JP6202486B2 - Iron oxide-protein complex, kit for immobilizing an object to iron oxide via a protein binding to iron oxide, and use thereof - Google Patents

Description

  The present invention relates to an iron oxide-protein complex in which a protein is immobilized on iron oxide, a kit for immobilizing an object on iron oxide via a protein that binds to iron oxide, and use thereof.

  As a method for isolating a target substance from a sample containing a plurality of types of substances such as proteins, nucleic acids, and antibodies, a method for isolating the target substance by immobilizing it on a support is known. As such a support, fine particles such as silicon oxide (silica), metal (for example, gold, silver, iron, etc.) are used (Patent Document 1 and Patent Document 2).

  As a method for recovering the support on which the target substance is immobilized, a centrifugal method is generally used, but fine particles having magnetism (hereinafter referred to as “magnetic support”) are used as the support. In some cases, such magnetic support can be recovered by capturing it in a magnetic field. In the centrifugal separation method, impurities are precipitated at the same time as the support, so that it is not easy to recover only the target substance. On the other hand, in the method of collecting the magnetic support by capturing it in the magnetic field, only the support can be recovered, so that only the target substance can be recovered easily and with high purity.

  Further, the magnetic support can be aggregated, dispersed, or moved in the liquid only by manipulating the magnetic field. Thus, magnetic supports are attracting attention because they are suitable for automated processes and may be used in a variety of applications that are not feasible with non-magnetic supports.

  The magnetic support is generally formed from iron, nickel, cobalt, etc. Among them, iron has strong magnetism, and its oxide iron oxide (eg, magnetite) is present stably in the liquid. Therefore, it is widely used as a magnetic support.

  Here, conventionally, when iron oxide fine particles are used as a support, in order to immobilize a target substance on the iron oxide fine particles, the surface of the iron oxide fine particles is coated with a specific polymer, or the iron oxide fine particles It is necessary to introduce a specific functional group on the surface.

  For example, in Patent Document 1, in order to immobilize nanoparticles such as iron oxide on a nucleic acid matrix, the first domain binds to the surface of the nanoparticle and the second domain binds to the nucleic acid. It is disclosed that a bifunctional protein is designed and the nanoparticles are coated with a monolayer that is functionalized to attach such a bifunctional protein.

JP 2006-505253 Gazette (announced February 16, 2006) International Publication No. 2007/055288 pamphlet (published on May 18, 2007)

  As described above, in the prior art, when iron oxide fine particles are used as a support, in order to immobilize the target substance on the iron oxide fine particles, at least a complicated operation of surface treatment of the iron oxide fine particles is required. For this reason, it has the subject that the cost and labor for preparing this iron oxide fine particle are required.

  Therefore, if a protein that can directly bind to iron oxide with a strong binding force can be used, the target substance can be directly immobilized on the iron oxide fine particles without any surface treatment of the iron oxide fine particles. It becomes possible. However, no protein capable of directly binding to iron oxide has been reported in the past.

  The present invention has been made in view of the above problems, and an object thereof is an iron oxide-protein complex capable of immobilizing a desired substance without any surface treatment of iron oxide. As well as the utilization of the iron oxide-protein complex.

The inventors have found a protein (L2; Si-tag, SBP) that binds strongly to silica (silicon dioxide, SiO ₂ ). Si-tag can be directly bonded to a silicon substrate or glass whose surface is not modified. For this reason, if a fusion protein of Si-tag and the target protein is prepared, the target protein is immobilized on the silicon substrate or the glass surface by allowing the Si-tag to function as an adhesion molecule to the silicon substrate or the glass surface. (Patent Document 2).

  As a result of intensive studies to achieve the above object, the present inventors have found that the Si-tag protein found by the present inventors as a protein that strongly binds to silica has a strong binding ability to iron oxide. The present invention was found for the first time, and the present invention was completed based on such new findings. As described above, no protein capable of directly and strongly binding to iron oxide has been reported in the past.

That is, the iron oxide-protein complex according to the present invention is an iron oxide-protein complex in which a protein is immobilized on iron oxide that is not surface-modified,
The protein is characterized by being a protein capable of binding to the iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate.

  The protein may be a protein containing at least an iron oxide binding domain.

In the iron oxide-protein complex according to the present invention, the protein may be the following protein (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;
(B) The amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted and / or added, and at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate A protein that can bind to iron oxide under heating conditions.

  In the iron oxide-protein complex according to the present invention, the protein may be a protein having a site that can be adsorbed to an object.

  In the iron oxide-protein complex according to the present invention, the protein may be a protein containing at least part of a protein that binds to an immunoglobulin.

  In the iron oxide-protein complex according to the present invention, the iron oxide may be magnetic.

  The kit for immobilizing an object according to the present invention on iron oxide contains at least a protein capable of binding to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. It is characterized by.

  A method for immobilizing an object according to the present invention on iron oxide includes a step of bringing the iron oxide-protein complex according to the present invention into contact with the object.

The method for recovering an object according to the present invention includes a first step of bringing the iron oxide-protein complex according to the present invention into contact with the object,
A second step of recovering the iron oxide-protein complex that has undergone the first step;
It is characterized by including.

In the method for recovering an object according to the present invention, the iron oxide has magnetism,
In the second step, it is preferable to collect the iron oxide-protein complex by capturing the iron oxide-protein complex that has undergone the first step in a magnetic field.

A method for killing a target cell according to the present invention includes a first step of bringing the iron oxide-protein complex according to the present invention into contact with the target cell,
A second step of heating the iron oxide-protein complex by applying an alternating magnetic field to the iron oxide-protein complex after the first step;
Including
The iron oxide is characterized by having magnetism.

In the method for immobilizing an object according to the present invention on iron oxide, a protein capable of binding to iron oxide under conditions of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate is oxidized without surface modification. A process of fixing to iron;
Immobilizing an object on the protein;
It is characterized by including.

  The present invention is a surface modification treatment of iron oxide, which is required in the conventional method, by immobilizing an object on iron oxide via a protein that can be directly bound to iron oxide, which the inventors have found for the first time. There is an effect that the object can be easily fixed to the iron oxide without performing any of the above.

  Furthermore, such a protein can maintain its ability to bind to iron oxide even under harsh conditions where the protein is denatured, usually under conditions of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. That is, such a protein has a strong binding ability to iron oxide. For this reason, the iron oxide-protein complex according to the present invention can be stably used even after various operations. Moreover, according to this invention, there exists an effect that a target object can be fixed to iron oxide easily and strongly. Therefore, according to the present invention, it is possible to easily collect an object and kill a target cell.

It is a figure which shows roughly an example of a structure of the iron oxide-protein complex concerning this invention. It is a figure showing the result of having compared stability of Si-tag fix | immobilized to the silica particle or the iron oxide particle by SDS-PAGE. It is a figure showing the detection result of H-FABP using the iron oxide particle which fix | immobilized the antibody.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” indicating a numerical range means “A or more and B or less”.

[1. (Iron oxide-protein complex)
The iron oxide-protein complex according to the present invention (hereinafter also referred to as “the iron oxide-protein complex of the present invention” or “the complex of the present invention”) is immobilized on iron oxide that is not surface-modified. The protein is a protein that can bind to the iron oxide under the conditions of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate (hereinafter referred to as “iron oxide-binding protein”). It is a certain configuration.

