WO2020194968A1 - Method for producing extracellular membrane vesicle-binding carrier - Google Patents

Abstract

The purpose of the present invention is to provide a method for efficiently producing an extracellular membrane vesicle-binding carrier. A method for producing an extracellular membrane vesicle-binding carrier according to the present invention is characterized by comprising a step of obtaining transformed cells by transforming host cells using a nucleic acid comprising a nucleotide sequence coding for a phosphatidylserine-binding peptide that does not have a disulfide bond in the molecule; a step of culturing the transformed cells; a step of purifying the phosphatidylserine-binding peptide from the cultured transformed cells; and a step of immobilizing the purified phosphatidylserine-binding peptide on a water-insoluble carrier.

Description

Method for producing outer cell membrane vesicle-binding carrier

The present invention relates to a method for efficiently producing an outer cell membrane vesicle-binding carrier.

In recent years, outer cell membrane vesicles such as exosomes control cell-cell communication, and their relationship with cancer, central nervous system diseases, immunity, etc. is gradually becoming clear. Recently, due to the relationship between exosomes and diseases, development of diagnostic methods targeting exosomes and clinical trials of exosome therapy in which exosomes themselves are administered as drugs are being conducted. For example, a method of isolating exosomes from a blood sample of a subject, analyzing miRNA contained in the exosomes, and diagnosing cancer is being studied. In the past, image analysis such as PET and diagnostic methods using tumor markers had the problem that they would not become positive unless the cancer had grown considerably, but if the method uses exosomes, early cancer can be detected. Can be.

However, a method capable of recovering and purifying exosomes with high recovery rate and high purity has not yet been developed. The most widely used exosome purification method is the ultracentrifugation method, but it is difficult to apply it to clinical applications because of the need for a multi-step purification process and the viewpoint of recovery rate and purity. In addition to this, for recovering and purifying exosomes from biological samples, such as a method using magnetic beads in which an antibody that specifically recognizes an exosome surface antigen is immobilized on the surface of magnetic particles and a method using precipitation with a polymer. Although various methods have been developed, they are not sufficient in terms of recovery rate, degree of purification, throughput, and cost.

In addition, as a means for recovering exosomes, a carrier on which a T cell immunoglobulin / mucin domain-containing molecule (Tim) protein is immobilized has been developed (Patent Document 1). Specifically, 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.

Further, Patent Document 2 describes that Mastoparan-X, Hemolysin, and LL37 have exosome-binding activity, and these are immobilized on a carrier to purify exosomes.

International Publication No. 2016/088689 Pamphlet Japanese Unexamined Patent Publication No. 2017-38566

As described above, a technique for immobilizing a protein exhibiting binding to outer cell membrane vesicles on a carrier and using it for purification of outer cell membrane vesicles has been studied. However, the production efficiency may not be sufficient to carry out such a carrier industrially.
That is, in the case of mass production of proteins, it is generally possible to prepare transformed cells using a gene encoding the target protein, cultivate the cells, and then purify the target protein from a culture medium or cell disruption solution. It is done. However, although the protein forms a stable higher-order structure, the protein produced by the transformed cells may aggregate without forming the higher-order structure, and the production efficiency may be significantly reduced. For example, according to the experimental findings of the present inventors, when the outer cell membrane vesicle-binding proteins CD300A and Tim4 were produced by the transformation method, no expression of CD300A was observed, and Tim4 was contained in the insoluble fraction. It was observed only in the cells, and was considered to be aggregated in the transformed cells. In order to prevent such aggregation, it is conceivable to co-express the molecular chaperone to promote the folding of the target protein, but this increases the production cost and lowers the production efficiency due to the purification of the target protein from the molecular chaperone. There's a problem.
Therefore, 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. As a result, 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.
Hereinafter, the present invention will be shown.

[1] 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.
[2] The phosphatidylserine-binding peptide is a C2 region of synaptotagmin, a C2 region of protein kinases α, βI, βII, and γ, a C2 region of MFG-E8, and an I region, II region, and III region of annexin V. And 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.
[4] The method according to any one of [1] to [3] above, wherein the phosphatidylserine-binding peptide contains at least the C2A region of synaptotagmin.
[5] The method according to any one of [1] to [4] above, wherein the phosphatidylserine-binding peptide contains a C2A region and a C2B region of synaptotagmin.
[6] The method according to any one of [1] to [5] above, wherein the phosphatidylserine-binding peptide is the C2 region of synaptotagmin, and the nucleic acid has the nucleotide sequence of SEQ ID NO: 2.
[7] 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.

