JPWO2018163947A1 - Protein crystallization using porous protein crystals - Google Patents

Abstract

Disclosed are fusion proteins comprising R1-EN and a protein fused to the C-terminus or N-terminus of the R1-EN or having the R1-EN in a loop, crystallizing the fusion protein A protein crystal comprising the fusion protein, a protein crystal obtained by the method, or a protein structural analysis method comprising a step of performing X-ray crystal structure analysis using the protein crystal, the fusion protein , An expression vector containing the nucleic acid, a transformant into which the nucleic acid has been introduced, and a method of disposing an arbitrary protein in the space of a honeycomb-structured crystal lattice using the fusion protein.

Description

  The present invention relates to a fusion protein capable of efficiently producing crystals of any protein, a nucleic acid encoding the fusion protein, an expression vector containing the nucleic acid, and a transformant into which the nucleic acid has been introduced. The present invention also relates to a protein crystal, a method for producing a protein crystal, a method for analyzing the structure of a protein, and a method for arranging an arbitrary protein in a void of a crystal lattice.

  The three-dimensional structure of a protein provides a great deal of biochemical understanding, and is essential for elucidating the pathogenesis of disease and designing effective drugs. X-ray crystal structure analysis is widely used as a method for analyzing the three-dimensional structure of a protein.However, in order to crystallize a protein, it is necessary to screen a large number of crystallization conditions and it is not always possible to obtain crystals This is not always the case. As described above, the crystal structure analysis of a protein has a problem that it has to cross the crystallization wall. Although much research has been done to facilitate crystallization, no effective method has yet been found.

  In crystal structure analysis, protein crystallization is indispensable, but it is sufficient that protein molecules are in a state of being regularly arranged. Therefore, an approach of constructing a three-dimensional lattice in advance and binding specific molecules to it is conceivable. As such a method, Non-Patent Document 1 reports that a “crystal sponge” was developed, and a low molecular weight organic compound was bonded to the crystal sponge for structural analysis. However, this method cannot be applied to proteins due to their large size.

  Non-Patent Document 2 reports a method of designing a DNA sequence and assembling it three-dimensionally. However, this method has a problem that it is limited to DNA binding proteins that bind to a specific sequence.

  Other highly porous materials to be used for crystallization include highly porous materials made of low molecular weight compounds and highly porous materials made of artificially designed proteins. There are problems such as low and difficult design.

  As a technique for promoting crystallization of materials other than high-porosity materials, Patent Document 1 discloses that a polypeptide that promotes dimerization of a protein is fused with a target protein to effectively crystallize the target protein. It is reported to be promoted. However, in this method, it is necessary to determine crystallization conditions.

  R1 is one of the retrotransposons found in the silkworm, Bombyx mori, and ORF2p in the two open reading frames encodes reverse transcriptase (RT) and endonuclease (EN). Then, the present inventors have reported a crystal structure with a resolution of 2.0 ° (space group P321, unit cell a = b = 141.3 °, c = 37.5 °) for the EN domain of R1 (R1-EN) ( Non-patent document 3).

WO 2014/181786

Y. Inokuma et al., Nature, Vol. 495, 461-466 (2013) J. Zheng et al., Nature, Vol. 461, 74-77 (2009) N. Maita et al., Nucleic Acid Research, Vol. 35, NO.12, 3917-3927 (2007)

  An object of the present invention is to provide a fusion protein capable of efficiently producing crystals of any protein, a nucleic acid encoding the fusion protein, an expression vector containing the nucleic acid, and a transformant into which the nucleic acid has been introduced. I do. The present invention also provides a protein crystal containing the fusion protein, a method for producing a protein crystal using the fusion protein, a method for analyzing the structure of a protein using the protein crystal, and a crystal lattice using the fusion protein. An object is to provide a method for arranging an arbitrary protein.

  The present inventors have conducted intensive studies to achieve the above object, and as a result, the R1-EN crystal has a large number of solvent regions (69.7%), has a hollow honeycomb structure, and both the N-terminus and the C-terminus. Since it is exposed to the solvent region, it has been found that proteins fused to the ends can be placed in the solvent region, and that such fusion proteins can be applied to the same purification and crystallization conditions as R1-EN. Was.