  The above “protein is immobilized on iron oxide that is not surface-modified” means that the iron oxide-binding protein is bound or adsorbed to iron oxide that is not surface-modified.

  Moreover, the above-mentioned “iron oxide not surface-modified” means surface modification (for example, coating with a specific polymer on the surface of iron oxide, oxidation, etc.) conventionally performed to fix the target substance to iron oxide. This refers to iron oxide that has not been subjected to the introduction of specific functional groups onto the iron surface. In the present specification, “iron oxide not surface-modified” may be simply referred to as “iron oxide”.

  In the present specification, the term “protein” is used interchangeably with “polypeptide” or “peptide”. “Proteins” include partial fragments of proteins. The “protein” includes a fusion protein.

Examples of the “iron oxide” include iron oxide (II) (FeO) (also referred to as ferrous oxide), diiron (III) oxide (II) (Fe ₃ O ₄ ) (also referred to as triiron tetroxide). ), Α-iron (III) oxide (α-Fe ₂ O ₃ ) (also referred to as α-ferric oxide or ferric trioxide), β-iron (III) oxide (β-Fe ₂ O ₃ ) (β -Also referred to as ferric oxide or ferric trioxide), γ-iron (III) oxide (γ-Fe ₂ O ₃ ) (also referred to as γ-ferric oxide or ferric trioxide), ε-iron oxide ( III) (ε-Fe ₂ O ₃ ) (also referred to as ε-ferric oxide or ferric trioxide). In the composite of the present invention, the “iron oxide” may contain one kind of iron oxide alone, or may contain plural kinds of iron oxides in combination. The “iron oxide” may contain other elements. Elements other than iron oxide are not particularly limited.

In the composite of the present invention, the “iron oxide” preferably has magnetism. Such iron oxide is not particularly limited as long as it has magnetism. Specifically, for example, diiron (III) oxide (II) (Fe ₃ O ₄ , magnetite), γ-iron oxide (III) ) (Γ-Fe ₂ O ₃ , maghemite) and the like. In the composite of the present invention, the “iron oxide” may contain one kind of these iron oxides having magnetism, or may contain a plurality of kinds in combination.

  In the composite of the present invention, when the “iron oxide” contains iron oxide having magnetism, the composite of the present invention is changed to a magnetic field (for example, a magnetic field formed by a magnet). It can be captured and recovered. As a result, it is possible to easily recover only the complex of the present invention without mixing impurities. Further, in the complex of the present invention, when the “iron oxide” has a magnetic property, the aggregation, dispersion, or movement of the complex of the present invention in the liquid can be performed only by operating the magnetic field. It becomes possible and handling of the composite of the present invention becomes easy.

  The “iron oxide” is not particularly limited as long as the iron oxide-binding protein can be immobilized on the surface, and can be appropriately designed according to the purpose. For example, particles, plates, and rods are intended. Specifically, for example, iron oxide-containing particles having an average particle diameter of 10 nm to several tens of μm are contemplated.

  In the complex of the present invention, the “iron oxide binding protein” may be a fusion protein with another protein. The “fusion protein” refers to a protein in which a part (fragment) or all of two or more heterologous or homologous proteins are combined. The fusion protein can be prepared as a recombinant protein by using, for example, a known genetic engineering technique. In the present specification, a fusion protein of an iron oxide binding protein and another protein may be referred to as “iron oxide binding protein”.

  In the complex of the present invention, the iron oxide binding protein may be a protein having a site capable of binding to an object (hereinafter referred to as “object binding site”).

  In the complex of the present invention, the iron oxide binding protein may be a protein containing at least part of a protein that binds to an immunoglobulin. In other words, the iron oxide binding protein may be a protein containing at least part of a protein that binds to an immunoglobulin as a target binding site. If the iron oxide binding protein is a protein having a target binding site, the target can be easily immobilized on the complex of the present invention.

  Here, an example of the structure of the composite of the present invention will be described with reference to FIG. FIG. 1 is a diagram schematically showing the structure of the composite of the present invention. As shown in FIG. 1, the complex of the present invention is obtained by immobilizing iron oxide binding protein 1 having an object binding site 2 on iron oxide 4. As shown in FIG. 1, in one embodiment, the composite of the present invention can be used as a support for immobilizing an object. As described later, an iron oxide-binding protein 1 having an object binding site 2 is prepared by preparing a fusion protein of a protein having a site that can be adsorbed to the object 3 and the iron oxide-binding protein 1. Can be produced. As a result, the object 3 can be easily immobilized on iron oxide using the complex of the present invention. In FIG. 1, all black thick solid lines represent iron oxide-binding protein 1, all white circles lacking a part represent object binding site 2, and all black triangles represent object 3. Yes.

  As an example, in the iron oxide-protein complex prepared in the examples described later, (i) Si-tag (SEQ ID NO: 1) which is a protein capable of binding to iron oxide (Genbank, Accession No. AAC76342.1) And (ii) a known fusion protein with immunoglobulin binding domain B1 in protein G, the fusion protein is immobilized on iron oxide via Si-tag, and then a predetermined immunoglobulin is converted into protein Immobilization to iron oxide via immunoglobulin binding domain B1 in G. Furthermore, heart-derived fatty acid-binding protein (H-FABP) or Escherichia coli was immobilized on iron oxide via such immunoglobulin. In this case, Si-tag corresponds to iron oxide-binding protein 1, a complex of immunoglobulin binding domain B1 in protein G and a predetermined immunoglobulin corresponds to target binding site 2, and is derived from heart-derived fatty acid binding protein or It can be said that E. coli corresponds to the object 3.

  Alternatively, Si-tag corresponds to iron oxide binding protein 1, immunoglobulin binding domain B1 in known protein G corresponds to target binding site 2, and a complex or immunity between immunoglobulin and heart-derived fatty acid binding protein It can be said that a complex of globulin and Escherichia coli corresponds to the object 3.

  Here, in the present specification, the “object” specifically refers to, for example, a protein (for example, immunoglobulin), a nucleic acid (DNA, RNA), a sugar chain, an organism (for example, a microorganism, a cell). Etc.), ions, other supports and the like are contemplated, but the invention is not limited thereto. The “object” may be composed of a single substance, or may be a reversible or irreversible combination of two or more of the same or different substances.

  In the present specification, the above-mentioned “immobilize an object” means that the object is reversibly or irreversibly bound or adsorbed to the complex of the present invention, and the object is supported on the complex of the present invention. Is intended to.

  Next, the “iron oxide binding protein” and the “object binding site” will be described in detail below.

(1-1. Iron oxide binding protein)
The iron oxide binding protein constituting the complex of the present invention is a protein that can bind to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% (w / v) sodium dodecyl sulfate.

  Whether it is a protein that can be used as an iron oxide binding protein in the present invention can be confirmed, for example, as follows. However, the confirmation method is not limited to this.

  That is, as described in Example 1 to be described later, for example, iron oxide is added to a solution containing the protein to be confirmed, and a sufficient mixture of the protein and iron oxide is obtained (for example, at 4 ° C. for 15 to 30 minutes). ) After mixing by inversion, iron oxide is collected and washed with a washing solution (25 mM Tris-HCl [pH 9.0], 0.5 M NaCl, 0.5% [v / v] Tween 20).

  Thereafter, the iron oxide is heated at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate, and it is confirmed using SDS-PAGE or the like whether or not the protein is bound to the iron oxide even after the heating.

