METHOD FOR CRYSTALLIZING MEMBRANE PROTEIN AND METHOD FOR ANALYZING MEMBRANE PROTEIN STRUCTURE

Abstract

To provide a versatile technique that can be used commonly in membrane protein purification, crystallization, and structural analysis.SOLUTION: A protein structure for membrane protein structural analysis can be bound to fluorescent protein and can be bound to immunoglobulin binding protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a protein structure for membrane protein structural analysis, a method for producing the same, and a method for purifying, crystallizing, and analyzing a membrane protein using the protein structure for membrane protein structural analysis. [Background technology]

[0002] Since the 1980s, development of tags suitable for "affinity purification" of recombinant proteins has been progressing. Since the 2000s, their uses have become more diverse, and GFP (see, for example, Non-Patent Documents 1-3) has been reported as a "reporter technology" for evaluating the thermal stability of membrane proteins and the search for detergents, T4 lysozyme and bRIL (see, for example, Non-Patent Documents 4-6) have been reported as a "thermal stability improvement technology" for G protein-coupled receptors, and Maltose Binding Protein (MBP) (see, for example, Non-Patent Document 7) has been reported as a "crystallization promotion technology" for low molecular weight proteins. In addition, a technology using antibody fragments (Fab and Fv-clasp) has been proposed for protein crystallization (see, for example, Non-Patent Documents 8-9).

[0003] However, these technological developments were based on specific approaches, and each technique could not be used in multiple situations. In addition, it was necessary to prepare a protein structure for structural analysis corresponding to the target protein. Furthermore, membrane proteins account for approximately 30% of the proteome of living organisms, and approximately 50% of these are estimated to be targets for drug discovery. Thus, elucidating the detailed three-dimensional structure of membrane proteins is considered extremely important not only from the perspective of basic research in which their molecular mechanisms are revealed, but also from the perspective of drug discovery research. However, due to their hydrophobic properties, crystallization is difficult, and the selection of the type of surfactant is also important, resulting in various constraints and making it difficult to achieve labor-saving and high-speed purification, crystallization, and structural analysis of membrane proteins.

[0004] Therefore, there is a strong demand for a universal technology that can be used in common for the purification, crystallization, and structural analysis of membrane proteins, which are challenges in structural biology research, including X-ray crystal structure analysis of membrane proteins. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] NATURE METHODS 2016 3:303-313 [Non-Patent Document 2] Structure 2006 14:673-681 [Non-Patent Document 3] SCIENCE 2016 353:664-669 [Non-Patent Document 4] Structure 2012 20:967-976 [Non-Patent Document 5] SCIENC 2007 318:1258-1265 [Non-Patent Document 6] NATURE 2013 499:444-450 [Non-Patent Document 7] PROTEIN SCIENCE 2016 25:559-571 [Non-Patent Document 8] Structure 2017 25:1611-1622 [Non-Patent Document 9] NATURE 2007 450:383-388 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to solve the above-mentioned problems in the prior art and to achieve the following object: That is, the present invention aims to provide a versatile technique that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins. [Means for solving the problem]

[0007] As a result of intensive research conducted by the inventors to achieve the above-mentioned object, they discovered that by adopting a protein structure for membrane protein structural analysis that is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, it is possible to provide a versatile technology that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins.

[0008] The present invention is based on the above findings by the present inventors, and the means for solving the above problems are as follows. <1> The protein structure is for use in analyzing the structure of a membrane protein, and is characterized in that it is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein. <2> A membrane protein and the <1> and a protein structure for membrane protein structure analysis according to the present invention. <3> This is a method for producing a chimeric protein, characterized by including a step of expressing a nucleic acid that is a fusion of a gene encoding a membrane protein and a gene encoding a protein structure for membrane protein structural analysis that is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein. <4> The present invention relates to a method for purifying a membrane protein, the method comprising the step of purifying a chimeric protein in which a membrane protein is fused with a protein structure for membrane protein structural analysis, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, using the immunoglobulin-binding protein. <5> The present invention relates to a method for crystallizing a membrane protein, the method comprising the step of crystallizing a chimeric protein in which a membrane protein is fused with a protein structure for membrane protein structural analysis, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein. <6> The method for analyzing the structure of a membrane protein comprises the steps of: purifying a chimeric protein, in which a membrane protein is fused with a protein structure for analyzing membrane protein structure, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, by using the immunoglobulin-binding protein; and crystallizing the chimeric protein to obtain a crystallized chimeric protein. Effect of the Invention

[0009] According to the present invention, a versatile technique that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins can be provided. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the construct of Example 1-1. [Diagram 2] FIG. 2 shows the purification results of Examples 1-3. [Diagram 3] FIG. 3 is a photograph showing the crystals of Examples 1-6. [Figure 4] FIG. 4 shows the construct of Example 2-1. [Diagram 5] FIG. 5 shows the purification results of Example 2-3. [Figure 6] FIG. 6 is a photograph showing the crystals of Example 2-6. [Figure 7] FIG. 7 shows the construct of Comparative Example 1-1. [Figure 8] FIG. 8 shows the purification results of Comparative Example 1-3. [Figure 9] FIG. 9 shows the construct of Comparative Example 2-1. [Figure 10] FIG. 10 shows the purification results of Comparative Example 2-3. [Figure 11] FIG. 11 shows the construct of Comparative Example 3-1. [Figure 12] FIG. 12 shows the purification results of Comparative Example 3-3. [Figure 13]FIG. 13 is a photograph showing the crystals of Comparative Example 3-6. [Figure 14] FIG. 14 shows the construct of Comparative Example 4-1. [Figure 15] FIG. 15 shows the purification results of Comparative Example 4-3. [Figure 16] FIG. 16 shows the construct of Comparative Example 5-1. [Figure 17] FIG. 17 shows the purification results of Comparative Example 5-3. [Figure 18] FIG. 18 is a photograph showing the crystals of Comparative Example 5-6. [Figure 19] FIG. 19 shows the construct of Comparative Example 6-1. [Figure 20] FIG. 20 shows the purification results of Comparative Example 6-3. [Figure 21] FIG. 21 shows the construct of Comparative Example 7-1. [Figure 22] FIG. 22 shows the purification results of Comparative Example 7-3. [Figure 23] FIG. 23 is a photograph showing the crystals of Comparative Example 7-6. [Figure 24] FIG. 24 shows the construct of Comparative Example 8-1. [Diagram 25] FIG. 25 shows the purification results of Comparative Example 8-3. [Figure 26] FIG. 26 shows the construct of Example 3-1. [Figure 27] FIG. 27 shows the purification results of Example 3-3. [Figure 28] FIG. 28 is a photograph showing the crystals of Example 3-6. [Figure 29] FIG. 29 is a diagram showing an electron density map of Example 3-7. [Diagram 30] FIG. 30 shows the final structure of Example 3-7. [Diagram 31] FIG. 31 shows the construct of Example 4-1. [Diagram 32] FIG. 32 shows the purification results of Example 4-3. [Diagram 33] FIG. 33 is a photograph showing the crystals of Example 4-6. [Diagram 34] FIG. 34 is a diagram showing an electron density map of Example 4-7. [Diagram 35] FIG. 35 shows the final structure of Example 4-7. [Diagram 36] FIG. 36 shows the construct of Example 5-1. [Figure 37] FIG. 37 is a photograph showing the crystals of Example 5-6. [Figure 38] FIG. 38 is a diagram showing an electron density map of Example 5-7. [Figure 39] FIG. 39 shows the final structure of Examples 5-7. [Diagram 40] FIG. 40 shows the construct of Example 6-1. [Diagram 41] FIG. 41 is a photograph showing the crystals of Example 6-6. [Diagram 42] FIG. 42 is a diagram showing an electron density map of Example 6-7. [Diagram 43] FIG. 43 shows the final structure of Examples 6-7. [Diagram 44] FIG. 44 is a photograph showing the crystals of Example 7-6. [Diagram 45] FIG. 45 is a diagram showing an electron density map of Example 7-7. [Diagram 46] FIG. 46 shows the final structure of Example 7-7. [Figure 47] FIG. 47 shows the construct of Example 8-1. [Figure 48] FIG. 48 is a photograph showing the crystals of Example 8-6. [Figure 49] FIG. 49 is a diagram showing an electron density map of Example 8-7. [Figure 50] FIG. 50 shows the final structure of Example 8-7. [Figure 51] FIG. 51 shows the construct of Example 9-1. [Figure 52]FIG. 52 is a photograph showing the crystals of Example 9-6. [Diagram 53] FIG. 53 is a diagram showing an electron density map of Example 9-7. [Figure 54] FIG. 54 shows the final structure of Example 9-7. [Figure 55] FIG. 55 shows the construct of Example 10-1. [Figure 56] FIG. 56 is a photograph showing the crystals of Example 10-6. [Figure 57] FIG. 57 shows the construct of Example 11-1. [Figure 58] FIG. 58 is a photograph showing the crystals of Example 11-6. [Figure 59] FIG. 59 is a photograph showing the crystals of Example 12. [Figure 60] FIG. 60 is a photograph showing the crystals of Example 13. [Figure 61] FIG. 61 is a photograph showing the crystals in a microgravity environment in Example 14. [Figure 62] FIG. 62 is a photograph showing the crystals of Example 14 on the ground. [Figure 63] FIG. 63 is a diagram showing an electron density map (under a microgravity environment) of Example 14. [Figure 64] FIG. 64 shows the final structure of Example 14 (under microgravity environment). [Figure 65] FIG. 65 is a diagram showing an electron density map (ground) of Example 14. [Figure 66] FIG. 66 shows the final structure (on the ground) of Example 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] (Protein structure for membrane protein structure analysis) The protein structure for membrane protein structure analysis is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein.