  The present invention has been completed by further studies based on these findings, and includes the following fusion proteins, nucleic acids, expression vectors, transformants, protein crystals, methods for producing protein crystals, methods for analyzing the structure of proteins, and the like. Is provided.

Item 1. A fusion protein comprising R1-EN and a protein fused to the C-terminus or N-terminus of the R1-EN or a protein having the R1-EN in a loop.
Item 2. A step of crystallizing the fusion protein according to Item 1,
A method for producing a protein crystal.
Item 3. Item 7. A protein crystal comprising the fusion protein according to Item 1.
Item 4. Item 2, comprising a step of performing X-ray crystal structure analysis using the protein crystal obtained by the method according to Item 2 or the protein crystal according to Item 3.
Protein structure analysis method.
Item 5. Item 7. A nucleic acid encoding the fusion protein according to Item 1.
Item 6. Item 6. An expression vector comprising the nucleic acid according to Item 5.
Item 7. A transformant into which the nucleic acid according to item 5 has been introduced.
Item 8. Item 10. A method for disposing an arbitrary protein in a space of a crystal lattice having a honeycomb structure using the fusion protein according to Item 1.

  The fusion protein obtained by fusing any protein to R1-EN of the present invention can be purified and crystallized under the same purification conditions and crystallization conditions as R1-EN, so there is no need to screen for crystallization conditions, It is possible to efficiently crystallize any protein. Further, in the present invention, an arbitrary protein can be arranged in the solvent region of the R1-EN crystal, and the inside diameter of the solvent region is approximately 110 °, so that a protein having a large molecular weight can be crystallized.

  Although there are still many important proteins whose structures are unknown because they cannot be crystallized, the present invention is expected to solve the problem of crystallization and promote the structural analysis of proteins whose structures are unknown. Is done.

It is a figure which shows the crystal lattice of the protein crystal of this invention. It is a figure which shows the chromatogram of the cation exchange column of R1-EN_Ubi. It is a figure which shows the chromatogram of the gel filtration column of R1-EN_Ubi. FIG. 4 is a view showing an electron density (Fo-Fc / 0.7σ) of a ubiquitin portion.

  Hereinafter, the present invention will be described in detail.

  In this specification, the term “comprise” includes the meaning of “essentially consist of” and the meaning of “consist of”.

  In the present invention, the term “gene” includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof, unless otherwise specified. In the present invention, the term “gene” means a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.

  In the present invention, “nucleic acid”, “nucleotide” and “polynucleotide” are synonymous, and include both DNA and RNA, and may be double-stranded or single-stranded.

  The fusion protein of the present invention comprises (1) R1-EN, and (2) a protein fused to the C-terminus or N-terminus of the R1-EN or a protein having the R1-EN in a loop (hereinafter, referred to herein as the present specification). (Sometimes referred to as "protein A").

  “R1-EN” in the present invention means an EN domain encoded by ORF2p which is one of R1 open reading frames. The EN domain is a region at the 1-240th position in the amino acid sequence encoded by ORF2p. When crystallization is intended as in the present invention, R1-EN may have an amino acid sequence of a portion where a crystal lattice of a honeycomb structure is obtained when crystallized, and such an amino acid sequence may be used. Represents the sequence at position 5-223 of the amino acid sequence encoded by ORF2p. R1 is one of the retrotransposons found in silkworms (Bombyx mori).

  The amino acid sequence of ORF2p of R1 is registered on the UniProt web site as UniProt Accession No. Q7M4J4 (SEQ ID NO: 1). Further, the three-dimensional structure of R1-EN is registered in the PDB as PDB ID: 2EI9.

  The `` R1-EN '' in the present invention includes, besides those having an amino acid sequence registered in the database as described above, also includes variants thereof, and the variants include proteins having the above amino acid sequences. A protein that forms a crystal lattice with an equivalent honeycomb structure is desirable. As a variant, two cysteines (Cys55, Cys72) were substituted with other amino acids for the purpose of preventing aggregation, and the active residue (Glu44) was substituted with another residue for the purpose of eliminating DNA cleavage activity. Things.

