JP2024012970A - Conjugate, and method for controlling surface properties of target substance and method for recovering target substance using the same - Google Patents

Abstract

To provide a conjugate that can specifically control surface properties of a target substance (hydrophobicity or hydrophilicity) or can specifically adsorb and aggregate the target substance, and to provide a method for controlling the surface properties of a target substance and a method for specifically recovering the target substance using the conjugate.SOLUTION: A conjugate includes a peptide (A) that specifically adsorbs a target substance, a hydrophobic site (B), a first linker (C) that includes a first specific cleavage site, and a hydrophilic site (D) in this order.SELECTED DRAWING: None

Description

The present invention relates to a conjugate, a method for controlling the surface properties of a target substance using the same, and a method for recovering the target substance.

There is a need for a method of controlling the surface of a specific target substance to be hydrophobic or hydrophilic from a mixture. It is well known that it is extremely difficult to specifically collect target particles from a mixture of fine particles, especially when the target substance is so small that it is virtually impossible to handle each individual particle. be.

For example, Patent Document 1 discloses a method in which target inorganic fine particles are selectively adsorbed and precipitated by using a flocculant and a substance with specific adsorption properties such as zeolite. There is. However, with this method, it has not been possible to accurately recover only the target substance from a mixture of target substances and impurities that have similar characteristics such as particle size, specific gravity, and polarity.

Particularly in the mining field, in order to refine metal resources, a process (ore beneficiation) is required to separate minerals into minerals containing the target metal and other minerals. In ore beneficiation, the ore is first ground sufficiently finely so that all particles are separated to improve accuracy. Then, depending on the characteristics of the target substance and contaminants, for example, the specific gravity separation method that separates the target substance based on the difference in specific gravity, the magnetic separation method if the target substance is attracted by magnetic force, or the hydrophobicity and They are selected using a flotation method that uses differences in surface properties such as hydrophilicity.

Particularly, in the flotation method (so-called flotation method), crushed ore is put into a water tank and bubbles are generated by aeration, and hydrophobic particles are attached to the bubbles and separated by flotation. At this time, if the particle size of the valuable mineral becomes too small during pulverization, the flotation efficiency will decrease and the valuable mineral will be mixed into worthless mineral particles, resulting in loss. Furthermore, if the minerals lost in this way are toxic, the minerals that cannot be recovered and are discharged cause serious mineral pollution, which becomes an environmental problem.

As a method of dealing with such loss of valuable minerals, techniques for agglomerating and dispersing fine particles in a solvent such as water are used. For example, Non-Patent Documents 1 and 2 show that by using a specific surfactant, finely ground chalcopyrite can be agglomerated to increase the apparent particle size, increasing the recovery efficiency in flotation. is listed. However, with the methods described in Non-Patent Documents 1 and 2, it was not possible to specifically aggregate only specific valuable mineral particles.

Especially in such cases, the target mineral and the impurities are both derived from rocks and have very similar physical properties, and in the process of crushing the rock, the particle size and particle size distribution must be precisely controlled. Good control is also virtually impossible, and existing methods have not been able to select only the target substance with greater precision.

On the other hand, the so-called phage display method is known as a method for selecting peptides that specifically adsorb only to a target substance. This method is a method to select a specific peptide that specifically adsorbs only the target substance from a peptide library with a variety of peptide sequences. A feature of this method is that only peptides that exhibit extremely high specificity for a particular substance can be selected.

Patent Documents 2 and 3 describe a fusion protein that has a function of acting only on a specific substance by fusing a specific peptide selected by a phage display method with a peptide having other functions.

Japanese Patent Application Publication No. 2013-200304 Special Publication No. 2007-508246 Special Publication No. 2009-508870

VothyHornn et al., “Kinetic Analysis for Agglomeration-Flotation of Finely GroundChalcopyrite: Comparison of First Order Kinetic Model and Experimental Results” Materials Transactions, Vol.61, No.10(2020), pp.1940 to 1948 VothyHornn et al., “Agglomeration-Flotation of Finely Ground Chalcopyrite UsingEmulsified Oil Stabilized by Emulsifiers: Implications for Porphyry Copper OreFlotation” Metals 2020, 10, 912

The present invention relates to a conjugate that can specifically control the surface properties (hydrophobicity or hydrophilicity) of a target substance, or that can specifically adsorb and aggregate a target substance, and a conjugate that can be used to target a target substance using the conjugate. The present invention aims to provide a method for specifically controlling the surface properties of a substance and a method for specifically recovering a target substance.

As a result of intensive research, the present inventors discovered that a unique conjugate containing a peptide that specifically adsorbs to a target substance, a hydrophobic site, a linker having a specific cleavage site, and a hydrophilic site, in this order, enables The inventors have discovered that the above problems can be solved, and have completed the present invention.

That is, the present invention is as follows.
[1]
Contains in this order a peptide site (A) that specifically adsorbs to the target substance, a hydrophobic site (B), a first linker (C) containing a first specific cleavage site, and a hydrophilic site (D). , conjugate.
[2]
The conjugate according to [1], wherein the first specific cleavage site in (C) above is cleavable by an enzyme.
[3]
A second linker (E) including a second specific cleavage site is further included between the above (A) and the above (B), and the first specific cleavage site and the second specific cleavage site are connected to each other. The conjugate according to [1] or [2], wherein the conjugate has a different cleavage site.
[4]
A method for controlling the surface properties of a target substance, the method comprising the step of binding the conjugate according to any one of [1] to [3].
[5]
A step of adsorbing the conjugate according to any one of [1] to [3] to a target substance present in an aqueous medium, and
A method for recovering a target substance, comprising a step of cleaving the above (C) in the conjugate and aggregating the above (B) with each other to aggregate the target substance.
[6]
The recovery method according to [5], wherein the target substance is a fine particle, and the fine particle has an average particle diameter of 1 nm or more and 10 μm or less.

According to the present invention, a conjugate capable of specifically controlling the surface properties of a target substance or specifically aggregating the target substance, a method of controlling the surface properties of a target substance using the same, and A method for recovering a target substance can be provided.

