization solution may be one or more than one.

[0048] The DNA sequence may have a promoter for binding of RNA polymerase upstream of the protein-encoding sequence. The promoter may be one that has activity in a protein synthesis system, or may be an expression-inducible promoter whose activity can be induced by a drug or the like. The promoter is not particularly limited, and examples thereof include T3, T7, SP6 promoter, cytomegalovirus promoter (CMV promoter), CMV early enhancer / chicken beta actin (CAG promoter), and the like. An example of an expression-inducible promoter is a doxycycline-inducible promoter (TetO promoter), whose promoter activity can be artificially controlled, and the like.

[0049] The above-mentioned RNA may have a ribosome binding site upstream of the protein-encoding sequence. Examples of the ribosome binding site include, but are not limited to, the Shine-Dalgarno sequence, the Kozak sequence, and the internal ribosome entry site (IRES), and any sequence known to those skilled in the art may be used.

[0050] In the production method according to this embodiment, the protein encoded by the nucleic acid in the crystallization solution is expressed, and the crystallization solution is incubated for a predetermined period of time until the expressed protein completes the formation of crystals. Here, the predetermined time may be 10 minutes or more, 30 minutes or more, 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, 32 hours or more, or 48 hours or more. The predetermined time may be 64 hours or less, 48 ​​hours or less, 32 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, 1 hour or less, or 30 minutes or less. The upper and lower limits of the predetermined time can be selected arbitrarily.

[0051] In the production method according to this embodiment, the temperature of the crystallization solution may be, for example, 3° C. or more, 4° C. or more, 10° C. or more, 15° C. or more, or 20° C. or more. The temperature may be, for example, 35° C. or less, 30° C. or less, 25° C. or less, 20° C. or less, 15° C. or less, 10° C. or less, or 5° C. or less. The upper and lower limits of the temperature can be selected arbitrarily.

[0052] When the crystallization solution is incubated for a predetermined period of time, protein crystals are formed in the crystallization solution. The formed crystals can be easily collected by a procedure such as centrifugation.

[0053] (Cell-free protein synthesis system) In the production method according to this embodiment, the cell-free protein synthesis system refers to a system in which proteins are not synthesized within cells, but rather proteins are synthesized in vitro from template nucleic acids using ribosomes, transcription / translation factors, and the like that are derived from living cells or artificially synthesized.

[0054] In the production method according to this embodiment, the cell-free protein synthesis system may be a synthesis system including a transcription process in addition to a translation process. In the production method according to this embodiment, when the nucleic acid encoding the protein is DNA, it is necessary to synthesize RNA encoding the protein by transcribing the DNA.

[0055] The production method according to this embodiment may include a step of transcribing DNA. That is, in the production method according to this embodiment, the cell-free protein synthesis system may include a factor that enables transcription. Examples of factors that enable transcription include, but are not limited to, RNA polymerase, nucleotides, and the like, and factors known to those skilled in the art may be used.

[0056] Alternatively, the production method according to this embodiment may not include the process of transcribing RNA from the above-mentioned DNA. That is, RNA may be synthesized in advance using a DNA encoding a protein as a template, and the RNA may be added to a cell-free protein synthesis system. Alternatively, RNA encoding a protein that is artificially chemically synthesized may be used instead of using a DNA encoding a protein.

[0057] The cell-free protein synthesis system is not particularly limited, and examples thereof include synthesis systems using cell extracts obtained from wheat germ, yeast, insect cells, cultured mammalian cells, rabbit reticulocytes, Escherichia coli, etc.; synthesis systems in which factors necessary for translation are reconstituted, etc. In the production method according to this embodiment, the cell-free protein synthesis system is not limited to the above-mentioned method, and known methods can be used (for example, see JP 2019-083825 A).

[0058] The cell extract used in the cell-free protein synthesis system may contain factors involved in translation, such as tRNA, aminoacylated tRNA synthetase, translation initiation factors, translation elongation factors, and translation termination factors.

[0059] The efficiency of protein synthesis may be increased by continuously adding, for example, amino acids and energy molecules (ATP, GTP) to the cell extract. In addition, for example, energy regeneration systems, salts, creatine phosphate, enzymes, etc. may be added to the cell extract as necessary.

[0060] The synthesis system in which factors necessary for translation are reconstituted is not particularly limited, and for example, a known method can be used. (For example, Shimizu, Y. et al., Cell-free translation reconstituted with purified components, Nature Biotech., 19, 751-755, 2001). The reconstituted synthesis system may contain, for example, a translation initiation factor, a translation elongation factor, a termination factor, an aminoacyl-tRNA synthetase, a methionyl-tRNA formyltransferase, etc.

[0061] By adding a target substance to a cell-free protein synthesis system, the target substance can be incorporated into protein crystals. For example, the target substance can be encapsulated in polyhedra.

[0062] Furthermore, for example, a non-natural amino acid, a radioactively labeled amino acid, or the like can be added to a cell-free protein synthesis system, whereby the non-natural amino acid or the radioactively labeled amino acid can be incorporated into a protein.

[0063] Furthermore, compounds that are harmful or toxic to cells can also be added to the cell-free protein synthesis system.

[0064] Furthermore, by using a cell-free protein synthesis system, it is possible to synthesize a large amount of protein. As described above, for example, by adding a factor that enhances the efficiency of protein synthesis to the cell-free protein synthesis system, it is possible to synthesize a large amount of protein.

[0065] For example, the following operations may be performed using the cell-free protein synthesis system. As described above, factors that regulate translation may be added to the cell-free protein synthesis system as needed. In addition, the pH, salt concentration, etc. of the synthesis system may be changed as needed.

