Method for producing protein crystal and method for analyzing crystal structure

Abstract

This crystal production method comprises the step of inducing the expression of a crystalline protein in an E. coli transfected with an expression construct for the crystalline protein and incubating the E. coli for a predetermined time until crystals of the crystalline protein are formed within the E. coli. This crystalline structure analysis method comprises the step of subjecting crystals in the E.coli produced by the production method to X-ray crystalline structure analysis. These methods are useful as a technique to form and analyze protein crystals easily.

Description

Technical Field

[0001] The present invention relates to a method for producing protein crystals and a method for analyzing crystal structures. This application claims priority based on Japanese Patent Application No. 2020-027386 filed in Japan on February 20, 2020, the content of which is incorporated herein by reference.

Background Art

[0002] The structural analysis of proteins is important for determining the three-dimensional structure of proteins and studying the structure-function correlation. In order to perform the structural analysis of proteins, it is necessary to produce high-quality protein crystals. The quality of protein crystals has a great influence on the accuracy and reliability of protein crystal structure analysis. Therefore, the crystallization of proteins has become the most rate-limiting step in protein structure analysis.

[0003] Many factors are involved in protein crystallization, such as protein concentration, purity, type of buffer solution, pH, type and concentration of precipitant, temperature, organic solvent, metal ion, type and concentration of surfactant, etc. Therefore, in order to produce high-quality protein crystals and perform crystal structure analysis, it is necessary to consider many conditions and requires a great deal of labor (see, for example, Non-Patent Document 1).

[0004] By the way, on the surface of protein molecules, there are amino acid side chains having various functional functional groups and having specific chemical properties. And when a plurality of protein molecules are regularly arranged, a cage-shaped three-dimensional structure (protein cage) may be formed.

[0005] For example, the polyhedrosis virus is a virus that infects cells of insects such as silkworms. The polyhedrosis virus produces a large amount of inclusion bodies called polyhedra in infected cells in the late stage of infection, reaching about half of the total cell protein, and encapsulates a large number of virus particles therein. Polyhedrin, which is a polyhedron protein, is an example of a protein that forms the above-mentioned protein cage.

[0006] As proteins that form protein cages, in addition to polyhedrin proteins, DNA binding proteins from starved cells (DPS), capsids of viruses that encapsulate RNA, etc. are known. Protein cages formed from these proteins may form crystals by arranging regularly.

[0007] For example, Patent Document 1 describes that a modified polyhedrin lacking at least a part of the amino acid sequence has the ability to form polyhedra. Furthermore, it is described that crystal structure analysis could be performed using the polyhedra formed from this modified polyhedrin protein.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a technique for easily producing protein crystals.

Means for Solving the Problems

[0011] The present invention includes the following aspects. [1] A method for producing the crystal, comprising the step of inducing expression of the crystalline protein in Escherichia coli into which an expression construct of the crystalline protein has been introduced, and incubating the Escherichia coli for a predetermined time until crystals of the crystalline protein are formed inside the Escherichia coli. [2] The Escherichia coli has been further introduced with an expression construct of an amorphous protein, and in the step of incubating, expression of the amorphous protein is induced together with the crystalline protein, and the crystal formed inside the Escherichia coli is a co-crystal of the crystalline protein and the amorphous protein. The production method according to [1]. [3] The production method according to [1] or [2], wherein the crystalline protein is a protein described in any one of the following (i) to (iii). (i) Cytoplasmic polyhedrin protein, nuclear polyhedrin protein, cathepsin B, ferritin, DNA-binding proteins from starved cells (DPS), luciferase, reovirus nonstructural protein (μNS), fusolin protein (Fusolin), Crystalline inclusion protein A (CipA), or Crystalline inclusion protein B (CipB) (ii) A protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein of (i), and having the ability to form crystals (iii) A fusion protein of the protein of (i) or (ii) and a target peptide [4] The production method according to any one of [1] to [3], wherein the crystalline protein is a fusion protein in which a target peptide is inserted between the 66th and 67th amino acids of the amino acid sequence set forth in SEQ ID NO: 1, or between the amino acids corresponding to the 66th and 67th amino acids of the amino acid sequence set forth in SEQ ID NO: 1 in an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1 and having the ability to form crystals. [5] The production method according to any one of [1] to [4], wherein the predetermined time is 3 to 30 hours. [6] The production method according to any one of [1] to [5], wherein the incubating step is performed at 18 to 40°C. [7] A crystal structure analysis method comprising a step of subjecting the crystal produced by the production method according to any one of [1] to [6] to X-ray crystal structure analysis together with the Escherichia coli. [Advantages of the Invention]

[0012] According to the present invention, a technique for easily producing protein crystals can be provided. [Brief Description of the Drawings]

[0013]

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[0014] [Method for Producing Protein Crystals] In one embodiment, the present invention provides a method for producing the crystal, including a step of inducing expression of the crystalline protein in Escherichia coli into which an expression construct of the crystalline protein has been introduced, and incubating the Escherichia coli for a predetermined time until crystals of the crystalline protein are formed inside the Escherichia coli.

[0015] The crystalline protein is not particularly limited as long as it can form crystals in Escherichia coli. Even an amorphous protein that does not originally form crystals can be included in the crystalline proteins in this specification if it can form crystals in Escherichia coli by methods such as chemical modification of the protein, creation of mutants, or fusion proteins.

