WO2018163947A1 - Cristallisation de protéine à l'aide d'un cristal poreux protéinique - Google Patents

Cristallisation de protéine à l'aide d'un cristal poreux protéinique Download PDF

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WO2018163947A1
WO2018163947A1 PCT/JP2018/007697 JP2018007697W WO2018163947A1 WO 2018163947 A1 WO2018163947 A1 WO 2018163947A1 JP 2018007697 W JP2018007697 W JP 2018007697W WO 2018163947 A1 WO2018163947 A1 WO 2018163947A1
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protein
crystal
fusion protein
nucleic acid
crystallization
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宣夫 真板
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国立大学法人徳島大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • 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 a protein structure, and a method for arranging an arbitrary protein in a gap of a crystal lattice.
  • the three-dimensional structure of proteins gives a lot of biochemical understanding, and is essential for elucidating the pathogenesis of diseases and designing effective drugs.
  • X-ray crystal structure analysis is widely used as a method for analyzing the three-dimensional structure of proteins, but in order to crystallize proteins, it is necessary to screen many crystallization conditions, and crystals are always obtained. The problem is not always the case.
  • the crystal structure analysis of proteins has a problem that the crystallization barrier must be overcome. There have been many studies to facilitate crystallization, but no effective method has yet been found.
  • Non-Patent Document 1 reports that a “crystal sponge” was developed, and a low molecular organic compound was bonded to the structure analysis. However, this method cannot be applied because proteins are large molecules.
  • 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 a DNA binding protein that binds to a specific sequence.
  • high-porosity materials used for crystallization include high-porosity materials based on low-molecular compounds and high-porosity materials based on artificially designed proteins, but they cannot be applied to large proteins. There are problems such as low and difficult design.
  • Patent Document 1 As a technique for promoting crystallization other than a highly porous material, in Patent Document 1, crystallization of a target protein is effectively achieved by fusing a polypeptide that promotes protein dimerization with the target protein. It has been reported to be promoted. However, in this method, it is necessary to determine the crystallization conditions.
  • 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. To 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 protein structure analysis method using the protein crystal, and a gap in a crystal lattice using the fusion protein.
  • An object is to provide a method for arranging an arbitrary protein.
  • the present inventors have found that the R1-EN crystal has a large solvent region (69.7%) and has a hollow honeycomb structure, and both the N-terminal and C-terminal are 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 with the same purification and crystallization conditions as R1-EN. It was.
  • the present invention has been completed based on these findings, and has been completed.
  • the following fusion proteins, nucleic acids, expression vectors, transformants, protein crystals, protein crystal production methods, protein structure analysis methods, etc. Is to provide.
  • Item 1 A fusion protein comprising R1-EN and a protein fused to the C-terminus or N-terminus of R1-EN or a protein having R1-EN in a loop.
  • Item 2. Including the step of crystallizing the fusion protein according to Item 1. A method for producing protein crystals.
  • Item 3. A protein crystal comprising the fusion protein according to Item 1.
  • Item 4. Including a step of performing an X-ray crystal structure analysis using the protein crystal obtained by the method of Item 2 or the protein crystal of Item 3. Protein structure analysis method.
  • Item 5. A nucleic acid encoding the fusion protein according to Item 1.
  • Item 6. An expression vector comprising the nucleic acid according to Item 5.
  • Item 7. Item 6. A transformant into which the nucleic acid according to Item 5 has been introduced.
  • Item 8. A method of arranging an arbitrary protein in a void of a crystal lattice having a honeycomb structure using the fusion protein according to Item
  • the fusion protein in which any protein is fused 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 inner diameter of this solvent region is approximately 110 mm, so that a protein with a large molecular weight can be crystallized.
  • gene includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof unless otherwise specified.
  • gene refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.
  • 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 It may be referred to as “protein A”).
  • R1-EN in the present invention means an EN domain encoded by ORF2p, which is one of the open reading frames of R1.