[0012] The fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include GFP, YFP, mCherry, mRFP1, DsRed, CFP, BFP, Sirius, mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mPlum, mNeptune, and NirFP. Among these, GFP, YFP, mCherry, mRFP1, and DsRed are preferred. These may be mutants.

[0013] The GFP is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, a wild-type GFP or a mutant GFP such as eGFP_A206K (F64L, S65T, A206K, H231L) or sfGFP (F64L, S65T, H231L, S30R, Y39N, N105T, Y145F, M153T, V163A, I171V, A206V). In addition, it may be one having a sequence such as His-tag calcium-binding domain (CBM-eGFP) added to the N-terminus.

[0014] The immunoglobulin-binding protein is not particularly limited and can be appropriately selected depending on the purpose. Examples of the immunoglobulin-binding protein include protein A, protein A / G, and protein G. Among these, protein A is preferable.

[0015] The molecular weight of the protein structure for membrane protein structural analysis is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 200 kDa or less, more preferably 100 kDa or less, even more preferably 30 kDa or less, and particularly preferably 15 kDa or less.

[0016] The protein structure for membrane protein structure analysis preferably contains a VHH domain (nanobody) of a camelid antibody, which is known to bind to immunoglobulin-binding proteins (Molecular & Cellular Proteomics 2008 7:282-289). The VHH domain of the Camelidae antibody is not particularly limited and can be appropriately selected depending on the purpose, but is preferably a VHH domain of a Camelidae antibody that recognizes the fluorescent protein.

[0017] The VHH domain of the camelid antibody that recognizes the fluorescent protein is not particularly limited and can be selected appropriately depending on the purpose, and examples include those having the insert gene sequence of addgene #61838 (GFP nanobody) or the insert gene sequence of addgene #70696 (mCherry nanobody).

[0018] (chimeric protein) The chimeric protein comprises a membrane protein and a protein structure for analyzing a membrane protein structure.

[0019] The membrane protein is not particularly limited as long as it is a protein attached to a biological membrane such as a cell or an organelle, and can be appropriately selected depending on the purpose. The membrane protein may be an integral membrane protein or a surface membrane protein. The integral membrane protein is not particularly limited and can be appropriately selected depending on the purpose. Examples of the integral membrane protein include channels, transporters, receptors, and enzymes. The surface membrane protein is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be a supporting protein.

[0020] The molecular weight of the membrane protein is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 200 kDa or less, more preferably 100 kDa or less, and even more preferably 30 kDa or less.

[0021] Specific examples of the membrane protein include the voltage sensor domain (VSD) of voltage-dependent phosphatase (VSP), the sugar transporter SWEET family protein SemiSWEET, translocator protein (BcTSPO), riboflavin transporter (TmRibU), and the β domain of intimin.

[0022] The VSP-VSD is not particularly limited and can be appropriately selected depending on the purpose. Examples of the VSP-VSD include VSP-VSD derived from D. rerio (Dr-VSP-VSD) and VSP-VSD derived from C. intestinalis (Ci-VSP-VSD). VSP-VSD in a resting state (wild type) Rest , an activated intermediate mutant form of VSP-VSD Int , the active state mutant VSP-VSD Act may be also possible.

[0023] The VSP-VSD of the active state mutant Act There are no particular limitations to the mutant, and the mutant may be appropriately selected depending on the purpose. Examples include a mutant in which T156 and I165 in the S4 helix are mutated to arginine (derived from D. Rerio), and a mutant in which R217 is mutated to glutamic acid (derived from C. intestinalis).

[0024] The VSP-VSD of the intermediate activation mutant Int There are no particular limitations on the type of the mutant, and the mutant may be appropriately selected depending on the purpose. For example, a mutant (derived from D. Rerio) in which T156 and I165 in the S4 helix are mutated to arginine, and R153 is mutated to glutamic acid may be mentioned. Furthermore, the VSP-VSD may be a VSP-VSD in which amino acid residues at the N-terminus of VSP-VSD, which is an unstable region having no secondary structure, are deleted. The VSP-VSD in which amino acid residues at the N-terminus of the VSP-VSD have been deleted is not particularly limited and can be appropriately selected depending on the purpose. Examples include Dr-VSP-VSD in which 40 amino acid residues have been deleted at the N-terminus of VSP-VSD, Dr-VSP-VSD in which 29 amino acid residues have been deleted at the N-terminus of VSP-VSD, and Ci-VSP-VSD in which 101 amino acid residues have been deleted at the N-terminus of VSP-VSD.

[0025] The SemiSWEET is not particularly limited and can be appropriately selected depending on the purpose. For example, SemiSWEET derived from Escherichia coli can be mentioned. The BcTSPO is not particularly limited and can be appropriately selected depending on the purpose. For example, BcTSPO derived from Bacillus cereus can be mentioned. The TmRibU is not particularly limited and can be appropriately selected depending on the purpose. For example, TmRibU derived from the hyperthermophilic bacterium Thermotoga maritima can be mentioned. The intimin β domain is not particularly limited and can be appropriately selected depending on the purpose. Examples include the β domain of intimin derived from enterohemorrhagic Escherichia coli O157:H7.

[0026] The membrane protein may be tagged with a fluorescent protein for confirming the expression of the membrane protein, a protein tag that acts as a molecular chaperone to improve solubilization efficiency, a His tag, or the like.

[0027] The protein structure for membrane protein structural analysis is as described above.

[0028] (Method of Producing Chimeric Protein) The method for producing the chimeric protein includes a step of expressing a nucleic acid obtained by fusing a gene encoding a membrane protein with a gene encoding a protein structure for membrane protein structural analysis capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, and may further include other steps.

[0029] The membrane protein and the protein structure for membrane protein structural analysis are as described above.

[0030] The fusion is not particularly limited and can be appropriately selected depending on the purpose. For example, the fusion may be fusion of the N-terminus or C-terminus of a gene encoding a membrane protein with a gene encoding a protein structure for membrane protein structural analysis. The fusion terminal region of the membrane protein and the protein construct for membrane protein structural analysis preferably has a secondary structure. The length of the linker in the secondary structure is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 16 amino acid residues or less, more preferably 6 amino acid residues or less, even more preferably 4 amino acid residues or less, and particularly preferably 2 amino acid residues or less.

[0031] The expression is not particularly limited and can be appropriately selected depending on the purpose. Examples of the expression include a method of preparing a plasmid containing a fusion gene between the N-terminus or C-terminus of the gene encoding the membrane protein and a gene encoding the protein structure for membrane protein structural analysis, and expressing the fusion protein using a baculovirus expression system, a method of expressing the fusion protein using an Escherichia coli expression system, a method of expressing the fusion protein using insect cells, a method of expressing the fusion protein using mammalian cells, and a method of expressing the fusion protein using a cell-free protein synthesis system.

[0032] The other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, the other steps include a step of recovering the expressed fusion protein and a step of solubilizing the fusion protein.

[0033] The step of recovering the expressed fusion protein is not particularly limited and can be appropriately selected depending on the purpose. For example, a method can be used in which the cell culture medium in which the fusion protein is expressed is centrifuged, the cells are disrupted, and then the cells are further centrifuged.

[0034] The step of solubilizing the fusion protein is not particularly limited and can be appropriately selected depending on the purpose. For example, a method of solubilizing the membrane fraction obtained in the step of recovering the fusion protein by adding a surfactant can be mentioned. The surfactant is not particularly limited and can be appropriately selected depending on the purpose. Examples of the surfactant include CYMAL-based surfactants such as CYMAL-5, CYMAL-6, and CYMAL-7; NG class surfactants such as lauryl maltose neopentyl glycol (LMNG), decyl maltose neopentyl glycol (DMNG), and octyl glucose neopentyl glycol (OGNG); maltoside-based surfactants such as dodecyl maltoside (DDM) and decyl maltoside (DM); and glucoside-based surfactants such as octyl glucoside (OG) and sucrose monolaurate (SM).

[0035] (Membrane protein purification method) The method for purifying a membrane protein includes a step of purifying a chimeric protein in which a membrane protein is fused with a protein structure for membrane protein structural analysis, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, using the immunoglobulin-binding protein, and may further include other steps.

[0036] The purification method is not particularly limited and can be appropriately selected depending on the purpose. Examples of the purification method include affinity chromatography, ion exchange chromatography, gel filtration, and a combination of these.

[0037] The affinity chromatography is not particularly limited and can be appropriately selected depending on the purpose. Examples of the affinity chromatography include a method using a column packed with the immunoglobulin-binding protein and a method using a resin to which the immunoglobulin-binding protein is bound.

[0038] The other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, the other steps include a step of electrophoresing the purified chimeric protein and a step of concentrating the purified chimeric protein.

[0039] The electrophoresis method used in the step of electrophoresing the purified chimeric protein is not particularly limited and can be appropriately selected depending on the purpose. Examples of the electrophoresis method include SDS-PAGE, native-PAGE, isoelectric focusing, and two-dimensional electrophoresis.

[0040] The method for concentrating the purified chimeric protein in the step is not particularly limited and can be appropriately selected depending on the purpose. For example, the method may be concentration using an ultrafiltration membrane.

[0041] (Membrane protein crystallization method) The method for crystallizing a membrane protein includes a step of crystallizing a chimeric protein in which a membrane protein is fused with a protein structure for membrane protein structural analysis, which is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, and may further include other steps.

[0042] The crystallization method is not particularly limited and can be appropriately selected depending on the purpose. Examples of the crystallization method include the lipid cubic phase method (LCP method), the vapor diffusion method (VD method), the Bicelle method, the HiLiDe method, and the counter diffusion method.