[0016] More limited crystalline proteins include the proteins described in any of the following (i) to (iii). (i) Cytoplasmic polyhedrin protein, nuclear polyhedrin protein, cathepsin B, ferritin, DPS, luciferase, μNS, Fusolin, CipA or CipB, (ii) A protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein of (i) and having the ability to form crystals (iii) A fusion protein of the protein of (i) or (ii) and a target peptide

[0017] The nuclear polyhedrin protein is a polyhedrin protein derived from Nucleopolyhedrovirus (NPV), which is the pathogenic virus of nuclear polyhedrosis. The cytoplasmic polyhedrin protein is a polyhedrin protein derived from Cypovirus (CPV), which is the pathogenic virus of cytoplasmic polyhedrosis. The amino acid sequence of the cytoplasmic polyhedrin protein is shown in SEQ ID NO: 2.

[0018] Cathepsin B is a protease having endopeptidase activity and exopeptidase activity. Cathepsin B is a protein that forms crystals in insect cultured cells. The amino acid sequence of cathepsin B derived from Trypanosoma brucei is shown in SEQ ID NO: 4.

[0019] Ferritin is a protein synthesized by almost all organisms including algae, bacteria, plants, humans, and non-human animals. Ferritin forms a cage consisting of 24 subunits and encapsulates iron ions to be responsible for iron storage in vivo. The outer diameter is about 12 nm.

[0020] Ferritin is not particularly limited, and examples include horse-derived ferritin, human-derived ferritin, etc. The amino acid sequence of the L chain of horse-derived ferritin is shown in SEQ ID NO: 5, and the amino acid sequence of the L chain of human-derived ferritin is shown in SEQ ID NO: 6.

[0021] DPS is a protein belonging to the ferritin superfamily synthesized by many bacteria. DPS forms a cage consisting of 12 subunits and protects chromosomal DNA from oxidative stress and the like by encapsulating the chromosomal DNA. The outer diameter is about 9 nm. The amino acid sequence of E. coli-derived DPS is shown in SEQ ID NO: 7.

[0022] Luciferase is a general term for enzymes that catalyze the chemical reaction in which a luminescent substance emits light in bioluminescence such as luminescent bacteria and fireflies. Luciferase is a protein that forms crystals in insect cells. The amino acid sequence of firefly-derived luciferase is shown in SEQ ID NO: 8, and the amino acid sequence of Renilla reniformis-derived luciferase is shown in SEQ ID NO: 9.

[0023] μNS is a non-structural protein of a reovirus having crystallinity. The amino acid sequence of reovirus-derived μNS is shown in SEQ ID NO: 10.

[0024] Fusolin is a constituent protein of a crystalline protein inclusion body formed by an insect poxvirus in a host cell. The amino acid sequence of insect poxvirus-derived Fusolin is shown in SEQ ID NO: 11.

[0025] CipA and CipB are constituent proteins of a crystalline protein inclusion body formed by the entomopathogenic bacterium Photorhabdus luminescens in the cytoplasm. The amino acid sequence of CipA is shown in SEQ ID NO: 16, and the amino acid sequence of CipB is shown in SEQ ID NO: 17.

[0026] As long as the crystalline protein has the ability to form crystals, it may be a mutant having mutations with respect to the cytoplasmic polyhedrin protein, nuclear polyhedrin protein, cathepsin B, ferritin, DPS, luciferase, μNS, Fusolin, CipA, CipB, etc. described above. More specifically, the crystalline protein may be, for example, a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequences described in SEQ ID NOs: 2 to 11, 16, 17 above.

[0027] Here, one or more means, for example, it may be 1 to 50, for example, it may be 1 to 40, for example, it may be 1 to 30, for example, it may be 1 to 20, for example, it may be 1 to 10, for example, it may be 1 to 5, for example, it may be 1 to 3. As will be described later in the examples, the inventors have confirmed that even when 38 amino acids of the polyhedrin protein are deleted, it has the ability to form crystals.

[0028] In addition, as long as the crystalline protein has the ability to form crystals, it may be a fusion protein of the protein and a target peptide described above. That is, the crystalline protein may be a fusion protein of the cytoplasmic polyhedrin protein, nuclear polyhedrin protein, cathepsin B, ferritin, DPS, luciferase, μNS, Fusolin, CipA, CipB or a mutant of these crystalline proteins described above and a target peptide.

[0029] Here, the target peptide may be, for example, a peptide that is the object of three-dimensional structure analysis. As will be described later, by expressing a crystalline protein that is a fusion protein of the above-described crystalline protein and a target peptide in Escherichia coli to form crystals, and subjecting the crystals of the crystalline protein to X-ray crystal structure analysis together with the Escherichia coli, the three-dimensional structure of the target peptide can be easily analyzed.

[0030] In this case, the target peptide may be any peptide for which there is a need to analyze its three-dimensional structure. From the perspective of maintaining the crystal-forming ability of the crystalline protein, the amino acid length of the target peptide is preferably about 5 to 50 amino acids, for example.

[0031] In the fusion protein of the variant of the crystalline protein and the target peptide, the crystalline protein may be the amino acid sequence set forth in SEQ ID NO: 1, and the target peptide may be inserted between the 66th and 67th amino acids of the amino acid sequence set forth in SEQ ID NO: 1.