  Thereby, it can be easily confirmed whether or not the protein has the ability to bind to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. The iron oxide to be added in the above confirmation method is not particularly limited, but if iron oxide having magnetism is captured by a magnetic field and can be recovered, it is easy to recover the iron oxide after mixing with the protein solution.

  Moreover, in the said confirmation method, as a solution used when mixing protein and iron oxide, for example, the Tris buffer solution of pH 8-9 containing 0.5M NaCl and 0.5% [v / v] Tween 20 is used. A general phosphate buffer solution (PBS) or the like can be preferably used. However, the solution is not limited to this. Any substance that does not contain a substance that inhibits the binding between protein and iron oxide can be used.

Examples of the iron oxide binding protein that can be suitably used in the present invention include the following proteins (a) or (b), but the present invention is not limited to these:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;
(B) The amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted and / or added, and at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate A protein that can bind to iron oxide under heating conditions.

  Here, the above "one or several amino acids are substituted, deleted and / or added" means substitution, deletion and / or by known mutant peptide production methods such as site-directed mutagenesis. It means that as many amino acids as can be added (for example, 5% or less, 3% or less, or 1% or less of the total number of amino acids) are substituted, deleted, and / or added. The number of substitutions, deletions and / or additions of one or several amino acids is 5% or less, 3% or less or 1% or less of the total number of amino acids, and substitutions, deletions and / or additions. Any number of proteins may be used as long as they have the ability to bind to iron oxide under the conditions of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate.

  The site at which one or several amino acids are substituted, deleted and / or added is the protein after substitution, deletion and / or addition of amino acids at 95 ° C. in the presence of 2% sodium dodecyl sulfate, Any site in the amino acid sequence may be used as long as it has an ability to bind to iron oxide under the condition of heating for 5 minutes.

  Such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, but is a product obtained by isolating and purifying a similar naturally occurring mutant polypeptide. It may be.

  It is well known in the art that some amino acids in the amino acid sequence of a protein can be easily modified without significantly affecting the structure or function of the protein. Furthermore, it is also well known that there are variants that not only artificially modify, but also do not significantly alter the structure or function of the protein in the native protein.

  Preferred variants have conservative or non-conservative amino acid substitutions, deletions, or additions. Particularly preferred is conservative substitution. These do not cause the iron oxide binding protein to lose its ability to bind to iron oxide.

  Typically seen as conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile, exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

The iron oxide binding protein that can be suitably used in the present invention may be, for example, a polypeptide encoded by the following polynucleotide (c) or (d). That means
(C) a polynucleotide comprising the base sequence of SEQ ID NO: 2;
(D) Hybridizing under stringent conditions with a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 and heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate A polynucleotide encoding a polypeptide capable of binding to iron oxide.

  The polynucleotide consisting of the base sequence of SEQ ID NO: 2 is a polynucleotide encoding the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

  As used herein, the term “stringent conditions” means that there is at least 90% identity, preferably at least 95% identity, most preferably at least 97% identity between sequences. It means that only when hybridization occurs. Specifically, a hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhart solution, 10% dextran sulfate, And 20 μg / ml of denatured sheared salmon sperm DNA) after overnight incubation at 42 ° C., the filter is intended to be washed in 0.1 × SSC at about 65 ° C. Depending on the polynucleotide to be used, the washing conditions at high stringency are appropriately changed. For example, when DNA derived from a mammal is used, washing at 65 ° C. in 0.5 × SSC containing 0.1% SDS (preferably 15 times × 2 times) is preferable, and when E. coli-derived DNA is used, 0.1% SDS containing 0.1% Washing at 68 ° C. in XSSC (preferably 15 minutes × 2 times) is preferable. When RNA is used, washing at 68 ° C. in 0.1 × SSC containing 0.1% SDS (preferably Is preferably 15 minutes × 2 times). When oligonucleotides are used, washing at a hybridization temperature in 0.1 × SSC containing 0.1% SDS (preferably 15 minutes × 2 times) is preferred. The hybridization can be performed by a well-known method described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a polynucleotide with higher homology can be obtained. The identity of the base sequence can be determined by the Sanger method.

  As the iron oxide binding protein constituting the complex of the present invention, any protein capable of binding to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate may be used. Therefore, even a protein other than those exemplified above can be obtained by those skilled in the art by confirming whether or not the protein can bind to iron oxide under the above conditions. It can be easily determined whether or not it is used.

  It is well known to those skilled in the art that the above-mentioned “conditions for heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate” are conditions under which proteins denature and lose their functions. However, since the complex of the present invention uses, as the iron oxide-binding protein, a protein that can bind to iron oxide even under the above severe conditions and has a strong binding ability to iron oxide, Stable (eg, iron oxide-binding protein is oxidized under severe conditions (eg, 45-100 ° C.) or in the presence of a surfactant (eg, 0.1-2% sodium dodecyl sulfate). Without being released from iron). For this reason, for example, reaction at a high temperature by a complex in which a thermostable protein derived from thermophile is immobilized, reaction in a foaming agent or detergent containing a surfactant, and membrane fraction solubilized by the surfactant It can also be used in situations such as collecting objects, and it has a high utility value.

  In addition, since the iron oxide-binding protein can be directly immobilized on iron oxide, it is not necessary to modify the surface of iron oxide in order to immobilize the protein, unlike conventional iron oxide particles. That is, the composite of the present invention can be manufactured easily compared to conventional iron oxide particles because the cost and labor required for the surface treatment of iron oxide when manufacturing such a composite can be reduced.

  Further, the iron oxide binding protein can be a protein containing at least an iron oxide binding domain. That is, for example, if the iron oxide binding protein includes at least the iron oxide binding domain of Si-tag that is a protein capable of binding to iron oxide, the other site constituting the iron oxide binding protein is iron oxide binding. Any protein may be used as long as it does not inhibit the binding of protein to iron oxide. The above-mentioned “iron oxide binding domain” is determined by using a known method such as a method for analyzing the three-dimensional structure of a protein, a method for producing a partial deletion mutant of a protein, or a method for producing a fragment of a fragmented protein. can do.

  The origin of the iron oxide binding protein is not particularly limited, and may be a protein derived from any organism such as bacteria, yeast, plant, animal and the like. Alternatively, an iron oxide binding protein may be selected from an artificially designed peptide library that is not derived from a living organism.

  In addition, the iron oxide binding protein may be produced using, for example, a gene recombination technique, or may be chemically synthesized using an amino acid synthesizer or the like.

  Further, the iron oxide binding protein may contain an additional peptide. Examples of the additional peptide include poly-histidine tag (His-tag) and epitope-labeled peptides such as Myc and Flag.

(1-2. Object binding site)
The iron oxide binding protein having a target binding site is a fusion protein of a protein having a site capable of binding to a target (hereinafter referred to as “target binding protein”) and an iron oxide binding protein. Can be obtained. That is, the part of the target binding protein in the fusion protein corresponds to the “target binding part” of the iron oxide binding protein having the target binding part.

