WO2018047856A1 - Protéine fusionnée, corps structural, agent de piégeage, méthode de piégeage, adn et vecteur - Google Patents

Protéine fusionnée, corps structural, agent de piégeage, méthode de piégeage, adn et vecteur Download PDF

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WO2018047856A1
WO2018047856A1 PCT/JP2017/032092 JP2017032092W WO2018047856A1 WO 2018047856 A1 WO2018047856 A1 WO 2018047856A1 JP 2017032092 W JP2017032092 W JP 2017032092W WO 2018047856 A1 WO2018047856 A1 WO 2018047856A1
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protein
amino acid
monomer
dna
seq
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Japanese (ja)
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了史 川上
憲二 宮本
近藤　宏紀
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学校法人慶應義塾
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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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier

Definitions

  • the present invention relates to a fusion protein, a structure, a collecting agent, a collecting method, DNA, and a vector.
  • Patent Document 1 describes that a cage-type (cage-type) huge fusion protein structure can be prepared by a fusion protein of a protein that forms a trimer and a protein that forms a dimer. Yes.
  • the document describes that a structure having a cavity such as a cage structure can be used for drug or gene delivery.
  • Patent Document 1 when a protein structure is produced from a trimer and a dimer, a structure having a geometrically different structure is necessarily manufactured, and thus a structure having a desired structure. could not only get. Therefore, it has been demanded to produce a structure with high structure uniformity in which variations of the structure to be manufactured are reduced.
  • the present invention has been made in view of the above circumstances, and a fusion protein capable of easily producing a protein structure having a highly uniform structure, and a structure formed by such a fusion protein.
  • the purpose is to provide.
  • Another object of the present invention is to provide a collecting agent, a collecting method, DNA, and a vector using the structure.
  • the inventors of the present invention fused a protein capable of forming a homopentameric or homohexamer with a protein capable of forming an antiparallel coiled-coil structure, thereby producing a protein structure with high structural uniformity.
  • a protein capable of forming a homopentameric or homohexamer with a protein capable of forming an antiparallel coiled-coil structure, thereby producing a protein structure with high structural uniformity.
  • the present invention has been completed. More specifically, the present invention provides the following.
  • a fusion protein comprising a protein capable of forming a homopentamer or a protein capable of forming a homohexamer and a protein capable of forming an antiparallel coiled coil structure.
  • a method for collecting a substance having affinity for the structure according to (2) A method comprising the step of bringing the structure into contact with a target substance.
  • FIG. 1 is a schematic diagram of one embodiment of a fusion protein of the present invention formed from a homopentameric monomer 11 and an antiparallel coiled coil structure monomer 12.
  • FIG. It is a figure which shows the flow in which a structure is formed from the homopentameric monomer 11 and the antiparallel type coiled coil structure monomer 12. It is a figure explaining the fullerene structure of (c) of Drawing 2 on the assumption of a soccer ball.
  • (A) It is a figure which shows the flow in which a fullerene structure is formed from the homopentameric monomer 11 and the antiparallel type coiled coil structure monomer 12.
  • FIG. B It is a figure which shows the flow in which a graphene structure is formed from the homo hexamer monomer 16 and the antiparallel coiled coil structure monomer 12.
  • FIG. It is a figure which shows the result of SDS-PAGE about the fusion protein (The fusion protein containing a homopentameric monomer and an antiparallel coiled-coil structure monomer) which concerns on an Example.
  • FIG. 3 is a graph showing a residual ratio of a protein after heat treatment at 30 ° C. to 90 ° C. for a protein of a structure according to an example (a structure having a fullerene structure). It is a graph which shows the measurement result by the dynamic light scattering method (DLS) about the protein of the structure (structure which has a fullerene structure) which concerns on an Example. It is a graph of the light absorbency which shows that the support
  • DLS dynamic light scattering method
  • the fusion protein of the present invention has a protein capable of forming a homopentamer or a protein capable of forming a homohexamer and a protein capable of forming an antiparallel coiled-coil structure.
  • a protein capable of forming a homopentamer or a protein capable of forming a homohexamer is a subunit (monomer) constituting a homopentamer or a homohexamer by interaction.
  • the protein capable of forming an antiparallel coiled coil structure is a dimeric subunit (monomer) constituting the antiparallel coiled coil structure.