FIG. 2 is a diagram showing the results of SDS-PAGE of the conjugate (fusion protein) of Example. They are a photograph (A) and an optical microscope image (B) showing the results of aggregation of chalcopyrite fine particles by a conjugate in an example. It is a figure which shows the result of observing the surface of the chalcopyrite microparticle aggregated by the conjugate in an Example with the scanning electron microscope. These are the results of observing the aggregation of silica fine particles by the conjugate in Examples using an optical microscope. FIG. 3 is a diagram showing the results of optical microscopic observation of aggregation of chalcopyrite fine particles when a mixture of chalcopyrite fine particles and silica fine particles formed by a conjugate in an example is used.

Embodiments of the present invention will be described in detail below. Note that the present invention is not limited to the following embodiments.

<Conjugate>
The conjugate according to this embodiment includes a peptide site (A) that specifically adsorbs to a target substance, a hydrophobic site (B), a first linker (C) containing a first specific cleavage site, and a hydrophilic site. The sex sites (D) are included in this order.

Since the conjugate according to this embodiment has (A), it can be specifically adsorbed to a target substance, and furthermore, since it has (B) to (D), the target substance specifically adsorbed as described below The surface properties of the target substance can be controlled to be hydrophilic or hydrophobic at any timing, or the target substance can be specifically aggregated. Therefore, the conjugate according to this embodiment is effective in aggregating only the target substance in a state where the target substance is mixed with other substances (for example, a suspension containing a plurality of substances).

(Target substance)
In this specification, the target substance may be any substance, and is not particularly limited, and may be an organic material (organic substance) or an inorganic material (inorganic substance). Although there may be a plurality of target substances, it is preferable that there is only one target substance. The target substance may be a substance to which a specific peptide is adsorbed with specificity by a so-called phage display method or the like.

Examples of organic materials include aliphatic polyolefin vinyl (co)polymers such as polyethylene, polypropylene, and butadiene; vinyl aromatic hydrocarbon (co)polymers such as polystyrene; polyacrylotrile, polyvinyl acetate, and polyvinyl acetate. Substituted vinyl (co)polymers such as vinyl chloride and polyvinylidene chloride; (meth)acrylic acids such as poly(meth)acrylic acid, polymethyl (meth)acrylate, and polybutyl (meth)acrylate, and their esters (co)polymers; Polymers: Polyester-based (co)polymers such as polyethylene terephthalate, polyethylene naphthalate, polycaprolactone, polycarbonate, and polylactic acid; Polyamide-based (co)polymers derived from aliphatic and aromatic diamines and aliphatic and aromatic dicarboxylic acids Polymers; polyimides derived from aliphatic and aromatic tetraamines and aliphatic and aromatic tetracarboxylic acids; phenolic resins; epoxy resins; polyurethane resins, polyether ether ketone resins; ) Polymers; polyether (co)polymers such as polyoxymethylene and polyalkylene glycol; synthetic resins such as modified polysaccharides such as triacetylcellulose and nitrocellulose; cellulose, hemicellulose, chitin, chitosan, fucoidan, alginic acid , polysaccharides such as hyaluronic acid, dextran, agarose, and heparin; lignins; proteins; nucleic acids; crystals, glass bodies, coagulates, etc. of organic low molecular weight compounds such as phthalocyanine, quinacridone, isoindoline, and disazo yellow; Can be mentioned.

Examples of the inorganic material include minerals, metals, alloys, metal salts, ceramics, and silica, and minerals are preferred among them.

As used herein, mineral refers to naturally occurring inorganic and organic crystalline substances. Minerals include, for example, elemental minerals consisting of the following metals; sulfide minerals such as chalcopyrite, galena, sphalerite, pyrite, nickel sulfite, and bornite; quartz, hematite, magnetite, titanite, and spinel. , corundum, bauxite, cassiterite, scheelite, zeolite, kaolin, bentonite, and other oxidized minerals; halite, fluorite, and other halogenated minerals; calcite, dolomite, limestone, and other carbonate minerals; borax, etc. Examples include borate minerals; sulfate minerals such as alumite and gypsum; phosphate minerals such as apatite; silicate minerals such as silica and asbestos; graphite; and coals.

When the target substance is a mineral, it may be a crushed product of the mineral. In crushed minerals, the target substance and impurities may be mixed in one particle, but if the target substance is exposed on the surface of the crushed mineral, (A) in the conjugate of the present invention Recognized.

Examples of metals include gold, platinum, silver, copper, titanium, iron, lead, zinc, nickel, aluminum, cobalt, manganese, tungsten, cadmium, antimony, tin, indium, mercury, palladium, iridium, rhodium, ruthenium, Single metals such as molybdenum, tantalum, niobium, vanadium, and zirconium, brass, bronze, cupronickel, red copper, shakudo, steel, magnesium alloy, chrome-molybdenum steel, stainless steel, manganese-molybdenum steel, duralumin steel, Hastelloy steel, Inconel Examples include alloys such as steel, solder, and amalgam. These may exist in any form such as metal crystals, metal salt crystals, and metal ions.

Examples of metal salts include metal oxides such as silver oxide, tin oxide, iron oxide, copper oxide, zinc oxide, alumina, titania, zirconia, and ceria; alkali metals (lithium, sodium, potassium, etc.), or alkaline earths. Metal salts such as hydrochlorides, sulfates, phosphates, carbonates, borates, organic carboxylates, organic sulfonates, organic phosphates, etc. of similar metals (beryllium, magnesium, calcium, barium, etc.) can be mentioned.

The shape or form of the target substance is not particularly limited, and may be solid, semi-solid, or gel-like, or may be particulate (granule-like), plate-like, rod-like, amorphous, powdery (powder), or lump-like. There may be. Preferably, the target substance is solid and has a size large enough to be suspended or dispersed in an aqueous medium such as water, and more preferably in the form of fine particles.

In the case of fine particles, it is virtually impossible to selectively handle individual particles, and conventional separation methods, for example by precipitation, specific gravity or polarity, are difficult, but the conjugates of the present invention According to the method, the surface properties of even fine particles can be specifically controlled or specifically recovered.

In conventional methods, when the average particle diameter of fine particles is less than 10 μm, it becomes difficult to sort and separate them. Therefore, the size (average particle diameter) of the target substance (fine particles) at which the effects of the present invention are particularly exhibited is preferably 10 μm or less, more preferably 5 μm or less, and 1 nm or more, more preferably 10 nm or less. The thickness is particularly preferably 100 nm or more. The average particle diameter may be, for example, 1 nm or more and 10 μm or less, preferably 10 nm or more and 5 μm or less, and more preferably 100 nm or more and 5 μm or less. Here, the average particle diameter means the D50 value of the particle size distribution obtained from the image analysis result from an electron microscope image.