  Here, the “object binding protein” will be described below. The “target binding protein” is not particularly limited, and any protein can be appropriately selected according to the purpose. The object binding protein may be (i) composed of a single protein, (ii) a fusion protein, or (iii) a protein complex. In the present specification, the “protein complex” means a part (fragment) or a part of two or more heterologous proteins reversibly bound to each other, or a part (fragment) of at least one protein. ) Or all and a part (fragment) or all of a substance (eg, sugar chain, nucleic acid, etc.) other than at least one protein are reversibly bound reversibly. Such “protein complexes” are distinguished from “fusion proteins” in which some or all of two or more heterologous proteins are irreversibly bound.

  As described above, the target object to which the complex of the present invention is immobilized includes, for example, proteins (eg, immunoglobulins, antigens, etc.), nucleic acids (DNA, RNA), sugar chains, organisms (eg, microorganisms, cells). Etc.), ions, other supports and the like are contemplated. For this reason, the target binding protein is a protein having a site that can be adsorbed to the target so that the target can be immobilized on the complex of the present invention.

  Here, “having a site that can be adsorbed to an object” means that when the object binding protein is composed of a single protein, the protein itself can be adsorbed to the object. It is intended to have. That is, “a protein having a site that can be adsorbed to an object” is intended to be a protein having an adsorption ability for the object.

  For example, when the “object” is a specific immunoglobulin, for example, a protein such as protein G or protein A, or a protein specifically recognized by such an immunoglobulin. (So-called “antigen”) corresponds to this. Further, when the “object” is a sugar chain, a lectin (for example, concanavalin A (ConA)) corresponds to this. Further, when the “object” is a specific antigen, an immunoglobulin that specifically recognizes the antigen corresponds to this.

  For example, among immunoglobulins, single-chain antibodies can be easily produced as fusion proteins with other proteins using gene recombination techniques. The iron oxide binding protein has a strong binding ability to iron oxide. Therefore, by preparing a fusion protein of an iron oxide binding protein and a single chain antibody as an object binding protein and immobilizing the fusion protein on iron oxide, for example, an object immobilized on the complex of the present invention When eluting an object, there is no possibility that the antibody is eluted together with the object, so that only the object can be easily eluted and recovered. In the complex of the present invention, the protein used as the object binding protein is not limited to those exemplified above.

  In addition, when the target binding protein is a fusion protein, the “protein having a site that can be adsorbed to the target” means at least two of the two or more heterologous proteins constituting the fusion protein. One is intended to be a protein that has the ability to adsorb to an object.

  In addition, when the target binding protein is a protein complex, the “protein having a site that can be adsorbed to the target” refers to two or more heterologous proteins constituting the protein complex. It is intended that at least one of the proteins is a protein having the ability to adsorb to an object, or at least one protein constituting the protein complex or at least one substance other than the protein is the object It is intended to be a protein that has an adsorption capacity for.

  When a specific example of such a protein complex is given, for example, when the “object” is a specific antigen, for example, an immunoglobulin that specifically recognizes the antigen and a protein that binds to the immunoglobulin (for example, , Protein G, protein A, etc.). Further, for example, when the “object” is a specific immunoglobulin, for example, a complex of an antigen specifically recognized by the immunoglobulin and another immunoglobulin that specifically recognizes the antigen It corresponds to this. In this case, the above-mentioned “specific immunoglobulin” epitope is preferably different from the above-mentioned “other immunoglobulin” epitope.

  However, in the complex of the present invention, the protein complex used as the object binding protein is not limited to those exemplified above.

  In the complex of the present invention, the object binding protein is linked to the iron oxide binding protein, but is not linked to the iron oxide binding protein so as not to hinder the adsorption ability of the object binding protein to the target. Preferably it is. When a protein complex is used as the target binding protein, a protein complex is constructed after preparing a fusion protein in which one of the proteins constituting the protein complex and an iron oxide binding protein are linked. A protein complex can be formed by bringing the fusion protein into contact with other constituent components.

  Specifically, for example, when a complex of immunoglobulin and protein G is used as the target binding protein, after preparing a fusion protein of protein G and iron oxide binding protein, To form a protein complex of immunoglobulin and protein G.

  In the complex of the present invention, the iron oxide binding protein may be a protein containing at least a part of a protein that binds to an immunoglobulin. In other words, a fusion protein with an iron oxide binding protein may be prepared by selecting a protein containing at least part of a protein that binds to an immunoglobulin as the object binding protein. In the present specification, “immunoglobulin” refers to IgA, IgD, IgE, IgG, IgM, IgY, IgW, IgX and their subclasses (for example, IgG1 to IgG4, etc.) and their Fab fragments, F (ab ′) 2 fragment and Fc fragment, which can also be referred to as “antibody”. Examples include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, anti-idiotype antibodies, and humanized antibodies.

  Here, the “protein that binds to immunoglobulin” is not particularly limited as long as it has a binding ability to immunoglobulin. Specifically, for example, protein G, protein A, protein L, and other immunoglobulins that specifically recognize immunoglobulins are contemplated.

  In the complex of the present invention, any object can be immobilized on iron oxide by appropriately designing the object binding site according to the purpose.

  The origin of the target binding protein serving as the target binding site is not particularly limited, and may be a protein derived from any organism such as bacteria, yeast, plant, animal and the like. In addition, an object-binding protein may be selected from an artificially designed peptide library that is not derived from a living organism.

  In addition, the target binding protein may be produced using, for example, a gene recombination technique, or may be chemically synthesized using an amino acid synthesizer or the like.

  In addition, the object binding protein may contain an additional peptide. Examples of the additional peptide include poly-histidine tag (His-tag) and epitope-labeled peptides such as Myc and Flag.

[2. kit〕
The kit according to the present invention (also referred to as “kit of the present invention”) is a kit for immobilizing an object on iron oxide, and is heated at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. It is the structure containing at least the protein (iron oxide binding protein) that can bind to iron oxide under the conditions. The “iron oxide-binding protein”, the “target”, and the “iron oxide” are the same as described in the section “1. Iron oxide-protein complex”, and thus the description thereof is omitted.

  In the kit of the present invention, the “iron oxide binding protein” may be provided as a polypeptide form, or as a form of a polynucleotide encoding the polypeptide (for example, a transformation vector containing the polynucleotide). You may have.

  As used herein, the term “polynucleotide” can also be referred to as “nucleic acid” or “nucleic acid molecule”, and is intended to be a polymer of nucleotides. The “base sequence” can also be referred to as “nucleic acid sequence” or “nucleotide sequence”, and is shown as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T).

  The kit of the present invention may be provided with the above-mentioned “iron oxide binding protein” in a state of being immobilized on iron oxide, that is, as a complex of the present invention described above, or immobilized on iron oxide. You may prepare in the state which is not. When the above-mentioned “iron oxide-binding protein” is provided in a state where it is not immobilized on iron oxide, for example, iron oxide is added to a solution containing iron oxide-binding protein, and iron oxide-binding protein and iron oxide are mixed. An iron oxide-protein complex in which an iron oxide-binding protein is immobilized on iron oxide can be easily obtained by inverting and mixing the mixed solution sufficiently (for example, at 4 ° C. for 15 to 30 minutes).

  Moreover, it is preferable that the kit of this invention is a protein in which the iron oxide binding protein has the site | part (object binding site) which can be couple | bonded with a target object. The “object binding site” and the method for producing an iron oxide binding protein having an object binding site are as described in the above section “1. Iron oxide-protein complex”. Omitted.

  The kit of the present invention may further comprise components other than the above “iron oxide binding protein”. For example, when a protein complex is used as the “target binding site”, all of the components constituting the protein complex may be filled in the same container, or each component may be separated. The container may be filled.