[0043] The LCP method is not particularly limited and can be appropriately selected depending on the purpose. For example, the method described in M. Caffrey & V. Cherezov, Nat. Protoc. 2013 can be mentioned.

[0044] The VD method is not particularly limited and can be appropriately selected depending on the purpose. For example, the method described in Oxford University Press, 1992, Crystallization of nucleic acids and proteins. A practical approach; edited by A. Ducruix and R. Giegk, can be mentioned.

[0045] The Bicelle method is not particularly limited and can be appropriately selected depending on the purpose. For example, the method described in Methods 2011 55:337-341 can be mentioned.

[0046] The HiLiDe method is not particularly limited and can be appropriately selected depending on the purpose. For example, the method described in Cryst. Growth Des. 2011 11:2098-2106 can be mentioned.

[0047] The counter diffusion method is not particularly limited and can be appropriately selected depending on the purpose. For example, the method described in JM Garcia-Ruiz et al., Meth. Enzymol. 2002 368:130-54 can be mentioned.

[0048] The method for crystallizing a membrane protein can further include the step of adding a fluorescent protein to the chimeric protein.

[0049] The fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include GFP, YFP, mCherry, mRFP1, DsRed, CFP, BFP, Sirius, mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mPlum, mNeptune, and NirFP. Among these, GFP, YFP, mCherry, mRFP1, and DsRed are preferred. These may be mutants.

[0050] The GFP is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be a wild-type GFP or a mutant GFP such as eGFP_A206K (F64L, S65T, A206K, H231L) or sfGFP (F64L, S65T, H231L, S30R, Y39N, N105T, Y145F, M153T, V163A, I171V, A206V). Among these, eGFP_A206K or sfGFP is preferable. Furthermore, the GFP may have a sequence such as a His tag or a calcium-binding domain (CBM-eGFP) added to the N-terminus thereof. By adding a sequence such as a His tag or a calcium-binding domain, crystals can be obtained under different conditions.

[0051] (Methods for structural analysis of membrane proteins) The method for analyzing the structure of a membrane protein includes a step of purifying a chimeric protein in which a membrane protein is fused with a protein structure for analyzing membrane protein structure, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, using the immunoglobulin-binding protein, and a step of crystallizing the chimeric protein to obtain a crystallized chimeric protein, and may further include other steps.

[0052] The step of purifying a chimeric protein in which a protein structure for membrane protein structural analysis capable of binding to a fluorescent protein and to an immunoglobulin-binding protein is fused to the membrane protein, using the immunoglobulin-binding protein, is as described above. The step of crystallizing the chimeric protein to obtain a crystallized chimeric protein is as described above.

[0053] The method for analyzing the structure of a membrane protein can further include a step of adding a fluorescent protein to the chimeric protein.

[0054] The fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include GFP, YFP, mCherry, mRFP1, DsRed, CFP, BFP, Sirius, mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mPlum, mNeptune, and NirFP. Among these, GFP, YFP, mCherry, mRFP1, and DsRed are preferred. These may be mutants.

[0055] The GFP is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be a wild-type GFP or a mutant GFP such as eGFP_A206K (F64L, S65T, A206K, H231L) or sfGFP (F64L, S65T, H231L, S30R, Y39N, N105T, Y145F, M153T, V163A, I171V, A206V). Among these, eGFP_A206K or sfGFP is preferable. Furthermore, the GFP may have a sequence such as a His tag or a calcium binding domain added to the N-terminus thereof. By adding a sequence such as a His tag or a calcium binding domain (CBM-eGFP), crystals can be obtained under different conditions.

[0056] The other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, the other steps include a step of analyzing the structure of the crystallized chimeric protein.

[0057] The structural analysis step for the crystallized chimeric protein is not particularly limited and can be appropriately selected depending on the purpose. For example, diffraction data can be collected from the obtained crystals using the high-flux microbeam beamline (BL32XU) at the large synchrotron radiation facility (SPring-8), and further, an initial phase is determined by molecular replacement using a search model, and the initial structure is then determined, and then the final structure is obtained.

[0058] Factors that make structural analysis of membrane proteins difficult include: 1. limitations on protein tag fusion, 2. low thermal stability, 3. difficulty in purification, 4. difficulty in crystallization, and 5. low resolution.

[0059] Regarding the above point 1, in the conventional method, it was necessary to select a protein tag depending on the topology of the target protein, but when the protein construct for membrane protein structural analysis is used, there is no such restriction.

[0060] Regarding point 2, in the conventional method, it was necessary to take measures such as "introducing mutations," "searching for fusion protein tags," and "additives" that contribute to improving thermal stability, and searching for orthologs with higher thermal stability. However, when the protein construct for membrane protein structural analysis is used, the addition of a fluorescent protein makes it easy to perform micromeasurements using a fluorescence detector, enabling high-throughput screening of these conditions.

[0061] Regarding 3 above, since the protein has low thermal stability and is difficult to handle, in the conventional method, high affinity purification or the like was required in a short period of time. However, when the protein construct for membrane protein structural analysis is used, high affinity purification can be achieved by utilizing binding with an immunoglobulin-binding protein. Furthermore, the addition of a fluorescent protein enables high-throughput screening of purification conditions by micromeasurement using a fluorescence detector and visual confirmation of the presence or absence of protein, thereby simplifying the purification process.

[0062] Regarding point 4 above, in conventional methods, crystallization required measures such as protein design, such as the selection of fusion protein tags depending on the crystallization method, such as the vapor diffusion method, the Bicelle method, or the LCP method, but when using the protein construct for membrane protein structure analysis, the crystallization hit rate can be dramatically improved by adding a fluorescent protein, regardless of the crystallization method. In addition, since the crystals exhibit a color dependent on the fluorescent protein, crystal observation and crystal picking become easier, and human errors such as "overlooking crystals" can be reduced.

[0063] Regarding point 5 above, in the conventional method, it was necessary to improve the purification purity, optimize or reconsider the crystallization conditions, redesign the protein, create antibody fragments, etc., but when using the protein structure for membrane protein structure analysis, different crystallization conditions can be easily explored by simply screening the types of fluorescent protein variants, which increases the possibility of successfully improving the resolution. This significantly shortens the time required compared to protein redesign, etc., and accelerates research. EXAMPLES

[0064] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0065] Example 1: GFP-Nb-Dr-VSP-VSD Int > Example 1-1: Construction of construct (SEQ ID NO: 1) The cDNA of VSP-VSD from D. rerio was obtained from Professor Okamura of the Department of Integrative Physiology, Graduate School of Medicine, Osaka University. For structural analysis, we used the activation intermediate mutant (T156R, I165R, R153E), Dr-VSP-VSD Int The cDNA of GFP-Nb was obtained from GeneScript using artificial gene synthesis, with the amino acid sequence of PDBID: 3OGO as a reference. Dr-VSP-VSD IntA fusion protein in which GFP-nabobody (1-115) was linked to the N-terminus of the 41-188 amino acid sequence of GFP with a two-residue linker (Gly-Ser): GFP-Nb-Dr-VSP-VSD Int A construct for this purpose was designed (Figure 1). Furthermore, to confirm expression and perform affinity purification, cDNA was prepared with MBP, His tag, Myc tag, and TEV protease recognition sequence at the N-terminus, and the cDNA was inserted into pFastBac1 to prepare plasmid DNA.

[0066] Example 1-2: Protein expression and cell membrane preparation The P2 recombinant baculovirus was produced from the plasmids prepared according to the protocol of the Bac-to-Bac Baculovirus Expression System (Thermo Fisher Scientific). Protein expression was performed using Sf9 cells at a cell concentration of 4.0 × 10 6 An appropriate amount of P2 virus solution was added at 120 rpm at 28°C for 24 hours, and then the temperature was lowered to 20°C and cultured at 120 rpm for 48 hours. The collected insect cells were suspended in ice-cold Buffer A (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 5 mM Benzamidine) and disrupted using an ultrasonic homogenizer. The disrupted solution was centrifuged at 10,000g for 10 minutes at 4°C, and the supernatant was ultracentrifuged at 45,000 rpm (himac P45AT rotor) for 1 hour at 4°C to collect the cell membrane fraction. The cell membrane was suspended in Buffer A and homogenized using a Dounce homogenizer, and then ultracentrifuged at 45,000 rpm (himac P45AT) for 1 hour at 4°C to wash out water-soluble proteins and membrane-associated proteins. The cell membrane was washed again by the same procedure.

