JP2012096232A - Method for producing magnetic carrier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a magnetic carrier to be used when protein is purified from a new biological sample, which is made remarkably simpler than the conventional method and which is capable of automation and high throughput.SOLUTION: The magnetic carrier having a biological material of any of glucide, protein, peptide, nucleic acid, a cell and a micro-organism on its surface is produced by a production method for a magnetic carrier in which magnetic particles are added to a layer-like coating film formation treatment by silver or a coating film formation treatment by the biological material, and the layer-like coating film formation treatment by silver is carried out by using any of a silver mirror reaction method, an electroless plating method, an electroplating method, a sputtering method, a vacuum deposition method, an ion plating method and a chemical vapor deposition method.

Description

  The present invention relates to a method for producing a magnetic carrier that can be suitably used for treating a target protein and the like from a biological sample.

Conventionally, samples such as bacteria, yeast, insect cells, animal cells, animal tissues, plant tissues (including crushed liquids and extracts obtained from them), cell-free protein synthesis liquids (hereinafter collectively referred to as these) There are a wide variety of methods, materials and approaches for extracting and purifying a specific protein of interest in a biological sample (referred to as a “biological sample”) from other components.

  An example of a common approach is to take advantage of the nonspecific affinity of a protein for a carrier. As such an approach, for example, ion exchange chromatography based on the charge of a protein molecule is known. In ion exchange chromatography, a protein mixture is added to a chromatography matrix having a charge opposite to that of the protein, and various proteins are bound to the matrix by reversible electrostatic interactions. Proteins bound to the matrix can be eluted in order of weak binding to strong binding by increasing the ionic strength or changing the pH of the elution buffer.

  Another example of a general approach is one that utilizes the physical properties of a protein as a separation means. As such an approach, for example, gel filtration based on protein size is known. In gel filtration, the protein mixture is added to a gel filtration column packed with a chromatography matrix having pores of a predetermined size. Thereafter, elution is carried out using an elution solution (usually a buffer), which can be collected as individual chromatographic fractions for analysis.

  Yet another example of a general approach is to take advantage of the specific affinity of a protein for a purification reagent. As such an approach, for example, an antibody that can be specifically adsorbed to the target protein is used, or an antigen that can be specifically adsorbed to the antibody when the target protein is an antibody. Affinity chromatography is known. In affinity chromatography, an antibody or antigen is usually bound to a column base material, a solution containing the antigen or antibody that can be specifically adsorbed to the antibody or antigen is added to the column, and an immune complex ( Antigen-antibody complex) is formed. Subsequently, the immune complex is destabilized and eluted, for example, by exposing the immune complex to a buffer with very high ionic strength, or a buffer with very high or low pH. As described above, affinity chromatography is a highly effective protein purification method that utilizes a specific interaction between a target protein and a ligand immobilized on a solid phase. In affinity chromatography, solid phase ligands usually have some inherent scientific properties that selectively adsorb target proteins. Contaminant protein does not bind to the solid phase ligand or can be removed by washing the solid phase ligand with an appropriate solution.

  The possibility of preparing hybrid genes by genetic technology in recent years has opened a new path. That is, a combination having an affinity peptide suitable for the above separation by binding a gene sequence encoding a desired protein and a gene sequence encoding a protein fragment (affinity peptide) having high affinity for a ligand. The recombinant protein can be expressed, and the desired recombinant protein can be purified in the form of a fusion protein in one step of purification using the affinity peptide. It is also possible to introduce a specific chemical or enzymatic cleavage site at the point of attachment of the affinity peptide and the desired recombinant protein by site-restricted mutation. After purification, the affinity peptide can be cleaved chemically or enzymatically to recover the desired recombinant protein. Such purification methods are known, for example, from Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, and Patent Document 1 and Patent Document 2.

  The affinity peptide of the fusion protein can be bound directly or indirectly to the biochemically active polypeptide or protein. If a single affinity peptide is used, the affinity peptide can be linked to the amino-terminal or carboxyl-terminal amino acid of a biochemically active polypeptide or protein. When using two affinity peptides, one of the affinity peptides can be conjugated to the amino terminal amino acid of a biochemically active polypeptide or protein and the other affinity peptide is biochemically bound. It can be linked to the carboxyl terminal amino acid of the active polypeptide or protein.

  In the case of indirect binding, the affinity peptides contain an appropriate selective cleavage site through which they can be bound to the desired biochemically active polypeptide or protein. As a suitable selective cleavage site, preferably the amino acid sequence-(Asp) n-Lys- (wherein n represents 2, 3 or 4) or -Ile-Glu-Gly-Arg- is known. . These selective cleavage sites can be specifically recognized by the proteases enterokinase and aggregate FactorXa, respectively. Such an affinity peptide can be cleaved enzymatically at the selective cleavage site by a method known per se.

  Also in the case of direct binding, the affinity peptide remains bound to the desired biochemically active polypeptide or protein. That is, the affinity peptide does not have a selective cleavage site that can be cleaved chemically or enzymatically. Direct binding is advantageous when the activity of the desired polypeptide or protein is not adversely affected by the presence of the affinity peptide.

  Patent Document 2 discloses a method for producing a binding protein having an affinity peptide exhibiting specific affinity for a specific carbohydrate, in other words, a fusion protein using the sugar binding protein, and simple purification using the same. A method is disclosed. Examples of the sugar-binding protein include mono-, di-, and polysaccharide-binding proteins, and more specifically, maltose-binding protein and arabinose-binding protein. In particular, maltose-binding protein, which is the malE gene product of E. coli, is a periplasmic protein that can be affected by osmotic pressure, and shows a specific affinity for maltose and maltodextrin. The maltose-binding protein, a fragment thereof, or a fusion protein with the target protein can be purified by affinity column chromatography using amylose resin. It is known by those skilled in the art that maltose-binding protein is highly likely to be purified in a solubilized state even when the target protein is hardly soluble or easily insolubilized by using it as an affinity peptide of the fusion protein. ing.

  By the way, Patent Document 3 discloses a magnetic carrier that is suitably used for purifying the protein contained in a biological sample containing the target protein. This magnetic carrier has metal oxide particles exhibiting ferromagnetism and a carbohydrate layer covering the particles.

Japanese Patent No. 2686090 Japanese Patent No. 2703770 JP 2003-30995 A

Itakura et al., Science 198, 1056-1063 (1977) Germino et al., Proc. Natl. Acad. Sci. U.S.A. 80, 6848-6852 (1983) Nilsson et al., Nucleic Acids Res. 13, 1151-1162 (1985) Smith et al., Gene 32, 321-327 (1984)

  The magnetic carrier as described above is formed by a method in which a saccharide is added to a dispersion in which oxide particles are dispersed, heated, and then cooled. However, in such a method, for example, a metal atom, a metal ion, -O-, -OH, a compound used for the synthesis of the metal oxide or a part of the metal oxide, etc. exist on the surface of the metal oxide or the like. Therefore, it is difficult to obtain a uniform coating, and some of the properties of the internal particle surface exhibiting ferromagnetism may be developed.

  Therefore, the object of the present invention is to provide a more uniform coating of biological material with fewer exposed portions of particles exhibiting ferromagnetism, preferably substantially free of exposed portions, as compared with the magnetic carrier as described above. Another object is to provide a magnetic carrier. Another object of the present invention is to provide a method for producing such a magnetic carrier.

  As a result of intensive studies to develop a protein purification method in which the exposure of particles exhibiting ferromagnetism is suppressed, the biological material is more uniformly coated, and can be easily automated and increased in throughput. By using silver coating and biomaterial coating in combination with the magnetic particles, formation of exposed portions of particles exhibiting ferromagnetism is suppressed, and a magnetic carrier coated with the biomaterial more uniformly is obtained. I found out that The combined use of these coating treatments includes a case in which a coating treatment with silver is performed, and thereafter a coating treatment with a biological material is performed (that is, performed sequentially), and a case where these treatments are performed simultaneously.

  The coating treatment means a treatment for adhering (or bonding) silver or a biological substance to a predetermined surface, for example, adhering (or bonding) in the form of a spot, a layer, or the like. A coating is formed on the surface. The form of the coating is determined according to the processing conditions, and is, for example, a spot shape or a layer shape as described above, but the shape and size of the coating are not particularly limited. The predetermined surface is generally at least a part (ie, part or whole surface) of the surface of the magnetic particle, but if another material is already present on the surface of the magnetic particle, the surface of the material is Also good. For example, as described below, the magnetic particles may have other materials on their surfaces (eg, in layered form, spot-like form, etc.), in which case the coating process will adhere to such material (or A process of combining).