  • a protein capable of forming a homopentamer, a protein capable of forming a homohexamer, and a protein capable of forming an antiparallel coiled coil structure are referred to as “homopentameric monomer” and “homopentameric monomer”, respectively.
  • hexamer monomer or "antiparallel coiled coil structure monomer”.
  • FIG. 1 is an example of a schematic diagram of the fusion protein of the present invention.
  • the fusion protein in the embodiment shown in FIG. 1 has a homopentameric monomer 11, an antiparallel coiled coil structure monomer 12, and a linker 13.
  • FIG. 2 shows a flow in which a structure is formed by the fusion protein in FIG.
  • the homopentameric monomer 11 forms a pentamer by interaction
  • the central pentamer portion has a pentagonal structure as shown in FIG. 2 (a).
  • the antiparallel coiled coil structure monomer 12 in FIG. 2 (a) forms a dimer
  • the antiparallel coiled coil structure monomer 12 is bound by hand
  • a hexagonal cavity (15) is formed between the pentagonal structures, and finally a fullerene structure can be formed as shown in FIG. 2 (c).
  • a parallel coiled coil structure is used instead of an antiparallel coiled coil structure, such a structure cannot be formed.
  • a graphene structure can be produced when a homo hexamer monomer is used instead of a homo pentamer monomer.
  • the fusion protein of the present invention can be designed according to the same principle regardless of the type of pentamer or hexamer, and thus the structure can be easily produced.
  • FIG. 4A shows a flow of forming a fullerene structure from a homopentameric monomer and an antiparallel coiled coil structure monomer
  • FIG. 4B shows a homohexamer monomer and an antiparallel coiled coil structure.
  • the flow by which a graphene structure is formed from a monomer is shown.
  • 4 (A) and 4 (B) shows a case in which a homopentameric monomer 11 forms a pentamer by interaction or a homohexameric monomer 16 forms a hexamer by interaction. It is a thing.
  • (B) in (A) and (B) of FIG. 4 is a more specific image of the schematic diagram of (a).
  • FIG. 4 (A) and 4 (B), (c) shows a schematic diagram of a structure obtained by binding each fusion protein.
  • C) of FIG. 4 (A) shows a fullerene structure in the case of using a pentamer, and (d) is a three-dimensional structure of a general fullerene structure.
  • 4B shows a graphene structure in the case of using a hexamer, and
  • FIG. 4D shows a three-dimensional structure of a general graphene structure.
  • the fullerene structure and the graphene structure refer to a structure having the same or similar structure as the fullerene or graphene formed by only carbon atoms, and the fullerene or graphene itself (a structure composed of carbon). ) Is different.
  • the homopentameric monomer of the present invention is not particularly limited as long as it has an amino acid sequence capable of forming a homopentamer, and the purpose (for example, the affinity of the target substance when used as a collecting agent described later) Etc.) can be set as appropriate.
  • Specific examples include a protein having the amino acid sequence set forth in any of SEQ ID NOs: 1-42.
  • a protein encoded by a mutant or homologue of DNA encoding these proteins may be used. Examples of such a protein include those having an amino acid sequence encoded by the DNA described in any of (1) to (3) below.
  • DNA having a base sequence capable of hybridizing under stringent conditions with a base sequence complementary to the base sequence encoding the amino acid sequence of any one of SEQ ID NOS: 1-42 (2) having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1-42, and a homopentamer DNA that can be formed (3) a DNA comprising a base sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 42 and capable of forming a homopentamer
  • the homopentameric monomer of the present invention is a protein having the amino acid sequence set forth in SEQ ID NO: 1 or a protein having this amino acid sequence, for example, in terms of being suitable for recovery of metals or nucleic acids when considered for use as a collector. Proteins encoded by mutants or homologues of DNA encoding the protein are preferred.
  • the homo hexamer monomer of the present invention is not particularly limited as long as it has an amino acid sequence capable of forming a homo hexamer, and the purpose (for example, the affinity of the target substance when used as a collecting agent described later) Etc.) can be set as appropriate.
  • Specific examples include proteins having the amino acid sequence set forth in any of SEQ ID NOs: 43 to 48 or SEQ ID NOs: 58 to 72, and the like.
  • DNA variants and proteins encoded by homologues encoding these proteins may be used.
  • Such proteins include those having the amino acid sequence encoded by the DNA described in any of (4) to (6) below.
