WO2020194968A1 - Procédé de production d'un vecteur de liaison de vésicule à membrane extracellulaire - Google Patents

Procédé de production d'un vecteur de liaison de vésicule à membrane extracellulaire Download PDF

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WO2020194968A1
WO2020194968A1 PCT/JP2019/051128 JP2019051128W WO2020194968A1 WO 2020194968 A1 WO2020194968 A1 WO 2020194968A1 JP 2019051128 W JP2019051128 W JP 2019051128W WO 2020194968 A1 WO2020194968 A1 WO 2020194968A1
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phosphatidylserine
binding
carrier
cell membrane
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Japanese (ja)
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慶一 唐杉
正克 西八條
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株式会社カネカ
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

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  • the present invention relates to a method for efficiently producing an outer cell membrane vesicle-binding carrier.
  • Patent Document 1 a carrier on which a T cell immunoglobulin / mucin domain-containing molecule (Tim) protein is immobilized.
  • the cell membrane of the outer cell membrane vesicle contains phosphatidylserine, and the Tim protein adsorbs phosphatidylserine in the presence of calcium ions, so that the carrier can adsorb exosomes.
  • Patent Document 2 describes that Mastoparan-X, Hemolysin, and LL37 have exosome-binding activity, and these are immobilized on a carrier to purify exosomes.
  • an object of the present invention is to provide a method for efficiently producing an outer cell membrane vesicle-binding carrier.
  • the present inventors have conducted extensive research to solve the above problems.
  • peptides that have binding properties to phosphatidylserine present in the cell membrane of extracellular membrane vesicles and do not have disulfide bonds in the molecule are disrupted in the culture medium or in the cell even if they are produced by transformed cells.
  • the present invention has been completed by finding that it can be efficiently isolated without agglomeration in the liquid and that an extracellular membrane vesicle-binding carrier can be efficiently produced.
  • the present invention will be shown.
  • a method for producing an outer cell membrane vesicle-binding carrier A step of obtaining transformed cells by transforming a host cell with a nucleic acid containing a base sequence encoding a phosphatidylserine-binding peptide having no disulfide bond in the molecule. The step of culturing the transformed cells, A step of purifying the phosphatidylserine-binding peptide from the cultured transformed cells, and A production method comprising the step of immobilizing the purified phosphatidylserine-binding peptide on a water-insoluble carrier.
  • the phosphatidylserine-binding peptide is a C2 region of synaptotagmin, a C2 region of protein kinases ⁇ , ⁇ I, ⁇ II, and ⁇ , a C2 region of MFG-E8, and an I region, II region, and III region of annexin V.
  • the method according to [1] above which is one or more phosphatidylserine-binding peptides selected from the IV region.
  • [3] The method according to the above [1] or [2], wherein the phosphatidylserine-binding peptide is immobilized on the water-insoluble carrier via a linker.
  • a method for purifying outer cell membrane vesicles The step of producing the outer cell membrane vesicle-binding carrier by the method according to any one of the above [1] to [6], and It is characterized by including a step of contacting a liquid sample containing outer cell membrane vesicles with the outer cell membrane vesicle-binding carrier and adsorbing the outer cell membrane vesicles on the outer cell membrane vesicle-binding carrier. how to.
  • the method of the present invention can be used for detecting and purifying outer cell membrane vesicles because of a diagnostic method for detecting outer cell membrane vesicles and a treatment method using outer cell membrane vesicles themselves as an active ingredient.
  • a carrier that exhibits binding to outer cell membrane vesicles can be efficiently produced. Therefore, the present invention is extremely industrially excellent as it promotes the practical application of the technique for utilizing outer cell membrane vesicles.
  • FIG. 1 is a graph showing the response of a peptide-immobilized flow cell according to the present invention to a phosphatidylserine-containing liposome.
  • FIG. 2 is a graph showing the response of the peptide-immobilized flow cell according to the present invention to milk exosomes.
  • FIG. 3 is a graph showing the amount of milk exosomes recovered by magnetic beads on which the peptide according to the present invention is immobilized.
  • FIG. 4 is a polyacrylamide gel photograph showing the results of SDS-PAGE of the soluble fraction of homogenates of transformed cells and the precipitation suspension.
  • transformed cells are obtained by transforming a host cell with a nucleic acid containing a nucleotide sequence encoding a phosphatidylserine-binding peptide having no disulfide bond in the molecule.