[0032] Alternatively, in the fusion protein of the variant of the crystalline protein and the target peptide, the crystalline protein may be a mutant protein of the amino acid sequence set forth in SEQ ID NO: 1. More specifically, the mutant protein of the amino acid sequence set forth in SEQ ID NO: 1 may consist of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1, and is a protein having the ability to form crystals. Here, the one or more is the same as described above.

[0033] And the fusion protein may be a mutant protein of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, in which the target peptide is inserted between the amino acid corresponding to the 66th amino acid and the amino acid corresponding to the 67th amino acid of the amino acid sequence set forth in SEQ ID NO: 1.

[0034] The amino acid corresponding to the 66th amino acid and the amino acid corresponding to the 67th amino acid of the amino acid sequence set forth in SEQ ID NO: 1 can be identified, for example, by aligning the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence of the mutant protein using software such as ClastalW.

[0035] In the production method of the present embodiment, the Escherichia coli has been further introduced with an expression construct of an amorphous protein. In the step of incubating, the expression of the amorphous protein is induced together with the crystalline protein, and the crystal formed inside the Escherichia coli may be a co-crystal of the crystalline protein and the amorphous protein.

[0036] In this specification, an amorphous protein generally means a protein that does not form crystals in Escherichia coli. As the amorphous protein, any protein having a molecular weight of about 1,000 to 100,000 can be used without particular limitation.

[0037] For example, as the amorphous protein, an unstable protein such as a membrane protein may be used to produce a co-crystal. Thereby, an unstable protein such as a membrane protein can be easily purified as a co-crystal. In addition, an unstable protein such as a membrane protein can be stably stored in the form of a co-crystal.

[0038] One type of amorphous protein may be used alone, or two or more types may be mixed and used. When two or more types of amorphous proteins are used, those amorphous proteins may form a complex.

[0039] In addition, the amorphous protein may be a fusion protein with a part of the crystalline protein. Thereby, the amorphous protein tends to be incorporated into the crystal of the crystalline protein, and a co-crystal is likely to be formed.

[0040] The method for expressing the crystalline protein and the amorphous protein in Escherichia coli described above is not particularly limited, and may be expressed by a commonly used method. For example, by incubating Escherichia coli introduced with an expression construct of a crystalline protein or an amorphous protein in a medium, the expression of the crystalline protein or the amorphous protein may be induced.

[0041] Introduction of the expression construct into E. coli may be performed, for example, by introducing an expression vector for a crystalline protein or an amorphous protein into E. coli in the form of a plasmid or the like, or by inserting an expression construct for a crystalline protein or an amorphous protein into the genome of E. coli.

[0042] In addition, induction of expression of a crystalline protein or an amorphous protein can be carried out by an appropriate method according to the characteristics of the expression construct for the crystalline protein or the amorphous protein. For example, an expression control system using the lactose operon can be utilized, and expression of the crystalline protein or the amorphous protein may be induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to the medium.

[0043] Alternatively, an expression control system using the Tet-on / Tet-off system can be utilized, and expression of the crystalline protein or the amorphous protein may be induced by adding tetracycline or its derivative to the medium or by removing tetracycline or its derivative from the medium.

[0044] Alternatively, an expression mode may be adopted in which expression of the crystalline protein or the amorphous protein is started simultaneously with the start of culturing of E. coli without performing expression induction control.

[0045] In the production method of this embodiment, expression of the crystalline protein or the amorphous protein is induced in E. coli into which an expression construct for the crystalline protein or the amorphous protein has been introduced. Expression induction is preferably carried out, for example, after the OD of E. coli reaches 0.6 to 0.8. 600 Reaching 0.6 to 0.8 is preferred.

[0046] Culturing of E. coli may be carried out in a small amount of medium of about 10 mL. As will be described later in the examples, the inventors have clarified that even in such a small amount of medium, a large amount of protein crystals can be produced, and the structure of the protein can be analyzed by performing X-ray crystal structure analysis on the whole E. coli.

[0047] Subsequently, after inducing the expression of the crystalline protein or the non-crystalline protein, the E. coli is incubated for a predetermined time until crystals of the crystalline protein or co-crystals of the crystalline protein and the non-crystalline protein are formed inside the E. coli. Here, the predetermined time may be 3 to 30 hours, for example, 3 to 24 hours. As will be described later in the examples, the inventors have clarified that protein structure analysis can be performed by subjecting the whole E. coli, including the formed crystals, to X-ray crystallographic analysis after incubating for only 20 to 24 hours to form crystals.

[0048] Also, the temperature for incubation until crystals of the crystalline protein or co-crystals of the crystalline protein and the non-crystalline protein are formed may be 18 to 40 °C, 25 to 38 °C, or about 30 °C. When the temperature for incubation until crystals of the crystalline protein or co-crystals of the crystalline protein and the non-crystalline protein are formed is within the above range, the quality of the formed protein crystals tends to improve.

[0049] [Crystal Structure Analysis Method] In one embodiment, the present invention provides a crystal structure analysis method including a step of subjecting the crystals produced by the above-described production method to X-ray crystallographic analysis of the whole E. coli.