  • DNA capable of forming homohexamers (6) It consists of a base sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 43 to 48 or SEQ ID NO: 58 to 72, and can form a homo hexamer DNA
  • the homo hexamer monomer of the present invention is preferably a protein having the amino acid sequence set forth in SEQ ID NO: 43 or a protein encoded by a mutant or homologue of a DNA encoding the protein.
  • the amino acid sequence capable of forming the homo pentamer monomer or homo hexamer of the present invention includes an amino acid sequence for interaction.
  • SEQ ID NO: 50 and SEQ ID NO: By having a sequence such as 51 in combination, a homopentamer is formed.
  • amino acid sequences encoded by DNA variants and homologs encoding these amino acids are used. May be.
  • Examples of the mutant or homologue of DNA encoding the amino acid sequence of SEQ ID NO: 50 include the amino acid sequences encoded by the DNA described in any of (7) to (9) below.
  • Examples of the mutant or homologue of the DNA encoding the amino acid sequence of SEQ ID NO: 51 include the amino acid sequence encoded by the DNA described in any of (10) to (12) below.
  • DNA having a base sequence capable of hybridizing under stringent conditions with a base sequence complementary to the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 50 DNA having a base sequence encoding an amino acid sequence in which 1, 2 or 3 amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 50 (9) DNA comprising a base sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 50 (10) DNA having a base sequence capable of hybridizing under stringent conditions with a base sequence complementary to the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 51 (11) DNA having a base sequence encoding an amino acid sequence in which 1 or 2 amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 51 (12) DNA comprising a base sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 51
  • the antiparallel coiled coil structure monomer of the present invention is not particularly limited as long as it has an amino acid sequence capable of forming an antiparallel coiled coil structure, and a protein having a conventionally known antiparallel coiled coil structure can be used. Depending on the purpose (for example, affinity of the target substance when used as a collecting agent to be described later), it can be appropriately set. Specific examples include a protein having the amino acid sequence set forth in SEQ ID NO: 49. Alternatively, instead of the protein having the amino acid sequence set forth in SEQ ID NO: 49, a DNA variant or a homologous protein encoding these proteins may be used. Examples of such a protein include those having an amino acid sequence encoded by the DNA described in any of (13) to (15) below.
  • DNA having a base sequence capable of hybridizing under stringent conditions with a base sequence complementary to the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 49 (14) DNA having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence set forth in SEQ ID NO: 49 and capable of forming an antiparallel coiled-coil structure
  • the antiparallel coiled coil structure monomer of the present invention is not particularly limited, but those having a leucine zipper structure are preferred. More specifically, a unit sequence consisting of 6 to 8 amino acid residues, preferably 7 amino acid residues, and containing a hydrophobic amino acid (leucine, isoleucine, etc.) in the vicinity of the middle of the sequence is 2 to 9 The inclusion is preferred.
  • SEQ ID NO: 49 has a structure including five unit sequences as indicated by MENKQVEE "ILRLLEKE” "IEDLQRM” KEQQE "LSLTEAS" "LQKLQER” RDQE "LRRLEEEE".
  • a homo pentamer can be formed means that the polypeptide encoded by the base sequence is homo 5 It means having the ability to form a monomer, form a homohexamer or form an antiparallel coiled coil structure.
  • the “stringent conditions” include, for example, carrying out the reaction at 40 to 70 ° C. (preferably 50 to 67 ° C., more preferably 60 to 65 ° C.) in a normal hybridization buffer. The conditions include washing in a washing solution having a salt concentration of 15 to 300 mM (preferably 15 to 150 mM, more preferably 15 to 60 mM, and still more preferably 30 to 50 mM).
  • DNA encoding the amino acid sequence of the homopentameric monomer, homohexamer monomer, or antiparallel coiled-coil structure monomer in the present invention one or more of the amino acid sequences described in any of SEQ ID NOs: 1 to 49 are used.
  • DNA having a base sequence encoding an amino acid sequence in which amino acids are substituted, deleted, and / or added is also included.
  • “one or more” is usually within 20 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids (eg, within 3 amino acids, 2 Within 1 amino acid).