  For example, when the coating process is sequentially performed, silver adheres to at least a part of the surface of the magnetic particle during the coating process with the biological material. Therefore, in the coating treatment with the biological material, the biological material is attached to a part of the surface of the magnetic particle to which silver is not attached (when such a part is present), or the silver ( For example, the attachment of biological material to layered silver (possibly attached to the entire surface of the magnetic particles) is included. In addition, for example, the magnetic particles may have a layer of another material, for example, a nonmagnetic material layer (silica layer, etc.) around the magnetic particles. The biological material adheres to silver and a part of the nonmagnetic material layer that is optionally present by the coating treatment with the biological material.

  When the coating treatment with silver is performed as described above, the influence of silver is not completely absent, but the surface to which silver is attached is simple compared to a ferromagnetic material such as a metal oxide, and if there is any influence. Even if there is, there is an advantage that it can be easily guessed whether the cause is due to silver or not. In particular, focusing on the specific binding of amylose and maltose-binding protein to the magnetic carrier particles of the present invention obtained by coating with amylose as a representative carbohydrate that is a kind of biological material, maltose-binding protein As a result of attempting to purify a protein from a biological sample containing this protein by binding the protein, it was confirmed that the protein could be isolated efficiently, and the present invention was completed.

  Accordingly, in the first aspect, the present invention provides a method for producing a magnetic carrier having a biological material on its surface, which method applies magnetic particles to the coating treatment with silver and the coating treatment with biological material (that is, the method) The coating is performed on the magnetic particles.

  In one preferred embodiment of the production method of the present invention, the magnetic particles are subjected to a coating treatment with silver, and then the magnetic particles thus coated are subjected to a coating treatment with a biological substance. In another preferred embodiment of the production method of the present invention, the coating treatment with silver and the coating treatment with a biological material are performed simultaneously.

  In a second aspect, the present invention is a method for purifying a target protein, wherein a biological sample containing the target protein has a biological substance that specifically binds to the protein on the surface, and is obtained by the above-described manufacturing method. It is characterized by contacting with a carrier.

  According to the method of the present invention, it is possible to produce a magnetic carrier that can be used for the purification of a protein that does not expose particles exhibiting ferromagnetism and is uniformly coated with a biological material, and that can be easily automated and has high throughput. Become.

It is a scanning electron microscope (SEM) photograph of the magnetite particle | grains (comparative example 4) which is not silver-coated. It is a scanning electron microscope (SEM) photograph of the particle | grains (Example 2) which carried out 0.5% silver coating process to the magnetite particle | grains. It is a scanning electron microscope (SEM) photograph of the particle | grains (Example 11) which carried out 5% silver coating process to the magnetite particle | grains. A sample before purification (FIG. 4 (a)) and a fusion protein MBP-LacZα (FIG. 4 (b)) in which a maltose-binding protein is bound to the amino terminus of the β-galactosidase α chain purified by this method are analyzed by SDS- The result compared by PAGE is shown.

  In the present invention, magnetic particles include at least one metal having magnetism (may be an oxide), at least one alloy having magnetism (may be an oxide), and various combinations thereof. It means a particle composed of a selected magnetic material.

  The magnetic particles are preferably ferromagnetic particles, such as ferromagnetic oxide particles. This ferromagnetic oxide particle refers to a granular material having magnetic response (sensitivity to a magnetic field) obtained by an oxidation reaction of particles made of metal. Here, “having magnetic responsiveness” means that when an external magnetic field is present due to a magnet or the like, it is sensitive to a magnetic field, such as being magnetized by a magnetic field or adsorbed to a magnet. The ferromagnetic oxide is not particularly limited, and examples thereof include known metals, alloys, and oxides thereof such as iron, cobalt, nickel, etc. However, since it is particularly sensitive to magnetic fields, it is a ferromagnetic iron oxide. Is preferred. Ferromagnetic iron oxide is particularly preferable as described above. However, even if it has superparamagnetism (for example, FePt particles and Fe particles having a particle size of 5 nm), it can be used if it is sensitive to a magnetic field. is there.

Various known ferromagnetic iron oxides can be used as the ferromagnetic iron oxide. Among them, since it is excellent in chemical stability, maghemite (γ-Fe ₂ O ₃ ), magnetite (Fe ₃ O ₄ ), nickel zinc ferrite (Ni _1-X Zn _X Fe ₂ O ₄ ), manganese zinc ferrite (Mn _{1- X} Zn _X Fe ₂ O ₄₎ is preferably at least one selected from ferrite, such as. Among these, magnetite is particularly preferable because it has a large amount of magnetization and is excellent in sensitivity to a magnetic field.

The ferromagnetic iron oxide particles can be produced by, for example, a known method in which particles such as Fe (OH) ₂ are oxidized in water. In the examples described later, magnetite particles were produced as an example.

  In the present invention, it is also preferable to use metallic nickel particles as magnetic particles. Since metallic nickel exhibits ferromagnetism, it can be used in the same manner as ferromagnetic iron oxide particles.

  Use of magnetic particles such as ferromagnetic iron oxide particles and metallic nickel particles is preferable because particles having magnetic responsiveness that are practically less problematic can be obtained even when coating with silver and a biological material is performed.

  In another aspect, in the present invention, as the magnetic particles, nonmagnetic material particles having a coating of the above-described magnetic material, preferably a coating covering the entire particles can be used. In this case, the magnetic material may be one in which individual particles of nonmagnetic material are coated one by one, or may be one in which particles of a plurality of nonmagnetic materials are coated together. For example, non-magnetic plating method, electroplating method, sputtering method, vacuum deposition method, ion plating method on non-magnetic particles formed by inorganic materials such as alumina and colloidal silica, or organic polymers such as acrylic and polystyrene. A material coated with a magnetic material using a chemical vapor deposition method or the like can be used. The non-magnetic material particles having such a magnetic material coating may be produced by any known method.

  As described above, a coating of a magnetic material such as metallic nickel is formed on the nonmagnetic material particles by electroless plating, electroplating, sputtering, vacuum deposition, ion plating, chemical vapor deposition, or the like. Thus, magnetism can be imparted to the particles of the nonmagnetic material. Similar to the magnetic particles described above, such particles can be coated with silver or a biological material, and particles having magnetic responsiveness that are not problematic in practice can be obtained.

  In yet another aspect, in the present invention, as the magnetic particles, magnetic material particles having a non-magnetic material coating, preferably a coating covering the entire particle can be used. In this case, each particle of the magnetic material may be coated one by one, or a plurality of particles of the magnetic material may be coated together. Examples of such nonmagnetic materials include compounds containing aluminum such as aluminate, compounds containing silicon such as silicate, acrylic resins, polystyrene, polymers such as polymethyl methacrylate, and the like. Examples of the method of coating the magnetic material particles with a non-magnetic material include a deposition method using aluminate, silicate, etc., a sol-gel method using alkoxysilane, and a method of depositing an organic polymer. It is done. Examples of a method of making a plurality of magnetic particles into one particle with a non-magnetic substance include a microencapsulation method and a suspension polymerization method. Conventionally known methods can be used for the above methods.

  In other words, when the magnetic material particles are subjected to a coating treatment with silver, as long as the magnetic response of the magnetic carrier imparted by the magnetic material particles is not adversely affected, the formed silver coating and magnetic material particles Various non-magnetic materials may be present between them (for example, as an intermediate layer). For example, silica can be used as the nonmagnetic material. Specifically, magnetic silica beads or the like in which particles of a magnetic material are covered with a silica coating can be used as the magnetic particles, which may be subjected to a coating treatment with silver. In addition to this, a magnetic carrier formed using magnetic particles having metal layers such as zinc and nickel and organic polymer layers such as acrylic and polystyrene as an intermediate layer between particles of magnetic material and silver can also be exemplified. . Further, such an intermediate layer is not necessarily limited to only one layer, and a plurality of layers of the materials exemplified above may be stacked.

  The magnetic particle used in the present invention uses the term “particle” for the sake of convenience, but the shape is not particularly limited as long as it is a fine lump or element (structural unit), and is spherical, ellipsoidal, granular, Examples thereof include a plate shape, a needle shape, and a polyhedron shape such as a cubic shape, but a spherical shape, an ellipsoid shape, or a granular shape is preferable because it can be easily formed into a suitable shape when a magnetic carrier described later is used.