  • the EN domain is the 1-240th region of the amino acid sequence encoded by ORF2p.
  • R1-EN only needs to have a portion of an amino acid sequence from which a crystal lattice of a honeycomb structure can be obtained when crystallized. Includes the 5-223rd sequence of the amino acid sequence encoded by ORF2p.
  • R1 is one of the retrotransposons found in Bombyx mori.
  • R1 ORF2p The amino acid sequence of R1 ORF2p is registered on the UniProt web site as UniProt Accession No. Q7M4J4 (SEQ ID NO: 1).
  • the three-dimensional structure of R1-EN is registered in the PDB as PDB ⁇ ⁇ ⁇ ID: 2EI9.
  • R1-EN includes those having amino acid sequences registered in the database as described above, including modified forms thereof. Proteins that produce a crystal lattice with an equivalent honeycomb structure are desirable. In addition, 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 other residues for the purpose of eliminating DNA cleavage activity. Such as things.
  • one or more amino acid sequences registered in the database as described above 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 amino acids are 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.
  • Identities of amino acid sequences can be calculated using analysis tools that are commercially available or available through telecommunication lines (Internet). Amino acid sequence identity (%) can be determined using a program commonly used in the art (eg, BLAST, FASTA, etc.) by default. *
  • protein A is not particularly limited, and examples thereof include natural proteins and artificial proteins, and also include natural or artificial peptides.
  • Protein A a protein or peptide whose steric structure is unknown, and a protein or peptide that could not be crystallized 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, preferably 5 kDa or more, more preferably 5 to 50 kDa, even 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 mm, more preferably 10 to 40 mm.
  • the R1-EN crystal has a hollow honeycomb structure as shown in FIG. 1 (the honeycomb inner diameter is approximately 110 mm), the voids continue in the c-axis direction, and both the N-terminal and C-terminal are exposed in the solvent region. Therefore, protein A can be placed in the solvent region regardless of whether it is fused to either the N-terminus or the C-terminus. In addition, since the N-terminal and C-terminal are adjacent to each other in the R1-EN crystal, there is no change in the structure of protein A even if another protein is fused in a loop protruding outside protein A. The protein A can also be arranged in the solvent region of the crystal of R1-EN by inserting the sequence of R1-EN 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 preferably regular hexagonal columns.
  • R1-EN and protein A may be directly bound, or may be bound together by any amino acid sequence.
  • 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 by culturing a transformant introduced with a nucleic acid encoding the fusion protein as described later.
  • the transformant can be cultured in a nutrient medium suitable for the host cell under conditions that allow expression of the fusion protein, so that the transformant can produce the fusion protein in the cell or in the medium. .
  • the produced polypeptide can be purified by 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, crystallization, or the like. In addition, purification can be performed alone or in appropriate combination of two or more in any order.
  • 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 not necessary to study the purification conditions, and thus efficient purification is possible. It is desirable to apply the same or similar purification conditions as R1-EN.
  • the nucleic acid of the present invention encodes the above 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 the production of the fusion protein.
  • Nucleic acids encoding R1-EN and protein A can be obtained as commercial products, or using known nucleotide sequence information registered in public databases such as GenBank, and using a cDNA library as a template. It can also be produced by conventional methods such as PCR. In addition, the expression efficiency can be increased by changing the codon to be 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-mentioned nucleic acid.
  • the expression vector is not particularly limited, and known expression vectors can be widely used.
  • An appropriate expression vector can be appropriately selected in consideration of the type of host cell into which the nucleic acid is introduced.
  • the expression vector can contain a promoter, an enhancer, a terminator, a polyadenylation signal, a selection marker, an origin of replication, and the like.
  • any of a vector that replicates autonomously and a product 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.
  • a vector in which a nucleic acid encoding a tag is added to one or both ends of the nucleic acid can also be used.
  • the tag include Flag, Myc, HA (hemagglutinin), GST (glutathione S-transferase), histidine and the like.