  As a variant, in the amino acid sequence registered in the database as described above, one or more, for example, 1 to 50, 1 to 25, 1 to 12, 1 to 9, 1 to 5 A protein comprising an amino acid sequence in which the amino acid has been substituted, deleted, inserted and / or added. Techniques for substituting, deleting, inserting and / or adding one or more amino acids in a specific amino acid sequence are known.

  Amino acid sequence identity can be calculated using analysis tools that are commercially available or available through a telecommunications line (the Internet). The amino acid sequence identity (%) can be determined using a program commonly used in the art (eg, BLAST, FASTA, etc.) by default.

  The “protein A” in the present invention is not particularly limited, and includes natural proteins, artificial proteins, and the like, and also includes natural or artificial peptides. As “Protein A”, a protein or peptide whose tertiary structure is unknown and a protein or peptide whose crystallization has not been possible are particularly suitable. The molecular mass of protein A is not particularly limited as long as it can be arranged in the solvent region of the R1-EN crystal, and is preferably 5 kDa or more, more preferably 5 to 50 kDa, and still more preferably 5 to 25 kDa. . The size of protein A is not particularly limited as long as it can be arranged in the solvent region of the R1-EN crystal, and is preferably 10 to 60 °, more preferably 10 to 40 °.

  The R1-EN crystal has a hollow honeycomb structure as shown in Fig. 1 (honeycomb inner diameter is about 110 mm), voids continue in the c-axis direction, and both N-terminal and C-terminal are exposed to the solvent region. Therefore, protein A can be placed in the solvent region regardless of whether the protein is fused to the N-terminus or the C-terminus. In addition, since the N-terminus and C-terminus are adjacent to each other in the R1-EN crystal, when the structure of protein A does not change even if another protein is fused in the loop protruding outside of protein A, Can also place protein A in the solvent region of the R1-EN crystal by inserting the R1-EN sequence into the loop of protein A.

  The “honeycomb structure” in the present invention means a structure in which hexagonal columns as shown in FIG. 1 are spread without gaps, and the hexagonal columns are desirably regular hexagonal columns.

  In the fusion protein of the present invention, R1-EN and protein A may be directly bound, or may be bound by any amino acid sequence present therebetween. In addition to these two proteins, other proteins and peptides may be bound to the fusion protein of the present invention.

  The fusion protein of the present invention can be produced, for example, by culturing a transformant into which a nucleic acid encoding the fusion protein has been introduced as described below. By culturing the transformant in a nutrient medium suitable for host cells under conditions that allow expression of the fusion protein, the transformant can cause the fusion protein to be produced intracellularly or in the medium. .

  Purification of the produced polypeptide includes affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydroxyapatite column chromatography, hydrophobic interaction chromatography, membrane filter, ultrafiltration, microfiltration, ammonium sulfate salting-out method, It can be performed by distillation treatment, crystallization, or the like. Further, purification can be performed alone or in an appropriate combination of two or more kinds in any order.

  Since the fusion protein of the present invention can be purified under the same or similar purification conditions as R1-EN (for example, see Non-Patent Document 3), it is possible to purify efficiently without having to study the purification conditions. , R1-EN, it is desirable to apply the same or similar purification conditions.

  The nucleic acid of the present invention encodes the above-mentioned fusion protein. The nucleic acid can be prepared by a conventional method such as biochemical cleavage / recombination using a nucleic acid encoding each protein constituting the fusion protein. The nucleic acid can be used for producing the fusion protein and the like. The nucleic acid encoding R1-EN and protein A can be obtained as a commercial product, or using information of a known base sequence registered in a public database such as GenBank, using a cDNA library as a template. It can also be produced by a conventional method such as a PCR method. In addition, the expression efficiency can be increased by changing to codons frequently used in the host cell into which the nucleic acid is introduced.

  The expression vector of the present invention is characterized by containing the above nucleic acid. The expression vector is not particularly limited, and a known expression vector can be widely used. An appropriate expression vector can be appropriately selected in consideration of the type of the host cell into which the nucleic acid is introduced, and the like. The expression vector may contain a promoter, an enhancer, a terminator, a polyadenylation signal, a selection marker, an origin of replication, and the like, in addition to the nucleic acid. As the expression vector, any of an autonomously replicating vector and a vector that is integrated into the genome of the host cell when introduced into the host cell and replicated together with the integrated chromosome can be used. The nucleic acid can be inserted into an expression vector by a known method.