According to the method of the present invention, it 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. In addition, 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.

Hereinafter, the method of the present invention will be described for each step, but the present invention is not limited to the aspects shown below.

1. 1. Transformation Step In this step, 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. When 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. On the other hand, 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.

Examples of the phosphatidylserine-binding peptide include the C2 region of synaptotagmin, the C2 region of protein kinase α, the C2 region of protein kinase βI, the C2 region of protein kinase βII, the C2 region of protein kinase γ, 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.

Among the synaptotagmin family, 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. As a method for this, a method of binding the ligand peptide to phosphatidylserine on the surface of the outer cell membrane vesicle in the presence of calcium ions and then dissociating with a chelating agent can be considered. From the above viewpoint, among synaptotagmins, 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. In 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. For example, the base sequence is designed by back-translating the amino acid sequence of the desired C2 region. At this time, it is preferable to use a codon suitable for the host cell to be used. For example, when Escherichia coli is used as a cell, it is preferable to use an Escherichia coli optimized codon.

For example, the 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). In addition, these isozymes are classified into the following three types from the viewpoint of structure and control factors. The first is the conventional type (α, βI, βII, γ) activated by diacylglycerol (DAG), calcium ion, and phospholipid (particularly phosphatidylserine), and has two C1 regions (DAG binding domains) and C2 regions (DAG binding domain). It is composed of a calcium-binding domain), a C3 region (ATP-binding domain), and a C4 region (kinase domain), and binds to calcium and phosphatidylserine by the C2 region. Hereinafter, the roles of each area are the same. The second is composed of a new type (δ, ε, η, θ) 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 (ζ, λ, μ) 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 Cα (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) 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 αvβ3 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. In addition to the sequence, 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. The term 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 α-helices A, B, and D. , E bundled structure has a structure capped by one α-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. An array can be mentioned. In addition, a protease recognition site may be inserted between these tags and the peptide. In addition, 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.

After designing the base sequence encoding the above phosphatidylserine-binding peptide, the nucleic acid containing the sequence is artificially synthesized. The nucleic acid may be amplified by the PCR method. Using this nucleic acid, 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. Usually, 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. For example, when Escherichia coli is used as a host, pQE-based vectors (Qiagen), pET-based vectors (Merck), and pGEX-based vectors (GE Healthcare Bioscience) can be mentioned.

As the host, 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; and insect cells can be used. From the viewpoint of mass-producing peptides at low cost, 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. Examples of 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. Further, as 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.

2. 2. Culturing step In this step, the transformed cells obtained in the above transformation step are cultured. As a result, the target phosphatidylserine-binding peptide is considered to accumulate in the proliferated transformed cells.

Culturing of transformed cells is carried out according to the usual conditions used for culturing the host. For example, 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. Specifically, 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. In addition, inorganic salts such as potassium salt, sodium salt, phosphate, magnesium salt, manganese salt, zinc salt and iron salt are added as needed. When a auxotrophic host cell is used, a vegetative substance required for growth may be added. If necessary, antibiotics such as penicillin, erythromycin, chloramphenicol, and neomycin may be added. For example, 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. , Yeast extract 0.5%, NaCl 1%) and the like.

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.

3. 3. Purification Step In this step, the phosphatidylserine-binding peptide is purified from the transformed cells cultured in the above culture step. As described above, 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.

4. Immobilization step In this step, the purified target peptide is immobilized on a water-insoluble carrier. By immobilizing the phosphatidylserine-binding peptide on a water-insoluble carrier, it can be used as an affinity ligand having an affinity for outer cell membrane vesicles. In the present disclosure, "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. In addition, "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. Commercially available products include 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, and an agarose-based crosslinked carrier. Examples thereof include a certain Sepharose ^(R) CL4B and Cellulfine ^(R) which is a cellulosic cross-linking carrier. However, 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. As the form of the carrier, beads, monoliths, fibers, films (including hollow fibers) and the like can be used, and any form can be selected.