[0066] Furthermore, the cell-free protein synthesis system allows the amount of synthesis system to be freely set. The upper limit of the reaction solution volume in the cell-free protein synthesis system is not particularly limited, and can be a volume that can form protein crystals. The reaction solution volume can be, for example, 5 mL or less, 1 mL or less, 500 μL or less, 200 μL or less, 100 μL or less, 50 μL or less, 30 μL or less, or 20 μL or less.

[0067] When synthesizing a protein within a cell, it is necessary to synthesize the protein at a temperature and in an environment suitable for the proliferation and growth of the cell. On the other hand, when synthesizing a protein in a cell-free protein synthesis system, the temperature, environment, etc. can be appropriately set. For example, by synthesizing a protein using a cell-free protein synthesis system at a temperature lower than the general culture temperature of cells, the quality of the obtained crystals can be improved.

[0068] (Crystallization control agent) In the production method according to this embodiment, the crystallization solution contains a crystallization control agent. The crystallization control agent may be one that suppresses the formation of twin crystals of the protein to be crystallized. Alternatively, the crystallization control agent may be one that promotes the formation of crystals of the protein to be crystallized. Alternatively, the crystallization control agent may be one that improves the resolution in the three-dimensional structure analysis of the crystals of the protein to be crystallized. An example of the three-dimensional structure analysis is X-ray structure analysis.

[0069] The crystallization control agent is not particularly limited as long as it exerts the effects of the present invention, and examples thereof include alcohols, organic solvents, water-soluble cyclic compounds, polyalkylene glycols, amine-polyoxyalkylene adducts, water-soluble polysaccharides, cyclophanes, metal ions, metal compounds, heavy atom compounds, lipid molecules, and salts.

[0070] The alcohol is not particularly limited as long as it exerts the effects of the present invention. Examples of the alcohol include ethanol, methanol, and propanol, with ethanol being preferred. The organic solvent is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include acetone, chloroform, and the like.

[0071] The water-soluble cyclic compound is not particularly limited as long as it exerts the effects of the present invention. Examples thereof include dioxane, cucurbituril, crown ether, cyclodextrin, etc., with dioxane being preferred.

[0072] The polyalkylene glycols and amine polyoxyalkylene adducts are not particularly limited as long as they achieve the effects of the present invention, and examples thereof include the following: Examples of alkylene oxides from which the polyalkylene glycols and amine polyoxyalkylene adducts are derived include ethylene oxide, propylene oxide, butylene oxide, and the like.

[0073] Examples of polyalkylene glycols include polyalkylene glycols, polyoxyalkylene glyceryl ethers, polyoxyalkylene acetylene glycols, polyoxyalkylene alkyl ethers, polyoxyalkylene esters, polyoxyalkylene alkylamides, aliphatic alcohol polyoxyalkylene adducts, aromatic alcohol polyoxyalkylene adducts, and polyhydric alcohol polyoxyalkylene adducts. Polyalkylene glycols are preferred, and triethylene glycol or polyethylene glycol is more preferred.

[0074] Examples of the amine polyoxyalkylene adducts include terminal diamine polyalkylene glycols, aliphatic amine polyoxyalkylene adducts, aromatic amine polyoxyalkylene adducts, and polyvalent amine polyoxyalkylene adducts.

[0075] The water-soluble polysaccharide is not particularly limited as long as it exerts the effects of the present invention. Examples of the water-soluble polysaccharide include dextrin, agarose, and ficoll, with dextrin or ficoll being preferred. The cyclophane is not particularly limited as long as it exerts the effects of the present invention, and examples thereof include pillararenes, porphyrin, and calixarene. Alternatively, examples of the crystallization control agent include polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, and polyalkyleneimine.

[0076] By using polymers such as polyalkylene glycols, amine polyoxyalkylene adducts, and water-soluble polysaccharides as crystallization control agents, it is possible to improve the resolution of crystals of the target protein in three-dimensional structure analysis.

[0077] Examples of the metal ion and the metal ion contained in the metal compound include magnesium, calcium, potassium, iron, copper, nickel, zinc, and the like. By interacting with metal ions, the protein to be crystallized becomes easier to obtain, and the three-dimensional structure of the crystals obtained becomes easier to analyze.

[0078] The heavy atom compound may be, for example, a complex containing a heavy atom, such as a gold atom, a platinum atom, a mercury atom, a uranium atom, an osmium atom, or a lanthanoid atom. By interacting with the heavy atom compound, the protein to be crystallized becomes easier to obtain, and the three-dimensional structure of the crystals obtained becomes easier to analyze.

[0079] The lipid molecule may be, for example, a lipid molecule constituting a cell membrane, and examples of the lipid molecule include cholesterol, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, monoacylglycerol such as monoolein, and the like. The inclusion of lipid molecules in the crystallization solution makes it easier to crystallize membrane proteins. When the crystallization solution contains monoacylglycerol, cholesterol, etc., it becomes easier to obtain crystals of membrane proteins using the lipid cubic phase method (LCP method).

[0080] Examples of salts include sodium chloride, potassium chloride, and ammonium sulfate. The inclusion of salts in the crystallization solution makes it easier to crystallize the protein.

[0081] The crystallization regulator may be used alone or in combination of two or more. As the crystallization control agent, among the above, one or more selected from the group consisting of alcohols, water-soluble cyclic compounds, polyalkylene glycols, amine-polyoxyalkylene adducts, water-soluble polysaccharides, cyclophanes, metal ions, heavy atom compounds, lipid molecules, and salts are preferred, and one or more selected from the group consisting of alcohols, water-soluble cyclic compounds, polyalkylene glycols, and water-soluble polysaccharides are more preferred.