  • the phosphatidylserine-binding peptide is not particularly limited as long as it has an affinity for phosphatidylserine and does not have a disulfide bond in the molecule.
  • a peptide having a disulfide bond in the molecule is produced by a transformation method for mass production, it aggregates without being correctly folded in a homogenate solution of transformed cells, which makes purification difficult and significantly reduces production efficiency. There is.
  • the present inventors have clarified that a peptide having no disulfide bond in the molecule, even if it contains a cysteine residue, can be produced with good efficiency by the transformation method.
  • phosphatidylserine-binding peptide examples include the C2 region of synaptotagmin, the C2 region of protein kinase ⁇ , the C2 region of protein kinase ⁇ I, the C2 region of protein kinase ⁇ II, the C2 region of protein kinase ⁇ , and the C2 region of MFG-E8. , One or more phosphatidylserine-binding peptides selected from the I region of annexin V, the II region of annexin V, the III region of annexin V, and the IV region of annexin V.
  • Synaptotagmin is a membrane protein that is abundantly abundant on synaptic vesicles, and is present in various species such as plants as well as animals, and the existence of 17 types of isoforms has been reported in humans and mice.
  • Common synaptotagmin commonly has a lumen region, a transmembrane region, a spacer region, and a cytoplasmic region from the N-terminal side, and has two C2 regions in the cytoplasmic region.
  • the C2 region includes a C2A region and a C2B region from the N-terminal side, both of which show binding properties to phospholipids such as phosphatidylserine in the presence of calcium ions, but the C2B region further exhibits inositol polyphosphoric acid in the absence of calcium ions. Shows binding to acid, adapter complex AP-2, and neurexin.
  • synaptotagmin 1, 2, 3, 5, 6, 7, 9, and 10 bind to calcium ions, and other synaptotagmin do not bind to calcium ions.
  • These synaptotagmins that bind to calcium ions function as calcium ion sensors that release neurotransmitters in a calcium ion concentration-dependent manner. From the viewpoint of maintaining the structure and activity of the outer cell membrane vesicles to be recovered and the activity of the ligand peptide, it is preferable to dissociate the outer cell membrane vesicles from the carrier under mild conditions as much as possible.
  • synaptotagmins 1, 2, 3, 5, 6, 7, 9, and 10 capable of binding phospholipids such as phosphatidylserine in the presence of calcium ions are particularly preferable.
  • the C2 region of the synaptotagmin used in the present invention may be only the C2A region or the C2B region, or may be both the C2A region and the C2B region.
  • the C2A region is preferable from the viewpoint of specificity for phosphatidylserine, and both the C2A region and the C2B region may be used.
  • the nucleic acid containing the base sequence encoding the C2 region of synaptotagmin used in this step is not particularly limited as long as it contains at least the base sequence encoding the C2A region and / or C2B region of synaptotagmin.
  • the base sequence is designed by back-translating the amino acid sequence of the desired C2 region.
  • Escherichia coli is used as a cell, it is preferable to use an Escherichia coli optimized codon.
  • amino acid sequences of the C2A region and C2B region of synaptotagmin 1 were added to SEQ ID NO: 1, and the recognition site of the restriction enzyme BamHI was added to the N-terminal side and the recognition site of the restriction enzyme EcoRI was added to the C-terminal side.
  • the base sequence in which the amino acid sequence is back-translated based on the E. coli optimized codon is shown in SEQ ID NO: 2.
  • Protein kinase C is a phospholipid-dependent serine / threonine kinase that is activated by extracellular stimuli such as growth factors, hormones, and neurotransmitters.
  • Mammalian protein kinase C is composed of at least 11 isozymes and consists of four conserved domains (C1-C4).
  • these isozymes are classified into the following three types from the viewpoint of structure and control factors. The first is the conventional type ( ⁇ , ⁇ I, ⁇ II, ⁇ ) activated by diacylglycerol (DAG), calcium ion, and phospholipid (particularly phosphatidylserine), and has two C1 regions (DAG binding domains) and C2 regions (DAG binding domain).
  • the second is composed of a new type ( ⁇ , ⁇ , ⁇ , ⁇ ) that is activated by DAG but does not require calcium ions and is continuous with C1 region, C2-like region, C3 region and C4 region, but C2.
  • the acyclic region does not bind calcium.
  • the third is atypical ( ⁇ , ⁇ , ⁇ ) that does not require DAG or calcium ions for activation and consists of one C1 region, C3 region and C4 region.