  Moreover, you may provide the suitable buffer solution in order to fix a target object to iron oxide. The appropriate buffer solution is as described in the above section “1. Iron oxide-protein complex”, and thus the description thereof is omitted.

  It may also contain one or more containers (eg, vials, tubes, ampoules, bottles, etc.) for storing the components that make up the kit of the present invention, and instructions for using the kit of the present invention. May be provided.

  By using the kit of the present invention, an object can be directly bound to iron oxide that is not surface-modified through an iron oxide-binding protein, so that the object can be more easily compared with the conventional method. Can be immobilized.

[3. Method 1 of immobilizing an object on iron oxide]
The immobilization method according to the present invention (also referred to as “immobilization method of the present invention”) is a method of immobilizing an object on iron oxide, and contacting the complex of the present invention described above with the object. It includes a process (hereinafter referred to as “contact process”). The “complex of the present invention” and the “object” are the same as described in the above section “1. Iron oxide-protein complex”, and thus the description thereof is omitted.

(3-1. Contact process)
In the contact step, the object is fixed to the iron oxide via the iron oxide-binding protein by bringing the object into contact with the complex of the present invention, that is, the iron oxide on which the iron oxide-binding protein is immobilized on the surface. It is a process to convert. In addition, it is preferable that an iron oxide binding protein is a protein which has a site | part (object binding site) which can be couple | bonded with a target object. The “object binding site” and the method for producing an iron oxide binding protein having an object binding site are as described in the above section “1. Iron oxide-protein complex”. Omitted.

  In the contacting step, the method for immobilizing the object on the surface of the complex of the present invention is not particularly limited. For example, the method of immersing the complex of the present invention in a solution containing the object, A method of applying or spotting a solution containing an object on the surface of the composite can be used. In the contacting step, the condition of the complex of the present invention and the object is not particularly limited, but it is preferable that the complex is sufficiently brought into contact, for example, by incubating at 4 ° C. to room temperature for about several seconds to 30 minutes.

  According to the immobilization method of the present invention, an object can be directly bound to iron oxide that is not surface-modified through an iron oxide-binding protein, so that the object can be more easily compared with a conventional method. It can be immobilized on iron oxide.

(3-2. Others)
In the immobilization method of the present invention, steps other than the above may be provided, and the contents of the steps other than the above are not limited. For example, by contacting iron oxide with a protein having an ability to bind to iron oxide (iron oxide binding protein) under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate, A step of immobilizing iron oxide binding protein on the surface of the substrate (hereinafter referred to as “iron oxide binding protein immobilization step”), in other words, a step of producing the complex of the present invention may be provided before the contact step. Good.

  The “iron oxide-binding protein immobilization step” is a step of immobilizing the iron oxide-binding protein on the surface of iron oxide that has not been surface-modified by bringing the iron oxide-binding protein into contact with iron oxide. In the iron oxide-binding protein immobilization step, as a method of immobilizing iron oxide-binding protein on the surface of iron oxide, for example, a method of immersing iron oxide in a solution containing iron oxide-binding protein, oxidation on the surface of iron oxide A method of applying or spotting a solution containing iron-binding protein can be used.

  The iron oxide-binding protein is a protein that can bind to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. Conditions for immobilizing iron oxide and iron oxide may be performed under any conditions. For example, by adding iron oxide to a solution containing iron oxide-binding protein and incubating at 4 ° C. to room temperature for several seconds to 30 minutes, the iron oxide-binding protein and iron oxide can be sufficiently brought into contact with each other. .

  Moreover, it is preferable that an iron oxide binding protein is a protein which has a site | part (object binding site | part) which can be couple | bonded with a target object. The “object binding site” and the method for producing an iron oxide binding protein having an object binding site are as described in the above section “1. Iron oxide-protein complex”. Omitted.

[4. (Recovery method of object)
The recovery method according to the present invention (also referred to as “recovery method of the present invention”) is a method of recovering an object, and the iron oxide-protein complex (complex of the present invention) according to the present invention described above, That is, a first step in which an iron oxide having an iron oxide-binding protein immobilized on the surface and an object are brought into contact with each other, and a second step in which the iron oxide-protein complex that has undergone the first step is recovered. It is characterized by inclusion. The “complex of the present invention” and the “object” are the same as described in the section “1. Iron oxide-protein complex”, and thus the description thereof is omitted.

(4-1. First step)
In the first step, the target is brought into contact with iron oxide via the iron oxide-binding protein by contacting the target with the complex of the present invention, that is, iron oxide with the iron oxide-binding protein immobilized on the surface. It is a process of immobilization. The method for immobilizing the object on the surface of the complex of the present invention is as described in the section “3-1. Contacting step” in “3. Method for immobilizing the object on iron oxide”. Therefore, explanation is omitted. The iron oxide binding protein is preferably a protein having a site capable of binding to an object (object binding site). The “object binding site” and the method for producing an iron oxide binding protein having an object binding site are as described in the above section “1. Iron oxide-protein complex”. Omitted.

(4-2. Second step)
A 2nd process is a process of collect | recovering the said iron oxide-protein complex which passed through the said 1st process, ie, the iron oxide-protein complex by which the target object was fix | immobilized on the surface.

  The method for recovering the iron oxide-protein complex is not particularly limited, and the iron oxide-protein complex can be recovered using a conventionally known method such as centrifugation. When the iron oxide constituting the iron oxide-protein complex is magnetic, the iron oxide-protein complex is recovered by capturing the iron oxide-protein complex in a magnetic field. It is preferable to do. In the method of recovering the iron oxide-protein complex by capturing it in a magnetic field, it is possible to recover only the iron oxide-protein complex without mixing in impurities, so that only the object can be easily recovered. .

  A magnet can be used as means for generating the “magnetic field”. That is, the phrase “capturing the iron oxide-protein complex in the magnetic field” is rephrased as “capturing the iron oxide-protein complex in the magnet”. As the “magnet”, for example, an electromagnet, a permanent magnet, or the like can be used. Since the electromagnet can control the generation of the magnetic field by turning on and off the power source, it is easy to cause the iron oxide-protein complex to be captured or detached from the electromagnet. For this reason, the electromagnet is more convenient than the permanent magnet.

(4-3. Others)
In the recovery method of the present invention, steps other than the above may be provided, and the contents of the steps other than the above are not limited. For example, it is not surface-modified by contacting iron oxide with an iron oxide-binding protein capable of binding to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. The step of immobilizing iron oxide binding protein on the surface of iron oxide (hereinafter referred to as “iron oxide binding protein immobilization step”), in other words, the step of producing the iron oxide-protein complex according to the present invention is described above. It may be provided before one step. The “iron oxide-binding protein immobilization step” is the same as that described in “3-2. Others” of “3. Omitted.

  Furthermore, in the recovery method of the present invention, a step of eluting the object from the iron oxide-protein complex may be provided after the second step. The method for eluting the target from the iron oxide-protein complex is not particularly limited. For example, when protein A is used as the target binding protein and immunoglobulin is immobilized as the target, treatment with an acidic solution ( For example, the target can be eluted from the iron oxide-protein complex by elution with a pH 2-3 glycine buffer or a pH 3 citrate buffer.