As used herein, aqueous medium means water or an aqueous solution. Examples of water include deionized water, distilled water, etc., and examples of the aqueous solution include buffers such as Tris-HCl buffer and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). It will be done. The target substance may be suspended or dispersed in an aqueous medium.

(Specifically adsorbing peptide site (A))
As used herein, "peptide" means two or more amino acid residues connected by a peptide bond (amide bond), and may be a polypeptide or protein. The amino acid residue may be a natural amino acid or a non-natural amino acid. The length of the peptide is not particularly limited, but may contain about 2 to 1000 amino acid residues.

The specifically adsorbing peptide site (A) is a site (region) that specifically adsorbs to a target substance and is intended to impart substance specificity to the conjugate of the present invention, and has this function. There is no particular limitation as long as it is a peptide (in the case of a conjugate, it is a peptide residue).

The length of the peptide (A) is not particularly limited, as long as the number of amino acid residues is 2 or more, preferably 4 or more, more preferably 6 or more. Further, the number of amino acid residues in (A) may be 32 or less, more preferably 16 or less, still more preferably 12 or less. When the specifically adsorbed peptide site (A) is selected by phage display, it may have the length of a peptide in a peptide library generally used for phage display.

Specific adsorption to a target substance means adsorption to the target substance but not or even adsorption to other substances, especially other substances in the mixture or suspension in which the target substance is present. The ability is 1/10 or less, 1/20 or less, 1/50 or less, or 1/100 or less of the target substance. In addition, in this specification, "adsorption" may also be referred to as "bonding" and may be physical adsorption (physical bond) or chemical adsorption (chemical bond). Adsorption capacity can be determined by ELISA method, surface plasmon observation type molecular interaction measurement method (so-called Biacore method), isothermal titration type calorimetry type molecular interaction measurement method (so-called iTC method), immunoprecipitation method, target substance recovery rate, etc. can be evaluated.

Peptides that specifically adsorb to a target substance can be obtained, for example, by preparing a peptide library, then bringing it into contact with the target substance, and performing screening. Examples of such methods include methods using a phage display method, a ribosome display method, a cell surface display method using bacteria or yeast, an mRNA display method, or a cDNA display method. Here, the peptide library may be, for example, a peptide library consisting of 4 to 8 amino acid residues having any combination, and such a library can be created by artificial synthesis. As used herein, peptide libraries also include phage libraries.

As a method for selecting peptides that specifically adsorb to a target substance, a so-called phage display method is particularly preferably used. This method is widely known as a method for efficiently selecting peptides that specifically adsorb to a target substance from a library.

The phage display method is generally the following method. First, by integrating an arbitrary gene into each individual phage, a collection of phages containing various genes is obtained. Such a collection is called a phage library. Next, this library is adsorbed to a target substance and a selection operation (biopanning, affinity selection, etc.) is performed to concentrate the phage group adsorbed to the target substance.

(Bio)panning involves first incubating a phage library with a target substance, so that phages that have an affinity for the target substance are adsorbed to the target substance, while phages that do not have an affinity for the target substance are washed away. This process collects only the phages that have an affinity for the target substance. Next, the phage adsorbed to the target substance is eluted, the host E. coli is infected with it, and the amplified phage is amplified in the culture medium. By repeating the operation several times, a group of phages with higher affinity (adsorption activity) can be selected and concentrated. After cloning the phage selected in this way, the genomic DNA of the phage is analyzed to identify the amino acid sequence of the peptide that specifically adsorbs to the target substance. Can be selected.

The specifically adsorbing peptide site (A) can be selected by the above method and is not particularly limited, but for example, a peptide that specifically binds to chalcopyrite (chalcopyrite-binding peptide, DSQKTNPS, SEQ ID NO: 2) can be mentioned. In addition, conventionally known peptides that specifically bind to specific substances include, for example, a peptide that specifically binds to gold (MHGKTQATSGTIQS, SEQ ID NO: 7), a peptide that specifically binds to platinum (PTSTGQA, sequence No. 8), a peptide that specifically binds to silver (NPSSLFRYLPSD, SEQ ID NO: 9), a peptide that specifically binds to titanium (RKLPDAPGMHTW, SEQ ID NO: 10), a peptide that specifically binds to zeolite (VKTQATSREEPPRLPSKHRPG, SEQ ID NO: 11) ), a peptide that specifically binds to calcium phosphate (KDVVVGVPGGQD, SEQ ID NO: 12), etc. (Sarikaya M et al., “Molecular biomimetics: nanotechnology through biology.” Nat Mater. 2003 Sep;2(9):577-85 ).

(Hydrophobic site (B))
The hydrophobic site (B) is a site (region) whose purpose is to aggregate a target substance from an aqueous solvent by hydrophobic interaction. That is, after adsorbing the conjugate according to this embodiment to a target substance via the specifically adsorbing peptide site (A), a first linker (described later) containing a first specific cleavage site ( By cutting C), the hydrophilic site (D) described below is separated from the conjugate, and the hydrophobic site (B) is exposed to the outside. As a result, the surface of the target substance becomes hydrophobic, and the target substance aggregates in the aqueous medium due to the hydrophobic interaction between (B).

The hydrophobic moiety (B) is not particularly limited as long as it has hydrophobicity to aggregate the target substance, and may be a group of a hydrophobic substance. One indicator of the hydrophobicity of the hydrophobic moiety (B) is, for example, the equivalent of the hydrophobic moiety (B) to the sum of the anionic polar groups in the molecule, specifically carboxyl groups and phosphate groups. It is sufficient if it is 500 g/eq or more, and preferably 700 g/eq or more. In addition, the ionic polar group equivalent to the sum of the ionic polar groups in the molecule of the hydrophobic part (B), specifically the above-mentioned anionic polar groups and cationic polar groups such as amino groups and imino groups, is 245 g/eq. It is preferable that it is above. Another indicator of hydrophobicity includes, for example, HLB (hydrophilicity/hydrophobicity balance).

(B) is not particularly limited as long as it is hydrophobic, and may be a synthetic polymer or a natural polymer. In this specification, the synthetic polymer is a general term for polymers or oligomers synthesized using chemical methods. Furthermore, natural polymers are a general term for polymers and oligomers obtained by extracting and decomposing (lowering molecular weight) natural products. Here, since the hydrophobic site (B) is chemically bonded to other sites in the conjugate, it exists not as a polymer compound but as a group of these polymer compounds.