  • the amino acid sequence of the C2 region of the protein kinase C ⁇ (PDB: 3GPE_A) derived from Rattus norvegicus is added to SEQ ID NO: 5, the recognition site of the restriction enzyme BamHI on the N-terminal side, and the restriction enzyme EcoRI on the C-terminal side.
  • the base sequence in which the amino acid sequence to which the recognition site is added is back-translated based on the Escherichia coli optimized codon is shown in SEQ ID NO: 6.
  • MFG-E8 Milk fat globe EGF / factor VIII
  • MFG-E8 is a glycoprotein that is abundantly secreted from epithelial cells during the lactation period of mammals and is also called lactahedrin. From the N-terminus, it consists of a signal sequence, two EGF-equivalent domains, and a factor VIII-equivalent domain (C2 region), and the C2 region binds to the anionic phospholipid phosphatidylserine.
  • the RGD region present at the N-terminus binds to ⁇ v ⁇ 3 integrin.
  • MFG-E8 is also involved in phagocytosis of dead cells, and promotes phagocytosis by binding to phosphatidylserine, which is abundant on the surface of apoptotic cells, and to integrin, which is present on macrophages.
  • the amino acid sequence of the C2 region corresponding to the 269th to 426th positions of the amino acid sequence of the C2 region (GenBank: EDM1675.6.1) of the MFG-E8 (GenBank: BAA76386.1) derived from Mus musculus is set to SEQ ID NO: 7, and the relevant amino acid.
  • the amino acid sequence in which the restriction enzyme BamHI recognition site is added to the N-terminal side and the restriction enzyme EcoRI recognition site is added to the C-terminal side is back-translated based on the Escherichia coli optimized codon, and the nucleotide sequence is shown in SEQ ID NO: 8. ..
  • Anexin is present in various protists and higher eukaryotes including plants, and has the property of recognizing phosphatidylserine in a calcium ion-dependent manner and binding to the cell membrane.
  • Anexin consists of tetramers of homologous domains. It has been found in hominids, invertebrates, slime molds and fungi, plants, and protists, and is classified as annexins A, B, C, D, and E, respectively.
  • Annexin V refers to Annexin A5.
  • Anexin is composed of I region (domain I), II region (domain II), III region (domain III), and IV region (domain IV) from the N-terminal, and each region consists of four ⁇ -helices A, B, and D.
  • E bundled structure has a structure capped by one ⁇ -helix C.
  • There are three Ca 2+ binding sites in one region and one phosphatidylserine can be recognized in one region. That is, a maximum of 12 Ca 2+ and 4 phosphatidylserine are bound in 4 regions.
  • Anexin is involved in intracellular membrane transport, exocytosis, endocytosis, cell membrane-cytoskeleton interaction, regulation of membrane protein activity, and calcium ion channel activity.
  • It is normally expressed intracellularly, but it may also be expressed extracellularly, and it also functions as an anticoagulant and anti-inflammatory protein. It is used as an apoptosis detection probe, labeled with fluorescent dyes, radioactive substances, etc., and is used in cell molecular biology and immunology research.
  • the amino acid sequence of Anexin V domains I to IV (PDB: 1A8A_A) derived from Rattus norvegicus is added to SEQ ID NO: 9, and the restriction enzyme BamHI recognition site is on the N-terminal side and the restriction enzyme EcoRI is on the C-terminal side.
  • the base sequence in which the amino acid sequence to which the recognition site of is added is back-translated based on the Escherichia coli optimized codon is shown in SEQ ID NO: 10.
  • the nucleic acid used in this step may contain another base sequence as long as it contains the base sequence encoding the above phosphatidylserine-binding peptide.
  • Other base sequences include, for example, bases encoding glutathione-S-transferase (GST), maltose-binding protein (MBP), His tag, HA tag, myc tag, FLAG tag, etc., which facilitate the purification of the peptide.
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • His tag His tag
  • HA tag HA tag
  • myc tag myc tag
  • FLAG tag etc.
  • a linker sequence may be introduced at the N-terminus and / or C-terminus to facilitate immobilization of the peptide on a carrier.
  • the linker sequence may be a spacer region or a part thereof contained in the peptide, the tag may be used as the linker sequence, or an artificial structure that does not interfere with the binding of the peptide to phosphatidylserine. It may be an array. In the present invention, it is not necessary to use a molecular chaperone for folding the peptide.