[0082] More specifically, examples of the embodiment to which the manufacturing method according to the present aspect is applied include the following.

[0083] (First embodiment) In the production method according to the first embodiment, the above-mentioned crystalline protein may be one selected from the group consisting of the following (a1) to (a10):

[0084] (a1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted or added, and having the ability to form polyhedra. (a2) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and having the ability to form polyhedra.

[0085] (a3) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 in which one or more amino acids have been deleted, substituted or added, and having crystal-forming ability. (a4) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one or more amino acids have been deleted, substituted or added, and having crystal-forming ability.

[0086] (a5) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 5, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which one or more amino acids have been deleted, substituted or added, and having crystal-forming ability. (a6) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 in which one or more amino acids have been deleted, substituted or added, and having crystal-forming ability.

[0087] (a7) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 7, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 7 in which one or more amino acids have been deleted, substituted or added, and having crystal-forming ability. (a8) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 8, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8 in which one or more amino acids have been deleted, substituted or added, and having crystal-forming ability. (a9) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 9, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 9 in which one or more amino acids have been deleted, substituted or added, and having crystal-forming ability. (a10) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 10, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 10 in which one or more amino acids have been deleted, substituted or added, and having crystal-forming ability.

[0088] Second embodiment In the production method according to the second embodiment, the above-mentioned crystalline protein may be one or more selected from the group consisting of the following (b1) to (b2): In the production method according to the second embodiment, the crystallization solution contains a nucleic acid encoding the following protein:

[0089] (b1) A fusion protein having polyhedron-forming ability, in which the 67th to 104th amino acids of the amino acid sequence set forth in SEQ ID NO: 1 are replaced by a target peptide, or a fusion protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added, and having polyhedron-forming ability.

[0090] (b2) A fusion protein having polyhedron-forming ability, in which the 70th to 77th amino acids in the amino acid sequence set forth in SEQ ID NO: 1 are replaced by a target peptide, or a fusion protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added, and having polyhedron-forming ability.

[0091] The number of amino acids in the amino acid sequence of the target peptide may be, for example, 5 to 25 residues, 7 to 23 residues, or 9 to 18 residues.

[0092] According to the production method of the second embodiment, the crystal structure of the polyhedron obtained by crystallizing the fusion protein can be analyzed to easily analyze the crystal structure of the target peptide. In addition, the size of the polyhedron crystals can be controlled by appropriately designing the amino acid sequence of the target peptide.

[0093] Third embodiment In the production method according to the third embodiment, the crystallization solution may contain a nucleic acid encoding the following (c2), in addition to a nucleic acid encoding the following (c1). (c1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted or added, and having the ability to form polyhedra. (c2) A fusion protein having a target peptide bound to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence set forth in SEQ ID NO: in which one or more amino acids have been deleted, substituted or added.

[0094] The amino acid sequence set forth in SEQ ID NO:11 is the H1 α-helical region of the wild-type cytoplasmic polyhedrin protein. In this embodiment, when a fusion protein and a protein capable of forming polyhedra are expressed in a cell-free protein synthesis system, these proteins form polyhedra, and the fusion protein can form part of the polyhedra.

[0095] When the target peptide bound to the C-terminus of the fusion protein binds and the protein capable of forming polyhedra forms polyhedra, the target peptide is encapsulated in the polyhedra. For example, when the target peptide is GFP, the polyhedra can encapsulate GFP (for example, JP 2015-159731 A).

[0096] The sequence of the target peptide is not particularly limited. The number of residues in the amino acid sequence of the target peptide is preferably 3 to 500 residues, more preferably 3 to 400 residues, and even more preferably 3 to 300 residues.

[0097] (Fourth embodiment) In the production method according to the fourth embodiment, the crystallization solution may contain a nucleic acid encoding a modified polyhedrin protein having at least a portion of an amino acid sequence deleted and having the ability to form polyhedra, which is a protein (d1) or (d2) described below. (d1) Wild-type polyhedrin protein (d2) A protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of a wild-type polyhedrin protein and having the ability to form polyhedra.

[0098] In the production method according to the fourth embodiment, the wild-type polyhedrin protein may be a wild-type polyhedrin protein expressed by CPV or a wild-type polyhedrin protein expressed by NPV. More specifically, the wild-type polyhedrin protein may be, for example, a protein consisting of the amino acid sequence set forth in SEQ ID NO:1, or a protein consisting of the amino acid sequence set forth in SEQ ID NO:2.

[0099] In the production method according to the present embodiment, the polyhedra in which the modified polyhedrin protein is crystallized have internal spaces. This makes it easy to encapsulate larger molecules that have been difficult to encapsulate in the past. In the present specification, the term "peptide" encompasses proteins.

[0100] The total length of at least a portion of the deleted amino acid sequence may be 30 or more amino acids, 35 or more amino acids, or 40 or more amino acids. If more amino acids can be deleted while maintaining the ability to form polyhedra, the space inside the polyhedra can be made larger.

[0101] The upper limit of the total length of at least a portion of the deleted amino acid sequence may be any length that enables the modified polyhedrin protein to maintain its ability to form polyhedra, and may be, for example, 100 amino acids, for example, 80 amino acids, or for example, 50 amino acids.