Examples of synthetic polymers include aliphatic polyolefin vinyl (co)polymers such as polyethylene, polypropylene, and butadiene; vinyl aromatic hydrocarbon (co)polymers such as polystyrene; polyacrylotrile, polyvinyl acetate, Substituted vinyl (co)polymers such as polyvinyl chloride and polyvinylidene chloride; Acrylic acid ester (co)polymers such as polymethyl (meth)acrylate and polybutyl (meth)acrylate; Polyethylene terephthalate, polyethylene naphthalate, polycaprolactone , polyester-based (co)polymers such as polycarbonate and polylactic acid; polyamide-based (co)polymers derived from aliphatic and aromatic diamines and aliphatic and aromatic dicarboxylic acids; aliphatic and aromatic tetraamines and aliphatic and polyimides derived from aromatic tetracarboxylic acids; epoxy resins; polyurethane resins; cycloolefin (co)polymers; polyethers such as polyalkylene glycols having 3 or more carbon atoms in one repeating unit Examples include (co)polymers.

Natural polymers include cellulose, hemicellulose, chitin, chitosan, water-insoluble polysaccharides such as water-insoluble alginic acid and water-insoluble hyaluronic acid, lignins, oils and fats such as beeswax, palm wax, and carnauba wax, and hydrophobic Examples include peptides (including hydrophobic proteins). Among these, natural polymers, especially hydrophobic peptides, are preferably used in view of stability in dispersion media and ease of constructing the conjugate.

It is preferable that the hydrophobic site (B) in the conjugate according to this embodiment is a hydrophobic peptide. Hydrophobic peptides may have a hydrophobic helical structure in their three-dimensional structure, and hydrophobicity can be evaluated using, for example, the above-mentioned hydrophobicity index, the hydropathy plot value described below, or HLB.

The hydropathy plot (hydrophobicity plot) value is an index indicating the hydrophilicity or hydrophobicity of a peptide. Specifically, the average value of the hydropathic index of five amino acid residues, including two amino acid residues before and after each amino acid residue, is calculated using the hydropathic index presented for each amino acid species that constitutes a protein. It is used as an index of hydrophilicity or hydrophobicity. When this value is large, it indicates higher hydrophobicity, and conversely, when this value is small, it is evaluated as having high hydrophilicity.

In the conjugate according to the present embodiment, the ratio of the number of amino acid residues having a hydropathic index of -1.8 or less to all amino acid residues in the hydrophobic site (B) is preferably 25% or less, more preferably 15% or less. % or less, more preferably 10% or less. Hydropathy plot values and hydropathic indices are shown, for example, in J. Kyte, R. F. Doolittle, "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 1982, 157, 105-132.

Examples of the hydrophobic peptide include a hydrophobic peptide containing the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 has a structure in which four α-helices are assembled into a bundle, and the inside of the bundle is thought to be a structure in which hydrophobic residues are surrounded. In an aqueous medium, this structure allows hydrophilic residues to appear on the surface of the conjugate, but with the cleavage of the first linker (C), hydrophobic residues appear on the surface. , it is estimated that more favorable hydrophobic interactions occur.

When the hydrophobic site (B) is a polymer compound, its weight average molecular weight is not particularly limited, and may be, for example, 1,000 or more, preferably 3,000 or more, more preferably 8,000 or more. It may be 80,000 or less, preferably 40,000 or less, more preferably 20,000 or less.

When (B) is a hydrophobic peptide (in a conjugate, it is a peptide residue), the number of amino acid residues in (B) is not particularly limited, but may be, for example, 8 or more, preferably 20 or more, It is more preferably 60 or more, and may be, for example, 700 or less, preferably 400 or less, and more preferably 180 or less. The number of amino acid residues in (B) may be, for example, 50 to 200 or 80 to 150.

(First linker (C))
The first linker (C) containing the first specific cleavage site is a site (region) that is intended to cleave the hydrophilic site (D) described below at any timing by cutting as necessary. It is. Moreover, (C) may consist only of the sequence of the first specific cleavage site, or may further include a linking sequence for the purpose of improving connectivity with other sites.

(C) connects (B) and (D) through a chemical bond. The first linker (C) is not particularly limited as long as it can connect (B) and (D) through a chemical bond. When both (B) and (D) described below are peptides, the first linker (C) may be, for example, a peptide linker. The peptide linker may be a linker consisting of 2 to 50, preferably 2 to 20, 3 to 15 or 4 to 10 amino acid residues. In addition to peptide linkers, linkers containing ε-aminocaproic acid, β-aminoalanine, γ-aminobutyric acid, 7-aminoheptanoic acid, 12-aminolauric acid, glutamic acid, p-aminobenzoic acid, etc. may be used. If either or both of (B) and (D) described below are not peptides, (C) contains the first specific cleavage site and is capable of chemically bonding (B) and (D). Any linker is fine.

A “specific cleavage site” is a site that can be specifically cleaved by some kind of cleavage process, and “specifically cleavable” means a site that does not cleave at a site other than the intended cleavage site; This means that only the intended cutting site can be cut. The first specific cleavage site in (C) is not particularly limited as long as it is a site that can be specifically cleaved; The cleavage site may have an ester bond and/or an amide bond, and may be a cleavage site that can be cleaved by enzymatic or non-enzymatic treatment. is preferred. Furthermore, although those skilled in the art can select suitable specific cleavage sites as necessary, structures that already exist elsewhere in the conjugate cannot be used as cleavage sites.

The cleavage treatment at the first specific cleavage site in (C) is not particularly limited as long as (C) can be specifically cleaved, but may be enzymatic or non-enzymatic cleavage. good. Enzymatic cleavage may be, for example, cleavage with esterase, protease, glycosidase, or the like. Non-enzymatic cleavage may be, for example, cleavage by light, acidic substances, basic substances, or heat.

The protease may be any protease that recognizes and cleaves the first specific cleavage site in (C), and those commonly known to those skilled in the art can be used. In particular, the method of cleaving using proteolytic enzymes is particularly preferably used because the bond can be cleaved with minimal effect on the functions of other (A), (B), and (D). Can be done.