Regarding 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. As a coupling method, 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. A method of introducing a reactive functional group into a peptide and performing a coupling reaction with a peptide to immobilize it, a condensation reagent such as carbodiimide in a system in which a carrier and a peptide are present, or a plurality of condensation reagents in a molecule such as glutaaldehyde. 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. Examples of the linker group include a C _1-6 alkandyl group, an amino group (-NH-), an ether group (-O-), a carbonyl group (-C (= O)-), and an ester group (-C (=). O) O- or -OC (= O)-), amide group (-C (= O) NH- or -NHC (= O)-), urea group (-NHC (= O) NH-); 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. Further, the C _1-6 alkanediyl group may be substituted with a substituent such as a hydroxyl group. Further, the linker may be an amino acid residue useful for immobilization or a peptide. Examples of 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. In addition, the linker may be a complex of interacting molecules such as biotin and avidin or streptavidin.

5. Method of using the outer membrane vesicle-binding carrier 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. For example, after preparing a liquid sample containing outer cell membrane vesicles such as blood, serum, and plasma of a subject, 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. At this time, it is preferable to adjust the pH of the liquid sample to 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. At this point, 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. Furthermore, by disrupting the purified outer membrane vesicles and examining the type and distribution of miRNA and mRNA contained therein, it can be useful for diagnosing the disease or the possibility of its onset. It is also conceivable to use the outer cell membrane vesicle itself as the active ingredient of the drug.

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) and the like can be mentioned, with preference given to EDTA and EGTA, but not limited to those listed here.

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.

This application claims the benefit of priority based on Japanese Patent Application No. 2019-58141 filed on March 26, 2019. The entire contents of the specification of Japanese Patent Application No. 2019-58141 filed on March 26, 2019 are incorporated herein by reference.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples as well as the present invention, and appropriate modifications are made to the extent that it can be adapted to the gist of the above and the following. Of course, it is possible to carry out, and all of them are included in the technical scope of the present invention.

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. 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-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.

(2) Purification of GST-C2A-C2B The culture solution obtained in (1) above was centrifuged at 9,000 rpm for 10 minutes to remove the supernatant. The remaining cells were suspended in 20 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA (pH 7.5), the cells were crushed with an ultrasonic crusher, and again at 15,000 rpm for 5 minutes. Centrifugation was performed to obtain a crushed cell supernatant.
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.

(3) Preparation of C2A-C2B and GST To the GST-C2A-C2B solution (3 mL) prepared in (2) above, GST-fused rhinovirus 3C protease ("Precision phase" manufactured by GE) (50 μL) was added. The reaction was carried out at 4 ° C. for 15 hours. Then, the reaction solution was diluted 10-fold 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 Healthcare Life Sciences). The flow-through fraction was collected by loading the product. Then, 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.

(4) Immobilization of ligand on sensor chip Set the sensor chip (“CM5” manufactured by GE Healthcare Life Science Co., Ltd.) in the interaction analyzer (“Biacore 3000” manufactured by GE Healthcare Life Science Co., Ltd.) and equilibrate with PBS. After conversion, the surface of the sensor chip was activated by flowing an EDC solution (120 μL) and an NHS solution (120 μL) at a rate of 5 μL / min for 7 minutes. The sensor chip was then washed with PBS and a solution of GST-C2A-C2B or GST was run at a rate of 10 μL / min for 7 minutes to immobilize each peptide. Then, the 1M ethanolamine hydrochloride solution was flowed at a rate of 5 μL / 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. 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. Further, in order to prepare PS20% liposomes containing 20% of PS in the lipid bilayer membrane, PS chloroform solution (1.6 mL) and PC chloroform solution (6.1 mL) were added to the eggplant flask and homogenized. Then, 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.
When preparing PS0% liposomes, 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. 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 μm 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.