[0102] Furthermore, the deleted amino acid sequence may be at one position or at two or more positions, so long as the polyhedron-forming ability is maintained. That is, the deleted amino acid sequence may be at one continuous amino acid sequence region or at two or more separate amino acid sequence regions in the amino acid sequence of the wild-type polyhedrin protein from which the modified polyhedrin protein is derived.

[0103] In this embodiment, more specific examples of modified polyhedrin proteins include, for example, a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12, or a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12 in which one or more amino acids have been deleted, substituted or added, and which has the ability to form polyhedra. The amino acid sequence shown in SEQ ID NO:12 is a sequence in which 38 residues from the 67th to the 104th residues in the amino acid sequence shown in SEQ ID NO:1 are deleted.

[0104] Fifth embodiment In the production method according to the fifth embodiment, the crystalline protein may be a protein having a moiety containing an amino acid having a coordinate property to a metal atom and having polyhedron forming ability, and the target molecule may be a substance containing a metal atom. The moiety containing an amino acid having a coordinate property to a metal atom may be located at the C-terminus of the protein.

[0105] Examples of amino acids having a coordinating property for a metal atom include histidine, glutamic acid, aspartic acid, cysteine, serine, etc. Examples of sites containing an amino acid having a coordinating property for a metal atom include sites containing one or more amino acids selected from the above-mentioned amino acids, such as a site with about six consecutive histidines, a site in which an amino acid having a coordinating property for a metal atom is exposed in the space inside a polyhedron crystal, and a site in which an amino acid having a coordinating property for a metal atom is exposed on the surface of a polyhedron crystal (for example, JP 2015-159731 A).

[0106] Sixth embodiment In the production method according to the sixth embodiment, the crystallization solution may contain a target molecule having a molecular weight of 10 to 100,000.

[0107] By expressing the above-mentioned crystalline protein in a cell-free protein synthesis system containing a target molecule, the crystals of the above-mentioned crystalline protein can encapsulate the target molecule.

[0108] As described above, by modifying the amino acid sequence of a protein capable of forming polyhedra, the target molecule can also be made to bind to the surface of the polyhedron.

[0109] The molecular weight of the target molecule is preferably 10 to 100000, more preferably 10 to 60000, and even more preferably 10 to 30000. When the molecular weight of the target molecule is within the above range, the target molecule is easily encapsulated in the polyhedron.

[0110] The target molecule is not particularly limited, and examples thereof include inorganic substances, organic substances, substances containing metal atoms, etc. The organic substance may be, for example, a peptide, a protein, fluorescein, etc.

[0111] Examples of substances containing metal atoms include metal complexes, metal atom-containing proteins, and metal atom-containing organic compounds.

[0112] Examples of metal complexes include substances containing one or more metal atoms selected from copper, nickel, zinc, cobalt, manganese, iron, ruthenium, rhenium, etc., such as Mn(CO)5Br, Mn(CO)5Cl, Ru2(CO)6Cl4, Fe3(CO) 12 , Re(CO)5Cl, etc.

[0113] Metal atom-containing proteins include hemoproteins such as cytochrome P450 and hemoglobin, and non-heme proteins such as superoxide dismutase (SOD) and alcohol dehydrogenase.

[0114] Examples of the metal atom-containing organic compound include protoporphyrin and chlorophyll.

[0115] In one embodiment, the production method according to this aspect may be as follows. The manufacturing method according to this embodiment uses the above-mentioned crystallization solution 102, dialysis membrane 301, and external dialysis solution 202, all of which are contained in a container. The manufacturing method according to this embodiment will be described with reference to FIG.

[0116] Hereinafter, embodiments capable of implementing the technology according to the present disclosure will be described in detail with reference to the drawings. Note that in the drawings used in the following description, characteristic parts may be shown enlarged for convenience, and the dimensional ratios of each component are not limited to those shown in the drawings.

[0117] 1, the crystallization solution 102 is contained in one of the storage spaces 101 divided by the dialysis membrane 301. The dialysis external solution 202 is contained in the other storage space 201 divided by the dialysis membrane 301.

[0118] The crystallization solution 102 contained in one of the storage spaces (which may be referred to as the first storage space 101) and the dialysis external solution 202 contained in the other storage space (which may be referred to as the second storage space 201) are in contact with the dialysis membrane 301.

[0119] The dialysis external fluid 202 may contain, for example, amino acids, energy molecules (ATP, GTP), energy regeneration systems, salts, buffers, creatine phosphate, reducing agents, and the like. The external dialysis solution 202 may contain a crystallization control agent.

[0120] Low molecular weight compounds such as amino acids can move from the external dialysis solution 202 to the crystallization solution 102 through the pores of the dialysis membrane. The molecular weight cutoff of the dialysis membrane is not limited as long as the effects of the present invention are achieved, but may be 10,000 to 100,000.

[0121] Proteins are synthesized using nucleic acids and amino acids by a cell-free protein synthesis system in the crystallization solution 102 contained in the first storage space 101. The synthesized proteins form crystals.

[0122] The protein synthesized and the crystals formed in the crystallization solution 102 in the first storage space 101 cannot pass through the pores of the dialysis membrane and are retained in the crystallization solution 102 .

[0123] When amino acids are consumed in the crystallization solution 102 in the first storage space 101, the amount of amino acids moving from the crystallization solution 102 in the first storage space 101 to the external dialysis solution 202 in the second storage space 201 through the dialysis membrane 301 decreases.

[0124] On the other hand, the second storage space 201 contains a sufficient amount of amino acids. In addition, additional amino acids may be added to the second storage space 201. In this way, the amino acids are supplied from the dialysis external solution 202 in the second storage space 201 to the crystallization solution 102 in the first storage space 101 through the dialysis membrane 301.