Suitable combinations of proteolytic enzymes and amino acid sequences of specific cleavage sites are widely known. For example, precision protease derived from human rhinovirus 3C protease (HRV3C) has the amino acid sequence LEVLPQ/GP (SEQ ID NO: 13, the cleavage site is indicated by /, the same applies hereinafter), and precision protease derived from bovine blood coagulation factor Xa (factor Xa) The amino acid sequence IDGR/ (SEQ ID NO: 4) or IEGR/ (SEQ ID NO: 14) is derived from bovine blood coagulation factor IIa (thrombin), and the amino acid sequence LVPRK/GS (SEQ ID NO: 15) is derived from tobacco etch virus NIa protease (TEV). Protease) is known to cleave the amino acid sequence ENLYFQ/G (SEQ ID NO: 16). Those skilled in the art can appropriately select the conditions (eg, temperature, time) for carrying out the cleavage reaction. Further, a person skilled in the art can appropriately select a specific cleavage site, but in this case, the sequences present at the sites (A), (B), and (D) may be used as the cleavage site sequence. Can not.

(Hydrophilic site (D))
The hydrophilic site (D) is a site (region) whose purpose is to prevent the conjugates from aggregating in an aqueous medium. It is not particularly limited as long as it is a hydrophilic site that has this function. More specifically, since the conjugate according to the present embodiment has (D), it is possible to suppress aggregation due to hydrophobic interaction of (B), and by cutting (C), the conjugate according to the present embodiment can be suppressed. (D) is separated from the conjugate in the form, and (B) aggregates with each other. That is, (D) is a hydrophilic site that exhibits an effect of suppressing aggregation of (B) in the conjugate according to the present embodiment.

The hydrophilic moiety (D) is not particularly limited as long as it has the hydrophilicity to uniformly dissolve or disperse the conjugate of this embodiment in an aqueous medium, and may be a group of a hydrophilic substance. As an indicator of the hydrophilicity of the hydrophilic site (D), the equivalent to the sum of anionic polar groups, specifically carboxyl groups and phosphoric acid groups in the molecule of the hydrophilic site (D) is 450 g/eq. It may be below, and preferably below 400 g/eq. In addition, the ionic polar group equivalent to the sum of the ionic polar groups in the molecule of the hydrophilic moiety (D), specifically the above-mentioned anionic polar groups and cationic polar groups such as amino groups and imino groups, is 230 g/eq. It is preferable that it is above. Another index of hydrophilicity includes, for example, HLB (hydrophilicity/hydrophobicity balance).

The hydrophilic portion (D) may be a synthetic polymer or a natural polymer as long as the above characteristics are exhibited. Here, since the hydrophilic site (D) is chemically bonded to other sites in the conjugate, it exists not as a polymer compound but as a group of these polymer compounds. Examples of the hydrophilic moiety (D) include hydrocarbon vinyl (co)polymers such as polyvinylbenzenesulfonic acid and its salts; water-soluble substituted vinyl (co)polymers such as polyvinylpyrrolidone and its salts; poly( Water-soluble acrylic ester-based (co)polymers such as meth)acrylic acid and its salts; Water-soluble synthetic resins such as water-soluble polyether-based (co)polymers such as polyethylene glycol; Water-soluble chitosan salts; Fucoidan water-soluble alginic acid and its salts; water-soluble hyaluronic acid and its salts; water-soluble polysaccharides and their salts such as dextran, agarose, and heparin; water-soluble natural polymers such as water-soluble proteins, and the like. Among these, natural polymers, particularly hydrophilic peptides, are preferred in view of stability in a dispersion medium and ease of constructing a conjugate.

When the hydrophilic site (D) in the conjugate according to the present embodiment is a peptide, it may have a hydrophilic helix structure in its three-dimensional structure, and the hydrophilicity can be determined by, for example, the above-mentioned hydrophilicity index, hydropathy plot value, It can be evaluated using HLB value or the like.

In the conjugate according to this embodiment, the ratio of the number of amino acid residues in the hydrophilic site (D) with a hydropathic index of -1.8 or less to all amino acid residues is preferably more than 25%, more preferably 30%. Hydrophilic sites having a hydrophilic content of 35% or more are preferred.

Examples of the hydrophilic peptide include a peptide consisting of the amino acid sequence of SEQ ID NO: 5 (C-terminal domain of ice nucleoprotein InaK derived from Pseudomonas syringae).

When the hydrophilic moiety (D) is a polymer compound, its weight average molecular weight is not particularly limited, and may be, for example, 1,000 or more, preferably 2,000 or more, more preferably 3,000 or more. , and may be 50,000 or less, preferably 30,000 or less, more preferably 1,000 or less.

When (D) is a hydrophilic peptide (in the conjugate, it is a peptide residue), the number of amino acid residues in (D) is not particularly limited, but may be, for example, 10 or more, preferably 20 or more, It is more preferably 40 or more, and may be 400 or less, preferably 200 or less, and more preferably 100 or less. The number of amino acid residues in (D) may be, for example, 50 to 200 or 80 to 150.

(conjugate)
In one embodiment of the conjugate, (B) and (D) are peptide residues and (C) can be a peptide linker. In this case, the conjugate may be a fusion protein. When the conjugate according to this embodiment is a fusion protein, it may contain (A) to (D) in the order from the N-terminus side to the C-terminus side, and may contain in the order of (D) to (A). It's okay to stay.

When the conjugate according to this embodiment is a fusion protein, the number of amino acid residues in the fusion protein is not particularly limited, but may be 20 or more, preferably 40 or more, more preferably 80 or more, and , may be 1000 or less, preferably 800 or less, more preferably 400 or less, particularly preferably 240 or less.

The weight average molecular weight of the conjugate according to this embodiment is not particularly limited, but may be, for example, 3,000 or more, preferably 5,000 or more, more preferably 10,000 or more, and 100, 000 or less, preferably 50,000 or less, more preferably 30,000 or less.

In the conjugate according to this embodiment, (A) and (B) may be directly fused, or may be connected via a linker. When (A) and (B) are connected via a linker, the linker may be a second linker (E) containing a second specific cleavage site. The structure of the second linker (E) is similar to that of the first linker (C) except that it has a specific cleavage site different from that of the first linker (C). When (B) and (D) are both peptide residues, and the first linker (C) and the second linker (E) are both peptide linkers, the conjugate according to this embodiment is a fusion protein. could be.