  • the nucleic acid containing the sequence is artificially synthesized.
  • the nucleic acid may be amplified by the PCR method.
  • cells are transformed by a conventional method. For example, first the nucleic acid is inserted into the vector.
  • the vector contains a base sequence encoding the phosphatidylserine-binding peptide and a promoter capable of functioning in a host operably linked to the base sequence.
  • the base sequence encoding the phosphatidylserine-binding peptide is linked or inserted into an appropriate vector.
  • the vector is not particularly limited as long as it can autonomously replicate in the host, and a plasmid vector or a phage vector can be used.
  • a plasmid vector or a phage vector can be used.
  • pQE-based vectors Qiagen
  • pET-based vectors Merck
  • pGEX-based vectors GE Healthcare Bioscience
  • fungi such as yeast
  • bacteria such as Escherichia coli and Bacillus subtilis
  • animal cells such as Chinese hamster ovary (CHO) cells, BHK cells, COS cells, and human-derived cells
  • insect cells such as Chinese hamster ovary (CHO) cells, BHK cells, COS cells, and human-derived cells
  • eubacteria such as Escherichia coli, Bacillus subtilis, Brevibacillus, Staphylococcus, Streptomyces, Streptomyces, and Corynebacterium can be preferably used.
  • Transformed cells can be obtained by introducing a recombinant vector containing a nucleic acid containing a nucleotide sequence encoding the above phosphatidylserine-binding peptide into a host cell.
  • the method for introducing the vector into the host cell include a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, an Agrobacterium infection method, a particle gun method and a polyethylene glycol method. , Not limited to these.
  • a method for expressing the phosphatidylserine-binding peptide gene in a host a method for integrating the nucleic acid into the genome may be used.
  • Culturing of transformed cells is carried out according to the usual conditions used for culturing the host.
  • the medium used for culturing transformed cells is not particularly limited as long as it can produce the target peptide with high efficiency and high yield.
  • a medium containing a carbon source or a nitrogen source such as glucose, sucrose, glycerol, polypeptone, meat extract, yeast extract, and casamino acid can be used.
  • inorganic salts such as potassium salt, sodium salt, phosphate, magnesium salt, manganese salt, zinc salt and iron salt are added as needed.
  • a vegetative substance required for growth may be added.
  • antibiotics such as penicillin, erythromycin, chloramphenicol, and neomycin may be added.
  • 2 ⁇ YT medium tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%) or LB medium (tryptone 1%) is used as a medium for culturing transformed cells obtained using Escherichia coli as a host.
  • the culture temperature is, for example, 15 ° C. or higher and 42 ° C. or lower, preferably 20 ° C. or higher and 40 ° C. or lower, and the target peptide is cultured in cultured cells aerobically for 10 hours or longer and 1 week or shorter under aeration and stirring conditions. Accumulate in.
  • the phosphatidylserine-binding peptide is purified from the transformed cells cultured in the above culture step.
  • the target peptide is considered to be accumulated in the cell. Therefore, after culturing, the cultured cells and the culture solution are separated by centrifugation, filtration, or the like. Then, the obtained bacterial cells are crushed by an ultrasonic crushing method, a French press method, or the like, and / or solubilized by adding a surfactant or the like to obtain a crude solution of the target peptide.
  • the target peptide may be purified from such a crude solution by a conventional method.
  • Chromatography or the like may be used as a method for purifying the target peptide. Further, when a tag for purification such as GST is introduced, a high-purity target peptide can be efficiently obtained by adopting a purification method according to the tag. The used tag may be removed after purification using a protease or the like.
  • the purified target peptide is immobilized on a water-insoluble carrier.
  • affinity ligand is a term that refers to a substance or functional group that selectively binds and collects a target molecule from a set of certain molecules based on the specific intermolecular affinity. In the invention, it refers to a peptide that binds to an outer membrane vesicle.
  • extracellular vesicle is a general term for various membrane vesicles secreted from cells, and is classified into exosomes and shedding vesicles according to the difference in their synthetic pathways, but is once secreted extracellularly. And the distinction is not clear.
  • Outer membrane vesicles contain genetic substances such as miRNA and mRNA, alter the traits of received cells, and contribute to the control of various biological phenomena. For example, in recent years it has been shown that outer cell membrane vesicles released by cancer cells promote angiogenesis.
  • Examples of the water-insoluble carrier used in the present invention include an inorganic carrier, an organic carrier, and a composite carrier such as organic-organic and organic-inorganic.