  • the tag include Flag, Myc, HA (hemagglutinin), GST (glutathione S-transferase), histidine and the like.
  • the transformant of the present invention is characterized in that the nucleic acid is introduced.
  • Nucleic acid can be introduced by using an expression vector containing the nucleic acid as described above.
  • the expression vector can be introduced into the host cell by a known method such as an electroporation method, a calcium phosphate method, a lipofection method, a liposome method, a microinjection method, or a lithium acetate method.
  • Examples of the host into which the nucleic acid is introduced include bacteria (e.g., Escherichia coli, Streptomyces, Rhodococcus, Streptococcus, Staphylococcus), Fungi (e.g. yeast (genus Saccharomyces, Schizosaccharomyces, etc.), Aspergillus), insect cells (e.g. Drosophila S2, Spodoptera SF), mammalian animal cells And plant cells.
  • bacteria e.g., Escherichia coli, Streptomyces
  • the method for producing a protein crystal of the present invention includes a step of crystallizing the fusion protein.
  • crystallization solution contains a precipitating agent, a buffering agent, R1-EN and the like as necessary in addition to the fusion protein.
  • concentration of the fusion protein in the crystallization solution is not particularly limited, and is usually about 1 to 10 mg / mL.
  • the precipitating agent examples include lithium chloride, sodium chloride, magnesium chloride, potassium chloride, sodium acetate, ammonium acetate, ammonium sulfate, lithium sulfate, magnesium sulfate, sodium citrate and the like, polyethylene glycol, Jeffamine® (trademark) ® M-600. And high molecular compounds such as 2-methyl-2,4-pentanediol, methanol, 2-propanol and the like. These can be used alone or in combination of two or more.
  • the concentration of the precipitating agent is usually about 0.5 to 3 M for salts, 0.1 to 2% (v / v) for polymer compounds, and about 2 to 20% (v / v) for alcohols.
  • buffer examples 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, preferably water.
  • the crystallization solution preferably contains a seed crystal, and the presence of the seed crystal makes it possible to promote crystallization.
  • a known crystallization method can be used as a method for crystallizing protein.
  • a vapor diffusion method (hanging / drop method, sitting / drop method, etc.), batch method, liquid-liquid diffusion method, dialysis method, The seeding method etc. are 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 are obtained, and is usually 20 hours to 30 days.
  • the protein crystal of the present invention is characterized by containing the above fusion protein.
  • Such a protein crystal can be produced, for example, by the method described above.
  • the above fusion protein can be crystallized under the same or similar crystallization conditions as R1-EN, the same or similar crystallization conditions as R1-EN (see, for example, Non-Patent Document 3) can be applied. It is desirable because screening of crystallization conditions is unnecessary and efficient crystallization is possible.
  • the crystal lattice has a honeycomb structure, and the voids have pores having an inner diameter of about 110 mm extending in the c-axis direction (see FIG. 1).
  • any protein that is, protein A
  • the inner diameter of the honeycomb is approximately 110 mm, any protein having a large molecular weight can be put into the pores.
  • the protein A having a larger molecular weight can be crystallized by mixing a certain amount of R1-EN (without protein fusion) with the fusion protein and crystallization. Examples of the ratio of the fusion protein and R1-EN molecules in such crystals include 3: 1 to 1: 3.
  • the protein crystal of the present invention has a hollow tubular shape in the solvent region, the rod-like protein A can be crystallized even if it has a certain length.
  • ubiquitin is used as an example of protein A.
  • any protein other than ubiquitin can be placed in the solvent region, it can be crystallized in the same manner as ubiquitin. It is done.
  • the protein structure analysis method of the present invention includes a step of performing an 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, enabling efficient analysis. It is also possible to obtain high resolution diffraction data of 2.0 mm or less.
  • the general procedure for X-ray crystal structure analysis is outlined below.
  • X-ray diffraction data is obtained by irradiating a protein crystal with X-rays using a beam line of a large synchrotron radiation facility, an X-ray irradiation device, or the like.