[0067] Example 1-3: Solubilization and purification of membrane proteins The washed cell membranes were suspended in Buffer B (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM imidazole, 2 mM DTT) and homogenized using a Dounce homogenizer. Then, 20% (w / v) DDM (n-Dodecyl-β-D-Maltopyranoside) was added to a final concentration of 2% (w / v). The mixture was incubated at 4°C for 1 hour with gentle stirring to obtain GFP-Nb-Dr-VSP-VSD. Int The insoluble fraction from the solubilized sample was removed by ultracentrifugation, and Ni-NTA resin was added to the supernatant and incubated at 4°C for 1 hour with gentle stirring. Unbound components were removed using an Econocolumn (Bio-Rad), and the Ni-NTA resin was washed with Buffer C (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM imidazole, 2 mM DTT, 0.08% (w / v) DDM). Next, the surfactant was replaced and the resin was washed with Buffer D (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM imidazole, 2 mM DTT, 0.12% (w / v) DMNG (Decyl Maltose Neopentyl Glycol)), and the GFP-Nb-Dr-VSP-VSD was purified with Buffer E (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 250 mM imidazole, 2 mM DTT, 0.03% (w / v) DMNG). Int The eluted fraction was mixed with an appropriate amount of His-tagged TEV protease and transferred to a dialysis tube, and dialyzed overnight against Buffer F (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.03% (w / v) DMNG) at 4°C. To remove the tag fragment containing MBP cleaved by TEV protease, the sample was added to Ni-NTA resin and incubated at 4°C for 1 hour with gentle stirring. GFP-Nb-Dr-VSP-VSD was purified using an Econo column. IntThe flow-through fraction containing eGFP and the Ni-NTA resin washing solution with Buffer F were collected and concentrated using an ultrafiltration membrane. The concentrated sample was diluted 10-fold with Buffer G (20 mM MES-Na [pH 6.0], 2 mM DTT, 0.03% (w / v) DMNG) and adsorbed onto a HiTrap SP HP (GE Healthcare), followed by cation exchange chromatography using Buffer G and Buffer H (20 mM MES-Na [pH 6.0], 2 mM DTT, 0.03% (w / v) DMNG, 1 M NaCl). The collected fractions were determined from the chromatogram and the results of SDS-PAGE electrophoresis, and eGFP_A206K produced as described below was converted to GFP-Nb-Dr-VSP-VSD Int The complex was prepared by adding 2x the molar ratio of eGFP_A206K and leaving it on ice for 1 hour. The complex was then concentrated using an ultrafiltration membrane and loaded onto a Superdex200 Increase 10 / 300 GL (GE Healthcare) equilibrated with Buffer I (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT, 0.03% (w / v) DMNG) to remove excess eGFP_A206K and multimeric components. The final purified preparation was concentrated to 20 mg / mL using an ultrafiltration membrane.

[0068] The results of gel filtration chromatography are shown in Figure 2A. In Figure 2A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. Samples indicated by the black lines in Figure 2A were collected and subjected to SDS-PAGE electrophoresis (Figure 2B: CBB staining, lane 1: molecular weight marker (M), lanes 2-12: fractions indicated by the black lines in Figure 2A from the left, lane 13: molecular weight marker (M)). The fractions shown in Figure 2B were collected.

[0069] (eGFP Production 1: Construct: SEQ ID NO:2) The cDNA of eGFP, an improved GFP, was obtained from pEGFP-N1 (Clontech). The expression region of 2-239, which contains the A206K mutation that makes it a monomer in solution, was modified to have a TEV protease recognition sequence at the N-terminus, and the cDNA was inserted into pET28b(+) using the Bam HI and Xho I restriction enzyme sites to prepare a plasmid. Upstream of the TEV protease sequence is a His tag derived from pET28b(+).

[0070] (eGFP Production 2: Protein Expression and Purification) The prepared plasmid (SEQ ID NO: 2) was transformed into BL21-CodonPlus(DE3)-RIPL (Agilent Technologies) and cultured in large quantities. Protein expression of eGFP_A206K was induced by adding IPTG to a final concentration of 0.5 mM when the turbidity was approximately OD600 = 0.5, and then the cells were cultured overnight at 20°C with a rotary gyration at 120 rpm. The collected E. coli was suspended in ice-cold Buffer A (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 5 mM Benzamidine, 2 mM DTT, 25 mM Imidazole) and disrupted using an ultrasonic homogenizer. The disrupted solution was centrifuged at 10,000g for 10 minutes at 4°C, and the supernatant was centrifuged at 40,000g (Beckman Coulter JA-25.50 rotor) for 30 minutes at 4°C, and the supernatant fraction was collected. Ni-NTA resin was added to the supernatant and incubated at 4°C for 1 hour with gentle stirring. Unbound components were removed using an Econo column, and the Ni-NTA resin was washed with Buffer A. Next, eGFP_A206K was eluted with Buffer B (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT, 250 mM imidazole). An appropriate amount of His-tagged TEV protease was added to the eluted fraction, which was transferred to a dialysis tube and dialyzed overnight against Buffer C (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT, 1 mM EDTA) at 4°C. To remove the His-tag cleaved by TEV protease, the sample was added to the Ni-NTA resin and incubated at 4°C for 1 hour with gentle stirring. The flow-through fraction containing eGFP_A206K and the resin washing solution with Buffer C were collected using an Econocolumn, concentrated using an ultrafiltration membrane, and subjected to gel filtration chromatography using HiLoad 16 / 600 Superdex200 pg equilibrated with Buffer D (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT). The fraction containing monomeric eGFP_A206K was collected and concentrated to 10 mg / mL using an ultrafiltration membrane.

[0071] Example 1-4: Preparation of LCP Monoolein stored at -30°C was heated to 42°C until it became transparent, and then filled into a gas-tight syringe (Hamilton) using a micropipette and the weight was measured. Then, a plunger (Hamilton) was set and the monoolein was pushed up to the tip of the syringe and moved. A sample with a weight 0.67 times that of monoolein was filled into another syringe and pushed up to the tip of the syringe. The two syringes were connected with a coupler and the plungers were alternately pushed (about 100 times) to thoroughly mix the sample and monoolein and prepare an LCP. Finally, the entire amount of the solution was moved to one of the syringes and left to stand at 20°C for about 1 hour.

[0072] Examples 1-5: Crystallization Screening A needle was connected to the syringe containing the LCP, and the syringe was attached to a microdispenser. 50 nL of LCP was dispensed into each well of a 96-well glass plate with a double-sided seal attached, and then 800 nL of reservoir solution was added. When adding the reservoir solution, the operation was performed quickly by using an 8-series micropipette to prevent the LCP from drying. When dispensing 50 nL of LCP, the LCP was moistened with a humidifier to prevent lipid phase transition. The screening solution described in the reference (Methods Enzymol. 2015 557:417-37) was used as the reservoir solution. Immediately after adding the reservoir solution, the drop was covered with a slide glass to seal it and left at 20 ° C.

[0073] Examples 1-6: Crystallization of membrane proteins Crystals were obtained using the precipitant 0.1 M HEPES-Na [pH 7.5], 0.05 M ammonium acetate, and 29-32.5% (v / v) PEG 400 (Figure 3). The scale bar indicates 0.1 mm.

[0074] Examples 1-7: X-ray diffraction experiments and structural analysis Diffraction data were collected at SPring-8 BL32XU using the "ZOO system" for automated measurement, and the resolution was 2.45 Å (Table 1). The initial phase was determined by molecular replacement using MOLREP (A. Vagin & A. Teplyakov, Acta Crystallogr. D, 2000) with the structure of the GFP-nanobody-eGFP complex (PDBID = 3OGO) as a search model. The initial structure of almost the entire region was determined by molecular replacement using the Ci-VSP-VSD (PDBID = 4G7V) as a search model for the obtained model. Based on this structure, the initial structure of Ci-VSP-VSD was determined using REFMAC and Coot (P. Emsley and K Cowtan, 2004 Acta Crystallogr. D Biol. Crystallogr.). Int The amino acid sequence was revised and refined to obtain the final structure (Table 1).

[0075] [Table 1]

[0076] Example 2: Dr-VSP-VSD Int -GFP-Nb> Example 2-1: Construction of construct (SEQ ID NO: 3) Dr-VSP-VSD Int A fusion protein in which GFP-nabobody (1-115) was linked to the C-terminus of the 41-188 amino acid sequence of Dr-VSP-VSD via a two-residue linker (Gly-Ser): Int A construct was designed in the same manner as in Example 1-1, except that a construct for -GFP-Nb was designed (Figure 4).

[0077] Examples 2-2 to 2-3 The same procedures as in Examples 1-2 to 1-3 were carried out, except that sfGFP produced as described below was added to the sample instead of eGFP_A206K.

[0078] (sfGFP Production 1: Construct: SEQ ID NO: 4) The cDNA of sfGFP, one of the improved GFPs, was prepared by introducing mutations into the template sequence of SEQ ID NO: 2. A cDNA was prepared with a TEV protease recognition sequence at the N-terminus, and inserted into pET28b(+) using the restriction enzyme sites Bam HI and Xho I to prepare a plasmid. Upstream of the TEV protease sequence, there is a His tag derived from pET28b(+).

[0079] (Production of sfGFP 2: Protein expression and purification) Preparation of eGFP 2: Protein expression and purification were carried out in the same manner.

[0080] The results of gel filtration chromatography are shown in Figure 5A. In Figure 5A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. Samples indicated by black lines in Figure 5A were collected and subjected to SDS-PAGE electrophoresis (Figure 5B: CBB staining, lane 1: molecular weight marker (M), lanes 2-13: fractions indicated by black lines in Figure 5A from the left, lane 14: molecular weight marker (M)). The fractions shown in Figure 5B were collected.

[0081] Examples 2-4 to 2-7 As a result of carrying out the same procedure as in Examples 1-4 to 1-7, crystals were obtained using the precipitant 0.1 M HEPES-Na [pH 7.5], 0.1 M magnesium sulfate, and 29% (v / v) PEG 300 (FIG. 6). The results of processing the collected data are shown in Table 2.

[0082] [Table 2]

[0083] <Comparative example 1: bRIL-Dr-VSP-VSD Int > Comparative Example 1-1: Preparation of construct (SEQ ID NO: 5) A construct was designed in the same manner as in Example 1-1, except that the cDNA of GFP-Nb was replaced with the cDNA of bRIL (FIG. 7). The cDNA of bRIL was obtained from GeneScript using artificial gene synthesis, with reference to the amino acid sequence of PDBID:1M6T.

[0084] Comparative Examples 1-2 to 1-3 The same procedures as in Examples 1-2 to 1-3 were carried out, except that purification by ion exchange chromatography was not performed, and eGFP_A206K was not added to the purified chimeric protein.