  The size of the magnetic particles used in the present invention is not particularly limited, and usually one magnetic particle or an aggregate of a plurality of, for example, 2 to 100 magnetic particles is integrated by the coating treatment of the invention. One element constituting the magnetic carrier is constituted. Therefore, usually, 1 to 100 magnetic particles are provided as one magnetic carrier in a form of being coated with a silver layer or a silver and biological material layer. The coating does not necessarily need to cover the entire magnetic particle, and may cover only a part of the magnetic particle. Even when a plurality of particles are integrated, the coating is sufficient to ensure a minimum unity. It's okay.

  Each particle, i.e. element, of the magnetic support obtained by the method of the present invention preferably has a particle size (also referred to as “particle diameter”) of 0.005 μm to 60 μm as described below, and therefore In order to form a magnetic carrier, the magnetic particles preferably have an average particle size of 0.005 μm to 45 μm. The “particle size” means the maximum length among the lengths in all directions of the particle, and can be measured, for example, in a transmission electron micrograph of the particle. The “average particle size” can be obtained by measuring the particle size of each of 300 particles in a transmission electron micrograph and calculating the number average.

  In the method for producing a magnetic carrier of the present invention, the coating treatment of the magnetic particles with silver is not particularly limited as long as it can form a coating by attaching silver to at least a part of the surface of the magnetic particles. For example, it can process using methods, such as an electroless-plating method (a silver mirror reaction method is included), an electroplating method, sputtering method, a vacuum evaporation method, an ion plating method, a chemical vapor deposition method. Such a method can be carried out by a known method. In addition, when the coating process with silver is first performed and then the coating process with a biological material is performed, it is preferable that a coating layer can be formed on the entire surface of the magnetic particles.

  For example, for the electroless plating method, a plating solution containing silver in the form of a silver salt, for example, is used as the silver compound. Commercially available silver plating agents can be used, and when these solutions are used, a relatively simple and clean coating can be achieved. However, it is naturally possible to individually purchase and use various reagents used for silver plating, and the method known as the silver mirror reaction method is particularly simple. The temperature of the electroless plating method is preferably 5 to 90 ° C, more preferably 5 to 40 ° C. When this temperature is lower than 5 ° C., the reaction may be slow and time may be required. When the temperature is higher than 90 ° C., the reaction proceeds too quickly and a uniform film may not be formed.

  In the method for producing a magnetic carrier of the present invention, the amount of silver covering the magnetic particles (amount of the silver coating layer) is preferably 0.1% by weight to 30% by weight with respect to the magnetic particles. When the amount of silver is less than 0.1% by weight, the possibility that the magnetic particles cannot be uniformly deposited by silver increases. As a result, there is a portion where the surface of the magnetic particle is exposed without the coating, and as a result, the deposition of the biological material is likely to be uneven, and eventually a part of the surface of the magnetic particle is exposed. It is also possible. Also, if the amount of silver exceeds 30% by weight with respect to the particles, it may adversely affect the magnetic properties of the magnetic carrier, resulting in a decrease in magnetic responsiveness, which is disadvantageous in terms of handling or disadvantageous in terms of cost. There is. The amount of silver is more preferably 0.3% to 20% by weight, and further preferably 0.5% to 10% by weight.

  In the method for producing a magnetic carrier of the present invention, the magnetic particles are coated with a biological material. “Biological substance” means a substance derived from an organism, and examples of such biological substance include carbohydrates, proteins, peptides, nucleic acids, cells, microorganisms and the like.

  The carbohydrate is not particularly limited, and examples thereof include mono-, di-, and polysaccharides and mixtures thereof. For example, the carbohydrate is purified using a magnetic carrier and has sufficient affinity for the protein depending on the target protein. What is possessed may be appropriately selected and used. Examples of such saccharides include glucose, galactose, arabinose, mannose, maltose, maltodextrin, amylose, dextrin, soluble starch, etc., but since glucose is inexpensive and readily available, glucose is the main unit. Oligosaccharides or polysaccharides are preferred, and amylose is more preferred. In particular, when a target protein is a fusion protein containing part or all of a maltose-binding protein, it is preferable to use amylose as a carbohydrate.

  Examples of the protein or peptide include antigens, antibodies, biological receptors, substances having specific binding properties such as ligands, and enzymes having various functions such as enzymes, and other biological substances such as avidin are fixed. Those that can be used are also preferably used. As a nucleic acid, DNA, RNA double strand, single strand, etc. are mentioned, for example.

  Furthermore, in the present invention, the term “biological substance” is used to include not only the biological substance itself but also a substance having an interaction with the biological substance. For example, a drug or drug candidate substance, a harmful substance such as an environmental hormone, and a substance that can be used for fixing other biological substances such as biotin can be used.

  In the method for producing a magnetic carrier of the present invention, the coating treatment of the magnetic particles with the biological material can be performed by attaching the biological material to at least a part of the surface of the magnetic particles, or by using silver as described above. There is no particular limitation as long as it is a method capable of forming a coating by attaching a biological material to at least a part of the surface of the magnetic particles after the coating treatment. In the case where the silver coating process is performed first, and then the biological material coating process is performed, it is preferable to form a biological material coating layer on the entire surface of the magnetic particles subjected to the silver coating process. Such a coating treatment with a biological material can be selected according to the biological material to be attached.

  For example, when using a soluble, for example, water-soluble biological material, such as a saccharide, a method of depositing the saccharide on the surface of the magnetic particle using the difference in solubility of the biological material as a solute in a solvent (for example, water) Can be used. Specifically, the solvent containing the magnetic particles is heated to dissolve the saccharide, and then the saccharide is deposited on the surface of the magnetic particle by cooling to an amount exceeding the saturation solubility. Moreover, it can replace with cooling and can precipitate a carbohydrate similarly by evaporating a solvent.

  In addition, for example, when using proteins, peptides, nucleic acids, cells, microorganisms, etc. as biological materials, a sulfur-containing compound having a site that reacts with these is treated in advance so as to adhere to the silver coating surface, Proteins, peptides, nucleic acids, cells, microorganisms and the like are bound to sulfur-containing compounds. Alternatively, proteins, peptides, nucleic acids, cells, microorganisms and the like are provided with a site containing sulfur, and this is attached to the silver coating surface. Further, a method combining these two methods may be used. The Ag—S bond has similar properties to the Au—S bond, and various known methods using the Au—S bond can be used for the method using the Ag—S bond.

In particular,
(A) at least one selected from functional groups containing sulfur such as thiol groups, sulfide groups, disulfide groups, thioether groups, and (B) aldehyde groups, epoxy groups, azide groups, succinimide groups, maleimide groups, hydrazides Group, primary amino group, secondary amino group, tertiary amino group, carboxyl group, imide ester group, carbodiimide group, isocyanate group, iodoacetyl group, functional group derived from acid chloride, functional group such as double bond, Derived from substances that bind to biological materials, such as functional groups derived from compounds such as biotin and avidin, complex groups containing transition metal ions such as nickel, cobalt, and copper, and functional groups derived from organometallic compounds At least one of the functional groups (i.e. at least one functional group (A) and at least one functional group ( B)) is used to bind the compound to the silver coating surface by Ag-S bonding by at least one functional group (A), and the biological material is made particles by at least one functional group (B). Cover the surface.

  In addition, when performing the coating process by silver and the coating process by a biological material simultaneously, both the above-mentioned processing methods are implemented simultaneously. For example, the electroless plating reaction may be performed in a solvent while cooling the solvent in which the biological material is dissolved by heating.

  In the method for producing a magnetic carrier of the present invention, the amount of biological material (eg, carbohydrate) that coats the magnetic particles (the amount of biological material attached) is 0.1 wt% to 30 wt% with respect to the magnetic particles. Is preferable, and it is more preferable that it is 0.5 weight%-20 weight%. If the amount of the biological material is less than 0.1% by weight with respect to the magnetic particles, the binding property of the magnetic carrier to the protein tends to be low, and the amount of the biological material is less than the magnetic particles. If it exceeds 30% by weight, the magnetic properties of the magnetic carrier may be adversely affected, and the efficiency of protein extraction / purification using a magnetic field, which will be described later, tends to decrease.