[0050] As will be described later in the examples, the inventors have surprisingly clarified that protein structure analysis can be performed by subjecting the E. coli in which protein crystals are formed inside the cells to X-ray crystallographic analysis of the whole E. coli. That is, it is possible to perform X-ray crystallographic analysis of the whole E. coli without purifying the protein crystals. According to the method of the present embodiment, protein structure analysis can be performed much more simply compared to the prior art. [Examples]

[0051] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0052] [Experimental Example 1] (Crystallization of Protein in Escherichia coli 1) Genes encoding polyhedrin derived from CPV (the amino acid sequence is shown in SEQ ID NO: 2) and genes encoding polyhedrin derived from NPV were inserted into the pET29b vector (Merck Millipore), respectively, to prepare expression vectors. Subsequently, each expression vector was transformed into Escherichia coli BL21 strain.

[0053] Subsequently, each of the transformed Escherichia coli was inoculated into 10 mL of LB medium and cultured at 37 °C until the OD 600 reached 0.6 to 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of polyhedrin. Subsequently, the culture was continued at 30 °C for 20 to 24 hours to form polyhedrin crystals.

[0054] Figs. 1(a) and (b) are optical micrographs of Escherichia coli in which crystals (polyhedra) of polyhedrin derived from CPV were formed inside the bacterial cells. Also, Figs. 1(c) and (d) are optical micrographs of Escherichia coli in which crystals of polyhedrin derived from NPV were formed inside the bacterial cells. In Figs. 1(a) to (d), the scale bar indicates 10 μm.

[0055] Subsequently, each Escherichia coli was collected by centrifugation. Subsequently, the Escherichia coli was disrupted by ultrasonic waves, and polyhedrin crystals were recovered by centrifugation. Fig. 2(a) is a photograph showing the result of observing crystals of polyhedrin derived from CPV with a scanning electron microscope. Fig. 2(b) is a photograph showing the result of observing crystals of polyhedrin derived from NPV with a scanning electron microscope. In Figs. 2(a) and (b), the scale bar indicates 2 μm. As a result, cubic crystals were observed in all samples, and it was confirmed that polyhedron crystals could be formed in Escherichia coli.

[0056] Figure 3 is a photograph showing the results of subjecting the recovered crystals of CPV-derived polyhedrin and NPV-derived polyhedrin to SDS-polyacrylamide gel electrophoresis (PAGE) and staining with Coomassie Brilliant Blue. In Figure 3, "M" indicates the molecular weight marker, and "CPV(WT)" indicates the crystals of CPV-derived polyhedrin expressed in the insect cell Sf21 strain, which was subjected to SDS-PAGE as a control.

[0057] As a result, it was confirmed that both the CPV-derived polyhedrin and the NPV-derived polyhedrin expressed in E. coli had the expected molecular weights.

[0058] Figures 4(a) and (b) are graphs showing the results of MALDI-TOF MS analysis of the recovered crystals of CPV-derived polyhedrin and NPV-derived polyhedrin. Figure 4(a) shows the results of CPV-derived polyhedrin, and Figure 4(b) shows the results of NPV-derived polyhedrin. In Figures 4(a) and (b), "Obs" indicates the measured molecular weight, and "Cal" indicates the molecular weight of the predicted full-length protein.

[0059] As a result, it was confirmed that the CPV-derived polyhedrin and the NPV-derived polyhedrin had the expected full-length molecular weights.

[0060] [Experimental Example 2] (Protein Crystallization in E. coli 2) CPV-derived polyhedrin mutants were expressed in E. coli in the same manner as in Experimental Example 1. As mutants, in the amino acid sequence shown in SEQ ID NO: 2, a mutant in which asparagine (N29) at the 29th position was substituted with serine (hereinafter referred to as "N29S", and the amino acid sequence is shown in SEQ ID NO: 12), in the amino acid sequence shown in SEQ ID NO: 2, a mutant consisting of an amino acid sequence in which glycine (G192), serine (S193), and alanine (A194) at the 192nd, 193rd, and 194th positions were deleted (hereinafter referred to as "Δ3", and the amino acid sequence is shown in SEQ ID NO: 13), and in the amino acid sequence shown in SEQ ID NO: 2, a mutant consisting of an amino acid sequence in which alanine (A67) at the 67th position to alanine (A104) at the 104th position were deleted (hereinafter referred to as "Δ38", and the amino acid sequence is shown in SEQ ID NO: 1) were used. Genes encoding CPV-derived polyhedrin mutants were inserted into pET29b vectors (Merck Millipore), respectively, to prepare expression vectors. Subsequently, each expression vector was transformed into E. coli BL21 strain.

[0061] Subsequently, each of the transformed E. coli was inoculated into 10 mL of LB medium and cultured at 37 °C until the OD600 reached 0.6 - 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of polyhedrin mutants. Subsequently, the culture was continued at 30 °C for 20 - 24 hours to form crystals of polyhedrin mutants.

[0062] Subsequently, each E. coli was collected by centrifugation. Subsequently, the E. coli was disrupted by ultrasonic waves, and crystals of polyhedrin mutants were recovered by centrifugation. Figures 5(a) - (c) are photographs showing the results of observing crystals of polyhedrin mutants with a scanning electron microscope. Figure 5(a) is a photograph showing the results of observing crystals of the mutant (N29S) with a scanning electron microscope, Figure 5(b) is a photograph showing the results of observing crystals of the mutant (Δ3) with a scanning electron microscope, and Figure 5(c) is a photograph showing the results of observing crystals of the mutant (Δ38) with a scanning electron microscope. In Figures 5(a) - (c), the scale bar indicates 2 μm. As a result, crystals were observed in all samples, and it was confirmed that crystals of polyhedrin mutants can be formed in E. coli.