[0085] The results of gel filtration chromatography are shown in FIG. 8A. In FIG. 8A, the vertical axis indicates absorbance at a wavelength of 280 nm or 254 nm, and the horizontal axis indicates the elution volume. As a result of gel filtration chromatography, one peak was obtained at about 14 mL. The sample shown by the black line in FIG. 8A was collected and subjected to SDS-PAGE electrophoresis (FIG. 8B: CBB staining, lane 1: molecular weight marker (M), lanes 2-11: fractions shown by the black line in FIG. 8A in order from the left, lane 12: molecular weight marker (M)). The fractions shown in FIG. 8B were collected.

[0086] Comparative Examples 1-4 to 1-6 When the same procedure as in Examples 1-4 to 1-6 was carried out, no crystals were obtained.

[0087] <Comparative example 2: Dr-VSP-VSD Int -bRIL> Comparative Example 2-1: Preparation of construct (SEQ ID NO: 6) A construct was designed in the same manner as in Example 2-1, except that the cDNA of GFP-Nb was replaced with the cDNA of bRIL (FIG. 9). The cDNA of bRIL was obtained from GeneScript using artificial gene synthesis, with reference to the amino acid sequence of PDBID:1M6T.

[0088] Comparative Examples 2-2 to 2-3 The same procedures as in Examples 1-2 to 1-3 were carried out, except that purification by ion exchange chromatography was not performed, and eGFP_A206K was not added to the purified chimeric protein.

[0089] The results of gel filtration chromatography are shown in Figure 10A. In Figure 10A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. Samples indicated by black lines in Figure 10A were collected and subjected to SDS-PAGE electrophoresis (Figure 10B: CBB staining, lane 1: molecular weight marker (M), lanes 2-11: fractions indicated by black lines in Figure 10A, from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 10B were collected.

[0090] Comparative Examples 2-4 to 2-6 When the same procedure as in Examples 1-4 to 1-6 was carried out, no crystals were obtained.

[0091] <Comparative example 3: T4L-Dr-VSP-VSD Int > Comparative Example 3-1: Preparation of construct (SEQ ID NO: 7) A construct was designed in the same manner as in Example 1-1, except that the cDNA of GFP-Nb was replaced with the cDNA of T4L (FIG. 11). The cDNA of T4L was obtained from GeneScript using artificial gene synthesis, with reference to the amino acid sequence of PDBID:212L.

[0092] Comparative Examples 3-2 to 3-3 The same procedures as in Examples 1-2 to 1-3 were performed using Buffer G (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG) and Buffer H (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG, 1 M NaCl) and omitting the addition of eGFP_A206K to the purified chimeric protein.

[0093] The results of gel filtration chromatography are shown in Figure 12A. In Figure 12A, the vertical axis indicates absorbance at a wavelength of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. Samples indicated by black lines in Figure 12A were collected and subjected to SDS-PAGE electrophoresis (Figure 12B: CBB staining, lane 1: molecular weight marker (M), lane 2: filtrate from ultrafiltration filter concentrator, lanes 3-11: samples indicated by black lines in Figure 12A from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 12B were collected.

[0094] Comparative Examples 3-4 to 3-7 The same procedure as in Examples 1-4 to 1-7 was carried out, and crystals were obtained using a precipitant of 0.1 M HEPES-Na [pH 7.0], 0.268 M sodium phosphate dibasic dihydrate, and 30% (v / v) PEG400 (FIG. 13), but no diffraction points were obtained.

[0095] <Comparative example 4: Dr-VSP-VSD Int -T4L> Comparative Example 4-1: Preparation of construct (SEQ ID NO: 8) A construct was designed in the same manner as in Example 2-1, except that the cDNA of GFP-Nb was replaced with the cDNA of T4L (FIG. 14). The cDNA of T4L was obtained from GeneScript using artificial gene synthesis, with reference to the amino acid sequence of PDBID:212L.

[0096] Comparative Examples 4-2 to 4-3 The same procedures as in Examples 1-2 to 1-3 were performed using Buffer G (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG) and Buffer H (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG, 1 M NaCl) and omitting the addition of eGFP_A206K to the purified chimeric protein.

[0097] The results of gel filtration chromatography are shown in Figure 15A. In Figure 15A, the vertical axis indicates absorbance at a wavelength of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. The samples shown by the black lines in Figure 15A were subjected to SDS-PAGE electrophoresis (Figure 15B: CBB staining, lane 1: molecular weight marker (M), lane 2: filtrate from the ultrafiltration filter concentrator, lanes 3-11: samples shown by the black lines in Figure 15A from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 15B were collected.

[0098] Comparative Examples 4-4 to 4-6 When the same procedure as in Examples 1-4 to 1-6 was carried out, no crystals were obtained.

[0099] <Comparative example 5: eGFP-Dr-VSP-VSD Int > Comparative Example 5-1: Preparation of construct (SEQ ID NO: 9) A construct was designed in the same manner as in Example 1-1, except that the cDNA of GFP-Nb was replaced with the cDNA of eGFP (FIG. 16). The cDNA of eGFP, which is one of the improved GFPs, was obtained from pEGFP-N1 (Clontech), and the A206K mutation was introduced.

[0100] Comparative Examples 5-2 to 5-3 The same procedures as in Examples 1-2 to 1-3 were repeated, except that the ion exchange column in the ion exchange chromatography was changed from HiTrap SP HP to anion exchange chromatography HiTrap Q HP (GE Healthcare), Buffer G (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG) and Buffer H (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG, 1 M NaCl) were used, and eGFP_A206K was not added to the purified chimeric protein.

[0101] The results of gel filtration chromatography are shown in FIG. 17A. In FIG. 17A, the vertical axis indicates absorbance at a wavelength of 280 nm or 254 nm, and the horizontal axis indicates the elution volume. As a result of gel filtration chromatography, one peak was obtained at about 13.7 mL. The sample shown by the black line in FIG. 17A was collected and subjected to SDS-PAGE electrophoresis (FIG. 17B: CBB staining, lane 1: molecular weight marker (M), lanes 2-11: fractions shown by the black line in FIG. 17A from the left, lane 12: molecular weight marker (M)). The fractions shown in FIG. 17B were collected.

[0102] Comparative Examples 5-4 to 5-7 When the same procedure as in Examples 1-4 to 1-7 was carried out, crystals were obtained using a precipitant of 0.1 M HEPES-Na [pH 7.0], 0.1 M sodium nitrate, and 30% (v / v) PEG300 (FIG. 18), and the resolution was about 6 Å.

[0103] <Comparative example 6: Dr-VSP-VSD Int -eGFP> Comparative Example 6-1: Preparation of construct (SEQ ID NO: 10) A construct was designed in the same manner as in Example 2-1, except that the cDNA of GFP-Nb was replaced with the cDNA of eGFP (FIG. 19). The cDNA of eGFP, which is one of the improved GFPs, was obtained from pEGFP-N1 (Clontech), and the A206K mutation was introduced.

[0104] Comparative Examples 6-2 to 6-3 The same procedures as in Examples 1-2 to 1-3 were repeated, except that the ion exchange column in the ion exchange chromatography was changed from HiTrap SP HP to anion exchange chromatography HiTrap Q HP, Buffer G (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG) and Buffer H (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG, 1 M NaCl) were used, and eGFP_A206K was not added to the purified chimeric protein.

[0105] The results of gel filtration chromatography are shown in Figure 20A. In Figure 20A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. Samples indicated by black lines in Figure 20A were collected and subjected to SDS-PAGE electrophoresis (Figure 20B: CBB staining, lane 1: molecular weight marker (M), lanes 2-13: fractions indicated by black lines in Figure 20A, from the left, lane 14: molecular weight marker (M)). The fractions shown in Figure 20B were collected.

[0106] Comparative Examples 6-4 to 6-6 When the same procedure as in Examples 1-4 to 1-6 was carried out, no crystals were obtained.

[0107] <Comparative example 7:MBP-mutantE-Dr-VSP-VSD> Comparative Example 7-1: Preparation of construct (SEQ ID NO: 11) A construct was designed in the same manner as in Example 1-1, except that the cDNA of GFP-Nb was replaced with the cDNA of MBP-mutantE (PROTEIN SCIENCE 2010 19:901-913), a mutant of MBP that promotes crystallization (FIG. 21). The cDNA of MBP-mutantE was prepared by introducing a mutation into MBP of pMAL-c2x (New England Biolabs).

[0108] Comparative Examples 7-2 to 7-3 The same procedures as in Examples 1-2 to 1-3 were performed using Buffer G (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG) and Buffer H (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG, 1 M NaCl) and omitting the addition of eGFP_A206K to the purified chimeric protein.

[0109] The results of gel filtration chromatography are shown in Figure 22A. In Figure 22A, the vertical axis indicates absorbance at a wavelength of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. Samples indicated by black lines in Figure 22A were collected and subjected to SDS-PAGE electrophoresis (Figure 22B: CBB staining, lane 1: molecular weight marker (M), lanes 2-11: samples indicated by black lines in Figure 22A from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 22B were collected.

[0110] Comparative Examples 7-4 to 7-7 As in Examples 1-4 to 1-7, crystals were obtained using a precipitant of 0.1 M HEPES-Na [pH 7.0], 0.1-0.15 M lithium chloride, and 29-31% (v / v) PEG300 (FIG. 23), and the resolution was about 5 Å.