  • a three-dimensional structure of the fusion protein is constructed by obtaining an electron density map of molecules constituting the fusion protein based on the X-ray diffraction data, and further modeling the molecule based on the electron density map.
  • 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, an electron density diagram can be drawn. Based on the electron density map obtained in this manner, a protein A model can be constructed on three-dimensional graphics by using a known program. Next, the constructed molecular model is refined (refined) using a known program in order to bring it closer to a more accurate structure.
  • anti-freeze treatment of the protein crystals is performed using an anti-freeze solution containing a cryoprotectant such as polyethylene glycol, glycerol, or sucrose to prevent the protein crystals from freezing. It is desirable to do.
  • 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. . Furthermore, by using the fusion protein of the present invention, an arbitrary protein can be arranged in the solvent region of the R1-EN crystal, so that a protein with a large molecular weight can be crystallized.
  • This plasmid was transformed into Escherichia coli BL21 (DE3) pLysS strain, and the logarithmic growth phase (turbidity (turbidity (turbidity)) After vigorously shaking culture at 37 ° C. until OD 600 ) is about 0.8), add IPTG with a final concentration of 0.2 mM and leave it for about 20 hours with slow shaking at 20 ° C. Thereafter, the Escherichia coli cells are precipitated by a centrifuge and collected, and stored frozen at -80 ° C.
  • ammonium sulfate (Wako Pure Chemical Industries, Ltd.) is added so that it becomes 60% saturated, and after ammonium sulfate is completely dissolved, it is centrifuged at 6000 ⁇ g ⁇ 20 ⁇ min at 4 ° C. Discard the supernatant and add 10 ⁇ mL of Factor Xa buffer (Note 3) to the precipitate to gently suspend the precipitate.
  • the reaction solution is made again with 60% saturated ammonium sulfate, centrifuged at 4 ° C. at 6000 ⁇ g ⁇ 20 ⁇ min, and the supernatant is discarded.
  • the precipitate is suspended in 20 ⁇ mL of SP buffer A (Note 4), passed through a 0.45 ⁇ m filter (Merck), and then loaded onto HiTrap SP HP 1 mL (GE Healthcare). Subsequently, the column is subjected to a linear gradient of 50-800 mM mM NaCl. R1-EN is eluted in a fraction of approximately 500 mM NaCl.
  • Non-patent document 3 describes in detail the crystallization method of R1-EN .
  • Crystallization is performed by the hanging drop vapor diffusion method.
  • Precipitation agent 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 ⁇ L of 7-8 mg / mL R1-EN solution obtained by the above purification.
  • Plasmid vector design for fusion protein production (pET16b-R1EN) 1) Stabilization of R1-EN From the crystal structure, two free cysteine residues were found on the protein surface. In order to prevent aggregation, these residues were substituted with serine (C55S / C72S). Further, E44, which is an active residue, was substituted with alanine for the purpose of eliminating the original DNA cleavage activity of R1-EN.
  • ⁇ CATATG is the NdeI site, and the part before this is common with pET16b. -The part in bold is the 5-223 sequence 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 ⁇ Italic is a multi-cloning site.
  • Plasmid vector design for fusion protein production 2 The above is a design to fuse the protein to the C-terminus of R1-EN, but we also designed a plasmid to fuse to the N-terminus of R1-EN.
  • GGATCC is a recognition sequence for BamHI, and AAGCTT is a recognition sequence for HindIII. As a result, any protein can be introduced with these two restriction enzymes. Also, before GGATCC, it is common with pGEX6P-1. -The bold part is the R1-EN 9-222 sequence. -Gray taa is a stop codon. ⁇ CTCGAG the recognition sequence of the XhoI, after this is identical to pGEX6P-1.