  • Examples of the material of the inorganic water-insoluble carrier include glass and silica gel.
  • Examples of the material of the organic water-insoluble carrier include synthetic polymer carriers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide and crosslinked polystyrene, and polysaccharide carriers such as crosslinked cellulose, crosslinked cellulose, crosslinked agarose and crosslinked dextran. be able to.
  • GCL2000 which is a porous cellulose gel
  • Sephacryl (R) S-1000 which is a covalently crosslinked allyldextran and methylenebisacrylamide
  • Toyopearl (R) which is an acrylate-based carrier
  • an agarose-based crosslinked carrier examples thereof include a certain Sepharose (R) CL4B and Cellulfine (R) which is a cellulosic cross-linking carrier.
  • the water-insoluble carrier in the present invention is not limited to these exemplified carriers.
  • the water-insoluble carrier used in the present invention preferably has a large surface area for the purpose of adsorbing outer cell membrane vesicles, and is preferably porous with a large number of pores of an appropriate size.
  • adsorbing outer cell membrane vesicles preferably has a large surface area for the purpose of adsorbing outer cell membrane vesicles, and is preferably porous with a large number of pores of an appropriate size.
  • beads, monoliths, fibers, films (including hollow fibers) and the like can be used, and any form can be selected.
  • the method for immobilizing the peptide on a water-insoluble carrier for example, it may be bound to the carrier by a conventional coupling method using an amino group, a carboxy group or a thiol group present in the peptide.
  • the carrier is activated by reacting the carrier with cyanide bromide, epichlorohydrin, diglycidyl ether, tosilyl lolide, tresilk lolide, hydrazine, sodium periodate, etc., or the surface of the carrier.
  • An immobilization method by adding a reagent having a functional group, condensing and cross-linking can be mentioned.
  • a linker consisting of a plurality of atoms may be introduced between the peptide and the carrier, or the peptide may be directly immobilized on the carrier.
  • C 1 -6 A group to which 2 to 10 groups selected from the group consisting of an alkandiyl group, an amino group, an ether group, a carbonyl group, an ester group, an amide group and a urea group are linked; an amino group, an ether group and a carbonyl group. , C 1-6 alkandiyl group having a group selected from the group consisting of an ester group, an amide group and a urea group at one end or both ends.
  • the number of connections is preferably 8 or less or 6 or less, more preferably 5 or less, and even more preferably 4 or less.
  • the C 1-6 alkanediyl group may be substituted with a substituent such as a hydroxyl group.
  • the linker may be an amino acid residue useful for immobilization or a peptide.
  • amino acid residues useful for immobilization include amino acid residues having a functional group useful for the chemical reaction of immobilization in the side chain, for example, Lys containing an amino group in the side chain and the side chain. Examples include Cys containing a thiol group.
  • the number of amino acid residues of the peptide as a linker may be appropriately adjusted, and may be, for example, 2 or more and 20 or less.
  • the linker may be a complex of interacting molecules such as biotin and avidin or streptavidin.
  • the carrier on which the above-mentioned phosphatidylserine-binding peptide is immobilized can be used as a carrier that exhibits binding to the outer membrane vesicles.
  • the liquid sample is passed through an affinity column packed with the outer cell membrane vesicle-binding carrier of the present invention. Then, it is brought into contact with the outer cell membrane vesicle-binding carrier to adsorb the outer cell membrane vesicles.
  • the pH of the liquid sample is neutral or substantially neutral, specifically 6.5 or more and 7.5 or less.
  • the inside of the column is then washed by passing an appropriate amount of pure buffer through the affinity column.
  • the outer cell membrane vesicles are adsorbed on the outer cell membrane vesicle-binding carrier of the present invention in the column.
  • the outer cell membrane vesicles can then be purified to a high degree of purity by passing a buffer solution containing a protein denaturing agent or a calcium chelating agent through the column and eluting the outer cell membrane vesicles.
  • the protein denaturant may be any one generally used in the art, and for example, SDS (sodium dodecyl sulfate), urea, and guanidine are preferable, but the protein denaturing agent is not limited to those listed here.
  • the calcium chelating agent may be any compound capable of chelating calcium, for example, EDTA (ethylenediaminetetraacetic acid), EGTA (glycol ether diaminetetraacetic acid), NTA (nitrillotetraacetic acid), DTPA (diethylenetriaminetetraacetic acid), and the like.