By inserting the linker (E) between (A) and (B), it becomes possible to disperse the target substance aggregated after the cleavage of (C) in the aqueous solvent again. For example, when the conjugate according to this embodiment is used for specific collection of fine particles, after collecting and washing the aggregated target substance by aeration etc., the fine particles can be placed in an aqueous medium by cutting off the hydrophobic part (B). can be redistributed to

Further, in the conjugate according to the present embodiment, for example, when a plurality of (B)s are bonded to the site of (A), and (C) and (D) are bonded to each site in order, It may also include a case where a plurality of (C) are combined and each (D) is combined in sequence. That is, one molecule of the conjugate may contain a plurality of (B) to (D), but the bonding order must be in the order of (A) to (D). Moreover, a plurality of (B), (C), or (D) may be the same or different.

The conjugate according to this embodiment has hydrophilic properties that allow it to be uniformly dissolved or dispersed in an aqueous medium. The hydrophilicity/hydrophobicity of the conjugate according to this embodiment is influenced by both the hydrophobic site (B) and the hydrophilic site (D). For example, if the hydrophobic site (B) is too large in size or too hydrophobic compared to the hydrophilic site (D), the conjugate as a whole may tend to be hydrophobic; It is necessary to design the conjugate according to this embodiment to be hydrophilic by considering the balance between (B) and the hydrophilic site (D). The hydrophilicity of the conjugate according to this embodiment can be evaluated using the above-mentioned index.

The conjugate according to this embodiment is not particularly limited as long as it has hydrophilicity that allows it to be uniformly dissolved or dispersed in an aqueous medium. Examples of hydrophilicity indicators include HLB (hydrophilic/hydrophobic balance) and hydropathy plot values. In addition, the number of amino acid residues (strong hydrophilic The ratio of residues (residues exhibiting this) is lower in the hydrophobic site (B). Further, the difference in the ratio of the number of amino acid residues exhibiting strong hydrophilicity to the total number of amino acid residues between (B) and (D) is not particularly limited, but is preferably 10% or more, more preferably 20% or more. , more preferably 30% or more.

(Preparation of conjugate)
The conjugate according to this embodiment can be produced by a method known to those skilled in the art. When the conjugate according to this embodiment is not a fusion protein, (A), (B), and (D) are prepared respectively, and then (A), (B), ( It can be produced by connecting these in the order of C) and (D). Furthermore, when the conjugate according to this embodiment is a fusion protein, that is, when (B), (C), and (D) are peptides, (A), (B), (C), and (D) Regarding the production of each peptide, if the peptide is short, it can be produced by artificial synthesis using a known peptide synthesis method. Examples of such synthesis methods include solid phase synthesis methods such as the Fmoc method and Boc method, and liquid phase synthesis methods. If the peptide is long, it can be produced using genetic engineering techniques. Although the production method using genetic engineering techniques is not particularly limited, it can be obtained by inserting a nucleic acid encoding a peptide of interest into an appropriate expression vector and expressing it in a host cell.

When the conjugate according to this embodiment is a fusion protein, the full-length fusion protein can be produced by the genetic engineering method described above. Moreover, it can also be produced by artificial synthesis.

(Use of conjugate)
The conjugate according to this embodiment can be used in a method for controlling the surface properties of a target substance and a method for recovering a target substance, which will be described later. More specifically, according to the conjugate according to this embodiment, the surface properties (hydrophilicity or hydrophobicity) of the target substance can be controlled specifically and at any timing. In view of this feature, it is possible to use not only a method for controlling the surface properties of a target substance or a specific recovery method for a target substance in the form of fine particles from a mixture, but also a method for controlling the surface properties of a target substance on a composite surface where multiple substances exist in addition to the target substance on the surface. It can also be used for marking, patterning, imaging, qualitative and quantitative analysis, and as a means for specific anchoring of other materials to the surface of a target material. Further, the conjugate according to this embodiment can also be used as an adsorbent for a target substance.

<Method for controlling surface properties of target substance>
The method for controlling the surface properties of a target material according to the present embodiment includes a step of adsorbing the conjugate onto the surface of the target material (hereinafter also referred to as "adsorption step"). Since the above-mentioned conjugate has a hydrophobic site (B) and a hydrophilic site (D), the surface properties of the target substance can be controlled to be hydrophobic or hydrophilic at any timing.

The adsorption step is a step of adsorbing the conjugate onto the surface of the target substance. Since the hydrophilic conjugate is adsorbed on the surface of the target substance, the surface of the target substance can be controlled to be hydrophilic. The adsorption step may be set as appropriate depending on the type, properties, etc. of the target substance, but it may also be a step of adsorption by mixing the target substance and the above conjugate in an aqueous medium, and the above conjugate may be adsorbed on the surface of the target substance. The adsorption process may be performed by applying a gate solution or a dispersion liquid.

The target substance is not particularly limited, and may be an organic material or an inorganic material. Alternatively, a material containing both of these may be used. The organic materials and inorganic materials are as described above.

The method for controlling the surface properties of a target substance according to the present embodiment may further include a step of cleaving the above (C) in the conjugate (hereinafter also referred to as a "cutting step"). By the cutting process, the hydrophilic site (D) is separated and the hydrophobic site (B) is exposed on the surface. Thereby, the surface of the target substance can be controlled to be hydrophobic. The cutting process is as described above.

Each of the above steps may be performed in an aqueous medium in which the conjugate is dispersed, or may be performed under conditions where the target substance is exposed to the atmosphere. Under conditions of exposure to the atmosphere, the conjugate can be dispersed in an aqueous medium and applied to the target substance, sprayed, etc.

The method for controlling the surface properties of a target substance according to the present embodiment can specifically control the surface properties of either the target substance alone or the target substance in a mixture.

<Target substance recovery method>
The target substance recovery method according to the present embodiment includes a step of adsorbing the conjugate to the target substance in an aqueous medium (hereinafter also referred to as "adsorption step"), and cleaving the above (C) in the conjugate. However, it includes a step (hereinafter also referred to as "aggregation step") of aggregating the target substance by aggregating the above (B). In the target substance recovery method according to the present embodiment, the target substance is specifically aggregated by adsorbing the conjugate on the surface of the target substance through an adsorption process, and making the surface of the target substance hydrophobic through a cutting process. Furthermore, the timing of aggregation can be arbitrarily controlled.

In the adsorption step, the conjugate is adsorbed onto the surface of the target substance in an aqueous medium. The adsorption step may be set as appropriate, but may be a step of adsorbing the target substance and the conjugate by mixing them in an aqueous medium. Although the target substance is as described above, it is particularly preferable that it is a fine particle.