  • hydrophobic amino acids A, I, L, M, F, P, W, Y, V
  • hydrophilic amino acids R, D, N, C, E, Q, G, H, K, S, T
  • amino acids having aliphatic side chains G, A, V, L, I, P
  • amino acids having hydroxyl group-containing side chains S, T, Y
  • sulfur atom-containing side chains An amino acid (C, M) having a carboxylic acid and an amide-containing side chain (D, N, E, Q), an amino acid having a base-containing side chain (R, K, H), an aromatic-containing side chain
  • the amino acid (H, F, Y, W) which can be mentioned can be mentioned (all parentheses represent the single letter code of amino acids).
  • a DNA variant or homolog having a base sequence encoding the amino acid sequence described in any one of SEQ ID NOs: 1 to 49 is highly homologous to the base sequence encoding the amino acid sequence described in any of SEQ ID NOs: 1 to 49.
  • DNA consisting of a base sequence having sex is included. Such DNA is preferably 90% or more, more preferably 95% or more (96% or more, 97% or more, 98% or more) with the base sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 49 , 99% or more).
  • the homology between amino acid sequences and base sequences can be determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993) by Karlin and Altschul.
  • the fusion protein of the present invention may have a linker between the homopentameric monomer or homohexamer monomer and the antiparallel coiled coil structure monomer, as in the embodiment shown in FIG. It does not have to be. From such a linker, the angle between the hexagon and the antiparallel coiled coil structure monomer can be adjusted by a pentagon or a homohexamer composed of a homopentamer, and the structure can be easily formed.
  • the “angle” is described with reference to FIG. 2C as an example.
  • FIG. 2C The angle “r” formed by the side 142 passing through the center of the pentamer and the side 152 along the antiparallel coiled-coil structure monomer.
  • FIG. 3 shows the fullerene structure shown in FIG. 2C as if it were a soccer ball (middle, right side).
  • the fullerene structure can be explained as a soccer ball, but the above-mentioned r is as shown in the right soccer ball (a part of which is cut out of a continuous pentagon and hexagon). It is.
  • r is in the range of more than 0 ° and less than 180 ° (more preferably in the range of 130 ° to 160 °). It is preferable in that the structure can be easily formed.
  • FIG. 2 the case of a homopentameric monomer has been described.
  • a homohexameric monomer when the structure forms a graphene structure, it is within a range of 90 ° to 180 ° (more preferably Is preferably 180 ° from the viewpoint of easy formation of a structure.
  • the angle can be adjusted, for example, by using a linker that knows how much the angle formed by the elements attached to both ends of the linker can be changed or adjusted by the linker. For example, the angle can be changed by adjusting the number of such linkers.
  • the position of the homopentameric monomer or homohexamer monomer and the antiparallel coiled-coil structure monomer may be located on the N-terminal side, and is located on the C-terminal side. Also good.
  • the antiparallel coiled coil structure monomer may be located on the N-terminal side, and the homopentameric monomer or homohexamer monomer may be located on the C-terminal side, or the homopentameric monomer or The homo hexamer monomer may be located on the N-terminal side, and the antiparallel coiled coil structure monomer may be located on the C-terminal side.
  • the fusion protein of the present invention may or may not have an amino acid sequence other than the amino acid sequences described above, as necessary, to the extent that the effects of the present invention are not impaired.
  • a tag amino acid sequence may be added for purification of the fusion protein.
  • the “DNA” in the present invention may be either a sense strand or an antisense strand (for example, can be used as a probe), and the shape thereof may be either single-stranded or double-stranded. Further, it may be genomic DNA, cDNA, or synthesized DNA.
  • the method for obtaining the DNA of the present invention is not particularly limited, but a method for obtaining cDNA by reverse transcription from mRNA (for example, RT-PCR method), a method for preparing from genomic DNA, a method for synthesis by chemical synthesis, A known method such as a method of isolating from a genomic DNA library or a cDNA library (for example, see JP-A-11-29599) can be mentioned.
  • the fusion protein of the present invention can be produced by a known method for preparing a chimeric protein.
  • the chimeric protein can be prepared, for example, by using a transformant into which an expression vector containing DNA encoding the above-described chimeric protein is introduced. For example, first, the transformant is cultured under appropriate conditions, and a chimeric protein encoded by the DNA is synthesized. Then, the chimeric protein (the fusion protein of the present invention) can be obtained by recovering the synthesized protein from the transformant or the culture solution.