  • GLDA L-glutamate diacetic acid
  • HEDTA hydroxyethylethylenediaminetetraacetic acid
  • the extracellular membrane vesicle-binding carrier of the present invention is passed through an appropriate strongly acidic or strongly alkaline pure buffer solution to the extent that the phosphatidylserine-binding peptide or the base material of the carrier does not completely impair its function. It can be reused by cleaning.
  • An appropriate denaturing agent or organic solvent may be added to the above-mentioned buffer solution for regeneration.
  • Example 1 (1) Breeding and culturing of GST-C2A-C2B domain-expressing Escherichia coli phosphatidylserine-binding C2A-C2B domain (SEQ ID NO: 1: 96 of the amino acid sequence of the synaptotagmin 1) of synaptotagmin 1 (GenBank: EDM1675.6.1) derived from Rattus novegicus Eurofin Genomics for artificial synthesis of a gene (SEQ ID NO: 2) in which a restriction enzyme BamHI recognition site is added to the N-terminal side of a gene encoding (1st to 421st) and a restriction enzyme EcoRI recognition site is added to the C-terminal side. Outsourced to the company. The gene was composed of E.
  • This gene was cleaved with BamHI and EcoRI and introduced into a GST fusion protein expression vector (“pGEX-6p-1” manufactured by GE Healthcare Co., Ltd.) cleaved with the same restriction enzyme to introduce the GST-C2A-C2B expression plasmid pGEX-.
  • GST-C2A-C2B was prepared.
  • Escherichia coli (“E. coli JM109” manufactured by Takara Bio Inc.) was transformed with the obtained plasmid. Then, the transformant in which the introduction of the GST-C2A-C2B gene was confirmed was cultured in a flask at 37 ° C. using 2 ⁇ YT medium. Then, IPTG was added and the mixture was cultured overnight at 25 ° C. to obtain a culture solution of GST-C2A-C2B-expressing Escherichia coli.
  • the obtained cell disruption supernatant was equilibrated with 20 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA (pH 7.5), and a column for GST fusion protein purification (“GSTrap HP 5 mL” GE. After loading (manufactured by Healthcare Life Science Co., Ltd.), the column was washed with the buffer used for equilibration, and the target peptide was eluted with 50 mM Tris-HCl, 10 mM reduced glutathione, and 2 mM EDTA (pH 8.0). Then, it was concentrated by a centrifugal filter device (manufactured by "Macrosep 3K” Pall), and a buffer was replaced with PBS to prepare a GST-C2A-C2B solution.
  • the column was washed with the above buffer, and the washing liquid was mixed with the flow-through fraction. Furthermore, the target protein was eluted with 50 mM Tris-HCl, 10 mM reduced glutathione, and 2 mM EDTA (pH 8.0).
  • the C2A-C2B solution was prepared by concentrating the flow-through fraction with a centrifugal filter device (“Macrosep 3K” manufactured by Pall) and exchanging the buffer with PBS. Further, the eluted fraction was concentrated with a centrifugal filter device (“Macrosep 3K” manufactured by Pall), and the solution was exchanged for PBS to prepare a GST solution.
  • the 1M ethanolamine hydrochloride solution was flowed at a rate of 5 ⁇ L / min for 7 minutes to block the activating group on the surface of the sensor chip, and then washed with PBS.
  • Table 1 shows each peptide immobilized on the sensor chip as a ligand and its molar mass. Further, as a reference cell, a cell in which only ethanolamine was immobilized was prepared in the same manner.
  • Example 2 DNA encoding GST-C2A in which the C2B domain has been deleted by the PCR method using the gene of Example 1 (SEQ ID NO: 2) as a template and primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4). Fragments were amplified. This DNA fragment was cleaved with BamHI and EcoRI and introduced into a GST fusion protein expression vector (“pGEX-6p-1” manufactured by GE Healthcare Co., Ltd.) cleaved with the same restriction enzyme to introduce the GST-C2A expression plasmid pGEX-GST. -C2A was prepared.
  • pGEX-6p-1 manufactured by GE Healthcare Co., Ltd.
  • Example 1 Using the obtained plasmid, a transformant was prepared and cultured in the same manner as in Example 1 to obtain a culture solution of GST-C2A-expressing Escherichia coli. Then, a GST-C2A solution was prepared in the same manner as in Example 1 (2). Using the obtained GST-C2A solution, a cell in which these peptides were immobilized on a sensor chip was prepared in the same manner as in Example 1 (4). Table 1 shows each peptide immobilized on the sensor chip as a ligand and its molar mass.