The aqueous medium is as described above. In an aqueous medium, the target substance may be floating, dispersed or suspended on the surface of the aqueous medium, or both. The size of the target substance is not particularly limited as long as it is large enough to be suspended or dispersed in an aqueous medium, and is preferably, for example, the above-mentioned fine particles. In addition to the target substance, other substances may be present in the aqueous medium. The recovery method of this embodiment allows only the target substance to be specifically adsorbed, aggregated, and recovered even in the presence of other substances.

The amount of the conjugate added in the aqueous medium may be appropriately set depending on the molecular weight of the conjugate, properties such as hydrophilicity/hydrophobicity, and the mass of the target substance. It may be 0.1% by weight or more, preferably 1% by weight or more, more preferably 3% by weight or more, and may be 100% by weight or less, preferably 20% by weight or less, more preferably 10% by weight or less. % by weight or less.

In the target substance recovery method according to the present embodiment, only one type of the above-mentioned conjugate may be used, or a plurality of types may be used simultaneously.

In the aggregation step, (C) in the above conjugate is cleaved and (B) is exposed on the surface of the target substance, thereby making the surface of the target substance hydrophobic, and further, (B) becomes hydrophobic with each other in an aqueous solvent. The target substance is aggregated by aggregation due to sexual interaction. Since the timing of cleaving (C) in the conjugate can be arbitrarily determined, aggregation of the target substance can be controlled at any timing. The cutting process (C) in the conjugate is as described above.

The target substance recovery method according to the present embodiment may further include a step of recovering the target substance (recovery process). The recovery process is a process of recovering only the target substance and removing substances other than the target substance. The recovery step may be a step of recovering aggregates of the target substance and removing other substances and the aqueous medium. The method for recovering a target substance according to the present embodiment includes a recovery step, so that the target substance can be recovered with high purity.

The recovery step may be performed by promoting sedimentation of the aggregates produced in the aggregation step, for example, by applying centrifugal force and/or gravity to the solution containing the aggregates. Specifically, centrifugation is mentioned.

The recovery step may also be performed by filtering the aqueous solvent containing aggregates and the like. Filtration can be performed using a general filter, strainer, etc., although settings may be made as appropriate. Centrifugation and filtration may be combined.

The recovery step may further be performed by aeration. Aeration is a method in which air is bubbled in a medium to cause the hydrophobic target substance to be adsorbed by the air bubbles, floated on the surface of the dispersion, and recovered.

In the method for recovering a target substance according to the present embodiment, when the conjugate includes a second linker (E) containing a second specific cleavage site, the method further cleaves the second linker (E) to target the target substance. It may also include a step of redispersing the substance (hereinafter referred to as "redispersion step"). By including the redispersion step, the hydrophobic site (B) on the surface of the target substance can be separated, and the target substance can be redispersed in the aqueous solvent. If the target substance is soluble, it can be handled in a solution state. When the target substance is an insoluble fine particle, it can be handled in the form of a dispersion in an aqueous solvent.

The redispersion step can be carried out by cutting (E). Note that the cutting process (E) is as described above.

The target substance recovery method according to the present embodiment may use only one type of the above-mentioned conjugate, or may use a plurality of types.

Since the target substance recovery method according to the present embodiment can specifically aggregate and recover the target substance as described above, it can also be regarded as a target substance removal method including the above steps.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples.

<1. Manufacture of conjugate>
The conjugate was designed to be a fusion protein consisting of the amino acid sequence shown in SEQ ID NO:1. Specifically, from the N-terminal side to the C-terminal side, a peptide site (A) that specifically adsorbs to the target substance is a peptide that specifically adsorbs to chalcopyrite (chalcopyrite-binding peptide, ChalBP, SEQ ID NO: 2). ) a linker in which the hydrophobic site (B) has an air binding domain (ABD, SEQ ID NO: 3), the first linker (C) has a Factor Xa cleavage site (SEQ ID NO: 4), which includes a first specific cleavage site; A fusion protein was designed in which the hydrophilic region (D) was composed of the C-terminal domain (InaKC, SEQ ID NO: 5) of the ice nucleoprotein InaK (Genbank accession number: AF013159) derived from Pseudomonas syringae. A peptide linker (X) with the amino acid sequence GGGS (SEQ ID NO: 6) was inserted between (A) and (B), and a peptide linker (X) was inserted at the C-terminus of the fusion protein so that it could be purified with a Ni column. A 6×His tag is added.
(A) + (X) + (B) + (C) + (D) Sequence number 1:
DSQKTNPSGGGSMDPSMKQLADSLHQLARQVSRLEHADPSMKQLADSLHQLARQVSRLEHADPSMKQLADSLHQLARQVSRLEHADPSMKQLADSLHQLARQVSRLEHAIDGRFRLWDGKRYRQLVARTGENGVEADIPYYVNEDDDIVDKPDEDDDWIEVK
(A) Sequence number 2: DSQKTNPS
(B) Sequence number 3:
MDPSMKQLADSLHQLARQVSRLEHADPSMKQLADSLHQLARQVSRLEHADPSMKQLADSLHQLARQVSRLEHADPSMKQLADSLHQLARQVSRLEHA
(C) Sequence number 4: IDGR
(D) Sequence number 5: FRLWDGKRYRQLVARTGENGVEADIPYYVNEDDDIVDKPDEDDDWIEVK
(X) Sequence number 6: GGGS

The chalcopyrite-binding peptide (ChalBP) was obtained by the phage display method, and in this method, the N-terminus of the peptide is free, so the N-terminus of the chalcopyrite-binding peptide is free in this fusion protein. It was placed at the end.

The peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 in the hydrophobic region (B) has the amino acid sequence M(DPSMKQLADSLHQLARQVSRLEHA) ₄ , and the amino acid sequence MKQLADSLHQLARQVSRLEHA forming an α-helical structure has the amino acid sequence DPS. There are four connected through the wire.

Above 1. In the hydrophobic site (B) of the fusion protein produced in , the number of amino acid residues with a hydropathic index of -1.8 or less (that is, amino acid residues exhibiting strong hydrophilicity) is the total number of amino acid residues in the hydrophobic site (B). There are 4 residues out of 97 residues, and the ratio is 4%. That is, it exhibits hydrophobicity because there are few residues that exhibit strong hydrophilicity. On the other hand, above 1. In the hydrophilic site (D) of the fusion protein produced, the number of residues with a hydropathic index of -1.8 or less is 19 out of the total number of 49 residues in the hydrophilic site (D), The ratio was 39%. That is, since a relatively large number of residues exhibiting strong hydrophilicity are contained, the hydrophilic site (B) exhibits strong hydrophilicity.