[0125] As a result, the protein is synthesized more efficiently and the protein concentration increases in the crystallization solution 102 in the first accommodation space 101. This makes it easier to obtain protein crystals.

[0126] 2, in the production method according to the present embodiment, a plurality of crystallization solutions 102 may be accommodated in the first accommodation space 101. This makes it possible to simultaneously obtain protein crystals in a short time with little effort.

[0127] Different types of proteins may be synthesized in each crystallization solution 102 in the first storage space 101. In this case, crystals of different types of proteins can be obtained simultaneously in each crystallization solution 102.

[0128] Alternatively, the first storage space 101 may contain crystallization solutions 102 each containing the same specific protein. In this case, for example, by changing the content of the crystallization control agent in each crystallization solution 102, it is possible to verify the optimal concentration of the crystallization control agent for crystallizing the specific protein.

[0129] Alternatively, protein crystals may be obtained by using a plurality of crystallization solutions 102 contained in the first storage space and a single dialysis external solution 202 contained in the second storage space. In this case, it becomes easier to standardize the crystallization conditions such as temperature, humidity, and various components in the dialysis external solution 202. This makes it easier to consider the optimal conditions for obtaining protein crystals.

[0130] Alternatively, in one embodiment, the production method according to this aspect may be as follows. In the production method according to this embodiment, the above-mentioned crystallization solution is brought into contact with a substrate solution containing an amino acid to form a bilayer.

[0131] The substrate solution may contain substrates such as, for example, amino acids, energy molecules (ATP, GTP), energy regeneration systems, salts, buffers, creatine phosphate, reducing agents, and the like. The substrate solution may contain a crystallization control agent.

[0132] The multilayer may be formed, for example, as follows. First, a substrate solution is poured into a reaction vessel, and then a crystallization solution is placed on top of the substrate solution in the reaction vessel to form an interface between the substrate solution and the crystallization solution. Alternatively, the crystallization solution is first poured into a reaction vessel, and then the substrate solution is placed on top of the crystallization solution in the reaction vessel to form an interface between the substrate solution and the crystallization solution.

[0133] This allows the substrate to diffuse slowly from the substrate solution into the crystallization solution through the interface, resulting in a high protein yield and making it easier to obtain protein crystals.

[0134] According to the production method of this embodiment described above, it is possible to produce protein crystals whose three-dimensional structure can be analyzed in a short time and with little effort.

[0135] The most common method for crystallizing proteins is to purify the protein from cells and then crystallize it. This method requires more than two weeks to obtain crystals and requires a great deal of effort.

[0136] For example, when the protein to be crystallized is a polyhedron protein, a method of infecting cells with baculovirus and growing polyhedron crystals within the cells has been used to produce polyhedron crystals, which requires approximately two weeks to prepare the baculovirus.

[0137] On the other hand, in the case of the production method according to the present embodiment, it is possible to express a protein and obtain crystals in about one day. Furthermore, the production method according to the present embodiment requires very simple operations and little labor.

[0138] In addition, the use of a cell-free protein synthesis system allows for the synthesis of large amounts of protein. As described above, for example, by adding a factor that enhances the efficiency of protein synthesis to the cell-free protein synthesis system, it becomes possible to more easily synthesize large amounts of protein.

[0139] In the manufacturing method according to this embodiment, since the crystallization solution contains a crystallization inhibitor, it is possible to more easily increase the amount of crystals obtained. Furthermore, according to the manufacturing method according to this embodiment, it is possible to suppress the formation of twin crystals. Furthermore, it is possible to improve the resolution of the obtained crystals in three-dimensional structure analysis. As a result, it is possible to obtain protein crystals whose structure can be analyzed by X-ray crystal structure analysis.

[0140] [Manufacturing equipment] The production apparatus according to this embodiment is an apparatus for producing protein crystals, and can be suitably used in the method for producing protein crystals according to the above embodiment. Hereinafter, an embodiment of the manufacturing apparatus according to this aspect will be described.

[0141] (First embodiment) A manufacturing apparatus 1A according to an embodiment will be described with reference to the drawings. Fig. 3 is a cross-sectional view of the manufacturing apparatus 1A.

[0142] Hereinafter, embodiments capable of implementing the technology according to the present disclosure will be described in detail with reference to the drawings. Note that in the drawings used in the following description, characteristic parts may be shown enlarged for convenience, and the dimensional ratios of each component are not limited to those shown in the drawings.

[0143] 3, the manufacturing apparatus 1A has a first container 11 and a second container 21. The second container 21 accommodates the first container 11. The interior of the first container 11 corresponds to the first accommodation space 14. Moreover, the interior of the second container 21 corresponds to the second accommodation space 23.

[0144] The first container 11 has a dialysis membrane 11b on its bottom surface 11a. The first storage space 14 is a space surrounded by the circumferential surface 11c and the bottom surface 11a of the first container 11. The second storage space 23 is a space surrounded by the circumferential surface and the bottom surface of the second container 21.

[0145] The first storage space 14 stores the crystallization solution L1. The second storage space 23 stores the dialysis external solution L2. The dialysis membrane 11b is in contact with both the crystallization solution L1 and the dialysis external solution L2.

[0146] 3, the outer diameter of the bottom surface 11a of the first container 11 and the inner diameter of the bottom surface of the second container 21 are substantially the same. Thus, in the second container 21, the first container 11 is supported by the inner peripheral surface of the second container 21 and does not tip over or tilt. As a result, the crystallization solution L1 in the first storage space 14 does not spill out from the first storage space 14 to the outside.