  • Test Example 1 Confirmation test of binding ability of PS-binding protein to PS (1) Preparation of liposomes Chloroform was added to a chloroform solution (1 mL) of 10 mg / mL phosphatidylserine (PS) and diluted to 10 mL. Separately, 25 mg of phosphatidylcholine (PC) was dissolved in chloroform (25 mL). A PC chloroform solution (7.6 mL) was added to the eggplant flask to prepare PS0% liposomes containing no PS in the lipid bilayer membrane.
  • PS phosphatidylserine
  • PS chloroform solution 1.6 mL
  • PC chloroform solution 6.1 mL
  • chloroform was removed under reduced pressure with an evaporator, and each eggplant flask was placed in a desiccator and dried under reduced pressure for 12 hours.
  • 10 mL of 20 mM Tris-HCl, 0.15 M NaCl (pH 7.4) was added to an eggplant flask and mixed by shaking with a vortex mixer to obtain a suspension.
  • PS20% liposomes When preparing PS20% liposomes, 10 mL of 20 mM Tris-HCl, 0.5 M NaCl (pH 7.4) was added to an eggplant flask and mixed by shaking with a vortex mixer to obtain a suspension. Each of the above suspensions was transferred to a 15 mL centrifuge tube and sonicated to obtain a clear liposome dispersion. Then, PS0% liposome and PS20% liposome solution were prepared by filtering with a 0.2 ⁇ m filter. It was confirmed that liposomes having particle diameters of 49 nm and 39 nm were prepared by a dynamic light scattering particle size measuring device (“Zetasizer Nano ZS” manufactured by Malvern), respectively.
  • a dynamic light scattering particle size measuring device (“Zetasizer Nano ZS” manufactured by Malvern
  • the total amount was 500 ⁇ L. Then, the mixture was centrifuged at 15,000 g for 10 minutes, the filtrate was discarded, and 20 mM Tris-HCl, 0.15 M NaCl, and 2 mM CaCl 2 (pH 7.4) were added to bring the total volume to 0.5 mL. Then, it was centrifuged at 15,000 g for 10 minutes. The above operations of centrifugation, disposal of the filtrate, and addition of 20 mM Tris-HCl, 0.15 M NaCl, and 2 mM CaCl 2 (pH 7.4) were repeated three more times. Then, the solution was recovered from the centrifugal filter device, the total amount was adjusted to 200 ⁇ L, and then homogenized by pipetting to obtain an exosome solution.
  • Example 3 Biotinlation of PS-binding peptide A 4 mg / mL GST-C2A aqueous solution (1 mL) or a 0.4 mg / mL C2A aqueous solution (1 mL) was placed in a 1.5 mL tube, and the temperature of the solution was raised to 4 ° C. on ice. And said. Then, a 20 mM aqueous solution of biotin-PEG (manufactured by Thermo Fisher) was added to each peptide solution so as to have a molar amount 5 times that of the peptide, and the mixture was allowed to stand on ice for 2 hours.
  • biotin-PEG manufactured by Thermo Fisher
  • biotin-PEG was removed by purification on a desalting / buffer exchange column (manufactured by "Hitrap desalting” GE Healthcare) to prepare a biotinylated GST-C2A solution and a biotinylated C2A solution.
  • Test Example 2 Capturing exosomes with magnetic beads A 10 mM CaCl 2 aqueous solution (1.2 ⁇ L) was added to an exosome solution (600 ⁇ L) prepared in the same manner as in Test Example 1 (2). This solution (100 ⁇ L) was added to magnetic beads, the volume was adjusted to 500 ⁇ L with 20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl 2 (pH 7.3), and then the mixture was inverted and mixed at 4 ° C. for 3 hours. Then, the particles were centrifuged for a short time with a desktop centrifuge, left on a magnet stand for 1 minute, and then the supernatant was removed.
  • FIG. 3 shows the amount of exosomes recovered from each of the GST-C2A-immobilized magnetic beads and the C2A-immobilized magnetic beads. From this result, it was found that each magnetic bead was able to recover 1.1 ⁇ g and 0.5 ⁇ g of exosomes, respectively.