  • 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
  • the plasmid expressing R1-EN_hUbi prepared upon purification of R1-EN_hUbi was transformed into E. coli BL21 (DE3) pLysS strain and expressed by the method described in the section [Method for expressing R1-EN]. Further, purification was performed in the same manner as described in the section of [R1-EN purification method].
  • R1-EN_Ubi was eluted in a fraction of approximately 600 mM NaCl on a cation exchange column (FIG. 2) and eluted in an approximately 17.4 mL fraction on a gel filtration column (FIG. 3).
  • the R1-EN_hUbi fraction was recovered and concentrated to 2 mg / mL with a 10 kDa ultrafiltration membrane (Amicon Ultra).
  • R1-EN_hUbi purified on crystallization of R1-EN_hUbi is the same as [R1-EN crystallization] in the precipitating agent (1.8 M sodium acetate pH7.6, 10 mM ammonium sulfate, 0.8% Jeffamine M600 pH7.0). Crystallization was carried out by the method. At this time, when seeding the R1-EN crystal obtained by [crystallization of R1-EN] using a fine homogenizer as a seed crystal, a crystal was obtained with good reproducibility.
  • Crystals obtained by crystal structure analysis of R1-EN_hUbi were seeded with a nylon loop (Hampton Research), transferred to an anti-freezing solution (glycerin 1 ⁇ L + precipitant 5 ⁇ L), re-soaked and immersed in liquid nitrogen to freeze the crystals .
  • 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.

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Abstract

L'invention concerne : une protéine de fusion contenant un R1-EN et une protéine fusionnée à une extrémité C-terminale ou à une extrémité N-terminale dudit R1-EN ou d'une protéine présentant ledit R1-EN dans une boucle ; un procédé de production de cristal protéinique comprenant une étape de cristallisation de ladite protéine de fusion ; des cristaux protéiniques contenant ladite protéine de fusion ; un procédé d'analyse de structure protéinique comprenant une étape consistant à effectuer une analyse de structure de cristal par rayons X à l'aide des cristaux protéiniques obtenus au moyen du procédé susmentionné ou des cristaux protéiniques susmentionnés ; un acide nucléique codant pour ladite protéine de fusion ; un vecteur d'expression contenant ledit acide nucléique ; un transformant dans lequel ledit acide nucléique a été introduit ; et un procédé consistant à placer une protéine arbitraire dans un interstice d'un réseau cristallin présentant une structure en nid d'abeilles dans laquelle ladite protéine de fusion est utilisée.
PCT/JP2018/007697 2017-03-07 2018-03-01 Cristallisation de protéine à l'aide d'un cristal poreux protéinique WO2018163947A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085962A1 (fr) * 2000-05-05 2001-11-15 Imperial College Innovations Limited Procedes cristallographiques
JP2012125190A (ja) * 2010-12-15 2012-07-05 Kakei Gakuen 会合ユニット作製用リンカーペプチド
WO2014181786A1 (fr) * 2013-05-07 2014-11-13 国立大学法人北海道大学 Polypeptide favorisant la cristallisation
JP2014212722A (ja) * 2013-04-24 2014-11-17 学校法人加計学園 八面体構造を有するｂ型肝炎ウイルス様粒子結晶

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085962A1 (fr) * 2000-05-05 2001-11-15 Imperial College Innovations Limited Procedes cristallographiques
JP2012125190A (ja) * 2010-12-15 2012-07-05 Kakei Gakuen 会合ユニット作製用リンカーペプチド
JP2014212722A (ja) * 2013-04-24 2014-11-17 学校法人加計学園 八面体構造を有するｂ型肝炎ウイルス様粒子結晶
WO2014181786A1 (fr) * 2013-05-07 2014-11-13 国立大学法人北海道大学 Polypeptide favorisant la cristallisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAITA N. ET AL.: "Characterization of the sequence specificity of the RlBm endonuclease domain by structural and biochemical studies", NUCLEIC ACIDS RESEARCH, vol. 35, no. 12, June 2007 (2007-06-01), pages 3918 - 3927, XP055555163 *

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