  In the method for producing a magnetic carrier of the present invention, the above-described particles are coated with a biological material layer. This “coating” refers to the presence of biological material, preferably in the form of a layer, at least partly covering the outside of the particles constituting the magnetic carrier and at the outermost part of the magnetic carrier. Therefore, when the coating process with a biological material is performed after the coating process with silver, the biological material layer may be formed so as to completely cover the particles. In another aspect, the target protein and the biological material As long as the affinity binding is not inhibited, the material other than the biological material may be formed so as to be exposed on the surface of the particle. In the case where the coating treatment with silver and the coating treatment with a biological material are simultaneously performed, the biological material layer and the silver layer may be formed so as to completely cover the particles. As long as the affinity binding with the biological substance is not inhibited, the material other than silver and the biological substance may be formed so as to be exposed on the surface of the particle.

  Further, when the coating process with a biological material is performed after the coating process with silver, the above-described biological material layer only needs to be formed on the outermost part of the particles constituting the magnetic carrier. If the magnetic responsiveness is not lost, an intermediate layer made of various materials (for example, polymers such as silica, acrylic resin, polystyrene, polymethyl methacrylate, etc.) may be formed between the silver coating layer and the biological material layer. Good.

  Specifically, there can be exemplified a magnetic carrier in which magnetic particles are applied to a silver coating treatment, and then particles having a silver coating are coated with silica, for example, to form magnetic silica particles, and a biological material layer is formed thereon. . In this case, the silica forms an intermediate layer between the silver layer and the biological material layer. Producing a magnetic carrier that has the advantage of easily adsorbing biological materials, especially DNA, RNA, etc., when having an intermediate layer formed of silica between the silver coating layer and the biological material layer. Can do. In addition, a magnetic carrier in which an organic polymer layer such as an acrylic resin or polystyrene is formed as an intermediate layer between the silver layer and the biological material layer can be exemplified. Further, the intermediate layer may be formed by laminating not only one layer but also a plurality of materials including those exemplified above.

  In the method for producing a magnetic carrier of the present invention, the above-described silver coating and biological material coating are sequentially performed in this order (sequential processing), or these coating processes are simultaneously performed (simultaneous processing). In the examples described later, both sequential coating and simultaneous coating are described.

  The unit elements constituting the magnetic carrier obtained by the production method of the present invention (that is, individual constituent elements constituting the carrier) are not particularly limited in shape, and may have various shapes such as a needle shape, a spherical shape, and a plate shape. It's okay. However, when collecting a magnetic carrier using a magnetic field, which will be described later, and isolating a protein bound to the magnetic carrier from a biological sample, the balance between the collection and dispersibility of the magnetic carrier is good, and operability is improved. From the viewpoint of superiority, the unit element preferably has a spherical, ellipsoidal or granular shape. Here, “spherical” means that the aspect ratio (ratio between the maximum length and the minimum length when measured in any direction) is in the range of 1.0 to 1.2 (1.0 to 1.2). A certain shape is indicated, and “ellipsoidal shape” indicates a shape having an aspect ratio exceeding 1.2 and not more than 1.5. “Granular” means that the length of the particles is uniform in all directions, such as a spherical shape, and there is a difference in length depending on the direction other than the one having a large length in one direction, such as an ellipsoid. As a whole, the shape has no particular anisotropy.

  The size of the magnetic carrier produced by the method of the present invention is not particularly limited, but the average particle size is preferably 0.005 μm to 60 μm from the viewpoint of good operability like the above shape, and 0 More preferably, the thickness is 0.01 μm to 45 μm. When the average particle size of the magnetic carrier is less than 0.005 μm, the specific surface area of the magnetic carrier increases, so that the amount of binding with the target protein increases, but it tends to be difficult to collect the magnetic carrier. On the other hand, when the average particle size of the magnetic carrier exceeds 45 μm, the specific surface area of the magnetic carrier becomes small and the magnetic carrier tends to settle, so that the binding amount with the target protein tends to decrease. The “particle size” and “average particle size” of the magnetic carrier are as described above.

  Since the magnetic carrier produced by the production method of the present invention can be used for various treatments (for example, protein purification) by operating using a magnetic field, the magnetic properties of the magnetic carrier are important. As such magnetic properties, saturation magnetization and coercivity are particularly important. For example, the saturation magnetization is mainly related to the collection of a protein-bound magnetic carrier, and the coercivity is mainly related to the separation of the magnetic carrier and protein (protein elution).

  In general, the higher the saturation magnetization, the greater the sensitivity to the magnetic field. Therefore, when using a magnetic carrier with a high saturation magnetization, for example, the collection of the magnetic carrier in a protein-bound state in the purification of the protein using the magnetic field. However, if the saturation magnetization is too high, the magnetic particles will be magnetically aggregated.

When producing the magnetic carrier according to the method of the present invention, the saturation magnetization of the magnetic carrier ^{2A · m 2 / kg~100A · m} 2 / kg, more preferably ^{4A · m 2 / kg (emu} / g) ~90A · m ² / kg (emu / g). If the saturation magnetization of the magnetic carrier is smaller than ^{2 A} · m ² / kg, the magnetic carrier is less sensitive to the magnetic field, and the trapping property tends to decrease. On the other hand, if the saturation magnetization of the magnetic carrier exceeds 100 A · m ² / kg, the magnetic carrier tends to aggregate magnetically and the dispersibility of the magnetic carrier in the protein purification system tends to decrease. The saturation magnetization of the magnetic carrier is measured by, for example, using a vibrating sample magnetometer (manufactured by Toei Kogyo Co., Ltd.) and measuring the amount of magnetization when a magnetic field of 796.5 kA / m (10 kilo-Oersted) is applied. It can ask for.

For magnetic carrier obtained by coating treatment of the magnetic particles composed of only the magnetic material, the saturation magnetization is ^{20A · m 2 / kg~100A · m} 2 / kg, preferably ^{30A · m 2 / kg (emu} / g) ~ It is desirable to manufacture so that it may become 80A * m < ² > / kg (emu / g). Further, if the magnetic carrier obtained magnetic particles the magnetic material coating treatment to the particles of non-magnetic material to coating treatment, the saturation magnetization is ^{2A · m 2 / kg~100A · m} 2 / kg, preferably 4A · m ^It is desirable to manufacture so that it may become ² / kg (emu / g) -90A * m < ² > / kg (emu / g). The reason why the preferable range of the saturation magnetization varies depending on the type is that the magnetic response varies depending on the specific gravity of the magnetic particles. Magnetic particles made of only a magnetic material have a higher specific gravity, whereas magnetic particles obtained by coating nonmagnetic material particles with a magnetic material have a lower specific gravity. For this reason, even if the particle size is the same, the weight of the magnetic carrier is different. The greater the specific gravity, the worse the magnetic response, so the saturation magnetization needs to be increased.

  In addition, the magnetic carrier is magnetized to some extent by the magnetic field applied at the time of collection. However, as the coercive force increases, the cohesive force between the constituent elements of the magnetic carrier increases, and the magnetic force when protein is eluted from the magnetic carrier. The dispersibility of the carrier is reduced. As a result, the elution property of the bound protein in the solution tends to be low, and the extraction efficiency tends to decrease.

  When a magnetic carrier is produced by the method of the present invention, the coercive force of the magnetic carrier is preferably 0.079 kA / m to 15.93 kA / m (10 to 200 oersted), more preferably 1.59 kA / m to 11. It implements so that it may become 94 kA / m (20 Oersted-150 Oersted). The magnetic carrier has a coercive force, for example, using a vibrating sample magnetometer (manufactured by Toei Kogyo Co., Ltd.) and applying a magnetic field of 796.5 kA / m (10 kilo-Oersted) to achieve saturation magnetization. It can be obtained from the strength of the applied magnetic field that returns to zero and further increases the magnetic field in the opposite direction and gradually increases the magnetization value to zero.

The magnetic carrier of the present invention is obtained by coating magnetic particles made of only a magnetic material from the viewpoint of improving the balance between the collection and dispersibility of the magnetic carrier in the protein purification method using a magnetic field, which will be described later. In the case of a magnetic carrier, the saturation magnetization is 20 A · m ² / kg to 100 A · m ² / kg, preferably 30 A · m ² / kg (emu / g) to 80 A · m ² / kg (emu / g). as also the case of the magnetic carrier obtained magnetic particles the magnetic material coating treatment to the particles of non-magnetic material to coating treatment, the saturation magnetization is ^{2A · m 2 / kg~100A · m} 2 / kg, preferably 4 A · m ² / kg (emu / g) to 90 A · m ² / kg (emu / g), and in any case, the coercive force is 0.079 kA / m to 15.93 kA. / M (10-20 It is particularly desirable to produce such that it is 0 Oersted. Such a magnetic carrier preferably has a constituent element having a spherical, ellipsoidal or granular shape, and an average particle size of 0.01 μm to 45 μm. In particular, in such an embodiment, it is particularly preferable that the carbohydrate as the biological material is amylose.