  As the expression vector of the present invention, a vector in which a nucleic acid encoding a tag is added to one or both ends of the above nucleic acid can also be used. The tag includes Flag, Myc, HA (hemagglutinin), GST (glutathione S-transferase), histidine and the like. In this way, by tagging the fusion protein, purification using the tag can be performed. In addition, by adding a protease recognition sequence between the fusion protein and the tag, the tag can be cleaved after purification.

  Methods for constructing an expression vector and for introducing the expression vector into a host cell are well-known. Can be implemented.

  The transformant of the present invention is characterized in that the above nucleic acid has been introduced. Introduction of a nucleic acid can be performed by using an expression vector containing the nucleic acid as described above. Introduction of an expression vector into a host cell can be performed by a known method such as an electroporation method, a calcium phosphate method, a lipofection method, a liposome method, a microinjection method, and a lithium acetate method. As a host for introducing the nucleic acid, for example, bacteria (e.g., Escherichia coli, Streptomyces (Streptomyces), Rhodococcus (Rhodococcus), Streptococcus (Streptococcus), Staphylococcus (Staphylococcus)), Fungi (e.g., yeasts (Saccharomyces), Schizosaccharomyces, etc.), Aspergillus (Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF), mammalian cells And plant cells.

  The method for producing a protein crystal of the present invention includes a step of crystallizing the fusion protein.

  The solution used in the crystallization step (hereinafter, referred to as “crystallization solution”) contains a precipitant, a buffer, R1-EN, and the like, if necessary, in addition to the fusion protein. The concentration of the fusion protein in the crystallization solution is not particularly limited, and is usually about 1 to 10 mg / mL. By mixing R1-EN with the crystallization solution, larger proteins A can be crystallized as described later.

  As the precipitant, for example, salts such as lithium chloride, sodium chloride, magnesium chloride, potassium chloride, sodium acetate, ammonium acetate, ammonium sulfate, lithium sulfate, magnesium sulfate, sodium citrate, polyethylene glycol, Jeffamine (trademark) M-600 And alcohols such as 2-methyl-2,4-pentanediol, methanol and 2-propanol. These can be used alone or in combination of two or more. The concentration of the precipitant is usually about 0.5 to 3 M for a salt, about 0.1 to 2% (v / v) for a polymer compound, and about 2 to 20% (v / v) for alcohols.

  Examples of the buffer include acetate buffer, citrate buffer, borate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer, MES buffer and the like. These can be used alone or in combination of two or more.

  Examples of the solvent for the crystallization solution include water, methanol, ethanol, isopropanol, and the like, with water being preferred.

  The crystallization solution preferably contains a seed crystal, and the presence of the seed crystal can promote crystallization.

  As a method of crystallizing a protein, a known crystallization method can be used, for example, a vapor diffusion method (a hanging drop method, a sitting drop method, etc.), a batch method, a liquid-liquid diffusion method, a dialysis method, Seeding method and the like can be mentioned.

  The crystallization temperature is not particularly limited as long as crystallization is possible, and is usually 0 to 20 ° C. The time for crystallization is not particularly limited as long as crystals can be obtained, and is usually 20 hours to 30 days.

  The protein crystal of the present invention is characterized by containing the fusion protein. Such a protein crystal can be produced, for example, by the method described above.

  Since the fusion protein can be crystallized under the same or similar crystallization conditions as R1-EN, it is possible to apply the same or similar crystallization conditions as R1-EN (for example, see Non-Patent Document 3). This is desirable because there is no need to screen for crystallization conditions and efficient crystallization is possible.

  In the protein crystal of the present invention, the crystal lattice has a honeycomb structure, and the pores have pores with an inner diameter of about 110 ° extending in the c-axis direction (see FIG. 1). For example, an arbitrary protein (ie, protein A) can be arranged without limitation in shape and type. Since the inner diameter of the honeycomb is approximately 110 °, any protein with a high molecular weight can be put into the pores. It is also considered that by mixing a certain amount of R1-EN (without fusion of the protein) with the above-mentioned fusion protein and crystallizing the same, protein A having a larger molecular weight can be crystallized. The ratio of the fusion protein to the R1-EN molecule in such a crystal is, for example, 3: 1 to 1: 3. Further, since the solvent region of the protein crystal of the present invention has a hollow tubular shape, it is considered that rod-shaped protein A can be crystallized even if it has a certain length.