  According to the recovery method of the present invention, an object can be directly bound to iron oxide that is not surface-modified through an iron oxide-binding protein, so that the object can be more easily compared with conventional methods. It can be immobilized on iron oxide. Moreover, since the iron oxide binding protein has a strong binding ability to iron oxide, the complex of the present invention can be used stably after various operations. Therefore, the recovery method of the present invention can be performed stably.

[5. (Method of killing target cells)
The method for killing a target cell according to the present invention (also referred to as “the cell killing method of the present invention”) is a method for killing a target cell in vitro, and the iron oxide-protein complex of the present invention described above. And a first step of bringing the target cells into contact with each other, and a second step of heating the iron oxide-protein complex by applying an alternating magnetic field to the iron oxide-protein complex that has undergone the first step. In addition, the iron oxide is characterized by having magnetism. The “iron oxide-protein complex of the present invention” is the same as described in the section “1. Iron oxide-protein complex”, and the description thereof is omitted.

  In the cell killing method of the present invention, the cells to be killed are not particularly limited and can be appropriately selected according to the purpose. For example, specific cells in a mixed culture containing various cells or a tissue containing various cells (for example, a tissue after being removed from a living body) can be killed, but not limited thereto.

  As one embodiment of the use of the complex of the present invention, the complex of the present invention can be applied to a method for killing a target cell in vivo. In this case, for example, the complex of the present invention is specific to the target cancer tissue (tumor) by administering, in the body, an antibody that recognizes the target cancer cell to the complex of the present invention. To collect. Thereafter, by applying an alternating magnetic field to the complex of the present invention and heating the complex, the target cancer tissue (tumor) can be locally heated to kill the cancer cells. A method of killing cancer cells by heating only the affected part with an external magnetic field by accumulating magnetic particles in cancer tissue is called thermotherapy (hyperthermia) and is expected to be clinically applied. That is, the complex of the present invention can be applied to thermotherapy (hyperthermia).

(5-1. First step)
The first step may be a step of bringing the target cell into contact with the iron oxide-protein complex according to the present invention, that is, the iron oxide having an iron oxide-binding protein immobilized on the surface thereof. The iron oxide-protein complex according to the present invention and the target cell may be contacted to fix the iron oxide-protein complex to the surface of the target cell via the iron oxide-binding protein, or By bringing the iron oxide-protein complex of the present invention into contact with the target cell, the iron oxide-protein complex of the present invention is taken into the target cell and converted into the iron oxide-protein complex in the cell. The target intracellular substance may be immobilized via an iron oxide binding protein.

  The iron oxide binding protein is preferably a protein having a site (target binding site) that can bind to a target (in this case, “target cell” or “target intracellular substance”). The method for producing the “target binding site” and the iron oxide binding protein having the target binding site is as described in the section “1. Iron oxide-protein complex”. Is omitted.

  The “intracellular substance of interest” is not particularly limited, and examples thereof include ribosome, actin, and RNA.

  The method for immobilizing the target cells on the surface of the complex of the present invention is not particularly limited. For example, a method of immersing the complex of the present invention in a solution containing the target cells can be used. . In this case, in the first step, the condition of the complex of the present invention and the target cell is not particularly limited. For example, the complex can be sufficiently brought into contact by incubating at 4 to 37 ° C. for several seconds to 30 minutes. preferable.

  In addition, as a method of incorporating the iron oxide-protein complex of the present invention into a target cell and immobilizing the target intracellular substance on the iron oxide-protein complex via the iron oxide-binding protein in the cell. Although there is no particular limitation, for example, it can be performed by a method that utilizes uptake into cells by endocytosis. In this case, cells having phagocytic ability (for example, macrophages, neutrophils, etc.) can be suitably used as the target cells.

(5-2. Second step)
In the second step, the iron oxide-protein complex that has passed through the first step, that is, the iron oxide-protein complex in which the target cell is immobilized on the surface or the target intracellular substance is taken into the target cell. Is a step of applying an alternating magnetic field to the iron oxide-protein complex on which is immobilized and heating the iron oxide-protein complex. It is known that heat is generated when an alternating magnetic field is applied to magnetic particles. In general, cells are known to die when heated to about 43 ° C. For this reason, an alternating magnetic field is applied to the iron oxide-protein complex in which the target cell is immobilized on the surface or the iron oxide-protein complex in which the target intracellular substance is immobilized by being incorporated into the target cell. Then, the cells can be killed by heating the iron oxide-protein complex.

  The applied magnetic field only needs to be strong enough to heat the iron oxide-protein complex of the present invention, and can be appropriately set depending on the composition, shape, size, etc. of the iron oxide-protein complex to be used. . For example, when magnetite particles having a particle diameter of 10 to 30 nm are used as the iron oxide constituting the iron oxide-protein complex, the iron oxide-protein complex can be heated by applying a high frequency magnetic field of 100 to 1000 kHz. it can.

(5-3. Others)
In the cell killing method of the present invention, steps other than the above may be provided, and the contents of the steps other than the above are not limited. For example, it is not surface-modified by contacting iron oxide with an iron oxide-binding protein capable of binding to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. The step of immobilizing iron oxide-binding protein on the surface of iron oxide (hereinafter referred to as “iron oxide-binding protein immobilization step”), in other words, the step of producing the iron oxide-protein complex of the present invention is the first step. You may provide before a process. The “iron oxide-binding protein immobilization step” is the same as that described in “3-2. Others” of “3. Omitted.

  According to the cell killing method of the present invention, since the target cell or the target intracellular substance can be directly bound to the iron oxide not surface-modified through the iron oxide binding protein, compared with the conventional method. Thus, the target cell or the target intracellular substance can be immobilized on iron oxide more easily. Moreover, since the iron oxide binding protein has a strong binding ability to iron oxide, the iron oxide-protein complex of the present invention can be used stably after various operations. Therefore, the cell killing method of the present invention can be performed stably.

[6. Method 2 for immobilizing an object on iron oxide]
The immobilization method according to the present invention (also referred to as “immobilization method of the present invention”) is a method of immobilizing an object on iron oxide, and is carried out at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. A step of immobilizing a protein that can bind to iron oxide under heating conditions (iron oxide-binding protein) to iron oxide that is not surface-modified (hereinafter referred to as “iron oxide-binding protein immobilization step”), and a target And a step of immobilizing an object on the protein (hereinafter referred to as “target immobilization step”). The “iron oxide-binding protein”, “iron oxide”, and “target” are the same as described in the above section “1. Iron oxide-protein complex”, and thus the description thereof is omitted.

  In the immobilization method of the present invention, the order in which the iron oxide binding protein immobilization step and the object immobilization step are performed is not particularly limited. That is, the object immobilization step may be performed after the iron oxide binding protein immobilization step, and vice versa. More specifically, for example, in the case where the object immobilization step is performed after the iron oxide binding protein immobilization step, the iron oxide binding protein is immobilized on the iron oxide, and then the object is added to the iron oxide binding protein. May be fixed. In addition, for example, when the target immobilization step is performed before the iron oxide binding protein immobilization step, the target and the iron oxide binding protein are immobilized, and then the iron oxide binding protein is fixed to the iron oxide. May be used.

  Moreover, in the immobilization method of this invention, processes other than the above may be provided, and the content of processes other than the above is not limited.

