JP7657406B2 - METHOD FOR CRYSTALLIZING MEMBRANE PROTEIN AND METHOD FOR ANALYZING MEMBRANE PROTEIN STRUCTURE - Google Patents

Description

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.

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, with GFP (see, for example, Non-Patent Documents 1-3) being reported as a "reporter technology" for evaluating the thermal stability of membrane proteins and for exploring detergents, T4 lysozyme and bRIL (see, for example, Non-Patent Documents 4-6) being reported as a "thermal stability improvement technology" for G protein-coupled receptors, and Maltose Binding Protein (MBP) (see, for example, Non-Patent Document 7) being 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).

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. Although 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, there are various constraints due to the difficulty of crystallization due to their hydrophobic properties, and the selection of the type of surfactant is also important, so that labor-saving and high-speed purification, crystallization, and structural analysis of membrane proteins have not been achieved.

Therefore, there is still a lack of commonly applicable and versatile technology for the purification, crystallization, and structural analysis of membrane proteins, which are issues in structural biology research, including X-ray crystal structure analysis of membrane proteins, and there is a strong demand for such technology to be provided as soon as possible.

NATURE METHODS 2016 3:303-313 Structure 2006 14:673-681 SCIENCE 2016 353:664-669 Structure 2012 20:967-976 SCIENC 2007 318:1258-1265 NATURE 2013 499:444-450 PROTEIN SCIENCE 2016 25:559-571 Structure 2017 25:1611-1622 NATURE 2007 450:383-388

The present invention aims to solve the above-mentioned problems and achieve the following objectives. That is, the present invention aims to provide a versatile technology that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins.

As a result of intensive research by the inventors to achieve the above object, they discovered that by adopting a protein structure for membrane protein structural analysis that can bind to a fluorescent protein and 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.

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> A protein structure for analyzing the structure of a membrane protein, which is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein.
<2> A chimeric protein comprising a membrane protein and the protein structure for membrane protein structure analysis according to <1>.
<3> A method for producing a chimeric protein, comprising 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.
<4> A method for purifying a membrane protein, comprising 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, by using the immunoglobulin-binding protein.
<5> A method for crystallizing a membrane protein, comprising a 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> A method for analyzing the structure of a membrane protein, comprising the steps of: purifying a chimeric protein, in which a membrane protein is fused with a protein structure for analyzing a membrane protein structure 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.

The present invention provides a versatile technology that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins.

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

(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.

The fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose. Examples of the fluorescent protein 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.

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). It may also be one to which a sequence such as His-tag calcium-binding domain (CBM-eGFP) has been added to the N-terminus.

The immunoglobulin-binding protein is not particularly limited and can be appropriately selected depending on the purpose. Examples include protein A, protein A/G, and protein G, and among these, protein A is preferred.

There are no particular limitations on the molecular weight of the protein structure for membrane protein structural analysis and it can be selected appropriately depending on the purpose, but it 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.

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 camelid antibody is not particularly limited and can be appropriately selected depending on the purpose, but is preferably a VHH domain of a camelid antibody that recognizes the fluorescent protein.

The VHH domain of the camelid antibody that recognizes the fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose. For example, it may have the inserted gene sequence of addgene #61838 (GFP nanobody) or the inserted gene sequence of addgene #70696 (mCherry nanobody).

(Chimeric Protein)
The chimeric protein comprises a membrane protein and a protein structure for analyzing a membrane protein structure.

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.

The molecular weight of the membrane protein is not particularly limited and can be selected appropriately 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.

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.

The VSP-VSD is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include VSP-VSD derived from D. Rerio (Dr-VSP-VSD), VSP-VSD derived from C. intestinalis (Ci-VSP-VSD), etc. VSP-VSD may be a resting state (wild type) VSP-VSD ^Rest , an active intermediate state mutant type VSP-VSD ^Int , or an active state mutant VSP-VSD ^Act .

The active state mutant VSP-VSD ^Act is not particularly limited and can be appropriately selected depending on the purpose. Examples of the mutant include a mutant in which T156 and I165 of 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).