  In the examples described below, ubiquitin is used as an example of protein A, but it is considered that any protein other than ubiquitin can be crystallized in the same manner as ubiquitin if it can be arranged in the solvent region. Can be

  The method for analyzing the structure of a protein of the present invention is characterized by including a step of performing X-ray crystal structure analysis using the protein crystal obtained by the above method or the above protein crystal.

  X-ray crystal structure analysis can be performed according to a known method. X-ray crystal structure analysis can also be performed systematically based on the structure of R1-EN, so that efficient analysis is possible. It is also possible to obtain high-resolution diffraction data of 2.0 ° or less. The general procedure of X-ray crystal structure analysis will be outlined below.

  First, X-ray diffraction data is obtained by irradiating a protein crystal with X-rays using a beam line or an X-ray irradiator of a large synchrotron radiation facility. Based on the X-ray diffraction data, an electron density diagram of a molecule constituting the fusion protein is obtained, and the molecule is modeled based on the electron density diagram to construct a three-dimensional structure of the fusion protein.

  In order to derive an electron density diagram, it is necessary to determine a phase by a molecular replacement method. In the present invention, an optimal solution can be obtained by a molecular replacement method based on the structure of R1-EN. If a good initial phase is obtained by the molecular replacement method, the electron density diagram can be drawn. A model of protein A can be constructed on three-dimensional graphics by using a known program based on the electron density diagram thus obtained. Next, the constructed molecule model is refined (refined) using a known program in order to approach a more accurate structure.

  If X-ray irradiation is performed while the protein crystals are frozen, use antifreeze solutions containing cryoprotectants such as polyethylene glycol, glycerol, and sucrose to prevent protein crystals from freezing. It is desirable to do.

  Since the fusion protein of the present invention can be purified and crystallized under the same purification conditions and crystallization conditions as R1-EN, there is no need to screen for crystallization conditions, and any protein can be efficiently crystallized. . Further, by using the fusion protein of the present invention, any protein can be arranged in the solvent region of the R1-EN crystal, so that a protein having a large molecular weight can be crystallized.

  There are still many important proteins whose structures are unknown because they cannot be crystallized. However, by using the fusion protein of the present invention, the problem of crystallization is solved, and the structure of proteins whose structure is unknown is analyzed. Is also possible.

  Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these Examples and the like.

<Reference example>
R1-EN expression method (see Non-Patent Document 3)
The 5-243 sequence of R1-ORF2p and a stop codon were inserted immediately after the restriction enzyme NdeI / XhoI of pET16b (Novagen) to obtain an expression plasmid vector.

This plasmid was transformed into Escherichia coli BL21 (DE3) pLysS strain, and a logarithmic growth phase (turbidity (turbidity ()) was added to 800 mL of an LB medium containing ampicillin (Sigma-Aldrich) and chloramphenicol (Wako Pure Chemical Industries, Ltd.). After vigorous shaking culture at 37 ° C. until the OD ₆₀₀ reaches about 0.8), IPTG having a final concentration of 0.2 mM is added, and the mixture is left at 20 ° C. for about 20 hours with gentle shaking. Then, the Escherichia coli body is precipitated and collected by a centrifuge, and stored frozen at -80 ° C.

R1-EN Purification method E. coli cells were suspended in 50 mL of buffer A (Note 1) for 15 g of E. coli cells, 0.1% Triton X100 (Sigma-Aldrich), 1 mM PMSF (Wako Pure Chemical Industries, Ltd.), 10 μg / mL RNaseA (Sigma-Aldrich) and 10 μg / mL Lysozyme (Wako Pure Chemical Industries, Ltd.) (all in final concentration) and sonicate on ice.

  The lysate is centrifuged at 6000 g × 30 min at 4 ° C, and the supernatant is collected. Further, polyethyleneimine (Sigma-Aldrich) having a final concentration of 0.5% is added to the supernatant, and the suspension is centrifuged at 6000 g × 30 min at 4 ° C. The supernatant is passed through a 0.45 μm filter, and the filtrate is passed through a HisTrap HP 5 mL column (GE Healthcare).