(2) Preparation of exosome solution Cosmo Bio's domestically produced milk-derived milk exosome was used for evaluation of binding ability, and the solvent was 20 mM Tris-HCl, 0.15 M NaCl, 2 mM CaCl ₂ (pH 7.4) according to the following procedure. Exchanged for. Specifically, a milk exosome solution (200 μL) stored at −80 ° C. was melted at 25 ° C. Transfer the thawed milk exosome solution to a centrifugal filter device (“Amicon ultra-0.5 mL, 10 K” manufactured by Merck Millipore) and add 20 mM Tris-HCl, 0.15 M NaCl, 2 mM CaCl ₂ (pH 7.4). The total amount was 500 μL. 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 μL, and then homogenized by pipetting to obtain an exosome solution.

(3) Evaluation of binding of PS-binding peptide to PS-containing liposomes and exosomes Liposomes and exosomes were flowed through each flow cell on which a PS-binding protein was immobilized, and the binding property to each peptide was evaluated. The binding evaluation was performed according to the following procedure.
After flowing PBS at a rate of 20 μL / min for 2 minutes to the flow cell, a liposome solution diluted 100-fold with PBS or a prepared exosome solution was added at a rate of 20 μL / min over 3 minutes, and the binding response was measured. The dissociation response was then measured by running PBS at a rate of 20 μL / min over 6 minutes. Then, 10 mM EDTA was flowed at a rate of 20 μL / min for 1 minute to dissociate the liposomes and exosomes bound to the peptide surface. The response value when the PS20% liposome solution was flowed is shown in FIG. 1, and the response value when the exosome solution was flowed is shown in FIG.
As shown in the results shown in FIGS. 1 and 2, GST-immobilized cells and reference cells in which peptides are not immobilized do not react with liposomes and milk exosomes at all, whereas GST-C2A-C2B and GST-C2A are PS-containing liposomes. And it can be seen that it binds to milk liposomes. The response values of GST-C2A-C2B and GST-C2A to PS-containing liposomes were 3143 and 3540, respectively, and the response values to milk exosomes were 395 and 205, respectively.
On the other hand, in the PC liposomes containing no PS, no response was observed in the cells on which any of the peptides was immobilized.
From the above results, it was shown that GST-C2A-C2B and GST-C2A specifically bind to vesicles containing PS.

Example 3
(1) 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. Then, excess 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.

(2) Immobilization of biotinylated PS-binding protein on magnetic beads Streptavidin (SA) -immobilized magnetic beads (“Dynabeads MyOne Streptavidin” manufactured by Veritas) were homogenized with a vortex mixer, and 60 μL in a 1.5 mL tube. Dispensed. Then, the mixture was left on a magnet stand for 1 minute, the supernatant was discarded, 500 μL of 20 mM Tris-HCl, 0.15 M NaCl, and 2 mM CaCl ₂ (pH 7.4) was added, and the mixture was homogenized with a vortex mixer. This operation was repeated once more. The supernatant was then discarded and 20 mM Tris-HCl, 0.15 M NaCl, 2 mM CaCl ₂ (pH 7.4) (500 μL) and biotinylated GST-C2A solution (500 μL) or biotinylated C2A solution (500 μL) were added. Then, it was inverted and mixed at 4 ° C. for 10 minutes. Then, the particles were centrifuged for a short time with a desktop centrifuge and left on a magnet stand for 1 minute. The supernatant was discarded, 500 μL of 20 mM Tris-HCl, 0.15 M NaCl, 2 mM CaCl ₂ (pH 7.4) was added, and the mixture was homogenized with a vortex mixer. By repeating this operation twice more, a slurry of biotinylated PS-binding protein-immobilized magnetic beads was obtained.

Test Example 2: Capturing exosomes with magnetic beads A 10 mM CaCl ₂ aqueous solution (1.2 μL) was added to an exosome solution (600 μL) prepared in the same manner as in Test Example 1 (2). This solution (100 μL) was added to magnetic beads, the volume was adjusted to 500 μL 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. To this, 20 mM Tris-HCl, 0.15 M NaCl, 2 mM CaCl ₂ (pH 7.4) (1 mL) was added and homogenized with a vortex mixer. This operation was repeated twice more. Then, the particles were centrifuged for a short time with a tabletop centrifuge to remove the supernatant. After adding 10 mM EDTA (50 μL) and homogenizing with a vortex mixer, the particles were centrifuged for a short time with a tabletop centrifuge, and the supernatant was collected in a microtube. After adding 10 mM EDTA (50 μL) again and homogenizing with a vortex mixer, the particles were centrifuged for a short time with a tabletop centrifuge, and the supernatant was collected. The above two eluates were combined and homogenized with a vortex mixer.
The amount of exosomes contained in the eluate was quantified using a bovine milk exosome ELISA kit (manufactured by Cosmo Bio Co., Ltd.). The operation was performed according to the method attached to the kit.
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 μg and 0.5 μg 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 Cα (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-PCKαC2 expression plasmid pGEX-GST-. PCKαC2 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.