The activation intermediate mutant VSP-VSD ^Int is not particularly limited and can 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 can be mentioned.
Furthermore, the VSP-VSD may be a VSP-VSD lacking amino acid residues at the N-terminus of VSP-VSD, which is an unstable region having no secondary structure.
The VSP-VSD lacking amino acid residues at the N-terminus of VSP-VSD is not particularly limited and can be appropriately selected depending on the purpose. Examples of the VSP-VSD lacking amino acid residues at the N-terminus of VSP-VSD include Dr-VSP-VSD lacking 40 amino acid residues at the N-terminus of VSP-VSD, Dr-VSP-VSD lacking 29 amino acid residues at the N-terminus of VSP-VSD, and Ci-VSP-VSD lacking 101 amino acid residues at the N-terminus of VSP-VSD.

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 thereof include the β domain of intimin derived from enterohemorrhagic Escherichia coli O157:H7.

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, etc.

The protein structure for membrane protein structure analysis is as described above.

(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.

The membrane protein and the protein structure for membrane protein structure analysis are as described above.

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 structure analysis preferably has a secondary structure.
The length of the linker of 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.

The expression method is not particularly limited and can be appropriately selected depending on the purpose. For example, 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 is prepared, and the fusion protein is expressed using a baculovirus expression system, an E. coli expression system, an insect cell, a mammalian cell, or a cell-free protein synthesis system.

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.

The process for 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 centrifuged again.

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).

(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.

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 combinations of these.

The affinity chromatography is not particularly limited and can be appropriately selected depending on the purpose. For example, a method using a column packed with the immunoglobulin-binding protein, a method using a resin bound to the immunoglobulin-binding protein, etc. can be mentioned.

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.

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, and examples include SDS-PAGE, native-PAGE, isoelectric focusing, and two-dimensional electrophoresis.

The method of concentration in the step of concentrating the purified chimeric protein is not particularly limited and can be appropriately selected depending on the purpose, and examples include concentration using an ultrafiltration membrane.

(Method of crystallizing membrane proteins)
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.

The crystallization method is not particularly limited and can be appropriately selected depending on the purpose. Examples 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.

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.

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

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.

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.

The counterdiffusion method is not particularly limited and can be appropriately selected depending on the purpose. For example, the method described in J. M. Garcia-Ruiz et al., Meth. Enzymol. 2002 368:130-54 can be mentioned.

The method for crystallizing a membrane protein may further include a step of adding a fluorescent protein to the chimeric protein.

The fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose. Examples of the fluorescent protein 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.

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.

(Method of 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.

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.

The method for analyzing the structure of a membrane protein may further include a step of adding a fluorescent protein to the chimeric protein.

The fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose. Examples of the fluorescent protein 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.

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.

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

The structural analysis process for the crystallized chimeric protein is not particularly limited and can be appropriately selected depending on the purpose. For example, the method includes a method of collecting diffraction data for the obtained crystals using the high-flux microbeam beamline (BL32XU) of the large synchrotron radiation facility (SPring-8), and further a method of determining the initial phase by molecular replacement using a search model, determining the initial structure, and then obtaining the final structure.

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

Regarding point 1 above, in conventional methods, it was necessary to select a protein tag depending on the topology of the target protein, but when using the protein structure for membrane protein structure analysis, there is no such restriction.

Regarding point 2 above, in conventional methods, it was necessary to take measures such as "introducing mutations," "searching for fusion protein tags," and "searching for additives" that contribute to improving thermal stability, and searching for orthologs with higher thermal stability. However, when using the protein construct for membrane protein structure analysis, adding a fluorescent protein makes it easier to perform micromeasurements using a fluorescence detector, enabling high-throughput screening of these conditions.

Regarding point 3 above, due to low thermal stability and high handling difficulty, conventional methods required high affinity purification in a short period of time. However, when the protein structure for membrane protein structure analysis is used, high affinity purification can be achieved by utilizing binding with immunoglobulin-binding proteins. In addition, the addition of a fluorescent protein allows high-throughput screening of purification conditions by micromeasurement using a fluorescence detector and visual confirmation of the presence or absence of protein, thus simplifying the purification process.