[0063] Figures 6(a) to (c) are graphs showing the results of MALDI-TOF MS analysis of the crystals of the recovered polyhedrin mutants. Figure 6(a) shows the results of the mutant (N29S), Figure 6(b) shows the results of the mutant (Δ3), and Figure 6(c) shows the results of the mutant (Δ38). As a result, it was confirmed that each polyhedrin mutant had the expected full-length molecular weight.

[0064] [Experimental Example 3] (Protein Crystallization in E. coli 3) CPV-derived polyhedrin (the amino acid sequence is shown in SEQ ID NO: 2) was expressed in E. coli BL21 strain in the same manner as in Experimental Example 1 to form crystals. Here, the crystallization temperature was set to 30 °C or 37 °C.

[0065] Specifically, first, the transformed E. coli was inoculated into 10 mL of LB medium and cultured at 37 °C until the OD 600 reached 0.6 to 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of polyhedrin. Subsequently, the culture was continued at 30 °C or 37 °C for 20 to 24 hours to form polyhedrin crystals.

[0066] Subsequently, each E. coli was collected by centrifugation. Subsequently, the E. coli was disrupted by ultrasonic waves, and polyhedrin crystals were recovered by centrifugation. Figures 7(a) to (c) are photographs showing the results of observing the crystals of polyhedrin crystallized at 30 °C with a scanning electron microscope. Also, Figures 7(d) to (f) are photographs showing the results of observing the crystals of polyhedrin crystallized at 37 °C with a scanning electron microscope. As a result, it became clear that polyhedrin could be crystallized at any temperature. Also, by crystallizing at a lower temperature, a tendency for the quality of polyhedrin crystals to improve was recognized.

[0067] [Experimental Example 4] (Structural Analysis of Polyhedrin Crystals) E. coli BL21 strain having the expression vector of CPV-derived polyhedrin (the amino acid sequence is shown in SEQ ID NO: 2) prepared in Experimental Example 1 was inoculated into 10 mL of LB medium and cultured at 37 °C until the OD 600 reached 0.6 to 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of polyhedrin. Subsequently, the culture was continued at 30 °C for 20 to 24 hours to form polyhedrin crystals.

[0068] Subsequently, this E. coli was subjected to X-ray crystal structure analysis as a whole cell. SPring-8 BL32XU was used for the X-ray crystal structure analysis. As a result, the structure analysis was successfully performed at a resolution of 1.8 Å. Figure 8 is a diagram showing the result of analyzing the crystal of CPV-derived polyhedrin as an E. coli cell.

[0069] Subsequently, in the same manner as in Experimental Example 1, X-ray crystal structure analysis was performed using the crystals of polyhedrin purified from E. coli. SPring-8 BL32XU was used for the X-ray crystal structure analysis. Also, for comparison, X-ray crystal structure analysis was similarly performed on the crystals of polyhedrin prepared and purified in insect cell Sf21 strain. As a result, the purified crystals were successfully analyzed at a resolution of 1.9 Å.

[0070] Figure 9(a) is a diagram in which the three-dimensional structures of the crystals of polyhedrin prepared in E. coli and the crystals of polyhedrin prepared in insect cells are superimposed. As a result, it was confirmed that the two were identical.

[0071] Figures 9(b) and (c) are images showing the results of the three-dimensional structure analysis of the crystals of polyhedrin prepared in E. coli. In Figures 9(b) and (c), "GTP" indicates guanosine triphosphate, "ATP" indicates adenosine triphosphate, and "CTP" indicates cytidine triphosphate. As a result, as shown in Figures 9(b) and (c), it was confirmed that nucleic acids were bound to the three-dimensional structure of the crystals of polyhedrin prepared in E. coli. This result further supports that the crystals of polyhedrin prepared in E. coli are equivalent to the crystals of polyhedrin prepared in insect cells.

[0072] [Experimental Example 5] (Crystallization of Protein in Escherichia coli 4) A gene encoding polyhedrin derived from CPV (the amino acid sequence is shown in SEQ ID NO: 2) was inserted into the pET29b vector (Merck Millipore) to prepare an expression vector. In addition, a gene encoding the non-crystalline protein superfolder green fluorescent protein (sfGFP) was inserted into the pET21c vector (Merck Millipore) to prepare an expression vector.

[0073] sfGFP was used in the form of a fusion protein (the amino acid sequence is shown in SEQ ID NO: 15) between the H1 region of polyhedrin derived from CPV (the amino acid sequence is shown in SEQ ID NO: 14) and sfGFP. Subsequently, each expression vector was mixed and transformed into Escherichia coli BL21 strain.

[0074] Subsequently, the transformed Escherichia coli was inoculated into 10 mL of LB medium and cultured at 37 °C until the OD 600 reached 0.6 to 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of polyhedrin and sfGFP and co-express them. Subsequently, the culture was continued at 30 °C for 20 to 24 hours to form co-crystals of polyhedrin and sfGFP.