[0147] The crystallization solution L1 may be the crystallization solution described above in the [Production Method]. The crystallization solution may not contain a crystallization inhibitor.

[0148] The crystallization solution L1 contains a cell-free protein synthesis system and a nucleic acid encoding a protein. In the crystallization solution L1 contained in the first storage space 14, a protein 32 is synthesized by the cell-free protein synthesis system using nucleic acids and amino acids 31. The synthesized protein 32 forms a crystal 33.

[0149] The dialysis external solution L2 contains amino acid 31. The dialysis external solution L2 may contain a low molecular weight compound necessary for the translation reaction. Examples of the low molecular weight compound include an energy regeneration system, salts, and creatine phosphate. The dialysis external solution L2 may contain a low molecular weight compound necessary for the translation reaction.

[0150] The amino acid 31 is capable of moving between the first storage space 14 and the second storage space 23 through the pores of the dialysis membrane 11b.

[0151] In the crystallization solution L1 in the first storage space 14, the synthesized protein 32 and the formed crystals 33 cannot pass through the pores of the dialysis membrane 11b and are retained in the crystallization solution L1.

[0152] When the amino acid 31 is consumed in the crystallization solution L1 in the first storage space 14, the amount of the amino acid that moves from the crystallization solution L1 in the first storage space 14 to the dialysis external solution L2 in the second storage space 23 through the dialysis membrane 11b decreases. As a result, the amino acid 31 is supplied from the dialysis external solution L2 in the second storage space 23 to the crystallization solution L1 in the first storage space 14 through the dialysis membrane 11b.

[0153] As a result, the protein 32 is synthesized more efficiently in the crystallization solution L1 in the first storage space 14, and the concentration of the protein 32 increases. This makes it easier to obtain crystals 33 of the protein 32.

[0154] Second embodiment FIG. 4 illustrates a cross-sectional view of a manufacturing apparatus 1B according to one embodiment. 4, the production apparatus 1B has a plurality of first containers 11. The plurality of first containers 11 are supported by a support member 30 so that the dialysis membranes 11b are in contact with the dialysis external solution L2. The production apparatus 1B does not necessarily have to have the support member 30.

[0155] In the manufacturing apparatus 1B, the same components as those in the manufacturing apparatus 1A of the first embodiment are denoted by the same reference numerals and the description thereof will be omitted.

[0156] The shape of the support member 30 is not particularly limited as long as the support member 30 can be accommodated in the second accommodation space 23 and the dialysis membrane 11b can contact the dialysis external solution L2. The shape of the support member 30 may be, for example, rectangular or substantially circular in a plan view.

[0157] The specific gravity of the support member 30 may be smaller than the specific gravity of the dialysis external solution L2. In this case, as illustrated in Fig. 4, the support member 30 floats in the dialysis external solution L2. This makes it easier to hold the support member 30 and the first container 11 supported by the support member 30 at a constant position in the depth direction relative to the dialysis external solution L2.

[0158] The support member 30 has a plurality of through holes 30a. The inner diameter of the through holes 30a is equal to or larger than the outer diameter of the first container 11. This allows the first container 11 to be inserted into the through holes 30a.

[0159] The number of through holes 30a in the support member 30 is not particularly limited and may be two or more, for example, 2 to 100.

[0160] The first container 11 has a positioning portion 11d. The outer shape of the positioning portion 11d is larger than the inner diameter of the through-hole 30a. This makes it easy to position the first container 11 on the support member 30 in a state where the first container 11 penetrates the through-hole 30a.

[0161] The inner diameters of the multiple through holes 30a may all be the same, or some of them may differ.

[0162] According to the production apparatus 1B, crystals 33 of a protein 32 can be produced simultaneously in a plurality of crystallization solutions L1 contained in a plurality of first containers 11 using one type of dialysis external solution L2.

[0163] In the production apparatus 1B, one type of dialysis external solution L2 is used, so that it is easier to replace the dialysis external solution L2 with a fresh one or to add amino acids or the like to the dialysis external solution L2.

[0164] As described above, the production apparatus 1B allows the production process of the crystals 33 of the protein 32 to be parallelized, and allows a large number of crystals 33 to be produced at the same time. In addition, since one type of dialysis external solution L2 is used for a plurality of crystallization solutions L1, it becomes easier to control the crystallization environment. This makes it easier to consider the crystallization conditions of the protein.

[0165] (Modification) Fig. 5 shows a modified manufacturing apparatus 1C. Fig. 5 shows a cross-sectional view of the manufacturing apparatus 1C. The manufacturing apparatus 1C is a modification of the manufacturing apparatus 1A, in which the crystallization solution and the external dialysis solution are arranged in the horizontal direction.

[0166] According to the production apparatus 1C, even without a support member, a plurality of crystallization solutions L1 and an external dialysis solution L2 can be brought into contact with the dialysis membrane 11b.

[0167] [kit] In one embodiment, the present invention provides a kit for producing protein crystals, comprising a nucleic acid encoding a protein, a crystallization controlling agent, and a reagent for a cell-free protein synthesis system. The kit of the present embodiment can be suitably used in the above-mentioned production method.

[0168] By using the reagents included in the kit of this embodiment, a cell-free protein synthesis system can be easily prepared. Examples of the cell-free protein synthesis system include those described above in the [Production method]. The cell-free protein synthesis system may be a synthesis system using a cell extract, or a synthesis system in which factors necessary for translation are reconstituted.