  Next, wash the column with 30 mL of buffer A and elute with 30 mL of buffer B (Note 2).

  Ammonium sulfate (Wako Pure Chemical Industries, Ltd.) is added to the eluate to 60% saturation, and the ammonium sulfate is completely dissolved, followed by centrifugation at 6000 g × 20 min at 4 ° C. Discard the supernatant, add 10 mL of Factor Xa buffer (Note 3) to the precipitate, and suspend the precipitate gently.

  Measure the protein concentration from UV280 with an absorbance meter (nano-drop ND-1000 (Thermo Fisher Scientific)), add Factor Xa (New England Biolab) of 1/300 by weight of the protein amount, and add 10 ° C. And leave overnight.

  The reaction solution is again converted to 60% saturated ammonium sulfate, centrifuged at 6000 g × 20 min at 4 ° C., and the supernatant is discarded. The precipitate is suspended in 20 mL of SP buffer A (Note 4), passed through a 0.45 μm filter (Merck), and then loaded into 1 mL of HiTrap SP HP (GE Healthcare). Subsequently, the column is subjected to a linear gradient of 50-800 mM NaCl. R1-EN elutes in a fraction of approximately 500 mM NaCl.

Add ammonium sulfate to the R1-EN fraction so that it becomes 60% saturated, centrifuge at 6000 g × 20 min at 4 ° C, and discard the supernatant. The precipitate is dissolved in 1-2 mL of a gel filtration buffer (Note 5), passed through a 0.22 μm filter (Merck), and then subjected to gel filtration using HPLC (Superdex200 10/300 (GE Healthcare)). To separate. R1-EN elutes in approximately 18 mL fractions. The R1-EN fraction is collected and concentrated to 7-8 mg / mL using a 10 kDa ultrafiltration membrane (Amicon Ultra (Merck)).
(Note 1) Composition of buffer A: 30 mM Tris, 50 mM Imidazole, pH 7.5, 0.5 M NaCl
(Note 2) Composition of buffer B: 30 mM Tris, 350 mM Imidazole, pH 7.5, 0.5 M NaCl
(Note 3) Composition of buffer for Factor Xa: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM CaCl ₂ , 1 mM DTT
(Note 4) Composition of SP buffer A: 40 mM sodium phosphate pH 5.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT
(Note 5) Composition of buffer for gel filtration: 40 mM Hepes-NaOH pH 7.2, 180 mM NaCl, 1 mM DTT, 1 mM EDTA

The crystallization method of R1-EN is described in detail in Non-Patent Document 3.

  The crystallization is performed by a hanging drop vapor diffusion method. Precipitant solution (1.8 M sodium acetate (Wako Pure Chemical Industries, Ltd.) (pH 7.3), 10 mM ammonium sulfate, 1.0% Jeffamine) was added to 1.5 μL of the 7-8 mg / mL R1-EN solution obtained by the above purification. Add 1.5 μL of M-600 (Hampton Research) and leave at 10 ° C. The crystals grow in about 7-10 days.

  When microseeding is performed, crystals can be obtained with good reproducibility.

<Production Example>
Design of plasmid vector for fusion protein production (pET16b-R1EN)
1) Stabilization of R1-EN The crystal structure revealed two free cysteine residues on the protein surface. These residues were replaced with serine to prevent aggregation (C55S / C72S). The active residue E44 was replaced with alanine in order to eliminate the original DNA cleavage activity of R1-EN.

2) Removal of extra sequence In the crystal structure, only the 222nd glycine at the C-terminal side was visible. The unseen region has 21 residues, which physically hindered the inside of the hole, so portions 224-243 were removed.

3) Insertion of a multi-cloning site A multi-cloning site was introduced immediately after R1-EN to enable insertion of sequences encoding various proteins.