  The magnetic carrier obtained by the production method of the present invention can be used for various applications depending on the type of biological material covering the surface. For example, when it has a saccharide layer coating, it can be used to purify substances (proteins, enzymes, etc.) that specifically bind to the saccharide, and can be used as a carrier for enzymatic reactions as another application.

  Proteins that can be purified using a magnetic carrier coated with a carbohydrate as a biological material obtained by the method for producing a magnetic carrier of the present invention (that is, such a magnetic carrier, specifically, a biological material present on the surface as a coating) The protein that can be bound, and such a protein is also referred to as “target protein”) is not particularly limited as long as it can specifically bind to a carbohydrate.

  In the present specification, “protein” refers to a fusion protein having a fragment capable of specifically binding to a sugar as well as various known sugar-binding proteins (for example, a fusion protein as disclosed in Patent Document 2). In addition, so-called peptides, oligopeptides and polypeptides such as these fragments are also included as long as they can specifically bind to sugars.

  Specifically, a maltose binding protein, an arabinose binding protein, a glucose binding protein, a mannose binding protein, a saccharide binding protein such as a lectin, a fusion protein comprising a part or all of these and having an affinity for sugar (for example, A fusion protein of a sugar-binding protein and a protein that does not substantially exhibit sugar-binding properties (a fusion protein MBP-LacZα in which a maltose-binding protein is bound to the amino terminus of a β-galactosidase α chain described later, and an amino terminus of an Escherichia coli transposase described later) Examples include fusion protein MBP-TNP1 to which maltose-binding protein is bound, and fusion protein MBP-GFP1 to which maltose-binding protein is bound to the amino terminus of green fluorescent protein)). Among them, a maltose-binding protein which is a malE gene product of Escherichia coli is particularly preferable because it is a periplasmic protein affected by osmotic pressure and can specifically bind to maltose and maltodextrin.

  Each condition in the production method of the present invention can be appropriately selected by those skilled in the art according to the description above and below, and the magnetic particles and biological materials used. Hereinafter, magnetic particles of magnetite, which is a ferromagnetic oxide, are subjected to a coating treatment with silver using a silver mirror reaction method (hereinafter, also referred to as silver-coated beads), and amylose is used as a carbohydrate that is a biological material. The present invention will be described more specifically by taking as an example the case where the coating treatment is performed using the above. In addition, as an example of a method of using beads (hereinafter also referred to as amylose-coated beads) as a magnetic carrier obtained by coating amylose, a purification method using a protein that specifically binds to amylose will be described.

(Synthesis of silver-coated beads)
First, a silver salt solution is prepared. Silver salts used in this solution include silver nitrate, silver nitrite, silver chloride, silver sulfate, silver sulfite, silver acetate, silver oxide, silver cyanide, silver fluoride, silver bromide, silver iodide, silver carbonate, thiocyanic acid Examples thereof include inorganic silver salts such as silver, silver chlorate, silver perchlorate, silver iodate, silver sulfide and silver azide, and organic silver such as silver N, N-diethyldithiocarbamate. Silver nitrate is particularly preferable in consideration of the production cost. The solvent for dissolving the silver salt is not particularly limited as long as the silver salt is dissolved, but water is particularly preferable from the viewpoint of cost and safety during production.

  A complexing agent is added dropwise to the silver salt solution. Any complexing agent may be used as long as it has a complexable ligand capable of complexing with silver, but ammonia or thiocyanate is particularly preferred. At this stage, silver is considered to form complex ions coordinated by the complexing agent. By forming complex ions, silver can be prevented from being deposited as silver oxide, and the value of the redox potential of silver can be stabilized.

  Separately from the silver salt solution, a liquid in which magnetite as magnetic particles is dispersed is prepared. Magnetite is dispersed in a solvent, and the solvent used in this case is not particularly limited, and examples thereof include water, ethyl alcohol, isopropyl alcohol, and the like. It is preferable to use water for the reason that the production cost can be lowered. A compound showing basicity by dissolving a salt compound having a hydroxyl group such as sodium hydroxide, potassium hydroxide or lithium hydroxide is added to the dispersion. This addition increases later reactivity, but addition is not essential. Furthermore, a reducing agent is added to this. As the reducing agent, sugars having a reducing ability (aldehyde group, ketone group) such as monosaccharides such as glucose and fructose, disaccharides such as maltose and lactose, compounds having an aldehyde group such as formaldehyde and acetaldehyde, Examples thereof include a ketone group-containing compound and tartrate, but glucose is preferably used.

  When the previously prepared silver salt solution is dropped into such a magnetite dispersion, silver adheres to the surface of the magnetite by a silver mirror reaction to form a silver coating. The reaction temperature at this time is generally 0 to 90 ° C, but 5 to 40 ° C is preferable. If the reaction temperature is too low, a long time is required for the reaction. Conversely, if the reaction temperature is too high, the reaction rate is too high and uniform coating is difficult. From this dispersion, magnetite is filtered and washed, and then dried to obtain silver-coated beads.

  In the above description, a magnetite dispersion containing a silver salt solution and a reducing agent has been described as the liquid used for electroless silver plating. However, a dispersion in which magnetite is dispersed in a silver salt solution may be prepared, and this may be combined with a reducing agent solution. Regarding the dropping, either liquid may be dropped on the other liquid.

(Manufacture of amylose-coated beads)
First, silver-coated beads are dispersed in a dispersion medium at room temperature (20 ° C.). The dispersion medium is not particularly limited, and water, ethyl alcohol, isopropyl alcohol and the like can be exemplified, but it is preferable to use water for the reason that the production cost can be reduced. The amount of the silver-coated beads added to the dispersion medium is not particularly limited, but it is preferable to add the silver-coated beads to a concentration of 1% by weight to 50% by weight because a uniform dispersion is easily obtained.

  Next, amylose is further added to the dispersion while stirring at room temperature, and then heated to about 90 ° C. The amount of amylose added to the silver-coated beads is preferably 0.1% to 30% by weight based on the weight of the silver-coated beads, but the amount of amylose dissolved in water is usually about several percent (2% to 6%), it is preferable to select the amount of water in which the magnetite particles are dispersed so as to be less than this concentration. For example, 10 g of silver-coated beads may be dispersed in 50 g of water, and about 0.1 g to 3 g of amylose may be added. After addition of amylose, the mixture is stirred for 10 minutes to 1 hour at room temperature and then heated to the above temperature. When the mixture is further stirred for 10 minutes to 1 hour in a heated state, amylose is also uniformly dispersed and a uniform carbohydrate layer is obtained. Is preferable in that it is easy to form.

  Subsequently, the amylose dissolved dispersion is cooled to room temperature while stirring. As a result, the saturated solubility of amylose decreases, so that the dissolved amylose gradually precipitates and adheres to the surface of the silver-coated beads to form a coating, thereby obtaining the magnetic carrier of the present invention.

  By carrying out the method of the present invention in this way, the magnetic carrier of the present invention having silver-coated beads and a carbohydrate layer formed of amylose coating the silver-coated beads can be produced. In the magnetic carrier having both the saturation magnetization and the coercive force, the number of silver-coated beads is 1 to 100 for each component of the magnetic carrier, for example, in the process of manufacturing the magnetic carrier. Thus, the saccharide layer may be coated and the ratio of the saccharide to the silver-coated beads may be 0.1 wt% to 30 wt%.

  The magnetic carrier of the present invention is not limited to that obtained by the production method of the present invention described above and below, and may be obtained by other production methods as long as it has the same configuration. .

(Protein purification)
The magnetic carrier obtained by the production method of the present invention is excellent in storage stability and can be suitably used, for example, for protein purification. A preferred magnetic carrier retains 80% or more (preferably 90% or more) of the protein binding capacity before storage even when stored in a dispersion at 30 ° C. (particularly 4 ° C.) for 30 days.