  • the “appropriate vector” is not particularly limited as long as it can be replicated and maintained in various prokaryotic and / or eukaryotic hosts, and can be appropriately selected depending on the purpose of use. For example, when a large amount of DNA is desired, a high copy vector can be selected, and when a polypeptide (fusion protein) is desired, an expression vector can be selected. Specific examples thereof are not particularly limited, and examples thereof include known vectors described in JP-A-11-29599.
  • the method for introducing a vector can be appropriately selected according to the type of vector and host. Specific examples thereof are not particularly limited. For example, when a bacterium is used as a host, known methods such as a protoplast method and a competent method (for example, see JP-A-11-29599) can be mentioned.
  • the host into which the expression vector is introduced is not particularly limited as long as it is compatible with the expression vector and can be transformed. Specific examples thereof include known natural cells such as bacteria, yeast, animal cells, and insect cells. Alternatively, artificially established cells (see JP-A-11-29599), or animals such as humans and mice can be mentioned.
  • the culture of the transformant is appropriately selected from known nutrient media according to the type of transformant so that the chimeric protein can be easily obtained in large quantities, and the temperature, pH of the nutrient medium, culture time, etc. It can be carried out with appropriate adjustment (for example, see JP-A-11-29599).
  • the method for isolating and purifying the fusion protein is not particularly limited, and a known method such as a method utilizing solubility, a method utilizing a difference in molecular weight, a method utilizing charge or the like (for example, JP-A-11-29599). Gazette).
  • the structure of the present invention is formed by binding the molecules of the above-mentioned fusion protein of the present invention.
  • a fullerene structure as shown in (c) of FIG. 2 and (c) of (A) of FIG. 4 can be formed.
  • This fullerene structure can form one fullerene structure by 60 molecules of the above-mentioned fusion protein of the present invention.
  • a graphene structure as shown in (c) of (B) of FIG. 4 can be formed.
  • the number of molecules of the above-described fusion protein of the present invention per structure is not particularly limited.
  • the structure of the present invention may be a large structure by bonding one molecule of the structure.
  • the bonds between the structures can be performed by, for example, a conventional cross-linking agent (such as glutaraldehyde) used for protein cross-linking.
  • the structures can form an aggregate by adjusting the pH of the solution containing the structures to near the isoelectric point (isoelectric point ⁇ 1.5). Such an assembly dissociates into a structure when the pH is in the vicinity of neutrality (for example, pH 7.0 ⁇ 1.0).
  • neutrality for example, pH 7.0 ⁇ 1.0
  • any solvent can be used as long as it does not denature proteins, and examples thereof include methanol, ethanol and the like.
  • the structure of the present invention is suitable for use as a collecting agent described later.
  • the collection agent of this invention is a collection agent of the target substance which has affinity with respect to said structure of this invention, Comprising: This structure is contained.
  • “collection” may be based on any mechanism of action as long as it is collected as a result, for example, a fullerene structure having affinity. It may be such that it is confined inside, or it may be such that substances of similar size interact and aggregate.
  • the capturing agent of the present invention may be appropriately designed as a structure having affinity for the target substance to be collected.
  • a positively charged substance such as a metal
  • the structure may have a property having an affinity for a positively charged substance, that is, a negatively charged substance.
  • the above-described homopentameric monomer of the present invention is a protein having the amino acid sequence set forth in SEQ ID NO: 1, or a protein encoded by a mutant or homologue of DNA encoding this protein, such as a metal, protein (lysozyme, etc.) Suitable for enzyme or the like) or nucleic acid collection.
  • the collection agent of the present invention is also suitable for separation of optically active compounds and selective collection of the compounds.
  • the collecting agent of the present invention can form an aggregate together with the target substance when the target substance is collected.
  • the aggregate may be an aggregate of the collection agent and the target substance or a form in which the target substance is included in the space of the collection body.
  • the resulting aggregate can be dissociated into a scavenger and the target substance by adding a salt (NaCl or the like) or adjusting the pH. Therefore, according to the collection agent of the present invention, the target substance can be freely collected and released.
  • the collection agent may be used for various purposes. For example, it may be used for analysis or fractionation by using it as a column support, or it may be used as a filter for isolating a target substance (for example, environment). It may be used to isolate only the components of interest from a pollutant population).
  • the method of the present invention is a method for collecting a substance having an affinity for the structure of the present invention, and having a step of bringing the structure into contact with a target substance.
  • the structure and the target substance can be the same as the above-described collecting agent of the present invention.