Regarding point 4 above, in conventional methods, crystallization requires measures such as protein design, such as the selection of fusion protein tags according to the crystallization method, such as the vapor diffusion method, the Bicelle method, or the LCP method. However, 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 that depends on the fluorescent protein, crystal observation and picking up become easier, and human errors such as "overlooking crystals" can be reduced.

Regarding point 5 above, in conventional methods, it was necessary to address this issue by improving purification purity, optimizing or reconsidering crystallization conditions, redesigning the protein, creating antibody fragments, etc., but when using the protein construct for membrane protein structure analysis, different crystallization conditions can be easily explored simply by screening the types of fluorescent protein variants, thereby increasing the chances of successfully improving resolution. This allows for a significant reduction in time compared to protein redesign, etc., and accelerates research.

The following describes examples of the present invention, but the present invention is not limited to these examples.

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

Example 1-2: Protein expression and preparation of cell membrane According to the protocol of Bac-to-Bac Baculovirus Expression System (Thermo Fisher Scientific), P2 recombinant baculovirus was produced from the prepared plasmid. Protein expression was carried out by adding an appropriate amount of P2 virus solution to Sf9 cells at a cell concentration of 4.0 × 10 ⁶ cells / mL, and performing rotational culture at 28 ° C. and 120 rpm for 24 hours, and then lowering the temperature to 20 ° C. and performing rotational culture 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 by an ultrasonic homogenizer. The disrupted solution was centrifuged at 10,000 g 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 a 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 carrying out the same procedure.

Example 1-3: Solubilization and purification of membrane proteins The washed cell membrane was suspended in Buffer B (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM Imidazole, 2 mM DTT) and homogenized with a Dounce homogenizer, and then 20% (w/v) DDM (n-Dodecyl-β-D-Maltopyranoside) 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-Dr-VSP-VSD ^Int . The insoluble fraction contained in 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 further 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 GFP-Nb-Dr-VSP-VSD ^Int was eluted with Buffer E (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 250 mM Imidazole, 2 mM DTT, 0.03% (w / v) DMNG). An appropriate amount of His-tag fused 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. In order 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. The flow-through fraction containing GFP-Nb-Dr-VSP-VSD ^Int and the Ni-NTA resin washing solution with Buffer F were collected using an Econo column 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), adsorbed to HiTrap SP HP (GE Healthcare), and cation exchange chromatography was performed 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 recovered fraction was determined from the chromatogram and the results of SDS-PAGE electrophoresis, and the eGFP_A206K produced as described below was added in an amount twice the molar ratio of GFP-Nb-Dr-VSP-VSD ^Int , and the complex was prepared by leaving it on ice for 1 hour. The mixture was then concentrated using an ultrafiltration membrane and subjected to 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 sample was concentrated to 20 mg/mL using an ultrafiltration membrane.

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. The 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 in order from the left, lane 13: molecular weight marker (M)). The fractions shown in Figure 2B were collected.

(eGFP Production 1: Construct: SEQ ID NO:2)
The cDNA of eGFP, one of the improved GFPs, was obtained from pEGFP-N1 (Clontech). A cDNA was prepared by adding a TEV protease recognition sequence to the N-terminus of the expression region of 2-239, which contains the mutation A206K that forms a monomer in solution, and inserting the cDNA 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(+).

(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 at a turbidity of approximately OD600 = 0.5, and then rotating culture was performed overnight at 20 ° C. 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 with 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 to collect the supernatant fraction. 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 further 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-tag fused TEV protease was added to the eluted fraction, which was then 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 the 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 eGFP_A206K and the resin washing solution with Buffer C were collected using an Econocolumn, concentrated using an ultrafiltration membrane, and gel filtration chromatography was performed using HiLoad 16/600 Superdex200 pg equilibrated with Buffer D (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT). Fractions containing monomeric eGFP_A206K were collected and concentrated to 10 mg/mL using an ultrafiltration membrane.

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 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 and moved. The two syringes were connected with a coupler, and the plungers were alternately pushed (about 100 times), so that the sample and monoolein were thoroughly mixed to 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.

Example 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 liquid 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 and allowed to stand at 20 ° C.