[0169] The nucleic acid included in the kit of this embodiment is not particularly limited, and examples thereof include those mentioned above in the [Production Method]. Examples of the crystallization regulator included in the kit of this embodiment include those mentioned above in the [Production Method].

[0170] When the cell-free protein synthesis system is a synthesis system using a cell extract, the reagents of the cell-free protein synthesis system included in the kit may contain a cell extract. The biological species of cells from which the cell extract is derived is not particularly limited, and examples thereof include those mentioned above in the [Production method].

[0171] When the cell-free protein synthesis system is a synthesis system in which factors necessary for translation are reconstituted, the kit may contain the factors necessary for translation. The biological species from which the factors necessary for translation are derived is not particularly limited.

[0172] The kit of this embodiment may contain factors that enhance the efficiency of protein synthesis, such as amino acids, energy molecules, etc. Examples of factors that enhance the efficiency of protein synthesis include those mentioned above in the [Production method].

[0173] The nucleic acid contained in the kit of this embodiment may be RNA or DNA. The sequence of the nucleic acid encoding the above-mentioned protein may be one that has synonymous substitution so as to optimize the expression of the protein in a cell-free protein synthesis system.

[0174] When the nucleic acid is DNA, the DNA may be linear or circular. The circular DNA may be, for example, an expression vector such as a plasmid. The plasmid may have a promoter or a ribosome binding site upstream of the DNA sequence encoding the above-mentioned protein.

[0175] The kit of this embodiment may contain factors necessary for transcription. Examples of factors necessary for transcription include the factors mentioned above in the [Production method]. When the nucleic acid encoding the protein is DNA, RNA can be synthesized by transcription using the factors necessary for transcription included in the kit of this embodiment.

[0176] The kit of this embodiment may further include the production apparatus according to the above embodiment. EXAMPLES

[0177] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0178] [Experimental Example 1] (Crystallization using a crystallization control agent) Proteins were expressed in a cell-free protein synthesis system, and a crystallization inhibitor was added to the system to form protein crystals.

[0179] The target protein for crystallization was Crystalline inclusion protein A (CipA) protein, which is shown in SEQ ID NO:9. The crystallization control agents used were ethanol, 1,4-dioxane, dextran, polyethylene glycol, and triethylene glycol. The polyethylene glycols used were PEG400 (Merck, molecular weight 380-420), PEG3350 (Merck, molecular weight 3015-3685), and PEG8000 (Merck, molecular weight 7000-9000).

[0180] The wheat germ cell-free protein synthesis system was used as the cell-free protein synthesis system. Specifically, SUB-AMIX SGC, WEPRO 7240, and pEU-E01-MCS vector from Cell Free Sciences were used.

[0181] First, cDNA encoding the CipA protein represented by SEQ ID NO: 9 was inserted into the pEU-E01-MCS vector to obtain an expression vector. 2 μg of the obtained expression vector was prepared and added to a Transcription Premix LM tube, which was then incubated at 37° C. for 6 hours to synthesize mRNA and obtain a transcription reaction solution.

[0182] Crystallization of the CipA protein was carried out using a production apparatus equipped with a dialysis membrane. A schematic diagram of the production apparatus is shown in Figure 6. This production apparatus has an inner container equipped with a dialysis membrane and an outer container that houses the inner container. A dialysis membrane is provided on the bottom of the inner container.

[0183] Using the above manufacturing equipment, first, translation buffer solution (SGC) was poured into the outer container as an external dialysis solution, and then a crystallization inhibitor was added. Next, the inner container was set in the outer container. A crystallization solution containing wheat germ extract, translation buffer solution (SGC), transcription reaction solution, and each crystallization inhibitor was added to the inner container.

[0184] Low molecular weight compounds such as amino acids contained in the translation buffer (SGC) can move between the external dialysis solution and the crystallization solution through the pores of the dialysis membrane. Proteins and crystals synthesized in the crystallization solution cannot pass through the pores of the dialysis membrane, and are retained in the crystallization solution in the inner container.

[0185] The crystallization control agents were added to the crystallization solution and the external dialysis solution so that their final concentrations were, respectively, ethanol (3 v / v%), 1,4-dioxane (3 v / v%), dextran (2 w / v%), PEG400 (1 v / v%), PEG3350 (1 w / v%), PEG8000 (1 w / v%), and triethylene glycol (1 v / v%).

[0186] Next, the mixture was incubated at 20° C. for 72 hours to carry out a translation reaction and synthesize CipA. A portion of the solution after the translation reaction was analyzed by SDS-PAGE to confirm the expression of CipA protein. The results are shown in Figure 7. Figure 7 shows an electrophoretic image of CipA protein stained by CBB after subjecting it to SDS-PAGE.

[0187] One day after the start of the translation reaction, the crystals formed in the inner container were observed with a scanning electron microscope (SEM). The results are shown in Figure 8. Figure 8 is a photograph of CipA crystals obtained using a crystallization inhibitor in a cell-free protein synthesis system. In Figure 8, (-) is a photograph of CipA crystals obtained without adding a crystallization inhibitor.

[0188] The crystal structure was then analyzed by X-ray crystal structure analysis to evaluate the resolution and the rate of twin formation.

[0189] The rate of twin crystal formation was evaluated according to the following evaluations 1 and 2. (Rating 1) In rating 1, the Stanley factor (i.e. 2 > / 2 ) was calculated. If the crystal obtained is a perfect twin, this value is 1.5. If the crystal obtained does not contain a twin, this value is 2.0. (Rating 2) The overlap ratio of the two domains of the hemihedral twin (i.e., the twin fraction) was calculated by the CCP4 CTRUNCATE program. If the crystal obtained is a perfect twin, this value is 0.5. If the crystal obtained does not contain a twin, this value is 0. The results are shown in Table 1.