[0075] Subsequently, each Escherichia coli was collected by centrifugation. Subsequently, the Escherichia coli was disrupted by ultrasonic waves, and the crystals of polyhedrin were recovered by centrifugation. Figures 10(a) and (b) are representative photographs showing the results of observing the co-crystals of polyhedrin and sfGFP crystallized at 30 °C with a confocal fluorescence microscope. In Figures 10(a) and (b), the bright-field image and the fluorescence image of sfGFP were superimposed and displayed. In Figures 10(a) and (b), the regions where the fluorescence of sfGFP was detected are indicated by dots.

[0076] As a result, it was confirmed that the formed crystals emitted the fluorescence of sfGFP. This result indicates that co-crystals of polyhedrin and sfGFP were formed.

[0077] [Experimental Example 6] (Structural Analysis of Crystals of Polyhedrin Mutants) X-ray crystallographic analysis was performed on the crystals of the polyhedrin variant (Δ3) derived from CPV and the crystals of the polyhedrin variant (Δ38) derived from CPV, which were purified in Experimental Example 2. SPring-8 BL32XU was used for the X-ray crystallographic analysis.

[0078] The upper part of Fig. 11(a) is a photograph showing the result of observing the crystals of the variant (Δ3) with a scanning electron microscope, and the lower part of Fig. 11(a) is a diagram showing the result of structural analysis of the crystals of the variant (Δ3). As a result, the crystals of the purified variant (Δ3) were successfully subjected to structural analysis at a resolution of 2.3 Å.

[0079] Also, the upper part of Fig. 11(b) is a photograph showing the result of observing the crystals of the variant (Δ38) with a scanning electron microscope, and the lower part of Fig. 11(b) is a diagram showing the result of structural analysis of the crystals of the variant (Δ38). As a result, the crystals of the purified variant (Δ38) were successfully subjected to structural analysis at a resolution of 2.5 Å.

[0080] [Experimental Example 7] (Crystallization of Proteins in Escherichia coli 5) The gene encoding Crystalline inclusion protein A (CipA) was inserted into the pET29b vector (Merck Millipore) to prepare an expression vector. Subsequently, the expression vector was transformed into Escherichia coli BL21 strain.

[0081] Subsequently, the transformed Escherichia coli was inoculated into 10 mL of LB medium and cultured at 37 °C until the OD 600 reached 0.6 to 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of CipA. Subsequently, the culture was continued at 30 °C for 20 to 24 hours to form crystals of CipA. Fig. 12(a) is an optical micrograph of Escherichia coli in which crystals of CipA were formed inside the cells. The scale bar indicates 10 μm.

[0082] Subsequently, E. coli was collected by centrifugation. Subsequently, the E. coli was disrupted by ultrasonic treatment, and the crystals of CipA were collected by centrifugation. Fig. 12(b) is a photograph showing the result of observing the crystals of CipA with a scanning electron microscope. The scale bar indicates 2 μm.

[0083] Subsequently, X-ray crystallographic analysis of the purified CipA crystals was performed. SPring-8 BL32XU was used for the X-ray crystallographic analysis. As a result, diffraction data could be obtained at a resolution of 2.8 Å.

[0084] From the above results, it became clear that CipA crystals could be formed in E. coli.

[0085] [Experimental Example 8] (Protein Crystallization in E. coli 6) A gene encoding a fusion protein of ubiquitin (hereinafter sometimes referred to as "Ubq") and cytoplasmic polyhedrin protein (hereinafter sometimes referred to as "PhM") was inserted into the pET29b vector (Merck Millipore) to prepare an expression vector.

[0086] Expression vectors were prepared for two types of fusion proteins, a fusion protein with Ubq located on the N-terminal side (hereinafter sometimes referred to as "Ubq-PhM") and a fusion protein with PhM located on the N-terminal side (hereinafter sometimes referred to as "PhM-Ubq"). In both Ubq-PhM and PhM-Ubq, a linker "GGGS (SEQ ID NO: 18)" was inserted between Ubq and PhM. The amino acid sequence of Ubq-PhM is shown in SEQ ID NO: 19, and the amino acid sequence of PhM-Ubq is shown in SEQ ID NO: 20.

[0087] Subsequently, each expression vector was transformed into E. coli BL21 strain. Subsequently, each transformed E. coli was inoculated into 10 mL of LB medium and cultured at 37°C until the OD 600It was cultured until it reached 0.6 to 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of Ubq-PhM and PhM-Ubq, respectively. Subsequently, it was cultured at 30 °C for 20 to 24 hours to form crystals of Ubq-PhM and PhM-Ubq, respectively.

[0088] The left of Fig. 13(a) is an optical micrograph of Escherichia coli in which crystals of Ubq-PhM were formed inside the bacterial cells, and the left of Fig. 13(b) is an optical micrograph of Escherichia coli in which crystals of PhM-Ubq were formed inside the bacterial cells. The scale bar indicates 10 μm.

[0089] Subsequently, each Escherichia coli was collected by centrifugation. Subsequently, each Escherichia coli was disrupted by ultrasonic waves, and crystals of Ubq-PhM and PhM-Ubq were collected by centrifugation, respectively. The right of Fig. 13(a) is a photograph showing the result of observing the crystals of Ubq-PhM with a scanning electron microscope, and the right of Fig. 13(b) is a photograph showing the result of observing the crystals of PhM-Ubq with a scanning electron microscope. The scale bar indicates 2 μm.