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

Example 1-7: X-ray diffraction experiment and structure analysis Diffraction data was collected by automatic measurement of the "ZOO system" in SPring-8 BL32XU, 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 the search model. The initial structure of almost the entire region was determined by molecular replacement using Ci-VSP-VSD (PDBID = 4G7V) as the search model for the obtained model. Based on this structure, the amino acid sequence was modified and refined to Dr-VSP-VSD ^Int using REFMAC and Coot (P. Emsley and K Cowtan, 2004 Acta Crystallogr. D Biol. Crystallogr.) to obtain the final structure (Table 1).

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

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.

(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(+).

(Production of sfGFP 2: Protein Expression and Purification)
eGFP Production 2: Protein expression and purification were carried out in the same manner.

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. The samples indicated by the black line 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 the black line in Figure 5A in order from the left, lane 14: molecular weight marker (M)). The fractions shown in Figure 5B were collected.

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 a precipitant of 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.

<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.

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.

The results of gel filtration chromatography are shown in Figure 8A. In Figure 8A, the vertical axis indicates absorbance at wavelengths 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 approximately 14 mL. The sample indicated by the black line in Figure 8A was collected and subjected to SDS-PAGE electrophoresis (Figure 8B: CBB staining, lane 1: molecular weight marker (M), lanes 2-11: fractions indicated by the black line in Figure 8A in order from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 8B were collected.

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.

<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.

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.

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. The samples indicated by the black line 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 the black line in Figure 10A in order from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 10B were collected.

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.

<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.

Comparative Examples 3-2 to 3-3
The same procedures as in Examples 1-2 to 1-3 were carried out 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 except that eGFP_A206K was not added to the purified chimeric protein.

The results of gel filtration chromatography are shown in Figure 12A. In Figure 12A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. The samples indicated by the 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 the ultrafiltration filter concentrator, lanes 3-11: samples indicated by the black lines in Figure 12A from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 12B were collected.

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) PEG 400 (FIG. 13), but no diffraction points were obtained.

<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.

Comparative Examples 4-2 to 4-3
The same procedures as in Examples 1-2 to 1-3 were carried out 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 except that eGFP_A206K was not added to the purified chimeric protein.

The results of gel filtration chromatography are shown in Figure 15A. In Figure 15A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. The samples indicated 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 indicated by the black lines in Figure 15A from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 15B were collected.

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.

<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.

Comparative Examples 5-2 to 5-3
The same procedures as in Examples 1-2 to 1-3 were carried out 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.

The results of gel filtration chromatography are shown in Figure 17A. In Figure 17A, the vertical axis indicates absorbance at wavelengths 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 approximately 13.7 mL. The sample indicated by the black line in Figure 17A was collected and subjected to SDS-PAGE electrophoresis (Figure 17B: CBB staining, lane 1: molecular weight marker (M), lanes 2-11: fractions indicated by the black line in Figure 17A in order from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 17B were collected.

Comparative Examples 5-4 to 5-7
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 sodium nitrate, and 30% (v/v) PEG300 (FIG. 18), with a resolution of about 6 Å.

<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.

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

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. The samples indicated by the 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 the black lines in Figure 20A, from left to right, lane 14: molecular weight marker (M)). The fractions shown in Figure 20B were collected.

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.

<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).

Comparative Examples 7-2 to 7-3
The same procedures as in Examples 1-2 to 1-3 were carried out 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 except that eGFP_A206K was not added to the purified chimeric protein.

The results of gel filtration chromatography are shown in Figure 22A. In Figure 22A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. The samples indicated by the 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 the black lines in Figure 22A in order from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 22B were collected.

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), with a resolution of about 5 Å.

<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).

Comparative Examples 8-2 to 8-3
The same procedures as in Examples 1-2 to 1-3 were carried out 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 except that eGFP_A206K was not added to the purified chimeric protein.

The results of gel filtration chromatography are shown in Figure 25A. In Figure 25A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. The samples indicated 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 indicated by the black lines in Figure 25A from the left, lane 12: molecular weight marker (M)). The fractions shown in Figure 25B were collected.

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.