  • the method of bringing the structure into contact with the target substance may be appropriately set according to the method of use of the structure (usage method as a column carrier, use method as a filter, etc.).
  • the present invention may include DNA encoding the above-described fusion protein of the present invention.
  • the present invention may include a vector incorporating the above-described DNA of the present invention.
  • the vector may be an expression vector or another vector.
  • ⁇ Fusion protein production-1> Design of structure with fullerene structure
  • the truncated icosahedron having a fullerene structure is a so-called soccer ball type polyhedron composed of 12 pentagons and 20 hexagons.
  • a homopentameric monomer is placed on the pentagonal face of the truncated icosahedron, and the dimer peptide is inverted on the part corresponding to the vacant hexagonal side.
  • a molecular design was carried out to place a parallel coiled-coil monomer (two-fold symmetry).
  • ID 2LW9, SEQ ID NO: 49
  • Gene design An artificial protein obtained by fusing pentamer and dimer monomer molecules on a gene was subjected to codon optimization for expression in E. coli, and the gene sequence was designed (SEQ ID NO: 52). At this time, a restriction enzyme recognition sequence of BamH I was added to the N terminus and Hind III was added to the C terminus so that it could be recombined into a pET-Duet 1 vector that can add histidine-Tag to the N terminus. . SEQ ID NO: 52 is shown below.
  • the above gene sequence was transformed into Escherichia coli XL10-Gold by the heat shock method, cultured in LB + Ampicillin medium, and extracted with a plasmid (VIOGENE).
  • This amplified plasmid and the pET-Duet 1 vector were treated with restriction enzymes using BamHI and HindIII. Agarose gel electrophoresis was performed, and the target insertion sequence and the cleaved pET-Duet 1 vector were cut out from the gel and purified (VIOGENE). Each sequence was ligated using T4 DNA Ligase.
  • the prepared plasmid was similarly transformed, cultured and extracted with a plasmid.
  • linker In order to adjust the dihedral angle between the surfaces forming the pentamer, a linker was added. The sequence was obtained by random mutagenesis using NNK (degenerate) codons. The length of the linker sequence was set to 1 to 3 amino acid residues.
  • the ins RVL mutant was cultured and expressed, and the fusion protein was purified.
  • the ins RVL mutant was cultured in LB + Amp agar medium for 16 hours at 37 ° C. to obtain colonies. This colony was inoculated into 3 mL LB + Amp liquid medium and pre-cultured at 37 ° C. for 8 hours. Transfer 2 mL of the pre-cultured liquid medium to 200 mL LB + Amp liquid medium, and perform main culture at 37 ° C. for 2 hours, and then add isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. / N expression induction was performed.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • the culture solution after the induction of expression was centrifuged (10000 ⁇ g, 10 min, 4 ° C.) to collect the cells and temporarily stored at ⁇ 30 ° C.
  • 10 mL of 20 mM Tris-HCl (pH 8) was added to the microbial cells corresponding to 100 mL of the culture solution, and subjected to ultrasonic disruption (Output 3, Duty Cycle 30, 20 min). After crushing, the supernatant was centrifuged (10000 ⁇ g, 10 min, 4 ° C.) to recover the supernatant.
  • the supernatant was passed through a Ni-NTA affinity column (QIAGEN) to bind the expressed fusion protein, and then washed with 20 mM Imidazole + 20 mM Tris-HCl (pH 8) for 10 CV or more. Thereafter, 3 mL of 500 mM Imidazole + 20 mM Tris-HCl (pH 8) was added and incubated for 1 hour, followed by elution. By SDS-PAGE, a band having a molecular weight (17.8 kDa) corresponding to the amino acid sequence (SEQ ID NO: 54) derived from the fusion protein planned for the elution fraction was confirmed (FIG. 5). In FIG.
  • FT is the lane of the cell extract before purification
  • Wash is the lane of the fraction washed in the purification process
  • Elution is the eluted fusion protein after purification.
  • Lane. The purified protein was ultrafiltered and concentrated until the protein concentration reached 2 mg / mL, followed by Blue Native-PAGE.
  • FIG. 6 The left lane in FIG. 6 is a marker
  • the right lane is a protein complex lane. From this result, it was found that a structure having a fullerene structure (hereinafter, sometimes referred to as “structure according to Example”) could be produced by the fusion protein.