  Examples of the “dispersion liquid” herein include buffers, and specific examples include potassium phosphate buffer, sodium phosphate buffer, Tris hydrochloride buffer, PIPES buffer, borate buffer, acetate buffer, MES buffer, and the like. . Among these, 20 mM to 100 mM potassium phosphate buffer (pH 5.0 to 8.0) is preferable. “Protein binding capacity” is the amount of protein that can bind to 1 g of the magnetic carrier. The amount of protein can be determined by a quantitative method by sodium dodecyl sulfate-polyacrylamide gel electrophoresis described below (hereinafter referred to as “SDS-PAGE”) and absorbance (280 nm) measurement.

(Protein quantification method by SDS-PAGE)
Quantification of protein amount by SDS-PAGE is performed as follows. First, a standard protein of known concentration (for example, bovine serum albumin) and a dilution series of the target protein (for example, 2-fold, 4-fold, 8-fold, etc.) are prepared, and SDS-PAGE is performed. Next, the minimum detectable concentration (A) of SDS-PAGE is determined from the dilution series of the standard protein, while the dilution ratio at which the target protein can be detected is determined from the dilution series of the target protein, and the protein concentration at that time is determined. Considering the above (A), the concentration of the target protein is determined by multiplying by the dilution factor. The SDS-PAGE is performed using a known method such as a method described in an electrophoresis experiment method (edited by the Japan Electrophoresis Society, 1999). Protein quantification by SDS-PAGE can also be performed by measuring absorbance at 280 nm.

  Further, the magnetic carrier obtained by the production method of the present invention preferably comprises a part or all of the protein rather than the protein itself capable of specifically binding to the above-mentioned carbohydrate, and has an affinity for the carbohydrate. It has a relatively high affinity for a fusion protein having sex. A magnetic carrier having such properties includes a part or all of a protein capable of specifically binding to a carbohydrate as described later, and is obtained from a biological sample containing a fusion protein having affinity for the carbohydrate. The fusion protein can be preferably used for separating, that is, purifying.

  Preferably, the magnetic carrier obtained by the production method of the present invention comprises a protein capable of specifically binding to a carbohydrate, and a fusion protein comprising a part or all of the protein and having affinity for the carbohydrate. When a purification operation is performed on a disrupted solution containing the expressed recombinant (for example, when purification is performed according to the methods of Experimental Examples 61 to 64 described later), the fusion protein is substantially 100% bound.

  As used herein, “substantially 100% binding to the fusion protein” means that the amount of the protein other than the fusion protein containing the protein that can specifically bind to the carbohydrate is less than the detection limit. Say. As described above, the amount of protein bound to the magnetic carrier can be determined by the above-described quantitative method by SDS-PAGE and absorbance (280 nm) measurement. Therefore, “substantially 100% binds to the fusion protein” means that proteins other than the fusion protein bind only to such an extent that they cannot be detected by these protein quantification methods.

  Further, the magnetic carrier obtained by the production method of the present invention is preferably eluted more efficiently than the other buffer by using a phosphate buffer in the eluent when the protein bound to the carrier is eluted. It has the characteristic that The fact that elution exhibits specificity for a specific buffer type means that the presence or absence of elution can be controlled by the buffer used, and when using the magnetic carrier obtained by the production method of the present invention as a carrier for immobilized enzyme. A great effect can be expected in that the lifetime of the immobilized enzyme is improved.

  Preferably, the recovery rate of the protein bound to the magnetic carrier is 60% or more (preferably 80% or more) when using a phosphate buffer, and 20% or less (preferably 10% or less) when using another buffer. is there.

  Examples of the phosphate buffer herein include potassium phosphate buffer and sodium phosphate buffer, among which 20 mM to 100 mM potassium phosphate buffer (pH 6.0 to 8.0) is preferable, and in particular, 50 mM potassium phosphate. A buffer (pH 7.5) is preferred. Examples of other buffers herein include 20 mM to 100 mM Tris-HCl buffer, Tris-sulfate buffer, HEPES buffer, MOPS buffer, PIPES buffer, borate buffer, and the like, and in particular, 50 mM Tris-HCl buffer (pH 7.5). ), Tris sulfate buffer (pH 8.0), HEPES buffer (pH 7.7), MOPS buffer (pH 7.5), PIPES buffer (pH 7.5), and borate buffer (pH 8.0).

  For example, when the target protein is a maltose-binding protein, the protein is eluted with a buffer containing 1 to 100 mM (preferably 10 mM) maltose. The “protein recovery rate” here refers to the amount of (recovered) protein obtained by performing a purification operation using a magnetic carrier using the purified protein as a sample, and the amount of protein as the (original) sample. The ratio of quantities. In addition, protein amount can be calculated | required by the quantitative method by said SDS-PAGE, and a light absorbency (280 nm) measurement.

  The present invention provides a method for separating, that is, purifying a protein from a biological sample containing a protein capable of specifically binding to a carbohydrate, using the magnetic carrier obtained by the above-described method of the present invention. That is, the protein purification method of the present invention includes [1] a step of binding a protein to a magnetic carrier, [2] a step of isolating the protein bound to the magnetic carrier from a biological sample, and [3] a biological sample. And separating the protein bound to the magnetic carrier from the magnetic carrier, thereby purifying the protein.

  In the step [1], first, a biological sample containing a target protein and a magnetic carrier are mixed to bind the target protein and the magnetic carrier. The binding of the target protein and the magnetic carrier is not particularly limited as long as they are mixed in an appropriate buffer as a dispersion medium so that they can contact each other. For this mixing, for example, it is sufficient that the tube containing the biological sample and the magnetic carrier is lightly agitated or shaken. For example, a commercially available vortex mixer can be used.

  In performing the step [1], the magnetic carrier is preferably prepared in advance as a protein extraction solution by being dispersed in an appropriate dispersion medium. The dispersion medium for dispersing the magnetic carrier is not particularly limited, but since it is a buffer generally used for protein purification, a potassium phosphate buffer, a sodium phosphate buffer, a tris-HCl buffer, a PIPES buffer, A boric acid buffer or the like is preferable, and it is preferable to use 20 mM to 100 mM potassium phosphate buffer (pH 6.0 to 8.0).

  In preparing the protein extraction solution, the magnetic carrier is preferably added so that the concentration in the dispersion is 0.1 g / mL to 1.0 g / mL. If the amount is less than 0.1 g / mL, a large amount of protein cannot be bound and the magnetic collection of the magnetic carrier tends to deteriorate, and if it exceeds 1.0 g / mL, the dispersibility of the dispersion liquid This is because the storage stability tends to deteriorate.

  The mixing ratio of the protein extraction solution and the biological sample depends on the molecular weight of the target protein, but the weight ratio of the magnetic carrier to the target protein contained in the biological sample is 1: 0.001-1. : The ratio is preferably 0.1.

  In the subsequent step [2], the target protein bound to the magnetic carrier in the step [1] is isolated from the biological sample together with the magnetic carrier. The isolation may be performed by centrifugation, filter separation, etc., but is easy to operate and specific isolation is possible in a short time. From the viewpoint of facilitating automation, it is preferable to use a magnetic field, that is, a magnet. As a magnet to be used, for example, a magnet having a magnetic flux density of about 0.03 T (300 gauss) can be suitably used. Specifically, the above step [1] is performed in an appropriate tube, and after the magnetic carrier and the target protein are bound, the magnet is brought close to the side wall of the tube and the magnetic carrier bound to the target protein is collected near the side wall of the tube. In this state, the remaining liquid may be discharged from the tube and then isolated.

  In the step [3], the target protein isolated from the biological sample as described above is separated from the magnetic carrier. In this step, for example, an elution solution capable of eluting the protein is injected into the tube after the step [2], and the protein is eluted from the magnetic carrier. Thereafter, the target protein is separated from the magnetic carrier by collecting the magnetic carrier again with a magnet and removing it from the tube.

  As the elution solution capable of eluting the protein, a solution containing a carbohydrate having affinity for the protein is preferably used. For example, when the target protein is a maltose binding protein, a buffer containing 1 mM to 100 mM maltose is exemplified. Examples of the buffer include potassium phosphate buffer, sodium phosphate buffer, Tris hydrochloride buffer, PIPES buffer, boric acid buffer, etc. Among them, 20 mM to 100 mM potassium phosphate buffer (pH 6.0 to 8.0) is preferable. .

  The target protein can be purified from the biological sample through the purification method basically including the steps [1] to [3] as described above. Such a purification method of the present invention is a method in which convenience is greatly improved as compared with the conventional purification method, and can be automated and have a high throughput.