[0111] <Comparative example 8: Dr-VSP-VSD-MBP-mutantE> Comparative Example 8-1: Preparation of construct (SEQ ID NO: 12) A construct was designed in the same manner as in Example 2-1, except that the cDNA of GFP-Nb was replaced with the cDNA of MBP-mutantE, a mutant of MBP that promotes crystallization (FIG. 24). The cDNA of MBP-mutantE was prepared by introducing a mutation into MBP in pMAL-c2x (New England Biolabs).

[0112] Comparative Examples 8-2 to 8-3 The same procedures as in Examples 1-2 to 1-3 were performed using Buffer G (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG) and Buffer H (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG, 1 M NaCl) and omitting the addition of eGFP_A206K to the purified chimeric protein.

[0113] The results of gel filtration chromatography are shown in Figure 25A. In Figure 25A, the vertical axis indicates absorbance at a wavelength of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. The samples shown by the black lines in Figure 25A were subjected to SDS-PAGE electrophoresis (Figure 25B: CBB staining, lane 1: molecular weight marker (M), lane 2: filtrate from the ultrafiltration filter concentrator, lanes 3-11: samples shown by the black lines in Figure 25A from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 25B were collected.

[0114] Comparative Examples 8-4 to 8-6 When the same procedure as in Examples 1-4 to 1-6 was carried out, no crystals were obtained.

[0115] From the above results, it was found that when bRIL, T4L, GFP, or MBP-mutantE of Comparative Examples 1 to 8 were used, no crystals were obtained, or even if they were obtained, the resolution was low, whereas when the protein construct for membrane protein structural analysis of Example 1 was used, crystals were obtained and the resolution was excellent. As described above, the protein construct for membrane protein structural analysis of Example 1 also binds to Protein A, and therefore is a versatile technique that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins.

[0116] <Example 3: GFP-Nb-TmRibU> Example 3-1: Construction of construct (SEQ ID NO: 13) The riboflavin transporter (TmRibU, UniProtKB-Q9X1G6) from the hyperthermophilic bacterium Thermotoga maritima is a six-transmembrane protein with 183 amino acid residues. The cDNA was obtained by artificial gene synthesis using the GenPlus gene synthesis service of GenScript, Inc., with a gene sequence optimized for the E. coli expression system. For structural analysis, a fusion protein: GFP-Nb-TmRibU was designed in which GFP-nabobody (1-115) was linked to the N-terminus with a two-residue linker (Gly-Ser) in the 2-173 region. In addition, for expression confirmation and affinity purification, a cDNA was prepared with the red fluorescent protein mRFP1, His tag, Myc tag, and TEV protease recognition sequence in the N-terminus, and a plasmid was prepared by inserting it into pET32a(+) (Figure 26).

[0117] Example 3-2: Protein expression and cell membrane preparation The prepared plasmid (SEQ ID NO: 13) was transformed into BL21-CodonPlus(DE3)-RIPL and cultured in large quantities. Protein expression of GFP-Nb-TmRibU was induced by adding IPTG to a final concentration of 0.5 mM when the turbidity was approximately OD600 = 0.5, and then the cells were cultured overnight at 20°C with a rotary gyration at 120 rpm. The collected E. coli was suspended in ice-cold Buffer A (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 5 mM Benzamidine, 2 mM DTT) and disrupted using a pressure homogenizer. The disrupted solution was centrifuged at 10,000g for 10 minutes at 4°C, and the supernatant was ultracentrifuged at 45,000 rpm (himac P45AT rotor) for 1 hour at 4°C to recover the cell membrane fraction containing GFP-Nb-TmRibU. The cell membranes were suspended in Buffer A and homogenized using a Dounce homogenizer, then ultracentrifuged at 45,000 rpm (himac P45AT) for 1 hour at 4°C to wash out water-soluble proteins and membrane-associated proteins. The same procedure was repeated to wash the cell membranes.

[0118] Example 3-3: Solubilization and purification of GFP-Nb-TmRibU The washed cell membranes were suspended in Buffer B (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM imidazole, 2 mM DTT) and homogenized using a Dounce homogenizer. 20% (w / v) DDM was added to a final concentration of 2% (w / v) and incubated at 4°C for 1 hour with gentle stirring to solubilize GFP-Nb-TmRibU. The insoluble fraction in the solubilized sample was removed by ultracentrifugation at 45,000 rpm (himac P45AT) for 1 hour at 4°C, and Ni-NTA resin was added to the supernatant and incubated at 4°C for 1 hour with gentle stirring. Unbound components were removed using an Econocolumn, and the Ni-NTA resin was washed with Buffer C (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM imidazole, 2 mM DTT, 0.04% (w / v) DDM). The Ni-NTA resin was then washed with Buffer D (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM imidazole, 2 mM DTT, 0.12% (w / v) DMNG), and GFP-Nb-TmRibU was eluted with Buffer E (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 300 mM imidazole, 2 mM DTT, 0.03% (w / v) DMNG). An appropriate amount of His-tagged TEV protease was added to the eluted fraction, which was then transferred to a dialysis tube and dialyzed overnight against Buffer F (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.03% (w / v) DMNG) at 4°C. To remove the tag fragment containing mRFP1 cleaved by TEV protease, the sample was added to Ni-NTA resin and incubated at 4°C for 1 hour with gentle stirring. The flow-through fraction containing GFP-Nb-TmRibU and the Ni-NTA resin washing solution with Buffer F were collected using an Econo column and concentrated using an ultrafiltration membrane.A complex of GFP-Nb-TmRibU and eGFP_A206K was prepared by adding eGFP_A206K at a molar ratio of 1:1.5 and incubating on ice for 1 hour. The complex was then diluted 10-fold with Buffer G (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG), adsorbed onto HiTrap Q HP, and subjected to anion exchange chromatography using Buffer G and Buffer H (20 mM Tris-HCl [pH 7.5], 1 M NaCl, 2 mM DTT, 0.03% (w / v) DMNG). The fraction containing the complex was concentrated using an ultrafiltration membrane and subjected to Superdex200 Increase 10 / 300 GL equilibrated with Buffer I (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT, 0.03% (w / v) DMNG) to remove excess eGFP_A206K and oligomer components. The final purified preparation was concentrated to 20 mg / mL using an ultrafiltration membrane.

[0119] The results of gel filtration chromatography are shown in Figure 27A. In Figure 27A, the vertical axis indicates absorbance at a wavelength of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. Samples indicated by black lines in Figure 27A were collected and subjected to SDS-PAGE electrophoresis (Figure 27B: CBB staining, lane 1: molecular weight marker (M), lane 2: gel filtration input sample, lanes 3-13: fractions indicated by black lines in Figure 27A from the left, lane 14: molecular weight marker (M)). The fractions shown in Figure 27B were collected.

[0120] Examples 3-4 to 3-6 As a result of carrying out the same procedure as in Examples 1-4 to 1-6, crystals were obtained using the precipitants 0.1 M Hepes-Na [pH 7.0], 50-100 mM magnesium formate dihydrate, and 30-36% (v / v) PEG300 (FIG. 28).

[0121] Examples 3-7: X-ray diffraction experiments and structural analysis The obtained crystals were analyzed automatically by the "ZOO system" at SPring-8 BL32XU, and diffraction data with a resolution of 3.44 Å was collected (Table 3). The initial phase was determined by the molecular replacement method using MOLREP with the structure of the GFP-nanobody-eGFP complex (PDBID=3OGO) as a search model. The initial structure of almost the entire region of GFP-Nb-TmRibU-eGFP_A206K was determined by molecular replacement using TmRibU (PDBID=5KBW) as a search model for the obtained model. Based on this structure, refinement was performed using REFMAC and Coot to obtain the final structure (Table 3). The electron density map within the transmembrane domain is shown in Figure 29, and the final structure is shown in Figure 30.

[0122] [Table 3]

[0123] Example 4: GFP-Nb-Intimin β domain Example 4-1: Construction of construct (SEQ ID NO: 14) Intimin (UniProtKB-P43261) derived from enterohemorrhagic Escherichia coli O157:H7 has a β domain of about 240 amino acid residues that penetrates the outer membrane in the N-terminal region. cDNA was obtained by artificial gene synthesis using the GenPlus gene synthesis service of GenScript, Inc., with a gene sequence optimized for the E. coli expression system. For structural analysis, a β domain of a fusion protein: GFP-Nb-intimin was designed in the 208-449th region, in which GFP-nabobody (1-115) was linked to the N-terminus with a two-residue linker (Gly-Ser). Furthermore, cDNA was prepared with the periplasmic transport signal pelB signal, Myc tag, His tag, and TEV protease recognition sequence added to the N-terminus for protein expression and affinity purification, and a plasmid was prepared by inserting it into pET32a(+) (Figure 31).

[0124] Examples 4-2 to 4-3 The same procedures as in Examples 3-2 to 3-3 were repeated, except that Buffer D was not used and Buffer C (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM imidazole, 2 mM DTT, 0.16% (w / v) DDM) was used instead, and the surfactant contained in the buffer was 0.04% (w / v) DDM instead of 0.03% (w / v) DMNG.

[0125] The results of gel filtration chromatography are shown in Figure 32A. In Figure 32A, the vertical axis indicates absorbance at a wavelength of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. Samples indicated by black lines in Figure 32A were collected and subjected to SDS-PAGE electrophoresis (Figure 32B: CBB staining, lane 1: molecular weight marker (M), lane 2: gel filtration input sample, lanes 3-12: fractions indicated by black lines in Figure 32A from the left, lane 13: molecular weight marker (M)). The fractions shown in Figure 32B were collected.

[0126] Examples 4-4 to 4-6 As a result of carrying out the same procedure as in Examples 1-4 to 1-6, crystals were obtained using the precipitants 0.1 M Hepes-Na [pH 7.0-7.5], 200-500 mM magnesium nitrate hexahydrate, and 30-36% (v / v) PEG300 (FIG. 33).