(6-1. Iron oxide binding protein immobilization step)
In the iron oxide binding protein immobilization process, the iron oxide binding protein is immobilized on the surface of the iron oxide by bringing the iron oxide into contact with the iron oxide binding protein (or the iron oxide binding protein on which the target substance is immobilized). It is a process to do. Immobilizing iron oxide-binding protein on iron oxide is intended to bind or adsorb iron oxide-binding protein (or iron oxide-binding protein on which an object is immobilized) to iron oxide.

  In the iron oxide binding protein immobilization step, as a method of immobilizing the iron oxide binding protein (or the iron oxide binding protein on which the target substance is immobilized) on the surface of the iron oxide, for example, the iron oxide binding protein (or the target) A method in which iron oxide is immersed in a solution containing an iron oxide-binding protein on which an object is immobilized), or an iron oxide-binding protein (or an iron oxide-binding protein on which an object is immobilized) is contained on the iron oxide surface For example, a method of applying or spotting the solution that is flowing can be used.

  The iron oxide-binding protein is a protein that can bind to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. The conditions for immobilizing the iron oxide (or the iron oxide binding protein on which the target is immobilized) and the iron oxide may be performed under any conditions. For example, oxidation is performed by adding iron oxide to a solution containing iron oxide-binding protein (or iron oxide-binding protein on which an object is immobilized) and incubating at 4 ° C. to room temperature for several seconds to 30 minutes. An iron-binding protein (or an iron oxide-binding protein on which an object is immobilized) and iron oxide can be sufficiently brought into contact with each other.

(6-2. Object immobilization process)
The object immobilization step is a step of immobilizing the object on the iron oxide-binding protein by bringing the iron oxide-binding protein into contact with the object.

  By immobilizing an object on an iron oxide binding protein, it is intended that the object is reversibly or irreversibly bound or adsorbed on the iron oxide binding protein and the object is supported on the iron oxide binding protein.

  The iron oxide binding protein is preferably a protein having a site capable of binding to an object (object binding site). Thereby, a target object can be fix | immobilized to an iron oxide binding protein. The method for producing the above-mentioned “target binding site” and the iron oxide binding protein having the target binding site is as described above in the section “1. Iron oxide-protein complex”. The description is omitted.

  In the object immobilization step, the method for immobilizing the object on the iron oxide-binding protein is not particularly limited. For example, a method of adding the iron oxide-binding protein to a solution containing the object may be used. it can. In the target immobilization step, the iron oxide-binding protein and the target are not particularly limited, but it is preferable that the iron oxide-binding protein is sufficiently brought into contact, for example, by incubating at 4 ° C. to room temperature for several seconds to 30 minutes. .

  In the target immobilization step, when the target is a protein, the target may be fixed to the iron oxide binding protein by preparing a fusion protein of the iron oxide binding protein and the target.

  According to the immobilization method of the present invention, an object can be directly bound to iron oxide that is not surface-modified through an iron oxide-binding protein, so that the object can be more easily compared with conventional methods. Can be immobilized on iron oxide.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by an Example.

[Example 1]
The binding ability of Si-tag (Genbank, Accession No. AAC76342.1) (the amino acid sequence is shown in SEQ ID NO: 1 and the base sequence is shown in SEQ ID NO: 2) to iron oxide was examined.

(Method)
DNA encoding an amino acid sequence corresponding to immunoglobulin binding domain B1 in protein G which is an antibody binding protein of Streptococcus bacteria (corresponding to residues 1259 to 1425 of SEQ ID NO: Genbank, Accession No. M13825.1) Was synthesized. PET-SpA-Sitag (Ikeda, T., Y. Hata, K. Ninomiya, Y. Ikura, K. Takeguchi, S. Aoyagi, R. Hirota and A. Kuroda. 2009. Oriented immobilization of antibodies on a Ligated with NdeI-BamHI digested fragment of silicon wafer using Si-tagged protein A. Anal. Biochem. 385: 132-137.) Fusion protein of immunoglobulin-binding domain B1 in Si-tag and protein G , Referred to as “Si-tag fusion protein G”).

  The resulting plasmid was transformed into Escherichia coli Rosetta (DE3) pLysS strain by electroporation. The obtained transformant was inoculated into 2 × YT liquid medium and cultured at 37 ° C. When the OD600 of the culture solution reached 0.5, IPTG having a final concentration of 1 mM was added, followed by further culturing at 28 ° C. for 7 hours. E. coli cells were collected by centrifugation (6,000 × g, 10 minutes). The obtained microbial cells were suspended in Tris buffered physiological saline, and the microbial cells were crushed by ultrasonic treatment. The supernatant obtained by centrifuging the cell disruption solution (40,000 × g, 30 minutes) was applied onto a cation exchange chromatography column (Applied Biosystems, Poros HS / M), and the protein adsorbed on the column was measured from 0M. Elution was performed with a linear gradient of 1M NaCl. Fractions containing Si-tag fusion protein G were collected and used for the following experiments.

  The purified Si-tag fusion protein G solution was added to a final concentration of 50 μg / ml and mixed by swirling and shaking at 4 ° C. for 30 minutes. Manufactured, particle size 0.8 μm) or iron oxide particles (manufactured by Nacalai Tesque, product number: 19522-45). Thereafter, each particle was washed three times with a washing solution (25 mM Tris-HCl [pH 9.0], 0.5 M NaCl, 0.5% [v / v] Tween 20), and then SDS-PAGE sample buffer (composition: 62.5). mM Tris-HCl [pH 6.8], 5% [v / v] 2-mercaptoethanol, 2% [w / v] SDS, 5% [w / v] sucrose, 0.005% [w / v] bromophenol blue) Heated at 95 ° C. for 5 minutes. (i) Supernatant after heating, (ii) Supernatant after mixing with silica particles or iron oxide particles, (iii) Supernatant after each washing is subjected to SDS-PAGE by 20 μl each and is contained in each solution Protein analysis was performed.

(result)
FIG. 2 shows the result of comparison of the stability of Si-tags immobilized on silica particles or iron oxide particles by SDS-PAGE. In FIG. 2, each lane shows the results of the following samples.

Lane M: molecular weight marker Lane 1: Si-tag fusion protein G before immobilization (for comparison)
Lane 2: Supernatant after heating (silica particles)
Lane 3: Supernatant after mixing (silica particles)
Lane 4: supernatant after the first wash (silica particles)
Lane 5: Supernatant after the second wash (silica particles)
Lane 6: Supernatant after third washing (silica particles)
Lane 7: Supernatant after heating (iron oxide particles)
Lane 8: supernatant after mixing (iron oxide particles)
Lane 9: supernatant after the first wash (iron oxide particles)
Lane 10: Supernatant after the second wash (iron oxide particles)
Lane 11: Supernatant after the third wash (iron oxide particles)
As shown in FIG. 2, since no bands of Si-tag fusion protein G are observed in lanes 3-6 and lanes 8-11, Si-tag fusion protein G is present in both silica particles and iron oxide particles. It was confirmed that they were combined.

  In lane 2, a band of Si-tag fusion protein G was observed. This indicates that the Si-tag fusion protein G bound to the silica particles was dissociated by heat treatment at 95 ° C. for 5 minutes. On the other hand, no band of Si-tag fusion protein G was observed in lane 7. This indicates that the Si-tag fusion protein G bound to the iron oxide particles was hardly dissociated by heat treatment at 95 ° C. for 5 minutes.