Comparative Example 1
Homo sapiens-derived T-cell immunoglobulin / mucin domain-containing molecule (Tim) protein 4 (NCBI Reference Sequence: XP_01153296.1) IgV domain (NCBI Reference Sequence: sequence from position 103 to position 134, amino acid _0115322996. An artificial synthesis of a gene (SEQ ID NO: 12) having a BamHI recognition site added to the N-terminal side of the gene encoding No. 11) and an EcoRI recognition site added to the C-terminal side 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-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. overnight in vitro, subcultured in TB medium, and cultured in flasks at 37 ° C. Then, IPTG was added and the mixture was cultured at 25 ° C. overnight to obtain a culture solution of phosphatidylserine-binding protein-expressing Escherichia coli. After measuring the cell concentration of each culture solution at OD600 nm, the cell concentration was adjusted so that the OD600 nm was 20, and the cells were suspended in PBS. After crushing the cells by ultrasonic crushing, the cells were centrifuged, and the supernatant was separated as a soluble fraction. The precipitate was suspended in PBS, the soluble fraction and the precipitate suspension were subjected to SDS-PAGE, and the expression level of the target protein was evaluated. The results are shown in FIG. 4 and Table 2. In Table 2, "○" indicates that the target peptide could be confirmed, and "x" indicates that the target peptide could not be confirmed.

As shown in the results shown in FIGS. 4 and 2, all of GST-PKCα-C2 and GST-C2A-C2B, which do not have an intramolecular disulfide bond, are expressed in the soluble fraction, and the precipitation suspension. I could not confirm it inside. Similarly, GST-annexin V and GST-C2A, which do not have an intramolecular disulfide bond, were also observed in the precipitation suspension, but more than half of them were observed in the soluble fraction. GST-annexin V and GST-C2A were confirmed in the precipitation suspension because the peptides were cross-linked by calcium ions, and it is considered that the precipitation of these peptides is probably eliminated by a chelating agent or the like. ing.
On the other hand, for GST-Tim4IgV and GST-CD300AIgV having an intramolecular disulfide bond, no band of the target peptide was observed in the soluble fraction. Further, in the insoluble fraction, expression of a peptide presumed to be the target substance was observed in the vicinity of the molecular weight of the target product only in GST-Tim4IgV. This result shows that GST-Tim4IgV having an intramolecular disulfide bond is more likely to aggregate in the transformed cell than a peptide having no intramolecular disulfide bond, and the GST-CD300AIgV gene is a transformation method. It shows that the expression itself is difficult in some cases.
From the above results, it is demonstrated that the phosphatidylserine-binding peptide having no intramolecular disulfide bond can be efficiently produced by the transformation method as compared with the phosphatidylserine-binding peptide having an intramolecular disulfide bond. It was.

Claims (7)

1. 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.
2. The phosphatidylserine-binding peptide is a C2 region of synaptotagmin, a C2 region of protein kinases α, βI, βII, and γ, a C2 region of MFG-E8, and an I region, II region, III region, and IV region of annexin V. The method according to claim 1, which is one or more phosphatidylserine-binding peptides selected from.
3. The method according to claim 1 or 2, wherein the phosphatidylserine-binding peptide is immobilized on the water-insoluble carrier via a linker.
4. The method according to any one of claims 1 to 3, wherein the phosphatidylserine-binding peptide contains at least the C2A region of synaptotagmin.
5. The method according to any one of claims 1 to 4, wherein the phosphatidylserine-binding peptide contains a C2A region and a C2B region of synaptotagmin.
6. The method according to any one of claims 1 to 5, wherein the phosphatidylserine-binding peptide is the C2 region of synaptotagmin, and the nucleic acid has the nucleotide sequence of SEQ ID NO: 2.
7. 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 claims 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.

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