  Furthermore, the present invention provides a protein capable of specifically binding to a carbohydrate using the magnetic carrier described above, and a fusion protein fusion protein comprising a part or all of the protein and having affinity for sugar. A method for purifying the fusion protein from the contained biological sample is provided. The method for purifying the fusion protein of the present invention comprises [1] a step of binding the fusion protein to a magnetic carrier, [2] isolating the fusion protein bound to the magnetic carrier from a biological sample, and [3 And a step of separating the fusion protein bound to the magnetic carrier isolated from the biological sample from the magnetic carrier, thereby purifying the fusion protein. Each step of [1] to [3] in this purification method may be performed according to the purification method of the protein that can specifically bind to the carbohydrate. From a biological sample containing a protein that can specifically bind to a carbohydrate and a fusion protein comprising the protein through a purification method basically including the steps [1] to [3]. The target fusion protein can be purified. Such a purification method of the present invention is a method with greatly improved convenience as compared with the conventional method, and can be automated and have a high throughput.

  The magnetic carrier of the present invention is a reagent kit comprising the magnetic carrier, a dispersion medium for dispersing the magnetic carrier to prepare the protein extraction solution, and an elution solution capable of eluting the protein described above. Alternatively, the protein extraction solution containing the magnetic carrier and the above-described elution solution capable of eluting the protein are provided as reagent kits for protein purification, each contained in a separate tube or other container. Also good. Such a reagent kit of the present invention can perform the method of the present invention quickly and using only the necessary amount, without the need to prepare and prepare various reagents when purifying proteins. .

  In one production method of the present invention, the magnetic particles are subjected to a treatment for forming a silver coating with a silver compound, and then a coating treatment with a biological material is performed. The coating treatment may be performed when it is used for actual use. For example, a magnetic carrier that has been subjected to a silver coating process may be prepared, and the user may change the biological material to be coated on the surface as necessary. Alternatively, the biological material coating itself may be used as a means for extraction, purification, detection, etc., and sulfur-containing biological material is bound to silver-coated magnetic particles for use in highly uniform extraction, purification, detection, etc. Is possible.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1
(Sequentially coated bead manufacture)
(Manufacture of magnetite particles)
Magnetite particles to be coated with silver were produced by the following method. 100 g of ferrous sulfate (FeSO ₄ · 7H ₂ O) was dissolved in 1000 cc of pure water. 28.8 g of sodium hydroxide was dissolved in 500 cc of pure water so as to have the same molar ratio as that of ferrous sulfate. Next, while stirring the aqueous ferrous sulfate solution, an aqueous sodium hydroxide solution was added dropwise to the solution over 1 hour to produce a ferrous hydroxide precipitate. After completion of dropping, the temperature of the suspension containing the ferrous hydroxide precipitate was raised to 85 ° C. while stirring. After the temperature of the suspension reached 85 ° C., it was oxidized for 8 hours while blowing air using an air pump at a rate of 200 L / hr to generate magnetite particles. The magnetite particles were substantially spherical, and the average particle size (“particle size” in the table below) was 0.23 μm. The average particle size of the magnetite particles was determined as a number average by measuring 300 particle sizes on a transmission electron micrograph.

(Production of silver-coated beads by electroless plating method (silver mirror reaction))
Silver-coated beads for coating with amylose were produced using an electroless plating method (silver mirror reaction). A 25% aqueous ammonia solution was added dropwise to a solution obtained by adding 10 g of water to 0.16 g of silver nitrate to completely dissolve the silver nitrate. Separately, a solution in which 10 g of magnetite particles (average particle size 230 nm) are dispersed in 10 g of water is prepared, 8.5 g of glucose is added and dissolved therein, and then the pH is adjusted using a 1N aqueous sodium hydroxide solution. Adjusted to 9. The silver nitrate solution was dropped into the magnetite dispersion to deposit silver on the surface of the magnetite particles, thereby carrying out the coating treatment. From this dispersion, magnetite particles were filtered, washed and dried to obtain silver-coated beads. According to X-ray fluorescence measurements, the silver-coated beads contained 0.5% silver.

(Manufacture of amylose-coated beads)
10 g of the obtained silver-coated beads were dispersed in 50 cc of pure water. 0.05 g of amylose was added to this dispersion at room temperature (20 ° C.) and stirred for 30 minutes, and then heated to 90 ° C. with stirring. After further stirring for 1 hour at 90 ° C., the coating treatment was carried out by gradually cooling to room temperature while continuing stirring. Amylose is easy to dissolve when heated, and difficult to dissolve when cooled. Therefore, amylose was deposited on the surface of the silver-coated beads during this cooling process. In this way, a magnetic carrier (amylose-coated beads) in which silver-coated beads were coated with a saccharide layer formed of amylose was produced. The obtained amylose-coated beads had a spherical or granular shape, and the average particle size was 250 nm. When the magnetic properties of the amylose-coated beads were measured using a vibrating sample magnetometer manufactured by Toei Kogyo Co., Ltd., the saturation magnetization measured by applying a magnetic field of 796.5 kA / m (10 kiloOersted) was 84 A. M ² / kg (84 emu / g) and coercive force 5 kA / m (63 Oersted). The amylose coating amount was confirmed by subjecting the amylose-coated beads to IR spectrum measurement.

Examples 2-47
(Manufacture of other amylose-coated beads)
Other amylose-coated beads were produced in the same manner as in Example 1 by changing the kind of magnetic particles and the production conditions of silver coating amount, amylose coating amount, complexing agent, reducing agent, base and the like. The magnetic particles used were magnetite particles, maghemite particles, metallic nickel particles, acrylic particles coated with nickel-phosphorus plating according to a conventional method ("Ni-coated acrylic" in Table 2 described later), and magnetite was microencapsulated with silica according to a conventional method. Particles (microencapsulated silica beads, “MC-modified Si” in Table 2 described later), polymer beads (“MMA polymer” in Table 2 described later) obtained by microencapsulating magnetite with polymethyl methacrylate according to a conventional method, and usual Table 1 and Table 2 summarize the production conditions and the characteristics of the produced beads, which are beads coated with magnetite by silica by the sol-gel method (“silica coating” in Table 2 described later).

Example 48
(Manufacture of simultaneous coated beads)
The simultaneous coating with silver and the coating with amylose were carried out simultaneously to produce simultaneously coated beads. For the coating treatment, the same method as in the previous example was adopted.

A 25% aqueous ammonia solution was added dropwise to a solution obtained by adding 10 g of water to 1.6 g of silver nitrate to completely dissolve the silver nitrate (Liquid 1). Separately from this, a liquid in which 10 g of magnetite particles (average particle size 230 nm) are dispersed in 10 g of water is prepared, 8.5 g of glucose is added and dissolved therein, and then the pH is adjusted using a 1N aqueous sodium hydroxide solution. To 9 (Liquid 2). 0.1 g of amylose was added to Liquid 2 and stirred for 30 minutes, and heated to 90 ° C. with stirring. After stirring at 90 ° C. for another hour, heating was stopped while stirring was continued. When the solution 1 was dropped into the solution 2 while the temperature was lowered to room temperature, silver and amylose adhered to the surface of the magnetite particles, and these coatings were formed. Magnetite particles were filtered off from this dispersion, washed and dried to obtain amylose-silver coated beads. According to fluorescent X-ray measurement, the beads contained 5% silver. The obtained amylose-silver coated beads had a spherical or granular shape, and the average particle size was 250 nm. As before, when the magnetic properties of amylose-silver coated beads were measured, the saturation magnetization measured by applying a magnetic field of 796.5 kA / m (10 kiloOersted) was 84 A · m ² / kg (84 emu / g). And the coercive force was 5 kA / m (63 oersted). The amylose coating was confirmed by IR spectrum measurement.

Examples 49-52
Amylose-silver coated beads were produced in the same manner as in Example 48, with various production conditions such as silver coating amount, amylose coating amount, base and the like. Table 2 summarizes the manufacturing conditions and the characteristics of the manufactured beads.

Examples 53-56
(Manufacture of silver-coated beads using various silver deposition methods)
Silver-coated beads were produced by changing the coating treatment with silver to a method other than the electroless plating method. Magnetite particles were used as the magnetic particles, and sputtering, vacuum deposition, ion plating, and chemical vapor deposition were used as the coating treatment with silver. These methods were carried out according to conventional methods.

(Manufacture of amylose-coated beads)
The obtained silver-coated beads were subjected to the coating treatment with amylose of Example 1 to produce amylose-coated beads. Table 3 summarizes the manufacturing conditions and characteristics of the beads.