A pET-22b(+) vector into which a base sequence corresponding to the above fusion protein was inserted was prepared and transformed into Escherichia coli BL21 (DE3) strain. This transformed bacterial cell was cultured at 37°C in LB medium containing ampicillin. Thereafter, IPTG was added to the final concentration of 0.2 mM, and the cells were further cultured to induce expression of the fusion protein. After induction of expression, the bacterial cells were collected by centrifugation, disrupted by ultrasonic irradiation, and the expression of the fusion protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

As described above, the designed fusion protein has a His tag added thereto for purification with a Ni column. Therefore, using the soluble fraction of the disrupted bacterial cell solution obtained by the above procedure, affinity purification using a Ni column was performed according to a conventional method. Table 1 shows the composition of the buffer used in purification, and Table 2 shows the procedure for His tag affinity purification. Furthermore, FIG. 1 shows the results of SDS-PAGE for samples obtained at each step in the purification process of the fusion protein. The fusion protein of interest is indicated by an arrow in FIG.

As can be seen from the concentrated protein lane (lane C) in Figure 1, in the concentrated fusion protein solution after purification, some bands other than the target fusion protein were also observed, but the relative concentration was that of the target fusion protein. This fusion protein solution was used for the evaluation of aggregation properties described below.

<2. Evaluation of aggregation of conjugate (1)>
The above 1. The chalcopyrite aggregate fusion protein obtained in (final concentration 2.5 μM) was added. Next, Factor Xa (1 μg, manufactured by Merck & Co., Ltd.), which is a site-specific proteolytic enzyme, was added and mixed by inversion at 25° C. for 180 seconds to prepare a chalcopyrite fine particle dispersion solution. The prepared chalcopyrite fine particle dispersion solution was observed with an optical microscope to evaluate the agglomeration of the chalcopyrite fine particles by the above fusion protein. For comparison, a similar evaluation was performed using a system that does not contain the fusion protein. The results are shown in FIG.

As shown in Figure 2 (A), visual inspection revealed that the chalcopyrite fine particles remained dispersed in the system without the addition of the fusion protein, whereas in the system with the addition of the fusion protein, the chalcopyrite fine particles aggregated. Ta. Furthermore, as shown in Figure 2 (B), when the chalcopyrite fine particle dispersion solutions of the system without and the system with the addition of the fusion protein were observed using an optical microscope, no aggregation of chalcopyrite was observed in the system without the addition of the fusion protein. However, in the fusion protein addition system, large aggregates of about 50 μm were observed.

Above 2. Although the mechanism of action of the results shown in is not completely elucidated, it is assumed as follows. The peptide (air-binding domain) consisting of the amino acid sequence shown by SEQ ID NO: 3 has a structure in which four α-helix structures are assembled in a bundle, and the inside of the bundle is surrounded by hydrophobic residues. It is considered to be a highly hydrophobic peptide that has the property of oriented at the gas-liquid interface. On the other hand, above 1. The fusion protein (InaKC) produced in the above is highly hydrophilic, and the bundle structure of the air-binding domain is capped by the highly hydrophilic InaKC. In an aqueous medium, the air-binding domain has the above structure with hydrophilic residues exposed on the surface, but the air-binding domain has a first structure containing the first specific cleavage site by a site-specific proteolytic enzyme. When InaKC, a hydrophilic site, is removed from the fusion protein due to cleavage of the linker (C), the conformation of the air-binding domain, a hydrophobic site, changes and becomes oriented at the hydrophobic (or air-liquid) interface. It is thought that the chalcopyrite fine particles aggregated because the hydrophobic residues of the air-binding domain were exposed to the surface layer and a cohesive force was generated due to their hydrophobic interaction.

In addition, when the powder after the agglomeration evaluation experiment of chalcopyrite fine particles was freeze-dried and the surface analyzed using a scanning electron microscope (SEM), as shown in Figure 3, it was found that in the above fusion protein addition system, yellow It was confirmed that copper ore fine particles were agglomerated.

<3. Evaluation of aggregation of conjugate (2)>
2. above except that silica fine particles or a mixture of chalcopyrite fine particles and silica fine particles were used instead of chalcopyrite fine particles. In the same manner as above, each fine particle dispersion solution was prepared and observed under an optical microscope to evaluate the aggregation property of each fine particle due to the above fusion protein. The results are shown in Figures 4 and 5.

As shown in FIG. 4, in the microparticle dispersion solution prepared using silica microparticles, no aggregation was observed in either the system without the addition of the fusion protein or the system with the addition of the fusion protein.

As shown in FIG. 5, in a microparticle dispersion solution prepared using a mixture of chalcopyrite microparticles (black microparticles) and silica microparticles (transparent microparticles), in the system without the addition of the fusion protein, chalcopyrite microparticles (black microparticles) and silica microparticles In contrast, in the above fusion protein addition system, aggregates of chalcopyrite fine particles (black fine particles) were observed.

Evaluation of the aggregation properties of microparticles by these fusion proteins showed that the fusion proteins have a function of specifically aggregating chalcopyrite microparticles.

Claims (6)

Contains in this order a peptide site (A) that specifically adsorbs to the target substance, a hydrophobic site (B), a first linker (C) containing a first specific cleavage site, and a hydrophilic site (D). , conjugate. The conjugate according to claim 1, wherein the first specific cleavage site in (C) is enzymatically cleavable. Further comprising a second linker (E) containing a second specific cleavage site between said (A) and said (B), said first specific cleavage site and said second specific cleavage site. 2. The conjugate of claim 1, wherein the cleavage sites are different. A method for controlling the surface properties of a target substance, comprising the step of adsorbing the conjugate according to any one of claims 1 to 3. adsorbing the conjugate according to any one of claims 1 to 3 onto a target substance present in an aqueous medium, and
A method for recovering a target substance, comprising a step of cleaving the (C) in the conjugate and aggregating the (B) with each other to aggregate the target substance. The method for collecting a target substance according to claim 5, wherein the target substance is a fine particle, and the fine particle has an average particle diameter of 1 nm or more and 10 μm or less.