  • Example 4 A recognition site for the restriction enzyme BamHI was added to the N-terminal side of the gene encoding the C2 domain (SEQ ID NO: 5) of the protein kinase C ⁇ (PDB: 3GPE_A) derived from Rattus norvegicus, and a recognition site for the restriction enzyme EcoRI was added to the C-terminal side.
  • the artificial synthesis of the gene (SEQ ID NO: 6) was outsourced to Ginscript.
  • This gene was cleaved with BamHI and EcoRI and introduced into a GST fusion protein expression vector (“pGEX-6p-1” manufactured by GE Healthcare Co., Ltd.) cleaved with the same restriction enzyme to introduce the GST-PCK ⁇ C2 expression plasmid pGEX-GST-. PCK ⁇ C2 was prepared.
  • Example 5 A BamHI recognition site was added to the N-terminal side of a gene encoding domains I to IV of Anexin V (NCBI Reference Sequence: NP_307264.1, SEQ ID NO: 9) derived from Rattus norvegicus, and an EcoRI recognition site was added to the C-terminal side.
  • the artificial synthesis of the gene (SEQ ID NO: 10) was outsourced to Ginscript.
  • the gene was composed of E. coli optimized codons.
  • This gene was cleaved with BamHI and EcoRI and introduced into a GST fusion protein expression vector (“pGEX-6p-1” manufactured by GE Healthcare Co., Ltd.) cleaved with the same restriction enzyme to introduce the GST-A5 expression plasmid pGEX-GST-.
  • A5 was prepared.
  • This gene was cleaved with BamHI and EcoRI and introduced into a GST fusion protein expression vector (“pGEX-6p-1” manufactured by GE Healthcare Co., Ltd.) cleaved with the same restriction enzyme to introduce the GST-Tim4IgV expression plasmid pGEX-GST-. Tim4IgV was prepared.
  • Comparative Example 2 A gene in which a BamHI recognition site is added to the N-terminal side of a gene encoding the IgV domain (SEQ ID NO: 13) of CD300A (PDB: 2Q87_A) derived from Homo sapiens, and an EcoRI recognition site is added to the C-terminal side (SEQ ID NO: 14).
  • the artificial synthesis of was outsourced to Ginscript.
  • the gene was composed of E. coli optimized codons.
  • This artificially synthesized gene was cleaved with BamHI and EcoRI and introduced into a GST fusion protein expression vector (“pGEX-6p-1” manufactured by GE Healthcare Co., Ltd.) cleaved with the same restriction enzyme to introduce the GST-CD300AIgV expression plasmid pGEX-.
  • GST-CD300AIgV was prepared.
  • Test Example 3 Evaluation of expression of phosphatidylserine-binding protein in Escherichia coli Escherichia coli (“E. coli”) using 6 types of phosphatidylserine-binding protein expression plasmids prepared in Examples 1, 2, 4, 5 and Comparative Examples 1 and 2. JM109 "manufactured by Takara Bio Co., Ltd.) was transformed. As a control, Escherichia coli (“E. coli JM109” manufactured by Takara Bio Co., Ltd.) was transformed with a GST fusion protein expression vector (“pGEX-6p-1” manufactured by GE Healthcare Co., Ltd.). The obtained 7 types of transformants were cultured in a 2 ⁇ YT medium at 30 ° C.

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Abstract

Procédé de production d'un vecteur de liaison de vésicule à membrane extracellulaire. Le procédé de production d'un vecteur de liaison de vésicule à membrane extracellulaire selon la présente invention est caractérisé en ce qu'il comprend une étape d'obtention de cellules transformées par transformation de cellules hôtes à l'aide d'un acide nucléique comprenant une séquence nucléotidique codant pour un peptide de liaison à la phosphatidylsérine n'ayant pas de liaison disulfure dans la molécule ; une étape de culture des cellules transformées ; une étape de purification du peptide de liaison à la phosphatidylsérine à partir des cellules transformées cultivées ; et une étape d'immobilisation du peptide de liaison à la phosphatidylsérine purifié sur un vecteur insoluble dans l'eau.
PCT/JP2019/051128 2019-03-26 2019-12-26 Procédé de production d'un vecteur de liaison de vésicule à membrane extracellulaire WO2020194968A1 (fr)

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WO2016088689A1 (fr) * 2014-12-05 2016-06-09 和光純薬工業株式会社 Transporteur lié à la protéine tim, procédés d'obtention, d'élimination et de détection de vésicules membranaires extracellulaires et de virus à l'aide dudit transporteur et kit comprenant ledit transporteur
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