Comparative Example 1
When performing the coating treatment with silver, the coating treatment with silver was performed in the same manner as in Example 1 except that ammonia as a complexing agent was not added. As a result of fluorescent X-ray analysis of the coated beads, no silver was detected and no silver coating was observed. Subsequently, the beads were subjected to a coating treatment with amylose in the same manner as in Example 1 so that the coating amount of amylose was 1%.

Comparative Example 2
When performing the coating treatment with silver, the coating treatment with silver was performed in the same manner as in Example 1 except that glucose as a reducing agent was not added. As a result of fluorescent X-ray analysis of the coated beads, no silver was detected and no silver coating was observed. Subsequently, the beads were subjected to a coating treatment with amylose in the same manner as in Example 1 so that the coating amount of amylose was 1%.

Comparative Example 3
The coating treatment with silver was not carried out, and the coating treatment with amylose was carried out in the same manner as in Example 1 so that the amylose coating amount was 1%.

Comparative Example 4
The silver coating treatment and amylose coating treatment in Example 1 were not performed. That is, the magnetic particles themselves.

  The conditions and results of Comparative Examples 1 to 4 are summarized in Table 4.

Experimental Examples 1-60
(Evaluation of coating uniformity of beads produced in Examples 1 to 56 and Comparative Examples 1 to 4)
Whether or not the silver coating was uniformly formed was evaluated by observation with a scanning electron microscope (SEM). Specifically, it was evaluated by comparing with the SEM image whether or not the shape of the base, which is the surface of the particles before the silver coating, was changed to a smooth shape of the silver coating. The following criteria were adopted in the evaluation:
◎: The base shape is almost unrecognized and the surface is smooth. ○: The rough base shape is recognized, but the fine shape is smooth. ×: The base shape is clearly recognized in whole or in part.

  This evaluation result was combined with Tables 1-4, and was shown. As an example, magnetite particles that have not been coated with silver (corresponding to Comparative Example 4, FIG. 1), magnetite particles with 0.5% silver coating (corresponding to Example 2, FIG. 2) The SEM photograph of the magnetite particle | grains (corresponding to Experimental example 11 and FIG. 3) on which a 5% silver coating is formed is shown.

  As shown in FIGS. 1 to 3, the base shape having irregularities on the surface of the magnetite particles, which was clear in the magnetite particles not subjected to the coating treatment with silver, has almost the base shape due to an increase in the amount of silver covered. It can be seen that the surface is unacceptably smooth. In addition, the same effect was acquired by any coating processing method without the difference by the coating processing method.

Experimental Examples 61-65
Using the amylose-coated beads produced in Examples 10 to 13 (corresponding to Experimental Examples 61 to 64) and Comparative Example 3 (corresponding to Experimental Example 65), the target protein is extracted from the biological sample by the following procedure. -Purified. As a biological sample for isolating the protein, plasmid pMALc2E (a plasmid expressing the fusion protein MBP-LacZα in which maltose-binding protein is bound to the amino terminus of β-galactosidase α chain (available from New England Biolab) Escherichia coli JM109 (sold by Toyobo Co., Ltd.)))) was maintained for 20 hours at 37 ° C. in a 50 mL TB medium / 500 mL flask. The cells are suspended in 50 mM potassium phosphate buffer (pH 7.5) so that the cell turbidity (OD660 nm) is 20, and intermittently disrupted with ultrasound for 9 minutes, and then the supernatant is centrifuged. Was used as a biological sample for protein purification.

  First, each of the amylose-coated beads was dispersed in 50 mM potassium phosphate buffer (pH 7.5) so as to be 0.2 g / mL. Each amylose-coated bead dispersion was mixed with 100 μL and 1 mL of a biological sample to prepare a mixed solution. After solid-liquid separation, it was washed with a washing solution (50 mM potassium phosphate buffer, pH 7.5). A 50 mM potassium phosphate buffer (pH 7.5) containing 10 mM maltose was used as an elution solution for recovering the protein bound to the amylose-coated beads.

Specific operations are as follows.
(1) The cell turbidity (OD 660 nm) of each mixed solution was measured, and the cells were centrifuged in a centrifuge tube. Next, the cells were suspended in 50 mM potassium phosphate buffer (pH 7.5) so that the cell turbidity was 20, and intermittently disrupted with ultrasound for 9 minutes, and then the supernatant was centrifuged.
(2) 1 mL of the supernatant was transferred to a 1.5 cc Eppendorf tube to obtain a biological sample, to which 100 μL of amylose-coated bead dispersion was added and mixed for about 5 minutes.
(3) The amylose-coated beads were collected on the magnet side by placing the tube on a magnet stand that matched the shape of the 1.5 cc Eppendorf tube.
(4) The solution was sucked with a filter chip and discharged.
(5) The tube was removed from the magnet stand, and 1 cc of a cleaning solution (50 mM potassium phosphate buffer, pH 7.5) was injected.
(6) After sufficiently mixing with the amylose-coated beads, it was placed on the magnet stand again, and the solution was discarded in the same manner as described above.

  50 mM potassium phosphate buffer (pH 7.5) containing 50 μL of 10 mM maltose was added to the amylose-coated beads to which proteins were bound by the above method, and mixed for about 5 minutes. Next, it was placed on a magnetic stand, and the solution to be collected was sucked with a filter chip and transferred to another new tube. The recovered amount was 40 μL. The collected protein was measured for absorbance (OD: 280 nm) with an absorptiometer to determine the concentration. The protein recovery amount was calculated by multiplying the protein concentration by the recovery volume.

  The results are shown in Table 5.

  4 shows a biological sample (FIG. 4 (a)) that is a mixture of various proteins before purification, and the fusion protein MBP purified by the above-described purification method (using the beads produced in Example 11). It is a figure which shows the result of having measured -LacZ (alpha) (FIG.4 (b)) by SDS-PAGE. As is apparent from FIG. 4, it can be seen that this purification method can obtain a highly pure protein by an extremely simple operation.

  When the amylose-coated ones as in Experimental Examples 61 to 65 were used, the protein could be purified.

  By performing silver coating on the magnetic particles as described above, and then amylose coating, it is possible to uniformly coat amylose so that the surface of the magnetite particles exhibiting ferromagnetism is not exposed, Purification could be performed without impairing the function of the biological material. By such a method for producing a magnetic carrier, it is possible to produce amylose-coated beads that can be used in a simple, automated and high-throughput apparatus.

Claims (11)

A method for producing a magnetic carrier having a biological material of any one of carbohydrates, proteins, peptides, nucleic acids, cells and microorganisms on its surface,
Applying magnetic particles to the layered coating treatment with silver and the coating treatment with biological material,
The layered coating treatment with silver is performed using any of the silver mirror reaction method, electroless plating method, electroplating method, sputtering method, vacuum deposition method, ion plating method, and chemical vapor deposition method. A method for producing a magnetic carrier. 2. The method for producing a magnetic carrier according to claim 1, wherein the protein is an antibody, an antigen, or a substance having specific binding properties such as a biological receptor or a ligand. The method for producing a magnetic carrier according to claim 1 or 2, wherein the magnetic particles are applied to the coating treatment with a biological material after the silver coating. 3. The method for producing a magnetic carrier according to claim 1, wherein the magnetic particles are simultaneously applied to the silver coating and the biological material coating. The magnetic particles are made of at least one material selected from the group consisting of metals and oxides thereof, alloys and oxides thereof, and any combination thereof. A method for producing the magnetic carrier according to claim 1. 6. The method for producing a magnetic carrier according to claim 5, wherein the magnetic particles are made of ferromagnetic iron oxide. 6. The method for producing a magnetic carrier according to claim 5, wherein the magnetic particles are made of metallic iron or metallic nickel. The saturation magnetization of the magnetic carrier to be produced is 2 A · m ² / Kg ~ 100A ・ m ² The method for producing a magnetic carrier according to claim 1, wherein the magnetic carrier is in a range of / kg. The method for producing a magnetic carrier according to any one of claims 1 to 8, wherein the magnetic carrier to be produced has a coercive force in the range of 0.079 kA / m to 15.93 kA / m. The method for producing a magnetic carrier according to any one of claims 1 to 9, wherein the magnetic carrier to be produced has an average particle size in the range of 0.005 to 60 µm. The method for producing a magnetic carrier according to any one of claims 1 to 10, wherein a substantially exposed portion of the magnetic particles does not exist due to the layered coating treatment with silver.