[0190] [Table 1]

[0191] This result indicates that the addition of the crystallization inhibitor suppressed the formation of twin crystals. Furthermore, the obtained crystals were analyzable by X-ray structural analysis with high resolution.

[0192] [Experimental Example 2] (Improvement of resolution in three-dimensional structure analysis) Proteins were expressed in a cell-free protein synthesis system, and a crystallization inhibitor was added to the system to form protein crystals.

[0193] Crystals of the target protein were produced in the same manner as in Experimental Example 1, except that the target protein to be crystallized and the crystallization controlling agent were changed as follows.

[0194] The target protein for crystallization was Crystalline inclusion protein A (CipB) protein, which is shown in SEQ ID NO:10.

[0195] The crystallization control agents used were ethanol, 1,4-dioxane, dextran, polyethylene glycol, triethylene glycol, and ficoll. The polyethylene glycols used were PEG400 (Merck, molecular weight 380-420), PEG3350 (Merck, molecular weight 3015-3685), and PEG8000 (Merck, molecular weight 7000-9000).

[0196] The crystallization control agents were added to the crystallization solution and the external dialysis solution so that their final concentrations were, respectively, ethanol (3 v / v%), 1,4-dioxane (3 v / v%), dextran (2 w / v%), PEG400 (1 v / v%), PEG3350 (1 w / v%), PEG8000 (1 w / v%), triethylene glycol (1 v / v%), and Ficoll (0.5 w / v%).

[0197] The crystals formed in the inner container were observed with a scanning electron microscope (SEM). The results are shown in Figures 9A and 9B. Figure 9A is an SEM image of CipB crystals obtained in a cell-free protein synthesis system without adding a crystallization inhibitor. Figure 9B is an SEM image of CipB crystals obtained in a cell-free protein synthesis system with the use of a crystallization inhibitor.

[0198] As shown in Figures 9A and 9B, the CipB crystals obtained with the addition of a crystallization inhibitor were larger, had less variation in crystal size, and had a more regular crystal shape than the CipB crystals obtained without the addition of a crystallization inhibitor. [Industrial Applicability]

[0199] According to the present invention, it is possible to provide a method for producing protein crystals whose three-dimensional structure can be analyzed in a short time with little effort. The present invention can be suitably used for high-throughput production of protein crystals. [Explanation of symbols]

[0200] 101: first storage space; 102: crystallization solution; 201: second storage space; 202: dialysis solution; 301: dialysis membrane Reference Signs List 11: first container, 11a: bottom surface, 11b: dialysis membrane, 11c: peripheral surface, 11d: positioning portion, 14: first storage space, 21: second container, 23: second storage space 30: Support member, 30a: Through hole 31 Amino acid, 32 Protein, 33 Crystal, L1 Crystallization solution, L2 External dialysis solution ​

Claims

1. A method for producing protein crystals, comprising a step of incubating a crystallization solution containing a cell-free protein synthesis system, a crystallization control agent, and a nucleic acid encoding the protein.

2. The method according to claim 1 , wherein the crystallization control agent comprises a compound that suppresses the formation of twin crystals of the protein.

3. The method according to claim 1 or 2, wherein the crystallization control agent comprises a compound that promotes the formation of the crystals.

4. The method according to any one of claims 1 to 3, wherein the crystallization control agent comprises a compound that improves the resolution of the crystal in three-dimensional structural analysis.

5. The method according to any one of claims 1 to 4, wherein the crystal is a crystal amenable to X-ray crystal structure analysis.

6. In the above process, The crystallization solution is contained in one of the container spaces divided by a dialysis membrane; The dialysis solution containing amino acids is accommodated in the other accommodation space, The crystallization solution contained in the one storage space and the dialysis external solution contained in the other storage space contact the dialysis membrane, The method according to any one of claims 1 to 5, wherein the amino acid is capable of moving from the external dialysis solution to the crystallization solution through pores in the dialysis membrane.

7. The method according to claim 6 , wherein a plurality of the crystallization solutions are contained in the one of the containing spaces.

8. The method according to any one of claims 1 to 5, wherein in the step, the crystallization solution is contacted with a substrate solution containing an amino acid to form a bilayer.

9. An apparatus for producing protein crystals, comprising: a container having a first storage space and a second storage space; a dialysis membrane facing both the first storage space and the second storage space; The first storage space contains a crystallization solution containing a cell-free protein synthesis system and a nucleic acid encoding a protein, The second storage space contains an external dialysis solution containing an amino acid, the dialysis membrane divides the internal space of the container into the first storage space and the second storage space, and is in contact with both the crystallization solution contained in the first storage space and the dialysis external solution contained in the second storage space; The amino acid is capable of moving from the external dialysis solution to the crystallization solution through pores in the dialysis membrane.

10. The manufacturing apparatus according to claim 9 , wherein a plurality of the crystallization solutions are contained in the first containing space.

11. The manufacturing apparatus according to claim 9 or 10, wherein the first accommodation space is accommodated in the container by being supported by a support member.

12. The manufacturing apparatus according to claim 11 , wherein the first accommodation space is positioned relative to the support member by a positioning portion.

13. A kit for cell-free crystal production comprising a nucleic acid encoding a protein, a crystallization control agent, and a reagent for a cell-free protein synthesis system.

14. The cell-free crystal production kit according to claim 13, further comprising the production apparatus according to any one of claims 9 to 12.

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