The above results show that when bRIL, T4L, GFP, or MBP-mutantE in 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 structure for membrane protein structural analysis in Example 1 was used, crystals were obtained and the resolution was excellent. As described above, the protein structure for membrane protein structural analysis in Example 1 also binds to Protein A, and therefore is a versatile technology that can be commonly used in the purification, crystallization, and structural analysis of membrane proteins.

Example 3: GFP-Nb-TmRibU
Example 3-1: Construction of construct (SEQ ID NO: 13)
The riboflavin transporter (TmRibU, UniProtKB-Q9X1G6) derived from the hyperthermophilic bacterium Thermotoga maritima is a six-transmembrane protein of 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 was designed in the 2-173 region, in which GFP-nabobody (1-115) was linked to the N-terminus via a two-residue linker (Gly-Ser): GFP-Nb-TmRibU. Furthermore, for expression confirmation and affinity purification, cDNA was prepared by adding the red fluorescent protein mRFP1, a His tag, a Myc tag, and a TEV protease recognition sequence to the N-terminal region, and a plasmid was prepared by inserting this into pET32a(+) ( FIG. 26 ).

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 at a turbidity of approximately OD600 = 0.5, and then rotating culture was performed overnight at 20 ° C. 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 10,000 g 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 a cell membrane fraction containing GFP-Nb-TmRibU. 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 same operation was performed again to wash the cell membrane.

Example 3-3: Solubilization and purification of GFP-Nb-TmRibU The washed cell membrane was suspended in Buffer B (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM Imidazole, 2 mM DTT) and homogenized with a Dounce homogenizer, and then 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 contained 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 further 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). 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), 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-tag fused TEV protease was added to the eluted fraction, 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. In order 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. eGFP_A206K was added so that the molar ratio was 1:1.5, and the mixture was incubated on ice for 1 hour to prepare a complex of GFP-Nb-TmRibU and eGFP_A206K. The complex was 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.

The results of gel filtration chromatography are shown in Figure 27A. In Figure 27A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. The samples indicated by the 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 the black lines in Figure 27A from the left, lane 14: molecular weight marker (M)). The fractions shown in Figure 27B were collected.

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 a precipitant of 0.1 M Hepes-Na [pH 7.0], 50-100 mM magnesium formate dihydrate, and 30-36% (v/v) PEG300 (FIG. 28).

Example 3-7: X-ray diffraction experiment and structure analysis The obtained crystals were collected with a resolution of 3.44 Å by automatic measurement of the "ZOO system" in SPring-8 BL32XU (Table 3). The initial phase was determined by molecular replacement using MOLREP with the structure of the GFP-nanobody-eGFP complex (PDBID = 3OGO) as the 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 the 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 in the transmembrane region is shown in Figure 29, and the final structure is shown in Figure 30.

Example 4: GFP-Nb-Intimin β domain
Example 4-1: Construction of construct (SEQ ID NO: 14)
Enterohemorrhagic Escherichia coli O157:H7-derived intimin (UniProtKB-P43261) 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 GenScript's GenPlus gene synthesis service to design a gene sequence optimized for the E. coli expression system. For structural analysis, a β domain of GFP-Nb-intimin was designed in the 208-449th region, in which GFP-nabobody (1-115) was linked to the N-terminus via a two-residue linker (Gly-Ser). Furthermore, for protein expression and affinity purification, cDNA was prepared by adding a periplasmic transport signal pelB signal, a Myc tag, a His tag, and a TEV protease recognition sequence to the N-terminal region, and a plasmid was prepared by inserting this into pET32a(+) (Figure 31).

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.

The results of gel filtration chromatography are shown in Figure 32A. In Figure 32A, the vertical axis indicates absorbance at wavelengths of 280 nm or 254 nm, and the horizontal axis indicates the elution amount. The samples indicated by the 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 the black lines in Figure 32A from left to right, lane 13: molecular weight marker (M)). The fractions shown in Figure 32B were collected.

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 a precipitant of 0.1 M Hepes-Na [pH 7.0-7.5], 200-500 mM Magnesium nitrate hexahydrate, and 30-36% (v/v) PEG300 (FIG. 33).