[0127] Examples 4-7: X-ray diffraction experiments and structural analysis The obtained crystals were analyzed automatically by the "ZOO system" at SPring-8 BL32XU, and diffraction data with a resolution of 2.85 Å was collected (Table 4). The initial phase was determined by the molecular replacement method using MOLREP with the structure of the GFP-nanobody-eGFP complex (PDBID=3OGO) as a search model. The initial structure of almost the entire region of the GFP-Nb-intimin β-domain-eGFP_A206K was determined by molecular replacement with the β-domain (PDBID=4EIS) as a search model for the obtained model. Based on this structure, refinement was performed using REFMAC and Coot to obtain the final structure (Table 4). The electron density map in the transmembrane domain is shown in Figure 34, and the final structure is shown in Figure 35.

[0128] [Table 4]

[0129] From the above results, it was found that various membrane proteins can be analyzed by using the protein construct for membrane protein structure analysis of the present application. It was also found that the crystallization conditions and resolution can be easily manipulated by using various GFPs. As described above, the protein construct for membrane protein structure analysis of the present application also binds to immunoglobulin-binding proteins, and is therefore a versatile technology that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins.

[0130] Example 5: GFP-Nb-Dr-VSP-VSD Rest > Example 5-1: Construction of construct (SEQ ID NO: 15) Wild-type Dr-VSP-VSD (Dr-VSP-VSD Rest The same procedure as in Example 1-1 was carried out, except that the 41-188 amino acid sequence of the following gene was used to prepare the construct shown in FIG. 36.

[0131] Examples 5-2 to 5-3 The same procedures as in Examples 1-2 to 1-3 were carried out except that sfGFP was used as the added GFP.

[0132] Examples 5-4 to 5-7 As in Examples 1-4 to 1-7, crystals were obtained using a precipitant of 0.1 M MES-Na [pH 5.5-6.0], 0.1-0.4 M sodium malonate, and 28-35% (v / v) PEG400 (Figure 37). The results of processing the collected data are shown in Table 5. The electron density map in the transmembrane domain is shown in Figure 38, and the final structure is shown in Figure 39.

[0133] [Table 5]

[0134] Example 6: GFP-Nb-Dr-VSP-VSD Act )> Example 6-1: Construction of construct (SEQ ID NO: 16) Dr-VSP-VSD, which is an activating mutant type (T156R, I165R) Act The same procedure as in Example 1-1 was carried out, except that the amino acid sequence of 41-188 of was used to prepare the construct shown in FIG.

[0135] Examples 6-2 to 6-3 The same procedures as in Examples 1-2 to 1-3 were carried out.

[0136] Examples 6-4 to 6-7 As in Examples 1-4 to 1-7, crystals were obtained using a precipitant of 0.1 M HEPES-Na [pH 6.6-7.0], 0.05-0.1 M sodium formate, and 29-31.5% (v / v) PEG300 (Figure 41). The results of processing the collected data are shown in Table 6. The electron density map in the transmembrane domain is shown in Figure 42, and the final structure is shown in Figure 43.

[0137] [Table 6]

[0138] Example 7: GFP-Nb-Dr-VSP-VSDInt > Example 7-1: Construction of construct (SEQ ID NO: 1) The same procedure as in Example 1-1 was followed.

[0139] Examples 7-2 to 7-3 The same procedures as in Examples 1-2 to 1-3 were carried out, except that CBM-eGFP produced as described below was used as the added GFP.

[0140] (Preparation 1 of CBM-eGFP: Construct: SEQ ID NO: 17) The cDNA of CBM-eGFP, one of the improved GFPs, was obtained from pEGFP-N1 (Clontech). The expression region of 2-239, which contains the A206K mutation that makes it a monomer in solution, was modified to have a TEV protease recognition sequence and a calcium binding sequence from human calmodulin 1 (UniProtKB-P0DP23) at the N-terminus, and the cDNA was inserted into pET28b(+) using the restriction enzyme sites Bam HI and Xho I to prepare a plasmid. Upstream of the TEV protease sequence, there is a His tag derived from pET28b(+). (CBM-eGFP Production 2: Protein Expression and Purification) Preparation of eGFP 2: Protein expression and purification were carried out in the same manner.

[0141] Examples 7-4 to 7-7 As in Examples 1-4 to 1-7, crystals were obtained using a precipitant of 0.1 M HEPES-Na [pH 7.0], 0.1 M potassium phosphate monobasic, and 30% (v / v) PEG 400 (Figure 44). The results of processing the collected data are shown in Table 7, the electron density map in the transmembrane domain is shown in Figure 45, and the final structure is shown in Figure 46.

[0142] [Table 7]

[0143] Example 8: mCherry-Nb-Ci-VSP-VSD Rest > Example 8-1: Construction of construct (SEQ ID NO: 18) The cDNA of VSP-VSD from C. intestinalis was obtained from Professor Yasushi Okamura, Department of Integrative Physiology, Graduate School of Medicine, Osaka University. For structural analysis, a fusion protein (mCherry-Nb-Ci-VSP-VSD) was prepared by linking mCherry-Nb (also known as LaM4, 1-124) with a two-residue linker (Gly-Ser) to the N-terminus of the voltage sensor domain (102-249) that shows the resting state in the wild type. Rest ) was designed (Figure 47). Furthermore, in order to confirm expression and affinity purification, cDNA was created with sfGFP, His tag, Myc tag, and TEV protease recognition sequence at the N-terminus, and a plasmid was prepared by inserting it into pFastBac1.

[0144] Examples 8-2 to 8-3 The procedures were the same as in Examples 1-2 to 1-3, except that the ion exchange column in the ion exchange chromatography was changed from HiTrap SP HP to anion exchange chromatography HiTrap Q HP, Buffer G (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG) and Buffer H (20 mM Tris-HCl [pH 7.5], 2 mM DTT, 0.03% (w / v) DMNG, 1 M NaCl) were used, and the fluorescent protein added was changed from eGFP_A206K to mCherry produced as described below.

[0145] (mCherry Production 1: Construct: SEQ ID NO: 19) mCherry cDNA was obtained from pmCherry-N1 (Clontech). A cDNA was created by adding a TEV protease recognition sequence to the N-terminus of the 1-236 expression region, and the cDNA was inserted into pET28b(+) using the restriction enzyme sites Bam HI and Xhol I to prepare a plasmid. Upstream of the TEV protease sequence, there is a His tag derived from pET28b(+).

[0146] (mCherry Production 2: Protein Expression and Purification) Preparation of eGFP 2: Protein expression and purification were carried out in the same manner.

[0147] Examples 8-4 to 8-6 As a result of carrying out the same procedure as in Examples 1-4 to 1-6, crystals were obtained using a precipitant of 0.1 M MES-Na [pH 6.0], 300-400 mM sodium chloride, and 26-32% (v / v) PEG300 (FIG. 48).

[0148] Example 8-7: X-ray diffraction experiment and structure analysis The obtained crystals were analyzed automatically by the "ZOO system" at SPring-8 BL32XU, and diffraction data with a resolution of 2.90 Å was collected (Table 8). The initial phase was determined by molecular replacement using MOLREP with the structure of the LaM4-mCherry complex (PDBID = 6IR1) as the search model. The obtained model was further analyzed by molecular replacement using Ci-VSP-VSD (PDBID = 4G7V and 4G80) as the search model to obtain mCherry-Nb-Ci-VSP-VSD. Rest The initial structure of almost the entire region of mCherry was determined. Based on this structure, refinement was performed using REFMAC and Coot to obtain the final structure (Tables 8 and 9). mCherry-Nb-Ci-VSP-VSD Rest The electron density map within the transmembrane domain is shown in FIG. 49, and the final structure is shown in FIG.

[0149] [Table 8]

[0150] Example 9: mCherry-Nb-Ci-VSP-VSD Act )> Example 9-1: Construction of construct (SEQ ID NO: 20) A mutant (Ci-VSP-VSD) in which the R217E mutation was introduced and fixed in the activated state Act The same procedure as in Example 8-1 was carried out, except that the amino acid sequence of 102-249 of was used to prepare the construct shown in Figure 51.

[0151] Examples 9-2 to 9-3 The same procedures as in Examples 8-2 to 8-3 were carried out.

[0152] Examples 9-4 to 9-7 The same procedure was carried out as in Examples 8-4 to 8-7. Crystals were obtained using 0.1M Hepes-Na [pH 7.0], 2.8% (w / v) Tacsimate [pH 7.0], and 30% (v / v) PEG400 as precipitants (FIG. 52). The results of processing the collected data are shown in Table 9. The electron density map in the transmembrane domain is shown in FIG. 53, and the final structure is shown in FIG. 54.

[0153] [Table 9]

[0154] Example 10: GFP-Nb-Dr-VSP-VSD Int (41-180)> Example 10-1: Construction of construct (SEQ ID NO: 21) The same procedure as in Example 1-1 was carried out, except that the 41-180 amino acid sequence of an activation intermediate mutant of Dr-VSP-VSD was used to prepare the construct shown in Figure 55.

[0155] Examples 10-2 to 10-3 The same procedures as in Examples 1-2 to 1-3 were carried out.

[0156] Examples 10-4 to 10-7 The same procedure as in Examples 1-4 to 1-7 was carried out, and crystals were obtained using a precipitant of 0.1 M HEPES-Na [pH 7.0], 0.1 M potassium phosphate monobasic, and 30% (v / v) PEG 300 (FIG. 56). The results of processing the collected data are shown in Table 10.