  From the above, Si-tag is very stably bound to iron oxide, and Si-tag has stronger binding ability to iron oxide than silica. It was revealed. Since the bond between Si-tag and iron oxide is very stable, the iron oxide particles on which the protein is immobilized by this method can be used even under severe conditions such as high temperature and the presence of a surfactant. It was revealed.

[Example 2]
An immunoassay using magnetic particles (without centrifugation) was examined.

(Method)
Magnetic particles (magnetite particles) (manufactured by Nacalai Tesque, product number: 19522-45) were used as iron oxide. After 20 μg of magnetite particles were once suspended in dH ₂ O, the magnetite particles were collected using a magnet and washed by removing the supernatant. Similarly, the magnetite particles were washed three times with phosphate buffered saline (PBS; pH 7.4). The magnetite particles after washing were washed again with 200 μl of a binding solution (25 mM Tris-HCl [pH 9.0], 0.5 M NaCl, 0.5% [v / v] Tween 20), and then suspended again in 200 μl of the binding solution.

  The Si-tag fusion protein G solution purified in Example 1 was added to a final concentration of 250 μg / ml, and mixed by swirling and shaking at 4 ° C. for 30 minutes, whereby Si-tag fusion protein G was magnetized. Immobilized on the particles.

Magnetite particles were collected with a magnet and washed three times with PBS. After collecting the magnetite particles using a magnet and removing the supernatant, an anti-H-FABP antibody solution diluted with PBS (5 μg / ml, manufactured by abcam, product number: ab8315) is added, and swirling is performed at 4 ° C. for 1 hour. The antibody was immobilized on magnetite particles by mixing at After mixing at 4 ° C. for 1 hour by rotary shaking, the magnetite particles were washed 3 times with PBST (PBS + 0.05% [v / v] Tween 20). Blocking treatment was performed by adding 1 ml of a PBS solution containing 1% (w / v) BSA to magnetite particles collected by a magnet and performing swirling and shaking at room temperature for 2 hours. After removing the supernatant, various concentrations of heart-derived fatty acid binding protein (H-FABP) (Cayman Chemical, product number: 10007432) and 1% (w / v) BSA 200 μl of PBS solution containing was added. After mixing by vortexing for 1 hour at room temperature, the magnetite particles were washed 3 times with PBST. Add 200 μl of HRP-labeled anti-H-FABP antibody (abcam, product number: ab24470) diluted 10,000 times with PBS containing 1% (w / v) BSA, and swirl at 4 ° C. for 1 hour. And mixed. The magnetite particles were washed 5 times with PBST, the supernatant was removed, and 200 μl of HRP chromogenic substrate (Promega TMB One Solution) was added to carry out a color reaction at room temperature. After adding 200 μl of 0.5 MH ₂ SO ₄ to stop the reaction, the absorbance at a wavelength of 450 nm was measured with a spectrophotometer.

(result)
The measurement results are shown in FIG. As shown in FIG. 3, color development corresponding to the H-FABP concentration was observed. From this, it was shown that the target antibody was functionally immobilized on the magnetite particles via the Si-tag fusion protein G.

  By this technique, iron oxide particles on which the target antibody was immobilized could be easily prepared. Iron oxide particles can be aggregated and dispersed in a liquid by manipulating a magnetic field. Usually, a centrifugal operation is required to collect the particles, but magnetic particles are suitable for an automated process because they do not require centrifugation, and can be used for various applications.

Example 3
Bacteria recovery method using magnetic particles with immobilized antibody was studied.

(Method)
The magnetite particles used in Example 2 were used as iron oxide. The antibody was immobilized on the magnetite particles by the same method as described in Example 2. However, an anti-E. Coli antibody (manufactured by abcam, product number: ab25823) was used as the antibody.

  The magnetite particles with the antibody immobilized thereon were washed 3 times with PBS. 200 μl of E. coli culture solution cultured overnight in LB liquid medium was added to the magnetite particles and mixed by swirling and shaking at room temperature for 1 hour. The particles were washed 5 times with PBST, then suspended in PBS and diluted as appropriate. 100 μl of the diluted solution was plated on an LB agar medium and cultured overnight at 37 ° C., and then the number of colonies formed on the agar medium was counted to evaluate the number of bacteria recovered by the magnetic particles.

(result)
The number of colonies formed is listed in Table 1.

  Compared to the case where the anti-E. Coli antibody was not immobilized, more colonies were formed when the anti-E. Coli antibody was immobilized. From this, it was shown that the target bacteria can be efficiently recovered by the iron oxide particles presenting antibodies against the target bacteria. In the recovery by centrifugation, other contaminants also precipitate at the same time. However, if the recovery is performed by magnetism, only the intended product can be recovered, which is highly useful.

  As described above, according to the present invention, there is an effect that an object can be easily and strongly fixed to iron oxide. Therefore, according to the present invention, it is possible to easily collect an object and kill a target cell. Therefore, the present invention can be used in a wide variety of industries that need to perform these operations.

1 Iron oxide binding protein 2 Object binding site 3 Object 4 Iron oxide

Claims (10)

An iron oxide-protein complex in which a protein is immobilized on iron oxide that is not surface-modified;
The protein is the following protein (a) or (b):
The iron oxide-protein complex characterized in that the binding between the iron oxide and the protein can be maintained under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;
(B) The amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted and / or added, and at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate A protein that can bind to iron oxide under heating conditions. The iron oxide-protein complex according to claim 1 , wherein the protein is a protein having a site capable of binding to an object. The iron oxide-protein complex according to claim 1 or 2 , wherein the protein comprises a protein that binds to immunoglobulin as at least a part thereof. The iron oxide-protein complex according to any one of claims 1 to 3 , wherein the iron oxide has magnetism. At least a protein capable of binding to iron oxide under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate,
The protein is a protein of the following (a) or (b) , wherein the target is surface-modified under the condition of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate. Kit for immobilization on not iron oxide :
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1 ;
(B) The amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted and / or added, and at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate A protein that can bind to iron oxide under heating conditions . A method for immobilizing an object on iron oxide, comprising a step of bringing the iron oxide-protein complex according to any one of claims 1 to 4 into contact with the object. A first step of bringing the iron oxide-protein complex according to any one of claims 1 to 4 into contact with an object;
A second step of recovering the iron oxide-protein complex that has undergone the first step;
A method for recovering an object, comprising: The iron oxide has magnetism,
The said 2nd process WHEREIN: The said iron oxide-protein complex is collect | recovered by making the said iron oxide-protein complex passed through the said 1st process capture | acquire by a magnetic field, The target object of Claim 7 characterized by the above-mentioned. Recovery method. A first step of bringing the iron oxide-protein complex according to any one of claims 1 to 4 into contact with a target cell;
A second step of heating the iron oxide-protein complex by applying an alternating magnetic field to the iron oxide-protein complex after the first step;
Including
The method of killing a target cell, wherein the iron oxide has magnetism. Immobilizing a protein capable of binding to iron oxide under conditions of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate to iron oxide not surface-modified;
Immobilizing an object on the protein;
Including
The protein is the following protein (a) or (b):
The method of immobilizing an object on iron oxide, wherein the binding between the iron oxide and the protein can be maintained under conditions of heating at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate :
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;
(B) The amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted and / or added, and at 95 ° C. for 5 minutes in the presence of 2% sodium dodecyl sulfate A protein that can bind to iron oxide under heating conditions .