Example 4-7: X-ray diffraction experiment and structure analysis The obtained crystals were collected with a resolution of 2.85 Å by automatic measurement of the "ZOO system" in SPring-8 BL32XU (Table 4). The initial phase was determined by molecular replacement using MOLREP with the structure of the GFP-nanobody-eGFP complex (PDBID = 3OGO) as the search model. The initial structure of almost the entire region of the β-domain-eGFP_A206K of GFP-Nb-intimin was further determined by molecular replacement with the β-domain (PDBID = 4EIS) as the 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 region is shown in Figure 34, and the final structure is shown in Figure 35.

The above results show that various membrane proteins can be analyzed by using the protein structure for membrane protein structure analysis of the present application. In addition, it was found that the crystallization conditions and resolution can be easily manipulated by using various GFPs. As described above, the protein structure 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.

<Example 5: GFP-Nb-Dr-VSP-VSD ^Rest >
Example 5-1: Construction of construct (SEQ ID NO: 15)
The same procedure as in Example 1-1 was carried out, except that the 41-188 amino acid sequence of wild-type Dr-VSP-VSD (Dr-VSP-VSD ^Rest ) showing a resting state was used to prepare the construct shown in FIG.

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.

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 (FIG. 37). The results of processing the collected data are shown in Table 5. The electron density map in the transmembrane domain is shown in FIG. 38, and the final structure is shown in FIG. 39.

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

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

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 format, and 29-31.5% (v/v) PEG300 (FIG. 41). The results of processing the collected data are shown in Table 6. The electron density map in the transmembrane domain is shown in FIG. 42, and the final structure is shown in FIG. 43.

<Example 7: GFP-Nb-Dr-VSP-VSD ^Int >
Example 7-1: Construction of construct (SEQ ID NO: 1)
The same procedure as in Example 1-1 was carried out.

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.

(Preparation of CBM-eGFP 1: Construct: SEQ ID NO: 17)
The cDNA of CBM-eGFP, one of the improved GFPs, was obtained from pEGFP-N1 (Clontech). A cDNA was prepared by adding a TEV protease recognition sequence and a calcium binding sequence of human calmodulin 1 (UniProtKB-P0DP23) to the N-terminus of the expression region of 2-239, which contains the mutation A206K that forms a monomer in solution, and inserting the cDNA 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(+).
(Production of CBM-eGFP 2: Protein Expression and Purification)
eGFP Production 2: Protein expression and purification were carried out in the same manner.

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 (FIG. 44). The results of processing the collected data are shown in Table 7, the electron density map in the transmembrane domain is shown in FIG. 45, and the final structure is shown in FIG. 46.

<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 Rest) was designed in which mCherry-Nb (also known as LaM4, 1-124) was linked with a two-residue linker (Gly-Ser) to the N-terminus of the voltage sensor domain up to 102-249, which shows a resting ^state in the wild type (Figure 47). Furthermore, for expression confirmation and affinity purification, cDNA was prepared 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.

Examples 8-2 to 8-3
The same procedures as in Examples 1-2 to 1-3 were carried out 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.

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

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

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).

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

<Example 9: mCherry-Nb-Ci-VSP-VSD ^Act >
Example 9-1: Construction of construct (SEQ ID NO: 20)
The same procedure as in Example 8-1 was carried out except that the construct in FIG. 51 was produced using the 102-249 amino acid sequence of a mutant (Ci-VSP-VSD ^Act ) in which the R217E mutation was introduced into Ci-VSP-VSD and fixed in an activated state.

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

Examples 9-4 to 9-7
The same procedure was followed as in Examples 8-4 to 8-7. Crystals were obtained using 0.1 M 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.

<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 FIG.

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

Examples 10-4 to 10-7
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 300 (FIG. 56). The results of processing the collected data are shown in Table 10.

<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 FIG.

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

Examples 11-4 to 11-7
As in Examples 1-3 to 1-7, 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.

The above results show that the wild type, intermediate activation state mutant type, and activation state mutant type of membrane proteins can be analyzed by using the protein structure 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 structure 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.

Example 12: VD method
The membrane protein GFP-Nb-Dr-VSP-VSD ^Int prepared in Example 1 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 to create a supersaturated state and crystallize (FIG. 59).