[0157] [Table 10]

[0158] Example 11: GFP-Nb-Dr-VSP-VSD Act (41-180)> Example 11-1: Construction of construct (SEQ ID NO: 22) The same procedure as in Example 1-1 was carried out, except that the 41-180 amino acid sequence of the activated mutant of Dr-VSP-VSD was used to prepare the construct shown in Figure 57.

[0159] Examples 11-2 to 11-3 The same procedures as in Examples 1-2 to 1-3 were carried out.

[0160] Examples 11-4 to 11-7 The same procedure as in Examples 1-3 to 1-7 was carried out, and crystals were obtained using a precipitant of 0.1 M HEPES-Na [pH 7.0], 0.1 M potassium chloride, and 30-34% (v / v) PEG 300 (FIG. 58). The results of processing the collected data are shown in Table 11.

[0161] [Table 11]

[0162] From the above results, it was found that the wild type, intermediate activation state mutant type, and activation state mutant type of membrane proteins can be analyzed by using the protein construct for membrane protein structure analysis of the present application. It was also found that the crystallization conditions and resolution can be easily manipulated by using various GFPs. As described above, the protein construct for membrane protein structure analysis of the present application also binds to immunoglobulin-binding proteins, and is therefore a versatile technology that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins.

[0163] <Example 12: VD method> Membrane protein GFP-Nb-Dr-VSP-VSD prepared in Example 1 IntThe protein was concentrated to 10 mg / mL using an ultrafiltration membrane and crystallized by the sitting drop vapor diffusion method (A. Ducruix and R. Giege, 1993 Oxford University Press). Specifically, a protein solution containing 1 μL of membrane protein solution and 1 μL of precipitant (0.1 M MES-Na [pH 6.0-6.5], 19-22% (w / v) PEG2000MME) was placed in a subwell of a protein crystallization plate, and 50 μL of precipitant solution was placed in a reservoir well, and the plate was sealed with a microplate seal. The crystallization plate was placed in a 20° incubator to evaporate the solvent of the protein solution using vapor diffusion, resulting in a supersaturated state and crystallization (Figure 59).

[0164] The diffraction data was collected from the obtained crystals at SPring-8 BL44XU, but in the same manner as in Example 1-7, and the results of processing the collected data are shown in Table 12.

[0165] [Table 12]

[0166] Example 13: Bicelle method Membrane protein GFP-Nb-Dr-VSP-VSD prepared in Example 1 Int The solution was concentrated to 10 mg / mL using an ultrafiltration membrane, and a bicelle was prepared in the same manner as in the general bicelle method (R. Ujwal and JUBowie, 2011 Methods), and crystallization was performed by the sitting drop vapor diffusion method. Specifically, 1 μL of bicelle was mixed with 1 μL of precipitant (0.05 M HEPES-Na [pH 7.5], 11% (w / v) PEG4000), and crystallization was performed in an incubator at 20 degrees as described above (Figure 60).

[0167] <Example 14: Counterdiffusion method> Membrane protein GFP-Nb-Dr-VSP-VSD prepared in Example 1 IntThe protein was concentrated to 10mg / mL using an ultrafiltration membrane, and crystallized in two different environments, on the ground and in a microgravity environment, using a common counterdiffusion method (JM Garcia-Ruiz et. al., Meth. Enzymol. 2002 368:130-54). For the microgravity environment, the membrane protein solution was transported to the Japanese Experiment Module "Kibo" of the International Space Station by SpaceX's Dragon supply ship, SpaceX-19, and crystallization was performed there. Specifically, 8μL of the membrane protein solution was filled into a capillary with an inner diameter of 0.5mm and immersed in 0.9mL of precipitant (0.1M MES-Na [pH5.4-5.6], 25% (w / v) PEG2000MME). The crystallization sample was placed in a 20°C incubator, and the protein solution was made supersaturated by liquid-liquid diffusion, and crystallized (Figure 61: Under microgravity environment) (Figure 62: on the ground).

[0168] The diffraction data from the obtained crystals was collected at SPring-8 BL45XU, and the collected data was processed in the same manner as in Examples 1-7. The results are shown in Table 13. The electron density map in the transmembrane domain of the crystal structure produced in a microgravity environment is shown in Figure 63, and the final structure is shown in Figure 64. The electron density map in the transmembrane domain of the crystal structure produced on the ground is shown in Figure 65, and the final structure is shown in Figure 66.

[0169] [Table 13]

[0170] The results of Example 14 show that the resolution of crystals grown in a microgravity environment is slightly improved compared to crystals grown on the ground. The reason for this is that in a microgravity environment, the density difference convection in the solution caused by gravity is suppressed, resulting in high-quality, large-sized crystals (Figure 61: Under microgravity environment) (Figure 62: On the ground).

[0171] Examples of aspects of the present invention include the following. <1> The protein structure is for use in analyzing the structure of a membrane protein, and is characterized in that it is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein. <2> The above-mentioned comprises a VHH domain of a camelid antibody. <1> The present invention relates to a protein structure for membrane protein structure analysis. <3> A membrane protein and the <1> from <2> and a protein structure for membrane protein structure analysis according to any one of the above. <4> This is a method for producing a chimeric protein, characterized by including a step of expressing a nucleic acid that is a fusion of a gene encoding a membrane protein and a gene encoding a protein structure for membrane protein structural analysis that is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein. <5> The present invention relates to a method for purifying a membrane protein, the method comprising the step of purifying a chimeric protein in which a membrane protein is fused with a protein structure for membrane protein structural analysis, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, using the immunoglobulin-binding protein. <6> The present invention relates to a method for crystallizing a membrane protein, the method comprising the step of crystallizing a chimeric protein in which a membrane protein is fused with a protein structure for membrane protein structural analysis, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein. <7> The method further comprises the step of adding a fluorescent protein to the chimeric protein. <6> This is a method for crystallizing a membrane protein according to the present invention. <8> The method for analyzing the structure of a membrane protein comprises the steps of: purifying a chimeric protein, in which a membrane protein is fused with a protein structure for analyzing membrane protein structure, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein, by using the immunoglobulin-binding protein; and crystallizing the chimeric protein to obtain a crystallized chimeric protein. <9> The method further comprises the step of adding a fluorescent protein to the chimeric protein. <8> This is a method for analyzing the structure of a membrane protein described in <10> The method further comprises a step of analyzing the structure of the crystallized chimeric protein. <8> or <9> The present invention relates to a method for analyzing the structure of a membrane protein according to any one of the above items.

Claims

1. A step of purifying a chimeric protein in which a protein structure for analyzing membrane protein structure, which contains a VHH domain of a camelid antibody capable of specifically recognizing and binding to a fluorescent protein and capable of binding to an immunoglobulin-binding protein, is fused to the N-terminus or C-terminus of a membrane protein having a molecular weight of 200 kDa or less; adding the fluorescent protein capable of specifically binding to the VHH domain to the chimeric protein to form a complex between the chimeric protein and the fluorescent protein; crystallizing the complex by a lipid cubic phase method, a vapor diffusion method, a Bicelle method, a HiLiDe method, or a counter diffusion method to obtain a crystallized chimeric protein; Including, A method for crystallizing a membrane protein, wherein the fluorescent protein in the purification step and the complex formation step is wild-type GFP, mutant GFP, wild-type mCherry, or mutant mCherry.

2. The method for crystallizing a membrane protein according to claim 1, wherein the membrane protein is any one selected from the group consisting of a voltage sensor domain of a voltage-dependent phosphatase, a sugar transporter SWEET family protein, SemiSWEET, a translocator protein, a riboflavin transporter, and the β domain of intimin.

3. The method for crystallizing a membrane protein according to claim 1 or 2, wherein the immunoglobulin-binding protein is Protein A, Protein A / G, or Protein G.

4. A method for crystallizing a membrane protein described in any one of claims 1 to 3, wherein the chimeric protein comprises a VHH domain of the camelid antibody, the membrane protein, and a linker that links the VHH domain of the camelid antibody to the membrane protein.

5. a step of purifying, by using an immunoglobulin-binding protein, a chimeric protein in which a protein structure for membrane protein structure analysis, which protein structure contains a VHH domain of a camelid antibody capable of specifically recognizing and binding to a fluorescent protein and capable of binding to an immunoglobulin-binding protein, is fused to the N-terminus or C-terminus of a membrane protein having a molecular weight of 200 kDa or less; adding the fluorescent protein capable of specifically binding to the VHH domain to the chimeric protein to form a complex between the chimeric protein and the fluorescent protein; and crystallizing the complex by a lipid cubic phase method, a vapor diffusion method, a Bicelle method, a HiLiDe method, or a counter diffusion method to obtain a crystallized chimeric protein. A method for analyzing the structure of a membrane protein, wherein the fluorescent protein in the purification step and the complex formation step is wild-type GFP, mutant GFP, wild-type mCherry, or mutant mCherry.

6. The method for analyzing the structure of a membrane protein according to claim 5 , further comprising a step of analyzing the structure of the crystallized chimeric protein.

7. The method for analyzing the structure of a membrane protein according to claim 5 or 6, wherein the membrane protein is any one selected from the group consisting of a voltage sensor domain of a voltage-dependent phosphatase, SemiSWEET, a sugar transporter SWEET family protein, a translocator protein, a riboflavin transporter, and the β domain of intimin.

8. The method for analyzing the structure of a membrane protein according to claim 5 , wherein the immunoglobulin-binding protein is Protein A, Protein A / G, or Protein G.

9. A method for analyzing the structure of a membrane protein described in any one of claims 5 to 8, wherein the chimeric protein comprises a VHH domain of the camelid antibody, the membrane protein, and a linker that links the VHH domain of the camelid antibody to the membrane protein.

Images