[0090] Fig. 13(c) is a graph showing the results of MALDI-TOF MS analysis of the collected crystals of Ubq-PhM and PhM-Ubq, respectively. The upper part of Fig. 13(c) shows the result of Ubq-PhM, and the lower part of Fig. 13(c) shows the result of PhM-Ubq. In Fig. 13(c), "Cal" indicates the molecular weight of each predicted fusion protein. As a result, it was confirmed that each of Ubq-PhM and PhM-Ubq had the expected molecular weight.

[0091] From the above results, it became clear that crystals of Ubq-PhM and PhM-Ubq can be formed in Escherichia coli.

[0092] [Experimental Example 9] (Protein Crystallization in Escherichia coli 7) A gene encoding a fusion protein of green fluorescent protein (hereinafter sometimes referred to as "GFP") and cytoplasmic polyhedrosis protein (hereinafter sometimes referred to as "PhM") was inserted into the pET29b vector (Merck Millipore) to prepare an expression vector.

[0093] In the prepared fusion protein, GFP was located on the N-terminal side (hereinafter sometimes referred to as "GFP-PhM"), and a linker "GGGS (SEQ ID NO: 18)" was inserted between GFP and PhM. The amino acid sequence of GFP-PhM is shown in SEQ ID NO: 21.

[0094] Subsequently, the expression vector was transformed into Escherichia coli BL21 strain. Subsequently, the transformed Escherichia coli was inoculated into 10 mL of LB medium respectively and cultured at 37 °C until the OD 600 reached 0.6 - 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of GFP-PhM. Subsequently, the culture was continued at 30 °C for 20 - 24 hours to form GFP-PhM crystals respectively.

[0095] Subsequently, the Escherichia coli was collected by centrifugation. Subsequently, the Escherichia coli was disrupted by ultrasonic waves, and the GFP-PhM crystals were recovered by centrifugation.

[0096] Figure 14(a) is an image merging the optical micrograph of the recovered GFP-PhM crystals and the photograph observing the fluorescence of GFP. The scale bar indicates 10 μm. Figure 14(b) is a photograph showing the result of observing the GFP-PhM crystals with a scanning electron microscope. The scale bar indicates 5 μm.

[0097] Figure 14(c) is a graph showing the result of MALDI-TOF MS analysis of the recovered GFP-PhM crystals. In Figure 14(c), "SfGFP-WTPhC 2+ " indicates the divalent ion of SfGFP-WTPhC (GFP-PhM), and "SfGFP-WTPhC +"」 represents the monovalent ions of SfGFP-WTPhC (GFP-PhM). Also, "Cal" represents the predicted molecular weight of the fusion protein. As a result, it was confirmed that GFP-PhM has the predicted molecular weight.

[0098] From the above results, it became clear that GFP-PhM crystals can be formed in E. coli.

[0099] [Experimental Example 10] (Protein Crystallization in E. coli 8) A gene encoding a fusion protein of the thalidomide-binding domain of the cereblon (CRBN) protein (hereinafter sometimes referred to as "TBD") and the cytoplasmic polyhedrin protein (hereinafter sometimes referred to as "PhM") was inserted into the pET29b vector (Merck Millipore) to prepare an expression vector.

[0100] Expression vectors were prepared for two types of fusion proteins: a fusion protein with TBD located on the N-terminal side (hereinafter sometimes referred to as "TBD-PhM") and a fusion protein with PhM located on the N-terminal side (hereinafter sometimes referred to as "PhM-TBD"). In both TBD-PhM and PhM-TBD, a linker "GGGS (SEQ ID NO: 18)" was inserted between TBD and PhM. The amino acid sequence of TBD-PhM is shown in SEQ ID NO: 22, and the amino acid sequence of PhM-TBD is shown in SEQ ID NO: 23.

[0101] Subsequently, each expression vector was transformed into E. coli BL21 strain. Subsequently, each transformed E. coli was inoculated into 10 mL of LB medium and cultured at 37 °C until the OD 600 reached 0.6 - 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of TBD-PhM and PhM-TBD, respectively. Subsequently, the culture was continued at 30 °C for 20 - 24 hours to form crystals of TBD-PhM and PhM-TBD, respectively.

[0102] Subsequently, each Escherichia coli was collected by centrifugation. Subsequently, each Escherichia coli was disrupted by ultrasonic waves, and the crystals of TBD-PhM and PhM-TBD were respectively collected by centrifugation.

[0103] Figure 15(a) is a diagram showing the three-dimensional structure of TBD (thalidomide-binding domain of CRBN protein). The upper part of Figure 15(b) is an optical micrograph of the recovered TBD-PhM crystal, and the upper part of Figure 15(c) is an optical micrograph of the recovered PhM-TBD crystal. Also, the lower part of Figure 15(b) is a photograph showing the result of observing the TBD-PhM crystal with a scanning electron microscope, and the lower part of Figure 15(c) is a photograph showing the result of observing the PhM-TBD crystal with a scanning electron microscope. The scale bar indicates 2 μm.

[0104] From the above results, it became clear that crystals of TBD-PhM and PhM-TBD can be formed in Escherichia coli.

[0105] [Experimental Example 11] (Protein Crystallization in Escherichia coli 9) A gene encoding a fragment of cytoplasmic polyhedrin protein was inserted into the pET29b vector (Merck Millipore) to prepare an expression vector.