The diffraction data was collected from the crystals obtained using SPring-8 BL44XU, but in the same manner as in Examples 1-7. The results of processing the collected data are shown in Table 12.

Example 13: Bicelle method
The membrane protein GFP-Nb-Dr-VSP-VSD ^Int prepared in Example 1 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 J. U. Bowie, 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 crystallized in a 20° incubator in the same manner as above (FIG. 60).

Example 14: Counterdiffusion method
The membrane protein GFP-Nb-Dr-VSP-VSD ^Int prepared in Example 1 was concentrated to 10 mg/mL using an ultrafiltration membrane, and crystallized in two different environments, on the ground and in a microgravity environment, using a common counter diffusion method (J.M. Garcia-Ruiz et. al., Meth. Enzymol. 2002 368:130-54). For crystallization in a microgravity environment, the crystal was transported to the Japanese Experiment Module "Kibo" of the International Space Station by SpaceX's Dragon supply ship operation No. 19 SpaceX-19, and crystallization was performed. Specifically, 8 μL of the membrane protein solution was filled into a capillary with an inner diameter of 0.5 mm, and immersed in 0.9 mL of precipitant (0.1 M MES-Na [pH 5.4-5.6], 25% (w/v) PEG2000MME). The crystallization sample was placed in an incubator at 20 degrees, and the protein solution was made supersaturated by liquid-liquid diffusion, and crystallized (Figure 61: Under microgravity environment) (Figure 62: on the ground).

The results of processing the collected data in the same manner as in Examples 1-7, except that diffraction data was collected from the crystals obtained on SPring-8 BL45XU, are shown in Table 13. The electron density map within the membrane-spanning domain in the crystal structure generated in a microgravity environment is shown in Figure 63, and the final structure is shown in Figure 64. The electron density map within the membrane-spanning domain in the crystal structure generated on the ground is shown in Figure 65, and the final structure is shown in Figure 66.

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

Examples of aspects of the present invention include the following.
<1> A protein structure for analyzing the structure of a membrane protein, which is capable of binding to a fluorescent protein and to an immunoglobulin-binding protein.
<2> A protein structure for membrane protein structure analysis according to <1>, comprising a VHH domain of a camelid antibody.
<3> A chimeric protein comprising a membrane protein and the protein structure for membrane protein structure analysis according to any one of <1> to <2>.
<4> A method for producing a chimeric protein, comprising a step of expressing a nucleic acid obtained by fusing a gene encoding a membrane protein with a gene encoding a protein structure for use in membrane protein structural analysis, the protein structure being capable of binding to a fluorescent protein and to an immunoglobulin-binding protein.
<5> A method for purifying a membrane protein, comprising 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, by using the immunoglobulin-binding protein.
<6> A method for crystallizing a membrane protein, comprising a 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 for crystallizing a membrane protein according to <6>, further comprising the step of adding a fluorescent protein to the chimeric protein.
<8> A method for analyzing the structure of a membrane protein, comprising the steps of: purifying a chimeric protein, in which a membrane protein is fused with a protein structure for analyzing a membrane protein structure 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 for analyzing a structure of a membrane protein according to <8>, further comprising the step of adding a fluorescent protein to the chimeric protein.
<10> The method for analyzing the structure of a membrane protein according to <8> or <9>, further comprising a step of analyzing the structure of the crystallized chimeric protein.

Claims (9)

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. 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 a β domain of intimin. 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. The method for crystallizing a membrane protein according to any one of claims 1 to 3, wherein the chimeric protein comprises the VHH domain of the camelid antibody, the membrane protein, and a linker that connects the VHH domain of the camelid antibody to the membrane protein. 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. 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. The method for structural analysis 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, a sugar transporter SWEET family protein, SemiSWEET, a translocator protein, a riboflavin transporter, and a β domain of intimin. The method for analyzing the structure of a membrane protein according to any one of claims 5 to 7, wherein the immunoglobulin-binding protein is protein A, protein A/G, or protein G. The method for analyzing the structure of a membrane protein according to 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 binds the VHH domain of the camelid antibody to the membrane protein.