  • ⁇ Particle size measurement> Before measuring the particle size of the structure according to the embodiment having a fullerene structure, first, after purifying the ins RVL mutant, a MWCO 3500 dialysis tube (Thermo Fisher Scientific) to remove small molecule compounds such as salts. Dialysis was performed at 4 ° C. using The outer solution was exchanged twice every 2 hours using 20 mM Tris-HCl (pH 8) 100 times the amount of the protein solution, and O / N incubation was carried out after the second exchange.
  • a MWCO 3500 dialysis tube Thermo Fisher Scientific was performed at 4 ° C. using The outer solution was exchanged twice every 2 hours using 20 mM Tris-HCl (pH 8) 100 times the amount of the protein solution, and O / N incubation was carried out after the second exchange.
  • the particle size was measured by dynamic light scattering (DLS), and cumulant analysis was performed. The result is shown in FIG. As shown in FIG. 8, it was shown that the Z average particle diameter was 11.75 nm (radius and diameter was 23.4 nm) and the molecular weight was 1070 kDa, and it was further confirmed that a 60-mer as planned was formed. It was done.
  • a cortexillin I / GCN4 hybrid peptide (PDB ID 1P9I) having an internal sequence in common with 4 residues was used.
  • the ⁇ -helix is 3.6 residues / 1 turn in order to align the direction in which the terminal ⁇ -helix extends with the ins RVL mutant, 7 residues (SEQ ID NO: 55) of the hybrid peptide Insertion was performed.
  • a nucleotide sequence encoding 7 amino acid residues (SEQ ID NO: 55) was inserted between the linker and the dimer by mutant PCR. Seven-residue insertion was performed using the template plasmid as an expression vector transduced with the ins RVL mutant. The PCR product was treated with Dpn I, agarose gel electrophoresis was performed, and it was confirmed that the product was amplified. E. coli XL10-Gold was transformed by the heat shock method. Colonies obtained by culturing at 37 ° C.
  • the ins LLASLEARVL mutant was cultured and expressed in the same manner as the above ins RVL mutant, and the protein was purified. After purification, SDS-PAGE confirmed a single band with a molecular weight (18.5 kDa) corresponding to the amino acid sequence derived from the fusion protein in the eluted fraction.
  • the purified protein was subjected to ultrafiltration and concentrated until the protein concentration reached 2 mg / mL, followed by Blue Native-PAGE.
  • the ins LLASLEARVL mutant had a higher molecular side than the ins RVL mutant. An increase in the molecular weight of the supramolecule corresponding to the number of amino acids inserted into was observed. From this result, it was found that the particle size and the like of the fullerene structure can be adjusted by adjusting the length of the linker.
  • the fusion protein in the ins RVL mutant aggregates by binding to metal ions, and resolubilizes by chelating metal ions with EDTA, so it can be used as a carrier to remove metal ions in water. investigated. In addition, the purification scale of the ins RVL mutant was expanded. In addition, in order to recycle the structures according to the examples in the ins RVL mutant, a carrier that is cross-linked with glutaraldehyde when combined with metal ions and aggregated, and does not re-solubilize even if the metal ions are removed with EDTA. was made.
  • the supernatant was passed through a Ni-NTA affinity column (fast flow) at a flow rate of 1.5 mL / min to bind the expressed protein, and then washed with 20 mM Imidazole + 20 mM Tris-HCl (pH 8) for 10 CV or more. Thereafter, 500 mM Imidazole + 20 mM Tris-HCl (pH 8) was allowed to flow at 5 CV for elution. A similar band was confirmed in the eluted fraction by SDS-PAGE. Further, after concentration until the protein concentration reached 2 mg / mL, Blue Native-PAGE was performed, and the same band was confirmed.
  • the prepared and washed carrier was dried in an incubator at 60 ° C., and 1 mL of 1 mM NiCl 2 was added and mixed. In order to remove Ni 2+ ions not bound to the carrier, the supernatant was removed by centrifugation (15000 rpm, 4 min, 4 ° C.). A total of three cycles of adding NiCl 2 to the carrier, removing the supernatant, and washing was performed. Washing was performed by adding 1 mL of 20 mM Tris-HCl (pH 8) and centrifuging (15000 rpm, 4 min, 4 ° C.) to remove the supernatant.
  • Graphene which is well known as a carbon material together with fullerene, has a so-called honeycomb structure composed of hexagons.