[0106] As fragments of cytoplasmic polyhedrin protein, a fragment consisting of the 1st to 114th amino acids of the amino acid sequence of cytoplasmic polyhedrin protein shown in SEQ ID NO: 2 (hereinafter sometimes referred to as "Fragment 1: M1~S114"), a fragment consisting of the 1st to 155th amino acids of the amino acid sequence of SEQ ID NO: 2 (hereinafter sometimes referred to as "Fragment 2: M1~R155"), a fragment consisting of the 116th to 248th amino acids of the amino acid sequence of SEQ ID NO: 2 (hereinafter sometimes referred to as "Fragment 3: S116~Q248"), and a fragment consisting of the 58th to 248th amino acids of the amino acid sequence of SEQ ID NO: 2 (hereinafter sometimes referred to as "Fragment 4: K58~Q248"), expression vectors were prepared for these four types.

[0107] Fragment 1: The amino acid sequence from M1 to S114 is shown in SEQ ID NO: 24, Fragment 2: The amino acid sequence from M1 to R155 is shown in SEQ ID NO: 25, Fragment 3: The amino acid sequence from S116 to Q248 is shown in SEQ ID NO: 26, and Fragment 4: The amino acid sequence from K58 to Q248 is shown in SEQ ID NO: 27.

[0108] Subsequently, each expression vector was transformed into Escherichia coli BL21 strain. Subsequently, each of the transformed Escherichia coli was inoculated into 10 mL of LB medium and cultured at 37 °C until the OD 600 reached 0.6 to 0.8. Subsequently, IPTG was added to a final concentration of 0.5 mM to induce the expression of Fragment 1 to Fragment 4, respectively. Subsequently, the culture was continued at 30 °C for 20 to 24 hours to form crystals of Fragment 1 to Fragment 4, respectively.

[0109] Subsequently, each Escherichia coli was collected by centrifugation. Subsequently, each Escherichia coli was disrupted by ultrasonic waves, and the crystals of Fragment 1 to Fragment 4 were collected by centrifugation, respectively. FIG. 16(a) is a photograph showing the result of observing the crystal of Fragment 1: M1 to S114 with a scanning electron microscope, and FIG. 16(c) is a photograph showing the result of observing the crystal of Fragment 2: M1 to R155 with a scanning electron microscope. The scale bar indicates 2 μm.

[0110] Further, Fig. 17(a) is a photograph showing the result of observing the crystal of Fragment 3: S116-Q248 with a scanning electron microscope, and Fig. 17(c) is a photograph showing the result of observing the crystal of Fragment 4: K58-Q248 with a scanning electron microscope. The scale bar indicates 5 μm. Also, Fig. 16(b) is a graph showing the result of MALDI-TOF MS analysis of the crystal of the recovered Fragment 1: M1-S114, Fig. 16(d) is a graph showing the result of MALDI-TOF MS analysis of the crystal of the recovered Fragment 2: M1-R155, Fig. 17(b) is a graph showing the result of MALDI-TOF MS analysis of the crystal of the recovered Fragment 3: S116-Q248, and Fig. 17(d) is a graph showing the result of MALDI-TOF MS analysis of the crystal of the recovered Fragment 4: K58-Q248. In Figs. 16 and 17, "Cal" indicates the molecular weight of each predicted fragment. As a result, it was confirmed that each of Fragments 1 to 4 has the predicted molecular weight.

[0111] From the above results, it became clear that crystals of fragments of cytoplasmic polyhedrin protein can be formed in E. coli.

Industrial Applicability

[0112] According to the present invention, it is possible to provide a technique for easily producing protein crystals.

Claims

1. Inducing the expression of the crystalline protein in Escherichia coli into which an expression construct of the crystalline protein has been introduced, incubating the Escherichia coli for a predetermined time until crystals of the crystalline protein are formed inside the Escherichia coli, and as a result, a step of producing the crystals; A step of performing X-ray crystal structure analysis on the crystals together with the Escherichia coli and determining the crystal structure at the resolution Å level; including A crystal structure analysis method, wherein the crystalline protein is a protein according to any one of the following (i) to (iii). (i) Cytoplasmic polyhedrin protein, nuclear polyhedrin protein, Crystalline inclusion protein A (CipA), or Crystalline inclusion protein B (CipB) (ii) A protein consisting of an amino acid sequence in which 1 to 50 amino acids are deleted, substituted or added in the amino acid sequence of the protein of (i) and having the ability to form crystals (iii) A fusion protein of the protein of (i) or (ii) and a target peptide of 5 to 50 amino acids

2. The crystalline protein is between the 66th and 67th amino acids of the amino acid sequence set forth in SEQ ID NO: 1, or in the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids are deleted, substituted or added, and a target peptide of 5 to 50 amino acids is inserted between the amino acid corresponding to the 66th amino acid and the amino acid corresponding to the 67th amino acid of the amino acid sequence set forth in SEQ ID NO: 1 of a protein having the ability to form crystals. The crystal structure analysis method according to claim 1, which is a fusion protein.

3. The crystal structure analysis method according to claim 1 or 2, wherein the predetermined time is 3 to 30 hours.

4. The crystal structure analysis method according to any one of claims 1 to 3, wherein the step of incubating is performed at 18 to 40°C.

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