・CATATG is an NdeI site, and the part before this is common to pET16b.
-The part in bold is the arrangement of 5-223 of R1-EN.
· GCA is to put a mutation in the original sequence gaa, · TCC that you are Glu44 → Ala44 is to put a mutation in the original sequence tgt, · TCG that you are Cys55 → Ser55 is to put a mutation in the original sequence tgc, Cys72 → Ser72 ・ Italics are multiple cloning sites, and recognition sequences for BamHI (GGATCC), SmaI (CCCGGG), SalI (GTCGAC), XhoI (TCTGAG), and SacI (GAGTCT) are aligned from the 5 'side.
・ The sequence after the gray part is the same as that after 5392 of pET16b.

Design of plasmid vector for production of fusion protein 2
Above is a design for fusing the protein to the C-terminal of R1-EN, but a plasmid for fusing to the N-terminal of R1-EN was also designed.

  Following 1) and 2) above, this time a construct was made based on pGEX6P-1.

GGATCC is a recognition sequence for BamHI, and AAGCTT is a recognition sequence for HindIII. This makes it possible to introduce an arbitrary protein with these two restriction enzymes. Before GGATCC, it is common with pGEX6P-1.
-The part in bold is the sequence of 9-222 of R1-EN.
• Gray taa is a stop codon.
・CTCGAG is a recognition sequence of XhoI, and is the same as pGEX6P-1 thereafter.

Preparation of R1-EN_hUbi Expression Vector A human ubiquitin (hUbi) sequence was inserted using BamHI / XhoI of pET16b-R1EN to prepare a plasmid expressing R1-EN_hUbi.

  The inserted sequence was as follows.

GGATCC: BamHI, CTCGAG: XhoI

Escherichia coli BL21 (DE3) pLysS strain was transformed with the plasmid expressing R1-EN_hUbi prepared in the purification of R1-EN_hUbi, and expressed by the method described in the section of [Method for Expression of R1-EN]. Purification was performed in the same manner as described in [R1-EN Purification Method].

  However, R1-EN_Ubi was eluted in a fraction of about 600 mM NaCl on the cation exchange column (FIG. 2), and was eluted in a fraction of about 17.4 mL on the gel filtration column (FIG. 3).

  The R1-EN_hUbi fraction was collected and concentrated to 2 mg / mL using a 10 kDa ultrafiltration membrane (Amicon Ultra).

<Test example>
R1-EN_hUbi purified on the crystallization of R1-EN_hUbi was treated with a precipitant (1.8 M sodium acetate pH 7.6, 10 mM ammonium sulfate, 0.8% Jeffamine M600 pH 7.0) as in [R1-EN crystallization]. Crystallization was performed by the method. At this time, when seeding was performed using the R1-EN crystal obtained by [crystallization of R1-EN] obtained by crushing with a microhomogenizer as a seed crystal, a crystal was obtained with good reproducibility.

The crystal obtained on the crystal structure analysis of R1-EN_hUbi was scooped with a nylon loop (Hampton Research), transferred to an anti-freezing solution (1 μL of glycerin + 5 μL of precipitant), again scooped, and immersed in liquid nitrogen to freeze the crystal . X-ray irradiation was performed at the synchrotron radiation facility SPring-8 BL44XU while frozen, and data with a maximum resolution of 1.8 mm was obtained.

  This data was in the space group P321, a = b = 141.21, c = 37.67 °, and was the same as the R1-EN space group (P321, a = b = 141.30, c = 37.51 °) within the error range. The optimal solution was obtained by the molecular replacement method based on the structure of R1-EN, and the electron density was calculated using the phase. As a result, the electron density was obtained in the void. A ubiquitin structure could be applied from the characteristics of the β-sheet (FIG. 4).

Claims (8)

  A fusion protein comprising R1-EN and a protein fused to the C-terminus or N-terminus of the R1-EN or a protein having the R1-EN in a loop. Crystallizing the fusion protein of claim 1,
A method for producing a protein crystal.   A protein crystal comprising the fusion protein according to claim 1. A protein crystal obtained by the method according to claim 2, or a step of performing an X-ray crystal structure analysis using the protein crystal according to claim 3,
Protein structure analysis method.   A nucleic acid encoding the fusion protein according to claim 1.   An expression vector comprising the nucleic acid according to claim 5.   A transformant into which the nucleic acid according to claim 5 has been introduced.   A method for arranging an arbitrary protein in voids of a crystal lattice having a honeycomb structure using the fusion protein according to claim 1.

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