  • fullerene structure as described above, marine microorganism-derived SM / LSM-like RNA binding proteins were used, but many LSM protein families form homohexamers (hexagons).
  • Gene design An artificial protein obtained by fusing hexamer and dimer monomer molecules on a gene was subjected to codon optimization for expression in E. coli, and the gene sequence was designed (SEQ ID NO: 56). At this time, a restriction enzyme recognition sequence of BamH I was added to the N terminus and Hind III was added to the C terminus so that it could be recombined into a pET-Duet 1 vector that can add histidine-Tag to the N terminus. . SEQ ID NO: 56 is shown below.
  • the above gene sequence was transformed into Escherichia coli XL10-Gold by the heat shock method, cultured in LB + Ampicillin medium, and extracted with a plasmid (VIOGENE).
  • This amplified plasmid and the pET-Duet 1 vector were treated with restriction enzymes using BamHI and HindIII. Agarose gel electrophoresis was performed, and the target insertion sequence and the cleaved pET-Duet 1 vector were cut out from the gel and purified (VIOGENE).
  • Each sequence was ligated using T4 DNA Ligase.
  • the prepared plasmid was similarly transformed, cultured and extracted with a plasmid.
  • E. coli BL21 Star (DE3) was transformed by the heat shock method.
  • the culture solution after the induction of the expression was centrifuged (10000 ⁇ g, 10 minutes, 4 ° C.) to recover the cells and temporarily stored at ⁇ 30 ° C. 10 mL of 20 mM Tris-HCl (pH 8) was added to the cells corresponding to 100 mL of the culture broth, and sonicated (Output 3, Duty Cycle 30, 20 minutes). After disruption, the supernatant was centrifuged (10000 ⁇ g, 10 minutes, 4 ° C.) to recover the supernatant.
  • the supernatant was passed through a Ni-NTA affinity column (QIAGEN) to bind the expressed protein, and then washed with 20 mM Imidazole + 20 mM Tris-HCl (pH 8) for 10 CV or more. Thereafter, 3 mL of 500 mM Imidazole + 20 mM Tris-HCl (pH 8) was added and incubated for 1 hour, followed by elution. By SDS-PAGE, a band having a molecular weight (16.3 kDa) corresponding to the amino acid sequence derived from the artificial protein (SEQ ID NO: 57) was confirmed in the eluted fraction. SEQ ID NO: 57 is shown below.

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Abstract

La présente invention concerne la production d'une protéine fusionnée capable de produire facilement une structure de protéine d'uniformité élevée, et la production d'un corps structural formé par une telle protéine fusionnée. Cette protéine fusionnée comporte une protéine capable de former un homopentamère ou une protéine capable de former un homohexamère, et une protéine capable de former une structure hélicoïdale du type hélicoïdal non parallèle. De plus, ce corps structural est formé par la liaison de molécules de la protéine fusionnée. La présente invention concerne un agent de piégeage d'une substance cible, ledit agent de piégeage ayant une affinité pour le corps structural indiqué ci-dessus et contenant le corps structural.
PCT/JP2017/032092 2016-09-06 2017-09-06 Protéine fusionnée, corps structural, agent de piégeage, méthode de piégeage, adn et vecteur WO2018047856A1 (fr)

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EP4103586A4 (fr) * 2020-02-14 2024-05-15 University of Washington Polypeptides et leur utilisation

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WO2010019725A2 (fr) * 2008-08-12 2010-02-18 Salemme Francis R Polypeptides nœuds pour un ensemble nanostructure
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WO2011162492A2 (fr) * 2010-06-23 2011-12-29 Korea Institute Of Science And Technology Protéine de fusion comprenant une petite protéine de choc thermique, protéine de cage formée ainsi et nouvelle utilisation associée
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WO2007037315A1 (fr) * 2005-09-29 2007-04-05 Riken Nouvelle nanostructure et son procede de construction
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WO2010132363A1 (fr) * 2009-05-11 2010-11-18 Imiplex Llc Procédé de fabrication d'une nanostructure protéique
WO2011162492A2 (fr) * 2010-06-23 2011-12-29 Korea Institute Of Science And Technology Protéine de fusion comprenant une petite protéine de choc thermique, protéine de cage formée ainsi et nouvelle utilisation associée
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4103586A4 (fr) * 2020-02-14 2024-05-15 University of Washington Polypeptides et leur utilisation

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